Podcasts about cd34

Host Dr. Davide Soldato and guest Dr. Harriet Kluger discuss the JCO article "Phase II Trial of Pembrolizumab in Combination With Bevacizumab for Untreated Melanoma Brain Metastases." Transcript The guest on this podcast episode has no disclosures to declare. Dr. Davide Soldato Hello and welcome to JCO After Hours, the podcast where we sit down with authors from some of the latest articles published in the Journal of Clinical Oncology. I am your host, Dr. Davide Soldato, Medical Oncologist at Ospedale San Martino in Genoa, Italy. Today, we are joined by JCO author Dr. Harriet Kluger. Dr. Kluger is a professor of medicine at Yale School of Medicine, Director of the Yale SPORE in Skin Cancer, and an internationally recognized expert in immuno-oncology for melanoma and renal cell carcinoma. She leads early-phase and translational trials that pair novel immunotherapies with predictive biomarkers to personalized care. Today, Dr. Kluger and I will be discussing the article titled "Phase 2 Trial of Pembrolizumab in Combination with Bevacizumab for Untreated Melanoma Brain Metastases." In this study, Dr. Kluger and colleagues evaluated four cycles of pembrolizumab plus the anti-VEGF antibody bevacizumab followed by pembrolizumab maintenance in patients with asymptomatic non-hemorrhagic melanoma brain metastases that had not previously received PD-1 therapy. Thank you for speaking with us, Dr. Kluger. Dr. Harriet Kluger Thank you for inviting me. The pleasure is really all mine. Dr. Davide Soldato So to kick off our podcast, I just wanted to ask if you could outline a little bit the biological and clinical rationale that led you to test this type of combination for patients with untreated brain metastases from metastatic melanoma. Dr. Harriet Kluger Back in approximately 2012, patients who had untreated brain metastases were excluded from all clinical trials. So by untreated, I mean brain metastases that had not received local therapy such as surgery or radiation. The reason for it was primarily because there was this fear that big molecules wouldn't penetrate brain lesions because they can't pass the blood-brain barrier. Turns out that the blood-brain barrier within a tumor is somewhat leaky and drugs sometimes can get in there. When PD-1 inhibitors were first identified as the next blockbuster class of drugs, we decided to conduct a phase 2 clinical trial of pembrolizumab monotherapy in patients with untreated brain metastases. We actually did it also in lung cancer, and we could talk about that later on. Responses were seen. The responses in the brain and the body were similar. They were concordant in melanoma patients. Now, at approximately that time, also another study was done by the Australian group by Dr. Georgina Long, where they did a randomized trial where patients who didn't require immediate steroid therapy received either nivolumab alone or nivolumab with ipilimumab, and the combination arm was substantially superior. Subsequently, also, Bristol Myers Squibb also conducted a large phase 2 multicenter trial of ipilimumab and nivolumab in patients with untreated brain metastases. And there, once again, they saw that the responses in the brain were similar to the responses in the body. Now, somewhere along the line there, we completed our anti-PD-1 monotherapy trial. And when we looked at our data, we still didn't have the data on ipilimumab and nivolumab. And our question was, “Well, how can we do better?” Just as we're always trying to do better. We saw two really big problems. One was that patients had a lot of perilesional edema. And the other one was that we were struggling with radiation necrosis in lesions that were previously Gamma Knifed. The instance of radiation necrosis was in excess of 30%. So the rationale behind this study was that if we added bevacizumab, maybe we could treat those patients who had some edema, not requiring steroids, but potentially get them on study, get that PD-1 inhibitor going, and also prevent subsequent radiation necrosis. And that was the main rationale behind the study. We had also done some preclinical work in mouse models of melanoma brain metastases and in an in vitro blood-brain barrier model where we showed that bevacizumab, or anti-VEGF, really tightens up those leaky basement membranes and therefore would be very likely to decrease the edema. Dr. Davide Soldato Thank you very much for putting in context the combination. So this was a phase 2 trial, and you included patients who had at least one lesion, and you wanted lesions that were behind 5 and 20 millimeters. Patients could be included also if the brain metastasis was higher in dimension than 20 millimeters, but it had to be treated, and it was then excluded from the evaluation of the primary objective of the trial. So regarding, a little bit, these characteristics, do you think that this is very similar to what we see in clinical practice? And what does this mean in terms of applicability of these results in clinical practice? Dr. Harriet Kluger So that's an excellent question. The brain metastasis clinical research field has somewhat been struggling with this issue of inclusion/exclusion criteria. When we started this, we showed pretty clearly that 5 to 10 millimeter lesions, which are below the RECIST criteria for inclusion, are measurable if you use MRIs with slices that are 1 to 2 millimeters. Most institutions in the United States do use these high-resolution MRIs. I don't know how applicable that is on a worldwide scale, but we certainly lowered the threshold for inclusion so that patients who have a smattering of small brain metastases would be eligible. Now, patients with single large brain metastases, the reason that we excluded those from the trial was because we were afraid that if a patient didn't respond to the systemic therapy that we were going to give them, they could really then develop severe neurological symptoms. So, for patient safety, we used 20 millimeters as the upper level for inclusion. Some of the other trials that I mentioned earlier also excluded patients with very large lesions. Now, in practice, one certainly can do Gamma Knife therapy to the large lesions and leave the smaller ones untreated. So I think it actually is very applicable to clinical practice. Dr. Davide Soldato Thank you very much for that insight, because I think that sometimes criteria for clinical trials, they have to be very restrictive. But then we know that in clinical practice, the applicability of these results is probably broader. So, going a little bit further in the results of the study, I just wanted a little bit of comment from you regarding what you saw in terms of intracranial response rate and duration of response among patients who obtained a response from the combination treatment. Dr. Harriet Kluger So we were actually surprised. When we first designed this study, as I said earlier, we weren't trying to beat out ipilimumab and nivolumab. We were really just trying to exclude those patients who wouldn't have otherwise been eligible for ipilimumab and nivolumab because of edema or possibly even previous radiation necrosis. So it was designed to differentiate between a response rate of 34%, and I believe the lower bound was somewhere in the 20s, because that's what we'd seen in the previous pembrolizumab study. What we saw in the first 20 patients that we enrolled was actually a response rate that far exceeded that. And so we enrolled another cohort to verify that result because we were concerned about premature publishing of a result that we might have achieved just by chance. The two cohorts were very similar in terms of the response rates. And certainly this still needs to be verified in a second study with additional institutions. We did include the Moffitt Cancer Center, and the response rate with Moffitt Cancer Center was very similar to the Yale Cancer Center response rate. Now, your other question was about duration of response. So the other thing that we started asking ourselves was whether this high response rate was really because the administration of the anti-VEGF will decrease the gadolinium enhancement and therefore we might actually just be seeing prettier scans but not tumor shrinkage. And the way to differentiate those two is by looking at the duration of the response. Median progression-free survival was 2.2 years. That's pretty long. The upper bound on the 95% confidence interval was not reached. I can't tell you that the duration is as good as the duration would be when you give ipilimumab. Perhaps it is less good. This was a fairly sick population of patients, and it included some who might not have been able to receive ipilimumab and nivolumab. So it provides an alternative. I do believe that we need to do a randomized trial where we compare it to ipilimumab and nivolumab, which is the current standard of care in this patient population. We do need to interpret these results with caution. I also want to point out regarding the progression-free survival that we only gave four doses of anti-VEGF. So one would think that even though anti-VEGF has a long half-life of three or four weeks, two years later, you no longer have anti-VEGF effect, presumably. So it does something when it's administered fairly early on in the course of the treatment. Dr. Davide Soldato So, in terms of clinical applicability, do you see this combination of pembrolizumab and bevacizumab - and of course, as we mentioned, this was a phase 2 trial. The number of patients included was not very high, but still you saw some very promising results when compared with the combination of ipilimumab and nivolumab. So do you see this combination as something that should be given particularly to those patients who might not be able to receive ipilimumab and nivolumab? So, for example, patients who are very symptomatic from the start or require a high dose of steroids, or also to provide a quicker response in terms of patients who have neurological symptoms, or do you think that someday it could be potentially used for all patients? Dr. Harriet Kluger The third part of your question, whether it can be used someday for all patients: I think we need to be very careful when we interpret these results. The study was substantially smaller than the ipilimumab/nivolumab trial that was conducted by Bristol Myers Squibb. Also going to point out that was a different population of patients. Those were all frontline patients. Here we had a mix of patients who'd had previous anti-CTLA-4 and frontline patients. So I don't think that we can replace ipilimumab and nivolumab with these results. But certainly the steroid-sparing aspect of it is something that we really need to take into consideration. A lot of patients have lesions in locations where edema can be dangerous, and some of them have a hard time coming off the steroids. So this is certainly a good approach for those folks. Dr. Davide Soldato And coming back to something that you mentioned in the very introduction, when you said that there were two main problems, which was one, the problem of the edema, and the second one, the problem of the radionecrosis. In your trial, there was a fair percentage of patients who received some type of local treatment before the systemic one. So the combination of pembrolizumab and bevacizumab. And most of the patients received radiosurgery. So I just wanted a brief comment regarding the incidence of radionecrosis in the trial and whether that specific component of the combination with bevacizumab was reduced. And how do you think that this fares in terms of what we see in clinical practice in terms of radionecrosis? Dr. Harriet Kluger I'm not sure that we really reduced the incidence of radiation necrosis. We saw radiation necrosis here. We saw less of it than in the trial of pembrolizumab monotherapy, but these were also different patients, different time. We saw more than we thought that we were going to see. It was 27%, I believe, which is fairly high still. We only gave the four doses of bevacizumab. Maybe to really prevent radiation necrosis, you have to continue to give the bevacizumab. That, too, needs to be tested. The reason that we gave the four doses of bevacizumab was simply because of the cost of the bevacizumab at the time. Dr. Davide Soldato Thank you very much for that comment on radionecrosis. And I really think that potentially this is a strategy, so continuing the bevacizumab, that really makes a lot of sense, especially considering that the tolerability of the regimen was really very, very good, and you didn't see any significant or serious adverse events related to bevacizumab. So just wondering if you could comment a little bit on the toxicities, whether you had anything unexpected. Dr. Harriet Kluger There was one patient who had a microperforation of a diverticulum, which was probably related to the bevacizumab. It was conservatively managed, and the patient did fine and actually remains alive now, many years later. We had one patient who had dehiscence of a previous wound. So there is some. We did not see any substantial hypertension, proteinuria, but we only gave the four doses. So it is possible that if you give it for longer, we would see some side effects. But still, relative to ipilimumab, it's very, very well tolerated. Dr. Davide Soldato Yeah, exactly. I think that the safety profile is really different when we compare the combination of ipilimumab/nivolumab with the pembrolizumab/bevacizumab. And as you said, this was a very small trial and probably we need additional results. But still, these results, in terms of tolerability and safety, I think they are very interesting. So one additional question that I think warrants a little bit of comment on your part is actually related to the presence of patients with BRAF mutation and, in general, to what you think would be the best course of treatment for these patients who present with the upfront brain metastases. So this, it's actually not completely related to the study, but I think that since patients with BRAF mutation were included, I think that this warrants a little bit of discussion on your part. Dr. Harriet Kluger So we really believe that long-term disease control, particularly in brain metastases, doesn't happen when you give BRAF/MEK inhibitors. You sometimes get long-term control if you've got oligometastatic disease in extracranial sites and if they've previously been treated with a lot of immune checkpoint inhibitors, which wasn't the case over here. So a patient who presents early in the course of the disease, regardless of their BRAF status, I do believe that between our studies and all the studies that have been done on immunotherapy earlier in the course of disease, we should withhold BRAF/MEK inhibitors unless they have overwhelming disease and we need immediate disease control, and then we switch them very quickly to immunotherapy. Can I also say something about the toxicity question from the bevacizumab? I have one more comment to make. I think it's important. We were very careful not to include patients who had overt hemorrhage from brain metastases. So melanoma brain metastases relative to other tumor types tend to bleed, and that was an exclusion criteria. We didn't see any bleeding that was attributable to the bevacizumab, but we don't know for sure that, if this is widely used, that that might not be a problem that's observed. So I would advise folks to use extreme caution and perhaps not use it outside of the setting of a clinical trial in patients with overt hemorrhage in the melanoma brain metastases. Dr. Davide Soldato Thank you very much. I think that one aspect that is really interesting in the trial is actually related to the fact that you collected a series of biomarkers, both circulating ones, but also some that were collected actually from the tissue. So just wondering if you could explain a little bit which type of biomarkers you evaluated and whether you saw any significant results that could suggest higher or lower efficacy of the combination. Dr. Harriet Kluger Thank you for that. So yes, the biomarker studies are fairly exploratory, and I want to emphasize that we don't have anything that's remotely useful in clinical practice at this juncture. But we did see an association between vessel density in the tumors and improved response to this regimen. So possibly those lesions that are more vascular are more fed by or driven by VEGF, and that could be the reason that there was improved response. We also saw that when there was less of an increase in circulating angiopoietin-2 levels, patients were more likely to respond. Whether or not that pans out in larger cohorts of patients remains to be determined. Dr. Davide Soldato Still, do you envision validation of these biomarkers in a potentially additional trial that will evaluate, again, the combination? Because I think that the signals were quite interesting, and they really make sense from a biological point of view, considering the mechanism of action of bevacizumab. So I think that, yeah, you're right, they are exploratory. But still, I think that there is very strong biological rationale. So really I wanted to congratulate you on including that specific part and on reporting it. And so the question is, really, do you envision validation of these biomarkers in larger cohorts? Dr. Harriet Kluger I would hope to see that, just as I'd like to see validation of the clinical results as well. The circulating biomarkers are very easy to do. It's a simple ELISA test. And the vessel density on the tumor is essentially CD34 staining and units per area of tumor. Also very simple to do. So I'd love to see that happen. Dr. Davide Soldato Do you think that considering the quality of the MRI that we are using right now, it would be possible to completely bypass even the evaluation on the tissue? Like, are we going in a direction where we can, at a certain point, say the amount of vessels that we see in these metastases is higher versus lower just based on MRI results? Dr. Harriet Kluger You gave me an outstanding idea for a follow-up study. I don't know whether you can measure the intensity of gadolinium as a surrogate, but certainly something worth asking our neuroradiology colleagues. Excellent idea. Thank you. Dr. Davide Soldato You're welcome. So just moving a step further, we spoke a lot about the validation of these results and the combination. And just wanted your idea on what do you think it would be more interesting to do: if designing a clinical trial that really compares pembrolizumab/bevacizumab with ipilimumab and nivolumab or going directly for the triplet. So we know that there has been some type of exploration of triplet combination in metastatic melanoma. So just your clinical impression: What would you do as an investigator? Dr. Harriet Kluger So it's under some discussion, actually. It's very difficult to compare drugs from different companies in an investigator-initiated trial. Perhaps our European colleagues can do that trial for us. In the United States, it's much harder, but it can be done through the cooperative groups, and we are actually having some discussions about that. I don't have the answer for you. It would be lovely to have a trial that compared the three drugs to ipi/nivo and to pembrolizumab/bevacizumab. So a three-arm trial. But remember, these are frontline melanoma patients. There aren't that many of them anymore like there used to be. So accrual will be hard, and we have to be practical. Dr. Davide Soldato Yeah, you're right. And in the discussion of the manuscript, you actually mentioned some other trials that are ongoing, especially one that is investigating the combination of pembro and lenvatinib, another one that is investigating the combination of nivolumab and relatlimab. So just wondering, do you think that the molecule in terms of VEGF inhibition, so bevacizumab versus lenvatinib, can really make a difference or is going to be just a mechanism of action? Of course, we don't have the results from this trial but just wondering if you could give us a general comment or your opinion on the topic. Dr. Harriet Kluger So that's a really great question. The trial of pembrolizumab and lenvatinib was our answer to the fact that bevacizumab is not manufactured by the same company as pembrolizumab, and we're trying to give a practical answer to our next study that might enable us to take this approach further. But it does turn out from our preclinical studies that bevacizumab and VEGF receptor inhibition aren't actually the same thing in terms of the effects on the blood-brain barrier or the perilesional tumor microenvironment in the brain. And these studies were done in mice and in in vitro models. Very different effects. The lenvatinib has stronger effect on the tumors themselves, the tumor cells themselves, than the bevacizumab, which has no effect whatsoever. But the lenvatinib doesn't appear to tighten up that blood-brain barrier. Dr. Davide Soldato Thank you. I think that's very interesting, and I think it's going to be interesting to see also results of these trials to actually improve and give more options to our patients in terms of different mechanism of action, different side effects. Because in the end, one thing that we discussed is that some combination may be useful in some specific clinical situation while others cannot be applicable, like, for example, an all immunotherapy-based combination. Just one final comment, because I think that we focused a lot on the intracranial response and progression-free survival. You briefly mentioned this but just wanted to reinforce the concept. Did you see any differences in terms of intracranial versus extracranial response for those patients who also had extracranial disease with the combination of pembro and bevacizumab? Dr. Harriet Kluger So the responses were almost always concordant. There were a couple of cases that might have had a body response and not an intracranial response and vice-versa, but the vast majority had concordant response or progression. We do believe that it's a biological phenomenon. The type of tumor that tends to go to the brain is going to be the type of tumor that will respond to whatever the regimen is that we're giving. In the previous trial also, we saw concordance of responses in the body and the brain. Dr. Davide Soldato Thank you very much. Just to highlight that really the combination is worth pursuing considering that there was not so much discordant responses, and the results, even in a phase 2 trial, were very, very promising. So thank you again, Dr. Kluger, for joining us today and giving us a little bit of insight into this very interesting trial. Dr. Harriet Kluger Thank you for having me. Dr. Davide Soldato So we appreciate you sharing more on your JCO article titled "Phase 2 Trial of Pembrolizumab in Combination with Bevacizumab for Untreated Melanoma Brain Metastases," which gave us the opportunity to discuss current treatment landscape in metastatic melanoma and future direction in research for melanoma brain metastasis. If you enjoy our show, please leave us a rating and review and be sure to come back for another episode. You can find all ASCO shows at asco.org/podcasts.   The purpose of this podcast is to educate and to inform. This is not a substitute for professional medical care and is not intended for use in the diagnosis or treatment of individual conditions. Guests on this podcast express their own opinions, experience, and conclusions. Guest statements on the podcast do not express the opinions of ASCO. The mention of any product, service, organization, activity, or therapy should not be construed as an ASCO endorsement.

There are incredible non-invasive or minimally invasive techniques to restore joint health and avoid surgery. We will explore Dr. Bianco's path and experience in this amazing field of joint restoration and offer you simple steps that you can engage to ease pain.Upfront summary: 1. Modern technology such as stem cells and exosomes allow us to accomplish results that weren't available 10 years ago.2. You can rebuild, restore and regenerate joint health in ways that surgery cannot, such as restoring cartilage. 3. Everyone over the age of 50 has some degree of arthritis but not everyone feels pain. Having arthritis and having pain are not synonymous.4. We are going to discuss the differences between these injection therapies:· PRP – Platelet Rich Plasma, an older technology using your own growth factors.· Stem cells & exosomes offer a huge magnitude of difference over PRP and supply true healing power.· Prolotherapy – stimulates the body's natural repair process.· Ozone stimulates 3 modes of action: mitochondrial energy, detoxification, and immune system stabilization and support. It also works to animate stem cells for repair.5. Adjuncts that support and amplify these injection therapies:· Peptides – BPC-157 is a natural occurring protein that stimulates a sharp rise in repair molecules VEGF, Factor VIII, collagen, and CD34.· Glucosamine & Chondroitin – stimulates chondrocytes to generate new cartilage and create hyaluronic acid for lubrication.· MSM – antioxidant and anti-inflammatory, makes cartilage stronger.· Boswellia & Curcumin – reduce inflammation like NSAIDS but without the side effect and danger.· Joint & Body Collagen (HippEvo) – Fortigel and Tendoactive are proven elements to strengthen joints and reduce inflammation.· Hyperbaric Oxygen chamber at Huber Personalized Medicine – doubles the speed of repair for a quicker return to action. · Red light laser therapy – mitochondrial stimulation to augment repair energy

Professor Adrian Martineau from the Queen Mary University London talks to us about the limitations of current latent TB infection diagnosis and how we can do better.  his research into whether we can use fragments of TB DNA found in bone marrow cells to better predict who should be treated with Tuberculosis Preventative Treatment.REFERENCESMartineau, Adrian R., et al. "Towards a molecular microbial blood test for tuberculosis infection." International Journal of Infectious Diseases (2024): 106988.Belay, Mulugeta, et al. "Detection of Mycobacterium tuberculosis complex DNA in CD34-positive peripheral blood mononuclear cells of asymptomatic tuberculosis contacts: an observational study." The Lancet Microbe 2.6 (2021): e267-e275.Repele, Federica, et al. "Detection of Mycobacterium tuberculosis DNA in CD34+ peripheral blood mononuclear cells of adults with tuberculosis infection and disease." International Journal of Infectious Diseases 141 (2024): 106999.

We all want to live as long as is possible, and for many years scientists have been exploring the subject of longevity to unlock its secrets with varying degrees of success, and so to explore ageing and metabolism I'm joined physiologist Prof Joseph Baur. We ask exactly what causes ageing and why there is no consensus on this question. We hear about calorie restriction and how it has been shown to increase the life of animals by up to 40%. Dr Baur busts the myth of antioxidants' ability to stave off ageing. Expect to hear about the molecule NAD, how it declines as we get older which seems to have an influence on the ageing process and we discover NAD's relationship with an enzyme called CD34. Dr Baur also tells us of the groundbreaking work he has done to discover an important NAD transporter that carries NAD from the cell cytoplasm into the mitochondria. We also ask how do we boost NAD levels and what significance does NMN have? Plus we hear about the market for anti-ageing NAD supplementation and why one needs to take anti-ageing assertions made by certain companies with a pinch of salt given the lack of clinical trials. Order my book Happy Habits for Mind and Body Audiobook: https://amzn.to/3QWulQf Order Kindle copy of Happy Habits for Mind and Body : https://amzn.to/3K8rlg5 Order US paperback of Happy Habits for Mind and Body : https://amzn.to/3UUEzlk Order UK paperback of Happy Habits for Mind and Body : https://rb.gy/jtfea5 Listen to all previous podcast episodes of the Happy Habit Podcast via these podcast platforms : Apple Podcasts https://podcasts.apple.com/ie/podcast/happy-habit-podcast Amazon https://www.amazon.com/Happy-Habit-Podcast/dp/B08K5887J8 Amazon music : https://music.amazon.com/podcasts/670836c2-ea4c-4a23-a67d-a54dd804ef61/happy-habit-podcast Spotify https://https://open.spotify.com/show/2VKIhQK6mYTzLCO8haUoRd Google Podcasts : https://t.ly/hTU8q ----- Follow the Happy Habit Podcast at: Website: https://happyhabitpodcast.wordpress.com/Facebook: Twitter: https://twitter.com/mathieunorry Instagram: https://www.instagram.com/happyhabitpodcast/ Newsletter:

In this podcast, we spoke with Margherita Neri, Director of Vector Process Development, Milan Site at AGC Biologics, Andrew Laskowski, Global Product Manager Bioreactors at Cytiva and Andreia Pedregal, Upstream Applications Specialist Manager at Cytiva about large-scale viral vector manufacturing. Our conversation included discussions around scalability, AAV (adeno-associated virus) and lentivirus production platforms, adherent culture, and next generation bioreactor improvements. I began the interview by asking Margherita about her work at AGC Biologics. She explained that as the Director of the Vector Process Development Unit, her team is responsible for process development of large scale viral vector production for gene therapy applications. Her team is also the first point of contact for new clients. Next, we talked about the types of viral vector platforms that AGC Biologics operates. Margherita described that at their Milan site, they offer AAV (adeno associated virus) and lentiviral vector production platforms in adhesion and in suspension, at 50-to-200-liter scale with expansion planned for up to 1,000 liters. I then asked her about some of the differences between adherent cell culture and suspension cell culture paths to commercial manufacturing. Margherita said that the first consideration is that most clinical trials in gene therapy have been sustained with vector produced from adherent cells, typically via processes performed using Cell Factory™ or Cell STACK®. Now that those gene therapy products are being commercialized, manufacturers need to increase scale and demonstrate comparability using a minimal comparability exercise. So, systems that allow adherent scale up are very useful in this process. Suspension processes are appealing from a scalability point of view because historically they were used for traditional protein bioproductions which can be scaled up to 20,000 – 30,000 liters. Of course, this scale still needs to be demonstrated for vector production that is performed mainly using transient transfection at 200-500 liter scale for lentivirus and between 500-to-1,000-liter maximum scale for AAV. Margherita went on to say that another important aspect in comparability between adherent and suspension systems is quality of the vector in terms of impurity profiles. She said that with adherent processes, cells are attached to the growth support, and the levels of host cell protein and cell DNA are lower when compared to suspension processes. This is very important for lentiviral vector production that is used in vivo where the requirements for impurity levels are very challenging, especially considering that for lentiviral vectors there is currently no affinity step for purification. I followed up by asking her how AGC Biologics can help customers that want to stay in adherent culture to scale up from current processes, for instance, from flatware to larger-scale production. She explained that when customers ask for a scale increase, they usually offer the iCELLis™ platform. First, they demonstrate at small scale the feasibility of the transition from flatware to the iCELLis bioreactor using the iCELLis Nano bioreactor. Using the iCELLis Nano bioreactor, AGC Biologics has developed a full upstream and downstream process that is highly representative of their process using the full-scale iCELLis bioreactor. AGC Biologics can then propose that customers use the vector produced in the iCELLis scale-down model to perform a comparability study between a clinical vector and the future commercial or large-scale vector. This comparability should be based not only on the comparison of titers, residuals, and all the CQA, but also AGC Biologics suggests performing a test of cell transduction on the target cells (i.e. CD34 or T cells) and evaluation on these cells of transfection efficiency – vector copy number, residuals and functionality. I followed up by asking Margherita about whether the iCEL...

“I think the most amazing thing we see is the softening of the skin, which can occur during the first two weeks of the conditioning regimen. The nurses on the floor see it, and I think it's just a tremendous gratification for them to see the results of something right before your eyes,” Tanya Helms, PA-C, from the division of hematological malignancies and cellular therapy at Duke University Medical Center in Durham, NC, told Lenise Taylor, MN, RN, AOCNS®, BMTCN®, oncology clinical specialist at ONS, during a discussion about what oncology nurses should know about transplantation for patients with non-oncologic conditions such as autoimmune disease, how the transplant process differs for non-oncology indications, and the clinical pearls oncology nurses should consider when caring for patients with autoimmune diseases during the transplantation process.  You can earn free NCPD contact hours after listening to this episode and completing the evaluation linked below.   Music Credit: “Fireflies and Stardust” by Kevin MacLeod  Licensed under Creative Commons by Attribution 3.0  Earn 0.5 NCPD contact hours of nursing continuing professional development (NCPD), which may be applied to the early post-transplant management and education, treatment modalities, diagnosis, staging and treatment planning, or coordination of care ILNA category, by listening to the full recording and completing an evaluation at myoutcomes.ons.org by September 29, 2025. The planners and faculty for this episode have no relevant financial relationships with ineligible companies to disclose. ONS is accredited as a provider of NCPD by the American Nurses Credentialing Center's Commission on Accreditation.  Learning outcome: The learner will report an increase in knowledge related to hematopoietic stem cell transplantation for scleroderma and other autoimmune diseases.   Episode Notes  Complete this evaluation for free NCPD.  Oncology Nursing Podcast:  Episode 173: Oncology Nurses' Role in Stem Cell Transplants for Pediatric Sickle Cell Disease  Episode 148: Stem Cell Transplant Nursing in the Home Setting  ONS Voice article: What Oncology Nurses Need to Know About Vaccination and Cancer (and other immunocompromised diseases)  Clinical Journal of Oncology Nursing article: Early Intervention With Transplantation Recipients to Improve Access to and Knowledge of Palliative Care  ONS course: Hematopoietic Stem Cell Transplantation  ONS Huddle Card™: Hematopoietic Stem Cell Transplantation  National Scleroderma Foundation  New England Journal of Medicine article about the SCOT trial: Myeloablative Autologous Stem-Cell Transplantation for Severe Scleroderma  Systemic Sclerosis as an Indication for Autologous Hematopoietic Cell Transplantation: Position Statement from the American Society for Blood and Marrow Transplantation  Autologous Hematopoietic Stem Cell Transplantation for Systemic Sclerosis: A Systematic Review and Meta-Analysis    To discuss the information in this episode with other oncology nurses, visit the ONS Communities.   To find resources for creating an ONS Podcast Club in your chapter or nursing community, visit the ONS Podcast Library.  To provide feedback or otherwise reach ONS about the podcast, email pubONSVoice@ons.org.    Highlights From Today's Episode  “The goal of treatment for patients with scleroderma is to reset the immune system, and there are three main components of the regimen used at Duke—that's total body radiation, cyclophosphamide, and ATG. This targets all the areas where the immune effector cells live. We also use CD34 selection, which is a process that separates CD34-positive cells from the stem cell product that's collected prior to transplant, to eliminate the possibility of reinfusing activated immune cells back into the patients.” TS 3:18  “For patients with diffuse scleroderma, you want to offer transplant when they have evidence of significant disease, but they're not so compromised that they can't tolerate or have increased risk of complications from the conditioning regimen. Understanding the patient's rate of disease progression is key when determining to transplant.” TS 6:45  “When a patient is referred, we call the patient, and we talk about how the transplant conditioning regimen works to reset the immune system and stop disease progression. We explain the workup visit and go over an example of the timeline needed to collect the cells, admit to the hospital for conditioning, and the recovery process as an outpatient. We want patients to understand the big picture before they ever come to Duke.” TS 7:57  “Some patients come to us significantly disabled by their scleroderma. They may be in a wheelchair, so they require special vehicles for travel. Patients whose hands are severely involved need assistance with their ADLs [activities of daily living].” TS 11:43  “There have been three clinical trials that show autologous transplant improves event-free survival and overall survival and has been shown to decrease all-cause mortality. But it does not repair damaged gastrointestinal, pulmonary, or cardiac tissue. Any fibrosis that has happened is permanent.” TS 12:22  “The most amazing thing we see is the softening of the skin, which can occur during the first two weeks of the conditioning regimen. The nurses on the floor see it, and I think it's just a tremendous gratification for them to see the results of something right before your eyes.” TS 13:01  “Social media has been a huge contributor towards patient self-referrals. Patients are telling their stories on Facebook; patients are asking other questions about how to get referred to a transplant center; and patients whose rheumatologists have not referred them will seek out transplant centers to learn more about transplant for scleroderma.” TS 13:48  “For people with hematologic malignancies, it's all about getting that patient to remission and then transplanting them. . . . These patients have experienced chemotherapy and the adverse effects. They know about low blood counts and fatigue and recovery. They know about central lines and transfusions. The scleroderma patients come to transplantation with progressive disease. They've typically not had blood transfusions, but they are now going to receive total body radiation, chemotherapy, and a stem cell transplant over the next six weeks. And it can be overwhelming. . . . Every day is something new for them to process and learn.” TS 14:56  “Patients become pancytopenic, and they are heavily immunosuppressed. They are on steroids during the conditioning regimen to prevent scleroderma flares during conditioning. These patients have a central line so monitoring for infections, such as assessing vital signs for signs and symptoms of infection, and being aware that steroids can mask a fever.” TS 16:49   

Dr. Paulo Garcia, CEO and Co-Founder of Kytopen, is developing systems to refine the types of payloads, including RNA, DNA, and CRISPR-Cas RMP, that can be delivered to primary T cells, primary Natural Killer cells, and CD34 stem cells. To maximize the number of successfully engineered cells, the Flowfect Discovery technology, using a non-viral approach, automates the process of cell engineering and gene delivery into cells while dramatically reducing the time to produce the cells. Paulo explains, "One of the key differentiating aspects of our technology is that we are using a continuous fluid flow format for the engineering of our cells. This fluid flow contributes to the delivery of these payloads to the different cells. And to put it into perspective, the flow rates that we use are quite large. They are in the tens of milliliters per minute. And if you translate that into a throughput of cells, you're talking about being able to process anywhere between 1 to 2 billion cells in 30 seconds. And these are large quantities of cells that have the possibility of being able to be then used not only in an autologous setting but also in an allogeneic setting in which many cells have to be administered to the patients in the different doses that are required." "No technology currently processes at the high-volume rates we're talking about. And generally, what is done today is a batch process. There are non-viral techniques, and there are viral techniques, but what is required is for these cells to be introduced into a bioreactor divide, let them grow to the relevant numbers, and then they can be administered to the patients. And with a process that leverages the throughput of the Flowfect technology that Kytopen is developing, you can really reduce the time these processes take. And that is the vein-to-vein time, and reduce it to numbers that are measured in days as opposed to in weeks or months." "Right now, our goal is to shrink the time that it takes from discovery to process development and into clinical manufacturing with the Flowfect technology that leverages different types of energy, mechanical energy, electrical energy, and buffer chemistry to give the best outcome and chance of the cells to be engineered and modified for the therapeutic applications of our partners." #Kytopen #CellTherapy #ClinicalManufacturing #CellEngineering Kytopen.com Listen to the podcast here

Dr. Paulo Garcia, CEO and Co-Founder of Kytopen, is developing systems to refine the types of payloads, including RNA, DNA, and CRISPR-Cas RMP, that can be delivered to primary T cells, primary Natural Killer cells, and CD34 stem cells. To maximize the number of successfully engineered cells, the Flowfect Discovery technology, using a non-viral approach, automates the process of cell engineering and gene delivery into cells while dramatically reducing the time to produce the cells. Paulo explains, "One of the key differentiating aspects of our technology is that we are using a continuous fluid flow format for the engineering of our cells. This fluid flow contributes to the delivery of these payloads to the different cells. And to put it into perspective, the flow rates that we use are quite large. They are in the tens of milliliters per minute. And if you translate that into a throughput of cells, you're talking about being able to process anywhere between 1 to 2 billion cells in 30 seconds. And these are large quantities of cells that have the possibility of being able to be then used not only in an autologous setting but also in an allogeneic setting in which many cells have to be administered to the patients in the different doses that are required." "No technology currently processes at the high-volume rates we're talking about. And generally, what is done today is a batch process. There are non-viral techniques, and there are viral techniques, but what is required is for these cells to be introduced into a bioreactor divide, let them grow to the relevant numbers, and then they can be administered to the patients. And with a process that leverages the throughput of the Flowfect technology that Kytopen is developing, you can really reduce the time these processes take. And that is the vein-to-vein time, and reduce it to numbers that are measured in days as opposed to in weeks or months." "Right now, our goal is to shrink the time that it takes from discovery to process development and into clinical manufacturing with the Flowfect technology that leverages different types of energy, mechanical energy, electrical energy, and buffer chemistry to give the best outcome and chance of the cells to be engineered and modified for the therapeutic applications of our partners." #Kytopen #CellTherapy #ClinicalManufacturing #CellEngineering Kytopen.com Download the transcript here

Professor Bart Kay0:00 Bart Kay Introduction2:40 Heart palpitations and tachycardia, are they due to potassium deficiency6:35 MS lesions healing but still get fatigued14:21 Waking up in the middle of the night thirsty and peeing19:45 What should be your maximum daily carb intake28:09 Any special requirement on carnivore or keto for those without a gallbladder31:29 More on belly fat and insulin resistance46:20 Why calories are a flawed metricBart has successfully taught aspects of physiology, anatomy, biochemistry, metabolism/nutrition, and research design/statistics since 2001: both in the UK and in Australasia. He has consulted regarding optimising physical performance to The New Zealand All Blacks, the National Rugby League (NRL) Referees Association, The Royal Australian Defence Force, and The Royal New Zealand Army. Bart has a number of publications in the areas of exercise physiology, biochemistry, metabolism, nutrition, statistics, and physiological performance modelling.  Research InterestPhysical performance, Health, Fitness, Exercise training, Nutrition, Hydration, Challenging environments, Inflammation, CD34+ adult stem cells, Atherosclerosis, Meta-analyses, Critical review writingThank you so much for listening to my podcast. I hope you enjoyed it. Your support means the absolute world to me. And if you're enjoying the show, I've got a small favor to ask you. I'd be incredibly grateful if you would consider becoming a supporter and make a small monthly donation. Your contribution will really help to improve the show. It's a small monthly contribution. You can cancel at any time, and the link is in the show notes. Support the showAll my links in 1 easy list, including booking and personal training workout plans at LINKTREE You can now download the carnivore experience appApple direct link for apple devices Google play store direct link to app for Android Coach Stephen's Instagram Book me for coaching My growing UK carnivore YouTube channel I have set up a community that is all about eating low-carb and specifically carnivore. CLICK HERE Support my podcast from just £3 per monthBECOME A SUPPORTER Success stories Optimal Health 5 Star reviews All my facebook and other reviews are here Thanks to www.audionautix.com for any music included. Ple...

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.04.531096v1?rss=1 Authors: Kong, W., Han, X., Zhu, X., Wang, H. Abstract: We previously identified three distinct pre-stem cell lineages that further develop into blood CD34-positive stem cells, and mesenchymal stem cells. The pre-CD34-positive stem cells are spore-like, while the pre-mesenchymal stem cells are fusiform-shaped. All of them originate or released from tube-shaped tissue structures, or niches. In the current study, we present two membrane-like pre-stem progenitors. One of them had red thin membrane structures that were from disconnection of bamboo-like tissues and developed into thin membrane-like multi-nucleated cells. The other membrane-like structure had geometric-shape and was the place of developing numerous c-kit-positive stem cell progenitors. One geometric-shaped membrane could produce dozens to hundreds of progenitors in a synchronized pattern. Our findings provide more evidence that postnatal stem cells are unipotent and self-renewed in sealed tube-shaped structures. The tube-shaped structures released their inclusions that have distinct morphological differences among the cellular lineages. Thus, the self-renewal of each lineage-distinct stem cell has its unique pattern. Further, our data suggests that postnatal stem cells are renewed via a recurrent pattern. Our findings again challenged the old concepts of stem cell niche components, the origins of stem cell lineages and the self-renewal of stem cells. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.17.528986v1?rss=1 Authors: Shi, J., Wang, J., Yin, S., Lin, S., Jiang, F., Zhang, M., Wu, X., Shen, L., Gu, X., Yang, R., Yang, J., Wen, J., Zhang, W., Chang, Q., Jiang, X. Abstract: Bone homeostasis and repair is a systematic progress with spatiotemporal interaction of multiple cell types involved in skeletal and immune system. Precise spatiotemporal regulation of cell type-specific functions in bone repair contributes to further development of tissue engineering and regenerative medicine. Here, we utilized single-cell RNA sequencing to illustrate a map of cell landscape and dynamics in the progression of rodent bone self-healing and a perturbation by lymphoid cell-deficiency. We identified different functions of myeloid cell and lymphoid cell to osteogenesis and angiogenesis during bone repair and their mutual complementation under lymphoid cell-deficient condition. Additionally, we used CD34+ humanized reconstituted mice to reveal further insights into the mechanism of human bone homeostasis and repair. Our integrated cellular analysis of bone repair explores the functional diversity and complementation between myeloid cells and lymphoid cells during bone healing process and provides further therapeutic implications for the treatment of bone disease and degeneration following ageing. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

3.18 Bartonella Henselae Microbiology review for the USMLE Step 1 exam Bartonella henselae is an intracellular gram negative rod that causes several different diseases Bartonella targets and lives inside specialized immune cells called CD34+ cells Bartonella creates a protective vacuole that protects it and helps it evade immune detection Bartonella henselae is a facultative intracellular bacteria Three different clinical syndromes associated with bartonella henselae: cat scratch disease, bacillary angiomatosis in immunocompromised patients, and bacterial endocarditis Cat scratch disease presents with cutaneous manifestations at the site of inoculation (warm, red, swollen, with a vesicle) and swollen lymph nodes near the site Bacillary angiomatosis symptoms include fever, multiple clustered red or violaceous papules or nodules on the skin and mucosa, and bone pain Bacterial endocarditis: Bartonella makes its way into the bloodstream and infects the inner surface of the heart Bartonella henselae is hard to culture from blood, usually diagnosed via serology Treatment depends on the clinical syndrome presented, cat scratch disease is usually self-limited and doesn't necessarily require antibiotics, bacillary angiomatosis treated with a long 4-month course of either erythromycin or doxycycline.  

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.12.05.519079v1?rss=1 Authors: Cao, Y., Bolam, S. M., Boss, A. L., Murray, H. C., Dalbeth, N., Brooks, A. E., Matthews, B. G. Abstract: Skeletal stem and progenitor cells are critical for bone homeostasis and healing, but their identity and diversity in humans are not well understood. In this study, we compared stromal populations in matched tissues from the femoral head and neck of 21 human participants using spectral flow cytometry of freshly isolated cells. High-level analysis indicated significant differences in marker distribution between periosteum, articular cartilage, endosteum and bone marrow stromal populations, and identified populations that were highly enriched or unique to specific tissues. Periosteum-enriched markers included CD90 and CD34. Articular cartilage, which has very poor regenerative potential, showed enrichment of multiple markers, including the PDPN+CD73+CD164+ population previously reported to represent human skeletal stem cells. We further characterized periosteal populations by combining CD90 with other strongly expressed markers. CD90+CD34+ cells sorted directly from periosteum showed significant colony-forming unit fibroblasts (CFU-F) enrichment, rapid expansion, and consistent multi-lineage differentiation of clonal populations. In situ, CD90+CD34+ cells include a perivascular population in the outer layer of the periosteum and non-perivascular cells closer to the bone surface. In conclusion, our study indicates considerable diversity in the stromal cell populations in different tissue compartments within the adult human skeleton, and suggests that periosteal stem cells reside within the CD90+CD34+ population. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.02.514828v1?rss=1 Authors: Fourneaux, C., Racine, L., Koering, C., Dussurgey, S., Vallin, E., Moussy, A., Parmentier, R., Brunard, F., Stockholm, D., Modolo, L., Picard, F., Gandrillon, O., Paldi, A., Gonin-Giraud, S. Abstract: Cell differentiation requires the integration of two opposite processes, a stabilizing cellular memory, especially at the transcriptional scale, and a burst of gene expression variability which follows the differentiation induction. Therefore, the actual capacity of a cell to undergo phenotypic change during a differentiation process relies upon a modification in this balance which favors change-inducing gene expression variability. However, there are no experimental data providing insight on how fast the transcriptomes of identical cells would diverge on the scale of the very first two cell divisions during the differentiation process. In order to quantitatively address this question, we developed different experimental methods to recover the transcriptomes of related cells, after one and two divisions, while preserving the information about their lineage at the scale of a single cell division. We analyzed the transcriptomes of related cells from two differentiation biological systems (human CD34+ cells and T2EC chicken primary erythrocytic progenitors) using two different single-cell transcriptomics technologies (sc-RT-qPCR and scRNA-seq). We identified that the gene transcription profiles of differentiating sister-cells are more similar to each-other than to those of non related cells of the same type, sharing the same environment and undergoing similar biological processes. More importantly, we observed greater discrepancies between differentiating sister-cells than between self-renewing sister-cells. Furthermore, a continuous increase in this divergence from first generation to second generation was observed when comparing differentiating cousin-cells to self renewing cousin-cells. Our results are in favor of a continuous and gradual erasure of transcriptional memory during the differentiation process. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Dr. Jana Dickter, MD (https://www.cityofhope.org/jana-dickter) is associate clinical professor in the department of medicine, division of infectious diseases, at City of Hope Comprehensive Cancer Center. She is board-certified in internal medicine, infectious diseases, and the American Academy of HIV Medicine. Dr. Dickter earned her undergraduate degree in cognitive sciences from the University of California, San Diego. She went on to receive her medical doctorate from Rush Medical College in Chicago. After an internal medicine residency at University of Pittsburgh Medical Center, Dr. Dickter began her fellowship at UCLA's Affiliated Program in Infectious Diseases. In her clinical work, she has focused on the management of infections in the immunosuppressed. At City of Hope, she is an on-site HIV specialist and has an interest treating people who are living with HIV and cancer. She was the principal investigator involved in presenting the case of The City of Hope patient: prolonged HIV-1 remission without antiretrovirals after allogeneic hematopoietic stem cell transplantation of CCR5-delta-32 mutation donor cells for acute myelogenous leukemia. She also serves as the HIV physician for the first-in-human trial to evaluate the feasibility, safety and engraftment of zinc finger nuclease genome edited CCR5 modified CD34+ hematopoietic stem/progenitor cells in HIV-1 infected patients. Additionally, Dr. Dickter has been involved in clinical trials for evaluating certain medications for difficult-to treat infections in immunosuppressed patients. She is also involved in antimicrobial stewardship, infection control, and has published papers on aspects of patient management with antimicrobial agents. These papers have dealt with nosocomial infections, cost assessment of antimicrobial use, and unusual case reports, all intended to teach practitioners who manage these difficult to treat patients. 

Emergencies happen in hematology and oncology. This is a fact. But how do we manage these emergencies? Look no further. In this episode, we'll talk about one of the key hematologic malignancies that you'll encounter as a fellow, one that requires immediate action to reduce mortality: acute promyelocytic leukemia (APL or APML)- Acute Promyelocytic leukemia (APL or APML):**Stay tuned for our upcoming “part two” and “chemotherapy basics” episodes for more information on non-acute management of this disease**APL is a true hematologic emergency! Although this is a very curable form of leukemia, it is associated with high rates of severe DIC and high mortality in the period immediately following diagnosis***Untreated, can see pulmonary or cerebrovascular hemorrhage in up to 40% of patients***10-20% incidence of hemorrhage-related mortality in the initial period***Statistically significant increase in mortality at 30 days with just a 12-hour delay in initial hematologist consultation- Disease basics:** Rare subtype of AML(

Marta Walasek and Danielle Nguyen Truong provide tips and tricks to lock down effective genome editing conditions for CD34+ cells, methods to evaluate genome editing efficiency, and optimal culture conditions to maintain Hematopoeitic stem cell function and long-term editing effects.

Marta Walasek and Danielle Nguyen Truong provide tips and tricks to lock down effective genome editing conditions for CD34+ cells, methods to evaluate genome editing efficiency, and optimal culture conditions to maintain Hematopoeitic stem cell function and long-term editing effects.

Immunohistochemistry PearlsIn Part 3 of our Heme Path series, we break down the basics of immunohistochemistry (IHC)1. What is Immunohistochemistry?A. A molecular technique that utilizes fluorescently-labeled probes or chromogenic reporter stains to identify populations of cells. The goal being to help identify clonalityB. Procedure entails taking thin slices of tissue and then using the probes to look for the presence (or absence) of cells specific to that probeC. Helps to identify specific proteins, the pattern and distribution of which can help identify what it is that we are looking at2. Pros of IHC: A. Can be performed on fixed specimen (vs. flow cytometry, which requires live cells) B. Provides an idea of micro-architecture of the sample (diffuse distribution of cells vs. clusters) in context3. Cons of IHC: A. More labor intensive than flow, therefore can take longer to result B. Need to have an index of suspicion for what you are looking for. Your search is only as good as the probes you use! 4. Examples of utility: A. Diagnosis of acute leukemia: For instance, staining for CD34, which is expressed on blasts, can help you determine the “blast percentage.” That is, you count up number of cells that stain for CD34 probe then divide by total number of cells in the sample, which gives blast percentage. B. Diagnosis of plasma cell dyscrasias: These disorders result in “sticky” bone marrows, such that it is hard to get a good aspirate. This means on flow, you may not get an accurate percentage. Using IHC helps you better estimate the numbers. C. Diagnosis of lymphoma: Staining for specific markers (for example CD20, CD19 for B-cell lymphomas) can help in diagnosis. Can also use IHC to determine KI67, which is the proliferation index. D. Diagnosis of metastatic cancers: For instance, if you see a lung mass but also note axillary lymphadenopathy, so you sample the lymph node because less invasive. How you do you know if the cells in the lymph node are representative of metastatic lung cancer cells? Use IHC to stain them!E. PDL-1 status: This is believed to be important marker to predict response to immunotherapy. References https://www.abcam.com/content/immunohistochemistry-the-complete-guide - Great description of process and images to showcase what IHC staining looks like (not endorsement of product) Please visit our website (TheFellowOnCall.com) for more information Twitter: @TheFellowOnCallInstagram: @TheFellowOnCallListen in on: Apple Podcast, Spotify, and Google Podcast

Oncotarget published "Carcinoma cells that have undergone an epithelial-mesenchymal transition differentiate into endothelial cells and contribute to tumor growth" which reported that the authors investigated whether EMT can confer endothelial attributes upon carcinoma cells, augmenting tumor growth and vascularization. Hypoxic regions, demarcated by HIF-1α staining, exhibited focal areas of E-cadherin loss and elevated levels of vimentin and the EMT-mediator FOXC2. Implantation of MCF-7 cells, co-mixed with human mammary epithelial cells overexpressing the EMT-inducer Snail, markedly potentiated tumor growth and vascularization, compared with MCF-7 cells injected alone or co-mixed with HMLE-vector cells. Intra-tumoral vessels contained CD31-positive cells derived from either donor cell type. FOXC2 knockdown abrogated the potentiating effects of HMLE-Snail cells on MCF-7 tumor growth and vascularization, and compromised endothelial transdifferentiation of mesenchymal cells cultured in endothelial growth medium. Hence, cells that have undergone EMT can promote tumor growth and neovascularization either indirectly, by promoting endothelial transdifferentiation of carcinoma cells, or directly, by acquiring an endothelial phenotype, with FOXC2 playing key roles in these processes. A fourth mechanism—termed vasculogenic mimicry—entails the de novo generation of microvessels, lined with highly invasive tumor cells embedded in a rich extracellular matrix, essentially mimicking a true vascular endothelium and, notably, lacking in the endothelial cell markers CD31 and CD34. Finally, newly formed blood vessels may emerge through transdifferentiation of neoplastic or tumor stem-like cells into CD31-positive endothelial-like cells, as has been documented in neuroblastoma, B-cell lymphoma, and glioblastoma. In addition, subcutaneous injection of B16 melanoma cells into Foxc2 haploinsufficient mice has been shown to lead to the impaired formation of tumor blood vessels and, accordingly, compromise tumor growth. Given the inherent plasticity of cells that have undergone EMT and the involvement of hypoxia in EMT and angiogenesis, the authors sought to ascertain whether cells, undergoing EMT in the hypoxic milieu, can acquire endothelial cell attributes and augment tumor growth by directly contributing to the tumor vasculature. These findings findings link the stemness, conferred through EMT, to the acquisition of endothelial cell traits and the augmentation of tumor angiogenesis in a FOXC2-dependent manner. The Sarkar Research Team concluded in their Oncotarget Research Output that their findings are consistent with the notion that the phenotypic attributes of cells within growing tumors are eminently pliable and that, as tumor size and the oxygen deficit increase, carcinoma cells become progressively dedifferentiated towards a mesenchymal, stem-like phenotype. DOI - https://doi.org/10.18632/oncotarget.27940

Oncotarget published "Carcinoma cells that have undergone an epithelial-mesenchymal transition differentiate into endothelial cells and contribute to tumor growth" which reported that the authors investigated whether EMT can confer endothelial attributes upon carcinoma cells, augmenting tumor growth and vascularization. Hypoxic regions, demarcated by HIF-1α staining, exhibited focal areas of E-cadherin loss and elevated levels of vimentin and the EMT-mediator FOXC2. Implantation of MCF-7 cells, co-mixed with human mammary epithelial cells overexpressing the EMT-inducer Snail, markedly potentiated tumor growth and vascularization, compared with MCF-7 cells injected alone or co-mixed with HMLE-vector cells. Intra-tumoral vessels contained CD31-positive cells derived from either donor cell type. FOXC2 knockdown abrogated the potentiating effects of HMLE-Snail cells on MCF-7 tumor growth and vascularization, and compromised endothelial transdifferentiation of mesenchymal cells cultured in endothelial growth medium. Hence, cells that have undergone EMT can promote tumor growth and neovascularization either indirectly, by promoting endothelial transdifferentiation of carcinoma cells, or directly, by acquiring an endothelial phenotype, with FOXC2 playing key roles in these processes. A fourth mechanism—termed vasculogenic mimicry—entails the de novo generation of microvessels, lined with highly invasive tumor cells embedded in a rich extracellular matrix, essentially mimicking a true vascular endothelium and, notably, lacking in the endothelial cell markers CD31 and CD34. Finally, newly formed blood vessels may emerge through transdifferentiation of neoplastic or tumor stem-like cells into CD31-positive endothelial-like cells, as has been documented in neuroblastoma, B-cell lymphoma, and glioblastoma. In addition, subcutaneous injection of B16 melanoma cells into Foxc2 haploinsufficient mice has been shown to lead to the impaired formation of tumor blood vessels and, accordingly, compromise tumor growth. Given the inherent plasticity of cells that have undergone EMT and the involvement of hypoxia in EMT and angiogenesis, the authors sought to ascertain whether cells, undergoing EMT in the hypoxic milieu, can acquire endothelial cell attributes and augment tumor growth by directly contributing to the tumor vasculature. These findings findings link the stemness, conferred through EMT, to the acquisition of endothelial cell traits and the augmentation of tumor angiogenesis in a FOXC2-dependent manner. The Sarkar Research Team concluded in their Oncotarget Research Output that their findings are consistent with the notion that the phenotypic attributes of cells within growing tumors are eminently pliable and that, as tumor size and the oxygen deficit increase, carcinoma cells become progressively dedifferentiated towards a mesenchymal, stem-like phenotype. DOI - https://doi.org/10.18632/oncotarget.27940

In this week’s podcast, articles “The Cardiac Late Sodium Channel Current is a Molecular Target for the Sodium-Glucose Co-Transporter 2 Inhibitor Empagliflozin” by Light et al (www.ahajournals.org/doi/10.1161/CIRCULATIONAHA.121.053350) and “Metabolic effects of empagliflozin in heart failure: A randomized, double-blind, and placebo-controlled trial (Empire HF Metabolic) by Jensen et al (www.ahajournals.org/doi/10.1161/CIRCULATIONAHA.120.053463) are discussed. Dr. Carolyn Lam: Welcome to Circulation on the Run, your weekly podcast, summary and backstage pass to the journal and its editors. We're your co-hosts, I'm Dr. Carolyn Lam, associate editor from the National Heart Center and Duke National University of Singapore. Dr. Greg Hundley: And I'm Dr. Greg Hundley, associate editor, director of the Pauley Heart Center at VCU Health in Richmond, Virginia, Dr. Carolyn Lam: Greg, it's double feature day. And guess what? Both papers that we're going to talk about are regarding the SGLT2 inhibitors, and really look at the mechanism of action of these amazing compounds, from both a pre-clinical and clinic point of view. That's all I want to say, because we've got to tune in, a very interesting discussion coming right up. Dr. Carolyn Lam: But first I'd like to ask you a question. What do you think is the association between health-related quality of life and mortality in heart failure around the world? Dr. Greg Hundley: Well, Carolyn, I would think that, actually, they might be linked. Dr. Carolyn Lam: That's a really clever answer. Thanks Greg. Well, the authors are actually going to tell you with this next paper. It's from Dr. Salim Yusuf from Population Health Research Institute and McMaster University in Hamilton, Canada, and colleagues, who looked at the global congestive heart failure, or GCHF study, which is the largest study that has systematically examined health-related quality of life, measured by the Kansas City Cardiomyopathy Questionnaire, which is the largest study that has systematically examined health-related quality of life and its association with outcomes in heart failure, across eight major geographic regions, spanning five continents. Dr. Greg Hundley: Wow, Carolyn. So what did they find here? Dr. Carolyn Lam: Health-related quality of life, as measured by the Kansas City Cardiomyopathy Questionnaire, or KCCQ, really differs considerably between geographic regions, with markedly lower quality of life related to heart failure in Africa compared to elsewhere. Health-related quality of life was also a strong predictor of death and heart failure hospitalization in all regions, irrespective of symptoms class, and with both preserved and reduced ejection fraction. Dr. Carolyn Lam: Indeed, this paper really highlighted a great need to address disparities that impact health-related quality of life in patients with heart failure in different regions of the world. Dr. Greg Hundley: Fantastic, Carolyn. Well, I have two studies to discuss, Carolyn, and they're kind of similar, so we're going to do them back to back. The first study reports the results of the Sort Out X Trial, a large scale randomized multi-center, single-blind, two-arm, non-inferiority trial, with registry based follow-up designed to evaluate the Dual Therapy Sirolimus-Eluting, and CD34 positive antibody coated combo stent or DTS versus the Sirolimus-Eluting Orsiro Stent or SES. Dr. Greg Hundley: And the study comes to us from Dr. Lars Jakobson, from Arhus University Hospital. The primary endpoint target lesion failure, or TLF was a composite of cardiac death, myocardial infarction, or target lesion revascularization within 12 months, all analyzed using intention to treat. Dr. Carolyn Lam: All right, Greg. So the DTS compared to the SES, what did they find? Dr. Greg Hundley: Thanks, Carolyn. So the DTS did not confirm non-inferiority to the SES stent for target lesion failure at 12 months. The SES was superior to the DTS, mainly because the DTS was associated with an increased risk of target lesion revascularization. However, rates of death, cardiac death, and myocardial infarction at 12 months did not differ significantly between the two stent groups. Dr. Greg Hundley: Now Carolyn, in this same issue, we have another study evaluating endothelial function and implantation of intercoronary stents. And this second study comes to us from Professor Alexandra Lansky, from the Yale University School of Medicine and Yale Cardiovascular Research Group. And Carolyn, the study evaluated whether implantation of an intercoronary stent that facilitated endothelialization after the four to six weeks smooth muscle anti-proliferative effects post-stent implantation would be non-inferior to traditional drug-eluting stents. Dr. Carolyn Lam: Okay, another interesting study. And so, how did they do that? What did they find? Dr. Greg Hundley: Yeah, so Carolyn, a total of 1,629 patients were randomly assigned in a two to one fashion to the supreme DES stent, so 1,086 patients, or the DPDES stent, which was 543 patients. And there were no significant differences in rates of device success, clinically driven, target lesion revascularization, or stent thrombosis at 12 months. Dr. Greg Hundley: And the safety composite of cardiovascular death and target vessel revascularization or myocardial infarction was 3.5 versus 4.6% with the supreme DES stent compared to the DPDES stent. But target revascularization for this new stent was two and a half fold higher. Dr. Greg Hundley: So Carolyn looking at these two papers, what have we learned? So first, the Jakobsen, et al, tested whether the stainless steel COMBO Sirolimus-Eluting Stent coated luminally with CD34 positive antibody could theoretically capture endothelial progenitor cells and regrow endothelium. Dr. Greg Hundley: And the investigators observed that this stent had higher, not lower or equivalent, target lesion revascularization relative to the current generation Cobalt-Chrome Stent that only eluted sirolimus. Dr. Greg Hundley: In the second study, Lansky and associates examined an approach which was touted as enhancing endothelial recovery, where the early erosion of material and release of drug was thought to allow earlier endothelial recovery enhancing vascular response. Non-inferiority of the rapid release was demonstrated, but rather than hints of superiority, there were signs of inferiority. Hereto, target lesion revascularization was problematic and was two and a half fold higher. Dr. Greg Hundley: And so, Carolyn, there's a wonderful editorial from Professor Elazer Edelman from the Massachusetts Institute of Technology entitled, “Karnovsky's Dictum that Endothelium is Good Looking and Smart,” where Dr. Edelman emphasizes that while some endothelial cells may have been present after deployment of these devices, perhaps a fully constituted functioning endothelium may not have been achieved. Dr. Greg Hundley: And as we know, it is a fully functioning endothelium with nitric oxide release, buried platelet adhesion that is most protective. It is a really provocative read that reflects on previous thoughts from Morris Karnovsky, who suggests preservation of endothelial function is optimized by minimizing injury to it. And so, Carolyn, these combined articles really highlight the current state of new developments within interventional cardiology to thwart re-stenosis and highly recommend them to our readers. Dr. Carolyn Lam: Wow, thank you, Greg. That was amazing. But you know what, so's this next paper, because it really provides novel insights into that enigma of the role that the epicardium plays in the pathogenesis of arrhythmogenic cardiomyopathy. Dr. Carolyn Lam: Now, to delineate the contributions of the epicardium to the pathogenesis of arrhythmogenic cardiomyopathy, doctors Marian from University of Texas Health Science Center at Houston, Texas and colleagues performed a series of elegant mouse experiments using conditional deletion of the gene encoding desmoplakin in the epicardial cells of mice. Mutations in genes and coding desmosome proteins, including desmoplakin are known to be major causes of arrhythmogenic cardiomyopathy. Dr. Greg Hundley: Wow, Carolyn, very interesting. So what did they find here? Dr. Carolyn Lam: Epicardial derived cardiac fibroblasts and epithelial cells expressed paracrine factors, including TGF-β1 and fibroblasts growth factors, which mediated epithelial mesenchymal transition and contributed to the pathogenesis of myocardial fibrosis, apoptosis, arrhythmias, and cardiac dysfunction in a mouse model of arrhythmogenic cardiomyopathy. These findings really uncover contributions of the epicardial derived cells to the pathogenesis of arrhythmogenic cardiomyopathy. Dr. Carolyn Lam: Greg, there's a whole lot of other interesting stuff in today's series, as well. There's an exchange of letters among doctors Mehmood, doctors Moayedi and Dr. Birks regarding the article “Prospective Multicenter Study of Myocardial Recovery Using Left Ventricular Assist Device.” There's an ECG challenge by Dr. Ezekowitz on a silent arrhythmia. How would you treat this patient? Go quiz yourself. Dr. Carolyn Lam: There is an AHA Update by Dr. Churchwell on how federal policy changes can advance the AHAs mission to achieve health equity. And finally, a Perspective by Dr. Talbert on rheumatic fever and the American Heart Association, The Nearly 100 hundred-Year War. Well, that wraps it up for the summaries. Let's go to the double feature, shall we? Dr. Greg Hundley: You bet. Dr. Carolyn Lam: Wow, today's feature discussion is really all about SGLT2 inhibitors, and that question that we're still asking, how do they work? And today, we are discussing two papers, very interestingly, looking at it from different aspects, one from a preclinical lens, finding a very novel target for SGLT2 inhibitors, and the other from a clinical lens, and really looking at the metabolic effects of the SGLT2 inhibitors in a way you've not seen before. Dr. Carolyn Lam: I'm very pleased to have with us the authors of these very exciting papers. We have Dr. Jesper Jensen from Herlev and Gentofte University Hospital in Denmark. We have Dr. Peter Light from University of Alberta, in Canada, and we have our associate editors, Dr. Thomas Eschenhagen from University Medical Center, Hamburg, and Dr. Justin Ezekowitz from University Alberta. Dr. Carolyn Lam: So, welcome gentlemen, thank you so much for joining us today. I suggest, let's start from the mice before we go to the men, and Peter, if you don't mind by starting us in, please tell us about this novel target you found, why you looked at it, how you found it, what it means. Dr. Peter Light: Hi, Carolyn, yeah, happy to discuss that. So, we all know that through numerous clinical trials, there's a very unexpected and exciting cardioprotective effect against heart failure with the SGLT2 inhibitors. And we decided to investigate some of the molecular mechanisms, which may underlie that protection. And in looking at the literature previously, and from my own lab's work, we're very interested in control of electrical excitability and ionic homeostasis in cells. Dr. Peter Light: So we investigated a known target or a known iron channel, which is involved in the etiology of heart failure as well as cardiac arrhythmias. And that would be the cardiac sodium channel. So, we investigated the effects specifically on a component of the cardiac sodium channel called the late sodium current, which is only induced in disease states, and they could be that during heart failure or ischemia, or can actually be in congenital conditions such as Long QT Syndrome Three, which involves certain mutations in this sodium channel. Dr. Peter Light: So we basically investigate the effects of empagliflozin, dapagliflozin and canagliflozin, in several different models of a sodium channel dysfunction, including mice with heart failure. And really what we've found is that this class of drug, and this is a class effect, it's not specific to just one of these SGLT2 inhibitors, what we found, they are very good inhibitors of this late current of the sodium channel. And in fact, they don't even affect the peak current at all. Dr. Peter Light: And when we did this and we analyzed the data, we found the IC 50s were in the low micromolar or even sub micromolar range for these drugs, which is exciting. And we extended those studies into cardiac myocytes and looked at calcium handling in those cardiac myocytes and saw that we get a very nice reduction in abnormal calcium handling in cardiac myocytes. Dr. Peter Light: We also used in silico molecular docking of these drugs to the cryo-EM structure of the NaV1.5, which is the cardiac sodium channel and identified that these drugs bind to a known region of that channel, which also binds the local anesthetics or anti-arrhythmic drug, Lidocaine, as well as the anti-anginal drug, Ranolazine. Dr. Peter Light: And finally, we showed that these drugs also reduce inflammation through the NLRP3 inflammasome in an isolated beating heart model. So collectively, we provide evidence that the late component of the sodium channel is a really important, or maybe a really important target for the molecular actions of this drug, and may underlie those observations received from the clinical trials relating both to heart failure, as well as sudden cardiac death. Dr. Carolyn Lam: Thomas, could you put this in context for us? Dr. Thomas Eschenhagen: Thanks, Carolyn. I mean, we immediately liked the story because as you said, and Peter as well, these drugs have amazing effects and every clinical paper and indeed some new ones, but it's really unclear how they do that. And what is, besides the established target, the SGL2 in the kidney, what could be the reason for all of this or some of this?   Dr. Thomas Eschenhagen: And then, of course, other examples proposed, like the sodium hydrogen exchanger, but this story didn't go so far. So we saw now this data from Peter showing that, and this is, of course, for a pharmacologist, just like me, very important, it's very potent binding. It's not a binding which happens in a millimolar or high micromolar, but as Peter said in low micromolar range. So that makes it a very realistic effect, for example, much more potent than ranolazine. Dr. Thomas Eschenhagen: And, of course, now the question is, to which extent could this, now I would say, establish the effect on the late sodium current, explain some of the findings which came out of the clinical studies, and actually, a question I would have to Peter, now that I think most of you know, the late sodium current is a reason for the increased sodium for LQT3 syndrome, very rare. Dr. Thomas Eschenhagen: But, of course it would be tempting to say, okay, maybe that would be a very good drug, particularly for people with LQT3. Did you think about that, Peter? Is it something on your list, mexiletine has been tried. Dr. Peter Light: Yeah, so I think that it's a certainly intriguing possibility. In fact, in our study, we did test out several different Long QT3 mutations and saw a reduction in the late component as also sodium channel. It's tempting to speculate that, indeed, these could actually be a rather effective anti-arrhythmic drug in patients with these LQT3 mutations or specific ones. I would love to be able to test that in at least some of the genetic mouse models of Long QT3 and to see whether this concept holds water or not. Dr. Carolyn Lam: Wow, this is incredible. SGLT2 inhibitors from anti-diabetic to now anti failure, and now anti-arrhythmic drugs? That's just amazing. Thank you, Peter. We should move on to this next paper, and this one all the way on the other spectrum, a clinical paper called Empire Heart Failure, Empire Heart Failure Metabolic, actually. Jesper, could you tell us about your trial and what you found? Dr. Jesper Jensen: Sure, thanks for the invitation to take part in the podcast, first of all. I'll tell you a little bit, we designed this study to try to get behind mechanisms, so the clinical benefits of the SGLT2 inhibitors in order to try to make a clinical outcome trial. But as you know, the DAPA-HF and the EMPEROR-Reduced were competed very fast, demonstrating the clinical benefits in HFrEF patients. Dr. Jesper Jensen: So, the data of our study provides some detailed mechanistic insights to these findings. And from the literature, we know that SGLT2 inhibitors improve glucose metabolism in patients with diabetes, and these changes might not be surprising in the diabetes population, but moreover, alterations in glucose metabolism may not be the main mechanism for the early occurring clinical benefits. Dr. Jesper Jensen: However, we know that many of our heart failure patients without diabetes are insulin resistant as a metabolic feature of the heart failure, and the insulin resistance is associated with an increased risk of developing future diabetes, which in turn reduces the long-term survival and quality of life. So, the targeting insulin resistance in HFrEF patients is, therefore, of clinical relevance to our patients. Dr. Jesper Jensen: So, the population of the Empire HF Metabolic consisted of patients with chronic HFrEF, with or without type two diabetes, who are on a stable guideline directed heart failure therapy, and have also indicated on anti-diabetic therapy. And we randomized patients to receive empagliflozin 10 milligrams once daily, or matching placebo as an-add on for 12 weeks. Dr. Jesper Jensen: And this was a modest sized randomized control trial, including 120 patients. A very large proportion of patients received guideline directed heart failure therapy, and they generally consisted of the best one third of atypical HFrEF population, and only 10% had concomitant history of type two diabetes. Dr. Jesper Jensen: We then, at baseline and after 12 weeks, we formed an oral glucose tolerance test to assess the hepatic and a peripheral insulin sensitivity and performed a whole body DXA scan to investigate alterations in body composition. We know that patients lose weight when they get an SGLT2 inhibitor with and without diabetes, but we don't know what it consists of in a HFrEF population. Dr. Carolyn Lam: Tell us what you found after 12 weeks. Dr. Jesper Jensen: Yeah, so a large proportion, actually half of the patients without a history of diabetes, had a new onset diabetes, or impact glucose tolerance at baseline. So even though few have no diabetes, this population were at very high risk of developing future diabetes. And the main finding was that empagliflozin improved insulin sensitivity. So the hepatic insulin sensitivity was improved by 13% and the peripheral insulin resistance was improved by 20% compared to placebo. Dr. Jesper Jensen: And moreover, both fasting and postprandial glucose were significantly reduced. And regarding the body composition, patients in a mean lost at 1.2 kilos, or 2.6 pounds, which mainly consisted of a loss in lean mass and no significant changes were observed in fatness, and this is from the DXA scan. Dr. Carolyn Lam: Hmm. Justin, could you shed some light on what the editors thought about this, and there's lots of questions still, huh? Dr. Justin Ezekowitz: Yeah, absolutely, Carolyn, and thanks Jesper for sharing this paper with Circulation. Thanks for summarizing it so well. I think the questions that come up and the reason why we liked it so much was we're all trying to understand mechanism of how these medications work so profoundly for our patients. Dr. Justin Ezekowitz: Now, in this predominantly non-diabetic population, the fact that the liver and the peripheral insulin sensitivity improves, how does that bear out for the fact that there is no fat loss in the early stages, yet that's all been linked to later improved exercise capacity and increased fat loss later on in life. Dr. Justin Ezekowitz: So, do you think those two are going to be linked if you went to say from 12 weeks beyond the 52 or two years down the road? Dr. Jesper Jensen: Yes, that is what we've seen from diabetes populations, at least. So you could imagine that the same would be the case also in the HFrEF primarily non-diabetic population, but again, we don't know. But early loss is the mass loss. Dr. Justin Ezekowitz: So Jesper, when you think about the peripheral insulin sensitivity improvement, is that largely indicating mostly muscle based insulin sensitivity improvement, and that would indicate that the muscles, perhaps, are functioning better in the short term with just a simple change in therapy. Dr. Jesper Jensen: Yeah, that could be a way to put it. I would agree upon that. Dr. Justin Ezekowitz: So thanks, Jesper, I think that may indicate the quality of life improvement that we may be seeing in the functional status there, Carolyn. Dr. Carolyn Lam: Yeah, but as you said, Justin, there just seems so many other questions. To Jesper, I want to know, what further might you want to do to find out what's happening with this? The loss of lean mass surprised me, frankly. I thought it would have been fat mass. So, what are you doing to look at that? And then to Peter, I want to go the other direction. What are you planning next that might bring this closer to humans and a clinical study? So maybe I'll ask Jesper to go first. Dr. Jesper Jensen: So, I definitely agree with you, Carolyn. We would also have to put our money on the fat from the beginning, before the study. So with respect to the weight loss, then a loss in lean mass is not preferable if it represents muscle. So however, the weight loss works to mediate the observed change in insulin resistance. And additionally, a significant loss in muscle would result in reduced insulin sensitivity. And we observed the opposite. Therefore, the observed loss in lean mass may be speculated to represent water and pointing towards the early diuretic effect SGLT2 inhibitors. So, the DXA scan is good at looking at body composition, but it has difficulties in separating lean mass from whether it's muscle or water, but combined with the findings on the insulin resistance, we speculate that the lean mass loss is more. Dr. Carolyn Lam: Thank you. And Peter, could you very quickly tell us what are next steps, in your view? Dr. Peter Light: Yeah, obviously we were studying mouse model of heart failure. We'd like to make a more of a translational step in the next experiments we do by studying human tissues. So getting access to ventricular tissue from ex-planted hearts, human hearts, too, and then measure electrical activity as well as some calcium imaging. Dr. Peter Light: Looking at some of these Long QT3 animal models would be another thing that we're going to do. And also start looking at to see whether we can get access to any electrophysiological data from electronic medical records to start looking for DCGs and measuring QT interval, for example, would be another nice step to that. Dr. Peter Light: And then, more of a drug development side of things, we are actively synthesizing new derivatives of these drugs and seeing whether we can enhance the cardio-protective effects on the late sodium current, but actually remove the ability to inhibit SGLT2. So we would no longer have a glucose-lowering drug, but we'd have a cardioprotective drug. So, it's all very exciting work going on right now. Dr. Carolyn Lam: You've been listening to Circulation on the Run. From Greg and I, don't forget to tune in again next week. Dr. Greg Hundley: This program is copyright of the American Heart Association, 2021. The opinions expressed by speakers in this podcast are their own and not necessarily those of the editors or of the American Heart Association. For more, visit ahajournals.org.  

pomeさんをゲストに迎え、ボストン界隈を中心としたCRISPR関連のバイオスタートアップについて話を伺いました。Show notes Top CRISPR Startup Companies Changing the Future of Biotech and Medicine … CRISPR関連スタートアップまとめ Researchat.fm, ep76 … ゲノム編集特集回 Researchat.fm, ep77 … ゲノム編集特集回 Researchat.fm, ep2 … CRISPR特集回 Researchat.fm, ep77 Researchat.fm, ep47 … SHERLOCKについて話しました。 Boston MIT … マサチューセッツ工科大学。Kendall Station近くに位置する。厳密にはボストン市ではなく、ケンブリッジ市である。 Harvard University … ハーバード大学。こちらもメインキャンパスはケンブリッジ市。Oxford Stに位置する建物もあるため、Oxford St., Cambridgeと住所的には何が何だかわからないところもある。 Boston University … BU University of Massachusetts … UMass Tufts University Berklee College of Music … いつもバークリーなのかバークレーなのかわからなくなる。有名な音楽学校。 NOVARTIS Takeda Biogen Bayer Cell Press … いわゆるCell誌。MIT,ハーバードの目と鼻の先にある。 カリフォルニア州 マサチューセッツ州 ニューヨーク州 ニュージャージー州 Akamai Feng Zhang David Liu Researchat.fm, Ep60 … 培養肉などについて話しました。 GMO …Genetically modified organism Plantedit FAD2 Solive … by Plantedit spCas9 Cas12 Cas13 Inscripta mad7 nuclease Unreal Engine Unity C4U BioPallete Cas3 モダリス Beam Therapeutics Researchat.fm, ep22 … Base editorやTarget AIDについて話しました。 in vivo ex vivo 鎌状赤血球 CRISPR Therapeutics 造血幹細胞 CD34+ ヘモグロビン BCL11A eGenesis Bio George Church 胚盤胞置換法 Living cell technologies (LCT) Diatranz Otsuka CAR-T レンチウイルス AAV HIV ゾルゲンスマ TLO … Technology License Organizationの略 Wyss Institute Wyss Instituteのポッドキャスト … めちゃくちゃ良い。tadasuはリピートしまくって聞いている時期が度々ある。 Peng Yin Researchat.fm, ep74 … DNA Origamiについて話しました。 Cambridge Innovation Center (CiC) Venture Cafe Venture Cafe Tokyo … 虎ノ門ヒルズ内にある。木曜日にイベントをやっているようです。 CiC Tokyo … 虎ノ門ヒルズ内にある。 Cambridge City, MA … ケンブリッジ市 スタンフォード大学のパテントに関する資料 … Stanford UniversityのOTLによる資料。 lab central Johnson & Johnson Roche Job Description 四行教授 … とあるブログ様を引用させていただきました。当時修士のtadasuに四行教授や「履歴書を汚せ」と説いた人物は黒川清先生です。 Researchat.fm, ep75 … I’m not sure though. Editorial notes とっても楽しいおしゃべりでした。仕事と関係ないマニアックな話をできる場はあんまりないので貴重です。またやりましょう。(pome) 後編もお楽しみに〜 (soh) 同じ地区に住んでいるのに何にも活かせていません…(tadasu) (coela)

In this episode of the ORS podcast Johnny and the fellas discuss a recent paper that has given additional support to the ability of repeated inflations of a cuff limiting muscle atrophy from disuse. Here's the paper: Kakehi, S., Tamura, Y., Kubota, A., Takeno, K., Kawaguchi, M., Sakuraba, K., Kawamori, R., & Watada, H. (2020). Effects of blood flow restriction on muscle size and gene expression in muscle during immobilization: A pilot study. Physiological Reports, 8(14), e14516. Other papers referenced: Du Bois, P., Pablo Tortola, C., Lodka, D., Kny, M., Schmidt, F., Song, K., Schmidt, S., Bassel-Duby, R., Olson, E. N., & Fielitz, J. (2015). Angiotensin II Induces Skeletal Muscle Atrophy by Activating TFEB-Mediated MuRF1 Expression. Circulation Research, 117(5), 424–436. Joshi, S., Wollenzien, H., Leclerc, E., & Jarajapu, Y. P. (2019). Hypoxic regulation of angiotensin-converting enzyme 2 and Mas receptor in human CD34+ cells. Journal of Cellular Physiology, 234(11), 20420–20431. Tipton, K. D., Hamilton, D. L., & Gallagher, I. J. (2018). Assessing the Role of Muscle Protein Breakdown in Response to Nutrition and Exercise in Humans. Sports Medicine , 48(Suppl 1), 53–64. Kubota, A., Sakuraba, K., Koh, S., Ogura, Y., & Tamura, Y. (2011). Blood flow restriction by low compressive force prevents disuse muscular weakness. Journal of Science and Medicine in Sport / Sports Medicine Australia, 14(2), 95–99. Kubota, A., Sakuraba, K., Sawaki, K., Sumide, T., & Tamura, Y. (2008). Prevention of disuse muscular weakness by restriction of blood flow. Medicine and Science in Sports and Exercise, 40(3), 529–534. What everyone wanted though…the squirrel story!! https://www.sciencemag.org/news/2019/10/here-s-how-hibernating-squirrels-live-months-without-water Credit to FoolBoyMedia on free sound.org for the sound byte: https://freesound.org/s/504687/

Big show today! We're back in business featuring a personal essay in dramatic form! The Time We Had, by Susana Ramirez. Susana Ramirez is a wife and mother of two. She is the Founder of Save The Sharks, a South Florida non-profit organization working to promote healthier shark populations through education, research and outreach. Susana immigrated from Cuba, living in Finland before settling in the U.S. She began writing at an early age, focusing on conservation work throughout the world. Today, she understands the power in having a voice and hopes her words can have an impact on others, leading to a global shift on how humans interact with wild animals and their environments. Find Susana on Twitter @susanaiswriting and check out Save The Sharks! This piece was originally published in The Selkie: https://theselkie.co.uk/the-time-we-had/ Brenda Zamora is an L.A. based actor, follow her adventures on Instagram! Produced by J. Alejandro. Visit idlewy.blog, a place for working class creatives! MUSIC FEATURED: Dreamsphere 7 by Sascha Ende Link: https://filmmusic.io/song/458-dreamsphere-7 License: http://creativecommons.org/licenses/by/4.0/ Calm In The Night by MusicLFiles Link: https://filmmusic.io/song/6232-calm-in-the-night License: http://creativecommons.org/licenses/by/4.0/ 5 vor 12 by Sascha Ende Link: https://filmmusic.io/song/2973-5-vor-12 License: http://creativecommons.org/licenses/by/4.0/ On the Edge of Forever - http://teknoaxe.com/Home.php Simple Piano Medley 1- http://teknoaxe.com/Home.php Freezing by Warm by Meydan: https://freemusicarchive.org/music/Meydan/Ambient_1860/Freezing_but_warm SOUND EFFECTS: https://freesound.org/people/florianreichelt/sounds/459977/ https://freesound.org/people/SpliceSound/sounds/369846/ https://freesound.org/people/borQue/sounds/336855/ https://freesound.org/people/AnthonyChan0/sounds/177956/ Additional sound effects obtained from https://www.zapsplat.com

Hyperstyles. CD34 | Full-On Fusion |  Tracklist can be found at: https://soundcloud.com/lentej/ Enjoy and dance on!

Dr. McKenna has successfully treated an incredible array of diseases, inside and outside of orthopedics with stem cells. McKenna has been on the front line of stem cells and their clinical application for decades. If you have ever wanted to know the vast array of proven applications for stem cells, do not miss this episode. What stem cells are, how they work, where they are and are not taken from, why cord blood and PRP are NOT the same as stem cells technology used today, the issues of the FDA, his association with RMI (Riordan, McKenna Institute for stem cells with Neil Riordan), treating Mel Gibson and other celebs & lawmakers, the future of research in stem cells, why all SC healthcare providers are not practicing equally (or ethically), why Panama & the Bahamas are used for advanced stem cell therapy and more.https://TheCellSpa.comhttps://drwademckenna.comAtrantilhttps://lovemytummy.com/spoonyProlon Fastinghttps://prolonfmd.com/isreferral.html?p=KBMD&w=FMDhttps://kbmdhealthhttps://gutcheckproject.comHey hi Mandy if you don't know me it's probably because I'm not famous but I did start a men's grooming company called Harry's the idea for Harry's came out of a frustrating experience I had buying razor blades most brands were overpriced overdesigned and out of touch and here is our approach is simple here's our secret we make sharp durable blades and sell them at honest prices for as low as two dollars each we care about quality so much that we do some crazy things by world-class German blade factory obsessing over every detail means were confident in offering 100% quality guarantee millions of guys have already made the switch to Harry's so thank you if you're one of them and if you're not we hope you give us a try with the special offer get a Harry starter set with a five blade razor weighted handle shave gel and a travel cover all for just three bucks plus free shipping just go to Harry's.com and enter 5000 at checkout that's Harry's.com code 5000 enjoy all life we are here with a gadget project this is episode number seven on marriage for you here with your host Dr. Kendra I was going on today good morning good morning how you doing I am doing well how are you doing this more well than what I am on the day number four of the fasting mimicking diet how about you I am also on day four the fasting mimicking diet made by prolonged made by prolonged so I want do a shout out to Dr. Joseph onto the CEO prolonged and Dr. Walter Longo who wrote the longevity diet they sent us some prolonged kits and were given a shot the fasting mimicking diet they are it is the fasting mimicking diet so I am comparing it to a previous experience of doing a water fast it's very interesting and this is far more tolerable quite honestly and just this morning I did check key tone levels I was it 2.2.1 date date three you're supposed to start doing it so by as this day goes on you probably start kicking up a whole lot more yeah so I found that to be beneficial it's only falling in line with what you want I did comparing this to a water fast at this point I was big meal for being in three nap days I feel like I was really ready to start eating the by day four that was enough for me I did dad it four full days of the water fast but with Pro line I feel actually feel really really good it's not too bad it is nice the way they can portion out every days meals what were going to be eating so my wife's doing with this also the only hard part was it earlier this early this week I had to cook dinner for the boys because they still gotta eat and family had some nice juicy steaks to sit there and you can have any of it I couldn't do anything with it was not the box about yourself how was yours will I'm you I'm doing well this is my fourth five day fast I guess is a little over year trying to do them every few months I first want to prolong which is not a big deal all second one I did kind of my own little fasting and keep mimicking style I didn't have that what I'm in a call that burst of energy right or possibly it was stimulation of stem cells listed in that in a few minutes here and then I did a water fast only my ketones went through the roof but I was quite miserable sore back to the prolonged given this a shot and we'll talk about that in the second but I mention the stem cells today's guest while this is going to be if you know anybody to have autoimmune disease if you know anybody that has back pain anybody has joint pain to an end because we have a stem cell expert Dr. Wade McKenna orthopedic surgeon skies a bad ass in this field and we were sitting there talking just outside I'm like holy cow I'm just considering take notes like we brought in a professor of stem cells may ease size pretty amazing how he actually played football at Oklahoma State went to med school I perform indices with him several years ago whenever he was still heavily just as he is today doing orthopedics he's a fantastic surgeon all of this is led to somebody's actually can remind me you he never wants to stop learning and so it's it's led to areas where he is today and he is I would say quite the expert with stem cells and where the future is going with them so this is so exciting that we have to time to fast for this episode because you know people throw around the word stem cell a whole lot and much like the CBD industry people throw around there's lots of misinformation there's lots of quality differences the people that are actually giving stem cells there's lots of differences with that so we could clarify all of it I really feel like there's some parallels you and I have brought in several CBD experts right and there's some parallels here and so you know this is a super exciting for that anything going on with the family anything going on socially well this last week whenever Morreale moved into preparing to do this fast I would say that the boys have the have enjoyed teasing us and other than that they've they thought back into all season basketball quite busy and is kind of the every day as usual at the round Renner household self typical tennis weekend both the Lucas and Karla were playing tennis and they both did really well it was kind of a little curb although I'm really proud of my team because as working to be launching the D hat health box Dr. Lisa Alvarez actually did a little commercial for she did so yeah and so do I get a chance to pop into the set see it the move honestly try to see what that happens I appreciate her taking her time to do that so another chemical thing I just go phone with Dr. Chang Ron Houston yeah great guy fantastic functional medicine doctor he actually has ties with prolonged him and him and Joseph were friends okay to be having a huge conference coming up on April 27 were he to talk about brain got issues where you have them on the show because he's got some incredible stuff on brain waves and its affect pre-and post trauma and its effect pre-and post-diet change and using hemp dry products so super cool I just have a phone with him so to get a chance go to his Facebook page a lot of really cool information so awesome I think the work on the move and all these unique directions right but what's the big deal stenciled what we want to talk about I think that it's it's the newest new frontier it's no different than the way we've been spending time talking about CBD why why just a few years ago the revelation that you had into polyphenols and how I could do these are it's a lot of what nature is giving us to work with and it's kind of amazing that it's it's all coming to fruition nowadays and you hit on it a little bit earlier I know that the Dr. McCann is going to address it as well the FDA it many times when we want this government entity to be on our side in helping us out can really be stymieing a lot of the progress that many of the citizens could be enjoying that they could be taken party to have a better quality of life and in a really odd yet when you get down to money reason you find out that the FDA is is hard to budge out of the way in in terms of progress so interesting because what he was talking about is exactly what I've been doing so in in all fields of medicine it's very hard to change the direction of this large Titanic like shipper people doing things and we have either many ways as he said as he described it to skin a cat or really none of them really working very well sure and then when you find something it's hard to get people to pay attention to much like trying to write I mean when we sit there when we came in without her until we know that mojo and 5.0 guys are talking there really trying to tell everybody about the bloating effects with it but we know that our trenches made up of polyphenols and we know those polyphenols are really good for you they actually are the antiaging and anti-inflammatory molecules in the Mediterranean diet so we need to expand that message a little bit more rhino people hey you can take these polyphenols which are in trying to and they can actually do some of the things Duncan that stem cells do and we can talk about this in some science but if you're curious about that if you looking at upfront Hills or any place that you go where they should go to love my tummy.com Ford/spoony that's love my tummy.com/spoony and then use the code spinning Sabal cash while you pick up your own polyphenols to be delivered right to your home and then keep listen to this because you do realize that these there's lots of overlap chain runs you to be doing a brain gut thing where he shows that you need to protect your gut I'll try to help with that we got Wade McKenna here talked about stem cells the body wants to rejuvenate itself you need to give it the things that can this is going to be so excited working a really geek out today I am the earliest I want to definitely I want to hang in there because what you learn is literally some of those cutting edge stuff you meant Dearborn stem cells person to make a circuit in the news and it's really odd the way the people began to report new science and health sometimes it can be this is the greatest thing ever or it can be a lot of scare tactics in our member the first time I heard about stem cells it was the unfortunate been taken from unborn babies etc. but that's not what's happening in all when you begin to get past that layer will guess what it's just like anything else you get past the first layer and then you find that there's a whole new world to discover and in terms of what Dr. McCann is going to talk about we have lawmakers here in the US to prohibit certain strip certain lines of stem cells being used but they are still incredibly beneficial and some of the culturing over the growth of those stem cells and do what the US would state would determine to be tissues they couldn't do it here they could do in Panama and oddly enough who found his work in Panama always couple lawmakers really kind of the good kind of weird and ironic that the same people there are part of that institution it doesn't allow us to do certain things will go out of the country to get that kind help is such a small world and its fate in whatever it is I think a lot of things that have happened in both our lives have been opportunities that we take advantage of and I love you and I were working one day and I was like dude did you see that Joe Rogan we had Mel Gibson on and some other guys some PhD knew it yeah I know those guys and I was like laughing because no Gibson said the same thing a lot of people think is like you think about stem cells you have a mouth going up the side of your face and now not at all as it turns out he took his dad down to Panama and his nonmaterial dad and he got better and that's what I was like whoa and you started saying to me this is a long time you're over your half your menu go to meet my buddy Wade because he's doing a lot of the same thing same parallel paths and since it is really scientific and is just trying to get people better that's it a year of your right on and just think about that so that being at least 18 months ago I believe that Mel's dad was started going down there maybe five years ago think of the advances in the tank and the technologies that have come along with stem cell research and send that's exactly why Wade Dr. Wei McCann is here to tell us a little bit more about where it's going how you going to measure what is authentic stem cell injection what is there the right protocol what you be looking for who are the imposters there's a there's a lot of information out there it's no different than learning about CBD and where to go get the right kind of CBD of its harvest of the Rahway producer away I just think it's it stinks I went to a doctor friend of mine Dr. Marlon Padilla and we are in his office and he just are talking about I'll check this out I'm now doing stem cell know the quote you have stencil expert on this week you go take a listen easier is it Hillcrest medical and University Park area… Having super great guy very innovative himself trying new things yeah and you he started do that like that small world some sort to pay attention to all these things so one of these lectures get caught up on everything that sewer pursuer at night to geek out a little bit so how I Titus altogether how to retire fasting together how to get stem cells and how we come full circle to discuss what's out there in the literature about what were passionate about also write well it's kind interesting because what were doing with that with that diet selection are trying to heal CBD fasting and learning that stem cells you find I think for all of our listeners as well as us this is all synergistic there's a reason why we're Gerber took the mail here with this kind of message so using our graduate student that always helps us out we've got some really cool articles kinds altogether, and with what I consider really geeky stuff I want you to hang in there for me okay so the first question is your on day four of the prolonged fast why the world even doing that well it has been shown that in cellular metabolism in July 2015 summarize whatever you and will fast it promotes stress resistance so basically when you're put on a fasting mimicking diet or I should back up we've always known that the caloric restriction diet has been shown in yeast and other animals to prolong life Walter Longo in his book figure out a way that you can eat a little bit and trick your body to believe that it is completely fast that's the fasting mimicking diet is so this study in 2015 looked at putting mice on the fasting limiting diet and they demonstrated that these mice decrease the size of multiple organs improved glucose control decreased visceral fat lower blood pressure improved bone mineral density rejuvenated the immune system and reduce cancer risk always too good to be true and Academy just five days three times a year and can accomplish this but wait there's more we got more here they also showed after they re-fed the mice solicit what this is the coolest thing about this when you listen to vaulter give lectures he said it's not so much the fast it's the recovery from the fast because when they re-fed him they showed that in older mice areas of the brain like the hippocampus showed neurogenesis and improved cognitive performance while yeah so it's pretty wild with the re-feeding that super important which makes it fun because on Monday for I'm really looking to some refuted yeah can't wait for some re-creating but any that's all part of the process I will say going through at my second fast that this is becomes easier it's it's not as hard as the first run of some that's with prolonged but it is the expectation is they are know what to expect I know struggle for so what would be contributing to better neural thinking that is you a question so the question is what's going on there so then we dug up an article in the Journal of stem cell research in 2016 what they showed is that fasting protects against immune system damage and induces regeneration by waking up stem cells or by catalyzing dormant stencils so all those what they realize is what this article describing is what vulture was figuring out right there what he had figured out that all comes down to stem cells at the refuting stage basically not with her not stenciled several times which is to find what is real quick stem cells are the body's raw material their pre-sells for all other cells stem cells are the only cells that can generate new cell types and they can divide into form what are called daughter cells which become specialized cells that eventually come specific organs that's all me to say about it because wage may come in here and blow our minds until us a whole lot more with what stem cells are so for my all intensive purposes on coming here knowing stem cells can become other cells – easier to tell us way more with that so basically after you fast and then you re-feed you flip a regenerative switch which promotes stem cell regeneration in the blood making organs so that the important thing so when you go into starvation mode the body will save energy and one ways to recycle immune cells and that causes autophagy so old and sick and dying cells are programmed to hate go away right and then the autophagy gets rid of the old site sells them when you re-feed the stem cells wake up and they go around there's a bunch of fallen soldiers they don't bother them but they go around and say we need new people to replace this over simplistic way to lead into a much cooler explanation of that and then with each cycle you re-feed you get rid of sick and dying cells and replace them is like a janitor it's exactly like a janitor so five day fast three times a year you just cannot quit you just doing some serious housecleaning I member Saturn panda when he talks about that that's the godfather of circadian rhythm fasting or intermittent fasting right he's got mouse models he still does a prolonged fast because he describes it as you brush your teeth every day and then you a couple times a year you going to get the deep cleaning from the dentist I like that analogy you're always doing maintenance which is you keep in the nine date of of what you're eating but every once while you get to do that deeply definitely and then trying to fast for the first time you you can look at you like why would I not want to eat but if you look at the history of time where people were in the movement nowadays to return to health where people are trying to get healthier and you look at things like Paleolithic's for instance they talk about new diets and ways to eat in a pale lifestyle some of that also includes fasting and the reason is the primal man also went a long time without having food and for a few days at a time they would have intermittent fasting org or a few days fasting themselves yeah and so basically you're just returning to what it is that we've all been programmed to do for a long time that it just so happens the last several generations we've had ubiquitous amount of food here in the US and so now we it'll thicker bigger let's okay so this is no doubt about it feet eating is awesome right but eating is an inflammatory process so when you eat your you to become inflamed a little bit and then you incorporate the nutrients and so will the way that were doing and how will we eat so much it's probably not the healthiest way so right now where the fast let's talk about was actually a lot going on with our bodies before talk about Howard to tie all this together two stencils. So day one this is basically the five defenses what's happened to you and I so day one just upon your body day to start doing some fat burning day three start doing some cellular recycling basically you're going to clean up start realizing were on day three now for backing cavemen times they one day to that's normal day three your body starts going up oh we better get ready for something because were you have to go out and get some food soon and that's when you start doing the cleanup and this is when a lot of people will reach ketosis day for you and I right now are in this this is the cell regeneration this is where autophagy started yesterday in autophagy is when those old sick and dying cells are programmed to go away much like we talked about with the polyphenols when they get in there in your list and causes my top a G5 in the foods we eat will do that then this starts artists are stem cell-based regeneration is starting to ramp up so when people talk about how I fascinated 24 hours 09 and identity a fast we've come this far when were this far into it tomorrow's what all the real magic happens all the magic day five regeneration continues now we've turned on our stem cells and the body is being rejuvenated from with it now the first time you and I did this did this are fast I think it was tonight will this happen to both of us we both slept what two hours yeah I even so every time that a fast so far I feel great whenever I go to bed and feel really rested but don't last night that of the of my fast I basically went to bed and then I thought I woke up again as a man what a great night sleep again and I looked over the clock I been asleep for two hours and 15 minutes and then I sat around the house like what am I going to do for the rest this morning about tired right now and then later I learned that was over Rex and it was being released telling me I always forget that all Rex and O Rex and so is released from hormone release my brain saying it's time for you to go and eat and I had in them and an abundant oriented interview time for you to get up have the energy to go kill the woolly mammoth so that you can eat and you can feel it a mere year manic and it was time and you saw me run when I quit the fast I mean I think of the time I my blood sugar was 54 and and and I felt fine with that and at key tones I think for 4.8 and the moment that we drew ride today the labs and that in the blood that one time it was it's time to eat yeah so both of us have ever similar expenses this is the third day that we done this so now let a geek out here for the last four minutes because this is where I think it comes in really cool and so we found an article that has a really long title and I just like I like saying it just because I realize that this is the kind of stuff I'm reading ditto for the show treatment of periodontal ligament stem cells with Emil Warren CBD promote cell survival and Ronald differentiation via the P 13 K a K TM tour pathway that would scare most people scared me off at first but our graduate student said this is a fantastic article hundred 11 so basically what this whole article shows is you can get stem cells from a few places Dr. McKenna will explain where bone marrow fat but one of the places periodontal ligament so these are known as mesenchymal stem cells meaning that they are the least differentiated cells and they can trying to become anything so one of the important things is quality of stem cells keep that in mind because we'll talk about that coming up quality stencils so one of the things about stem cells is that there are a lot of different qualities but once you get the stem sellout you have to keep the stencil healthy so you have to keep it alive and you have to keep it in the best environment possible so there's different mediums to do this this study looked at taking stem cells out in vitro meeting in a dish and they bathed them in CBD and more range in MO R which is a program to sign a day in which is a polyphenol which is the same stuff that upfront deals made of so they they bathed them in CBD and in a polyphenol blend amazing supercool they did it for $40 and what they showed is that they demonstrated longer survival less a pop ptosis or programmed cell death decrease the M tour pathway the M tour pathways the pathway that makes cells grow right so bodybuilders lots and tour pathway but guess what cancer enter pathway also so to growth pathway increased differentiation capacity meaning they can become more of something quicker they increase nesting and DDN after which neurogenesis or new nerves new brain tissue right and then it did a deep dive into the genes that these stem cells turned on that gets into the cool epigenetic stuff that we talked about before base will have these genes so they concluded that in the field of stem cell research it may be improved by bathing them in CBD and a polyphenol mix so if you not get injected anytime soon might not be a bad idea to start from the inside using some CBD and possibly some upfront it sounds to me like the research is probably on the on the cusp ears are going into that that's why they're bathing them in the point assignment in the CBD but it's weird that just three weeks ago when we had Mark on he was talking about his D differentiated contra sarcoma test stem cells that basically were released and they didn't know exactly where to go they begin to proliferate and he even said it could be found in an organ anywhere in your body even when you feel like you taken so it's critically important that your stem cells are differentiated they get to the place they're going to be and do what you need them to do you don't want them growing out of control so maybe possibly will find on the future that combining a polyphenol set with a plant a point of sanity and CBD along with stem cell therapy would be a protocol I be awesome to get people started here or should they go to get I would eagerly for you go to K BMT health.com and go to the store you can find both are trying to heal and the new KB MD CVD or you can always go to love my tummy.com/spooning and hearing about four minutes really joined by the Dr. Wayne McKenna so if you know anybody that has joint issues back issues knee issues and take it one step further autoimmune disease there's so many things that now look into the science of stem cells that is going to be supercool to geek out and this guy knows his stuff I am excited to have youth if you had any questions about stem cells is the witness of the main unit turn to Dr. Wade McCain is going to join us here we can take a break in about 10 seconds thing wrap up now just thank you guys for the prolonged high talk Elson if you are trying to quit drinking or doing too many drugs listen to me you don't know me and will never meet I had a problem like you want I drank and used a party a little too much till he got out of control and almost ruined my life I realize I needed help to fix my problem before it totally destroyed me if you tried to fix your drinking and drug problem and you know you can't do it alone you need to call the national treatment advisors they'll immerse you into a 30 day program to replace your old habits with new habits and totally change your life and if you have PPL private health insurance the entire program may be covered fix your problem right now before it gets any worse get clean call now and learn more 800-296-1252 800-296-1252 800-296-1252 800-296-1252 it looks like you're losing I am I losing weight I am losing my lost about 10 pounds how are you doing it funny name but I done it with review zone RAD use zone.com and the stuff works it's you you get it all that the molecule this that found in that all I can tell you is it it's a it makes you feel full and he keeps your mind off of wanting to overeat and also boost your metabolism yes you're done and more guys try it today it's gonna work for you like his work for Brad and countless other people read you zone.com are IDUs owners.com fast track student loans can get your student loans out of the vault stop any wage garnishments stop collection calls and stop seizure of your tax refund give yourself a break to stop the stress and get your student loan payments down to as little as $25 a month based on what you can afford to pay 800-709-4395 800-709-4395 800-709-4395 800-709-4395 okay were back it's now the second half hour episode number seven of the gut check project we are now joined with Dr. Wade McKenna but McKenna thanks so much for coming in today really appreciate absolutely so see what I do know him yeah you know whatever no word of her Rhonda Patrick goes on Joe Rogan's economy gets better paper out you'll be taking some notes I favor Gerardi, Patrick never feels more like absolutely just a caveman. Dr. she's a lot of knowledge of sausage yellow management so you since you do already went through a couple of small things in the last half-hour you played football look on the state you been orthopedic surgeon for several years but that's not really what you here to talk about today the cool part is I actually can you hear we good we are little my problem were going to get something fixed here real quick right area is going the cool part for me they spent is kind of been allowed to reinvent myself as a traditional surgeon when the science, caught up to what we do and figure out that a lot of what we would think of as traditional medical approaches were less than optimal from patients and point so exited a fellowship in trauma and post dramatic reconstruction after an orthopedic surgery residency and after a general surgery internship so in during a general surgery internship when I thought I was going to do transplant surgery because that was the coolest guys at the hospitalists on which Trina here in Dallas Fort Worth multiple hospitals with DFW medical service rendered him orthopedic residency I did my fellowship and trauma to the general I was Roy Sanders 2000 but hospital where as to trauma fellows panorama the program we had 10 residents in for helicopters and no sleep with a whole new episode on what lack of sleep will do to your stem cells well it lack of sleep and not healthiest UW not like lack of sleep induced by fasting where you feel like Superman lack of sleep induced of note falling asleep in the boat in the lounge chair waiting for the nurse to tapping the short essays time to go from zero to hero so but during the trauma fellowship we became very adept because we got stuck with a lot of the fractures that other people treated heel so the posttraumatic part of that is acutely we were stabilizing multiple extremity injuries but we also do those people over multiple extremity injuries and we would get referred a lot of the trauma patients the gentleman traumas what we called it which comes to visit you in clinic does it come to you in the middle night by helicopter gentlemen trauma that watching your clinic is like I get this knife stuck out as well hey I've had five surgeries on my femur fracture I still can't walk and still no bonus I have is soon healed and we had to find a way to not only promote tried to trick the body and the human something that already showed didn't want to heal but in the least invasive way possible to turn the table can help the patient generate new bone best way to do that early on was Boomer us were concentrate the very Baston first uses of bone roster concentrated in traditional surgery was in the treatment of nonunion when someone has a fracture that doesn't heal there is delayed union which is means takes forever that but there's nonunion wishes means it doesn't heal there's no bone if you have a leg doesn't have a hip fracture can't walk can't but wait on it given upper extremity with an unhealed fracture pretty much flail you can you have a non-nonfunctional extremity bone rest will concentrate in the treatment protocol this allowed us to be much less invasive instead of just it doesn't make a lot of sense to just take out all the other plates and stripped the blood supply room muscle replay fracture further destroying the blood supply to the fracture that already the numbness blood supply to heal so let's go ahead and revisit that really quick because as a surgeon it's interesting to dear somebody because that's typically that's a knee-jerk reaction all that last surged and worked when he operated as exactly so say one more time with re-operating guys will here's the problem with re-operating if you didn't heal the first time it's because of the formation of scar tissue not healed tissue so the healing gets stopped the fibrotic tissue begins scar tissue hurts scar tissue does not much blood supply and scar tissue is very functional it's fibrotic it it can take up some space for the most part the difference between the interface between healthy tissue and scar tissue continues to be painful forever every time you move something severe big fibrotic knot of scar with an attendant and you have some healthy tissue that generate the connects to it the mismatch in pliability that mismatch of you would never use metals that have different hardness when you put together an engine it is the soft metal in the hard metal caused threading and corrosion in significant problems and and metal mismatch soft tissue mismatches just as big a problem we create scar tissue and people hurts generates pain generates an inflammatory response or chronic inflammatory response from cytokines that without decent blood supply to scar tissue you want to get rid of so you end up with long-term and continued muscle that and a lot of our surgery approaches and a lot of surgery where you just strip off the blood supply to the bone that it needs to help heal don't work very well because were not focused on how the body needs to really heal this fracture were focused on making x-ray and I did just make you look nice so your I think it's fascinating because essentially I've done the same I've done the same thing in my practice rub to move from traditional gastroenterology become almost a functional orthopedist yeah I would like to think that that I I just come to the table with some of the extra tools that I need to kinda set the table for the patient to help them heal I the body has unique ability to overcome a lot of things and in our body actually wants to heal a lot of times I'm just try to help people get our own way the same way the gastro neurology diet you're trying to help people get their own way from being about health right with with orthopedics I have to help the body the body knows the triggers and mechanisms and has the entire growth factors to help your body heal as we get a little older as we have chronic injury you rely radiation cigarettes coffee alcohol late nights cortisol stress we impair our body's ability to respond appropriately to injuries what the stem cell does for you is that the cell that helps you respond injury the problem is is you get a little older you have less of them and they don't do as much as a used I would guess that a second I'm still I want to hear the history only here got half because you ended with during residency restarted down do bone marrow concentrate bone marrow aspirate concentrate for nonunion fracture not only fractions your worst people so you guys were willing to try some things other people work well we had the opportunity because these people had no other options so the best part about doing a trauma fellowship is we were there last Hope we were the the Island of misfit toys so to speak Ryland Mr. Toyo we will use the we stood especially around Christmas time when that movie comes out with Chris Pringle we we would literally collect the injured patients from all over South Florida South North Florida Alabama Georgia we were the only level I trauma center on the West Coast of Florida and so when people would fail multiple surgeries we they walk into the resident clinic and you had to come up with a way to solve the problem and a lot of times it was as easy as finding a way to put more stress on the fracture you did have some plate that was plated and distraction so last times was just taken some screws out sometimes it was loosening up a frame that was holding the fracture apart and then let the fracture heal we would compress the frame so that the P0 electric effect fractures need stress to generate bone so stress across bone generates electronegative charge calcium and phosphorus are positively charged the biomechanics of basic physiology which unfortunately is certain's only when were supposed to forget that but apparently we do is trained out of us is what I was told the residence when the witnesses don't let don't let basic science and in physiology be trained out of you it into a surgical approach but when you create electronegative chars from a compression fracture calcium goes in and she get some healing without blood supply there is the rule in an osteopathic physician so it's a deal medical school because our team Dr. local state was the and and did manipulated felt good and I want to know how to do that I didn't even notice a difference on your will to be an orthopedic surgeon I said can I be an orthopedic surgeon because the he said absolutely yes that's about how I made that decision Okemos they had a deal medical school oh use was MD the last thing the world I wanted was a red diploma so state fans of loyalty I got a mistake know you was an easy decision I do know what the deal was but one of the owners of the time give me a book called the difference of genomics and you trying to teach me about the school just decided to go to and they said that when it comes to healing that the rule of the artery is supreme rule of the rings have the right to rule the artery is supreme but lymphatics have veto power I never heard this so that was the it's it's the foundation of a T still creation illustrate your stupid mess was created by the write a few stills and indeed he started the first year medical school in Kansas but he did it because he was unhappy with traditional medical approaches mobilization the joint instead of letting it get all swollen up seem to make the patient's function will be better and execute the plan by creating with: Patty pump people would read out all the way to push down her chest and he let it would create open up the alveoli to get people over dramatic pulmonary effusions by crating the sink on the lymphatic pump widget side of my chylomicrons on the lung tissue will with what we are doing it literally goes back to the foundations of what created a lot of modern medical sciences that without blood spiders and healing and that's true for orthopedic fractures is true for muscles tendon injuries we first started doing Bomer go back to that point on the trauma surgeon we were real sure that if we put bone marrow into a tendon that would make that was her fear with that we thought when we took Bomer Asper concentrate were real careful to make sure we we kept it in the in the osseous chart of the animals yet or you got it in the mentor factor was that there some really good studies published a bummer go back to the mid-90s there is a there was there was actually a really great study but here's a little they knew about what we're doing in Israel 1520 years ago there was a study on product complex possible tibial fractures which is a disaster if you have a tibial plateau the base you need if it's a complex fracture more than one particle shot six rights of Shatzer's fracture if you have a Shatzer six we used to call foobar that was our classification so we we with the Shatzer six what they did in Israel is they treated half of them with bone browser concentrate and half of them which is plating without moments were concentrated but interestingly because it was so early they added PRP to the bone restaurant concentrate thinking that it made work better and really all it does is dilute down so PRP the machine I have developed our tears I called them on sale define what you okay so the machine that we work with the machine I've been working with for for quite a while and have actually helped hopefully without taking real credit for anything but but knowing that that a been a significant part of innovation the development of their kit I'm actually patented the bone Ross Britt Catherine Catherine comes like it is my my design PRP is when you take whole blood and spin it down the machine to concentrate the growth factors get rid of some of the white cells and so you create was called platelet rich plasma write an platelet rich plasma is generated from the centrifugation of whole blood into the growth factors and platelets there needed to help get rid of inflammatory change a lot of times getting rid of the inflammatory change is the way to start the healing cycle inflammation gets in the way you put out the fire before you can grow new graphs right and so with inflammatory change if you turn the inflammation off ligaments tissue tendons heal faster with Bomer Esper concentrate what was it really understood as well in the 90s early 2000 and it is now hopefully is that bone marrow is still 97% whole blood so when we spend down bone marrow you're getting platelet rich plasma so your you are actually doing PRP yeah but you're doing PRP with stem cells yet so bone respite concentrate has the stem cells needed to help you heal and we know that those stem cells is when went on to hold your but while I was in school there wasn't such a thing as a musical social musical stem cell was named the music will stem cell 1995 by Dr. Arnie Case Western and Arnie Kaplan named a cell that previously in medicine what we do is we name cells based on the characteristics right so before it was musical stem cell was in a plastic undifferentiated employee potential adherent so that I liked it way better back in that day. I call it Mrs. a couple of them so well and and I doubt that there's terms equals some so most commonly know what they're talking about right so there's a CD marker a surface cell marker verse 600 different types of the cells based on their surface marker so we talked about was equal stem cells people think there's like one time no there someone that we don't really need help you heal the sum of the week need crucially to help you heal and we know the difference between these based on their CD markers we've actually quantitated what cells we want was told we don't want found a way to concentrate the cells want we do the spends so with bone marrow you're getting platelet rich plasma but you're getting the best platelet rich plasma because you're spending it from the most immature blood when you spend on whole blood you're getting PRP we are getting them no stem cells alright so let's clarify because it's a definition is really that what you're saying is that saying just stem cells doesn't mean just themselves how stem cells we have these mesenchymal stem cells which is the earliest of the stencil correct you guys have markers where you can determine the type of one that you know which ones do you need what was preferentially help you grow Cartledge what was professorship tendon what was preferentially grow fat right so fat stem cells that if you make if you make fat graph or stem so graph from fat those cells grow fat really well yeah we know what they don't grow really well's cartilage because there's a peptide called Sox nine this are secreted by the frustum so so when we quantitate stem cells I'm not interested in what those cells could become a me to say right now is this my pet. If you get a stem cell lecture in the first slide they show is this one so become these five types of cells in the differentiation and the building of the cells become these five is what makes somatic that's completely wrong that's true in the lab that's not how it works in the body what happens in the body is your set your body season injury it secretes cells starting with the humor putting stem cell which is the CD34 right so thrive for all the one second I got a good message where it says I can get a little stressed out I feel like I'm producing the wrong stem cells because I putting on weight change my stem cells to get rid of the adipose tissue and so sent there just go I got lost that CD something or other yeah you are way more so than I thought I would be as a orthopedist at the document bone bent may make straight actually know the medical school the running joke at time UNIX was the hardest residency to get into W had to become stupid right away take the smartest guys we need only to talk about when I mean I mean it when I say don't unlearn medicine right you the orthopedic resident was the hardest residency get to but you are expected to never even look at an EKG again if you walked into the surgery patient and you're like looking at their head or their EKG the attendings walking to go one the how you do know that you talk about this is Eric little people to sleep we have a certain dog like no dying right thing will similarly used to put my patients asleep so you feel like Dr. McKenna the CD six and 07 mesenchymal cells ready there's a joke that was that he found an orthopedist and the radiologist near going opposite direction of the code blue running away from the seed that what we used to say you know if you want if you have a dollar and you want to hide from a surgeon or from any kind of position there different places you put it right 100 from radiologist to tape to patient if you want to hide it if you want to hide it from orthopedic surgeon you put it in a book if you want to hide up a plastic surgeon we can hundred dollars from plastics are you going at a rabbit hole there went out on CD34 what were your talk was initiation healing right so with the CD34 it secretes a peptide for PG to PGE2 is is one of those keys and starting new vessel growth will the way to grow hair the way to have ED go away way to have stress urinary incontinence go away the way to have your wrinkles go away the way to have your fracture healed way to have your tendon remote you had me at wrinkles ED hair okay so so now we start out treating nonunion fractures right what we figure out pretty quick that if you put Bomer Asper concentrate it was it wasn't and is a great study published it was a limb salvage patient in Japan where 15 surgeries big proximal defect it's possible to be a and the the general surgeon was was livid at the orthopedic surgeons want to to put bone restaurant concentrate a fracture because he was proving that there was no vascularity to the way so to back door the orthopedic surgeon the supposed case report that a vascular surgeon does this arteriogram a lower extremity and it shows that there's literally no collateral flow around the fracture site is kid basically has a limb salvage frame on his leg this big proximal defect two years out multiple fascia economies nonmusical leg mask were really high needs needs annotation orthopedic surgeon is busy all the stuff about Mercer concentrate wants to inject bone marrow before he takes frame off eventually as a surgeon you become kind of emotionally connected to to your work right the guy does not want to cut the slide off the vascular surgeon try to prove needs come off he does arteriogram family Stone will it cut off so they have bone marrow injected in the fracture site at eight weeks they redo the arteriogram because the orthopedic surgeon the arterial was ordered by him but you start to see new bone kids have less pain from weight on lag you get new bone formation but they have this arteriogram set out to the shows no blood flow so they redo the arteriogram and there's all this collateral circulation on the fracture site so basically forever listening arteriogram is a study were it actually shows the arteries they put Diana vessel and shows up on x-ray and there is no blood flow going below over the fracture risers no.the die stops and there's a little bitty pattern this will trickle that I injected something into the bone not into the are not in the artery into the Perry steel sleeve the covering of the bone with the with the board there was no bone produces big bony defect there but Bomer us for concentrating their the way the bone roster concentrate work it didn't become bone which is what we thought what it does is it secretes the peptides and proteins necessary to bring new blood flow which allowed the bone to heal now there's a certain paragraphs that out there shows no collateral flow say that saying again that blood flow the rule of the artery is supreme the arteries and she still give him credit for that from the 1800s rule the arteries supreme lymphatics have veto power and that's a Dr. Graham from local states manipulation class, add on that if everything so swollen of blood flow can't get to it okay yeah so you guys injected this is the first time you saw that Ballmer answer because this can be a great segue will be go to the next half hour more we really do jump into the stem cell we got a little will try and keep it as it at a level that we can help people because I get a lot of back to let me make sure I'm being asked questions like it doesn't help with back yes to the health of the components were next on but on the great papers published read everyone says oh there's no literature published there's been 3500 papers published with my little Catherine the kit was designed for bummer aspirate 3500 papers published there's never been in toward report there's never been a tumor there's never been you can't reject your own bone marrow so this is the bone marrow aspirate injections you guys are doing this is the very beginning of Stansberry Reese is the only sell the US are allowed to cost himself you cannot you guys really literally were the first people playing around stems the trauma surgeon department: bone marrow yet not not knowing what we were doing we are using Bomer Asper concentrate for the fatty component of marrow that seem to help fracture so faster which is where microfracture surgery the knee all this comes in my mind from we would we do niece go there's an uncovered Carla Jerry would put a couple holes in the bone where in the bone the lesson bone marrow leak into the knee thinking that I hope the cards losing hills, microfracture doesn't work very well it creates a cartilaginous Good Cartilage but It Does Heal Something But My Thought Was When I Credit This Catheter If a Couple Drops of Bone Marrow Makes a Difference What Would What Would 60 ML Concentrated on the 45 That Was Where We Started with This During Joliet Harlan's Injuries at Work That's Where That Slow Beginning That's That's Only for Mia Do You Think I Do Think That Today's Bone Marrow in the Stem Cells Come from Bone Marrow Are Really Adapt to This Type of Healing to the Because That's Where We Release Our Red Blood Cells for Your Body Does Yeah Okay so This Is How Your Body Is Ready Right Right That You Were Not Were Inventing a New Way to Make Something He'll This Is How the Body Heals This Is Where Those You Know It This How God Does It Right He Sends the Cell There Is Secretes As Protein Vessel Grows You Get Your Butts by Tenant's Right This Is How It Works Already Is Just As We Get Older Or If You Get Your Lymphedema Swelling Only for the Lymphatic Flow Attacker Has Veto Power Is a Big Swollen Leg Give a Big Swollen Foot Good Books I Can't Get to It Right so It's All about Mobilizing Ankle Fracture and Also so That Blood Flow Can Get to It Because Official Swollen You Can't Put Any Extra Water in a Full Glass So New Water Can't Get to It That All Contaminated Dead Water Sits There yet to Pour the Glass out a Little Bit to Put Some New Healthy Water Back in That's How Bloodflow Work Which Is Why the Lymphedema and Lymphedema Is so Dangerous and Has It Has Absolute Control over Blood Flow to the to the Injured Tissue before We Dive Deep into Stem Cells Does PRP Work I Love You so Here's the Deal so Peer Peas like Boomer Light Okay so PRP Is Bone Marrow Announced Himself It's It's a Good Growth Component It's Great at It's a One-Time Shot Right so You When You Pop European Something You Get a One-Time Shot Growth Factors That Limits and Stops the Inflammatory Response from Cytokines You Undergo Tissue There's No Stem Cells You're Not Getting a Stem Cell Injection Which Is One of the Things He Pushes Me over the Edges Somewhere with I Went Got Stem Cells from My Blood Union You Got an Injection Visitor Was a Stem so That PRP Player Was Positive Now PRP Is Also a Bummer With Our Stem Cells in Bone Marrow so When You Say PRP Splenda If You Got It from Your Whole Blood It's Just Pure. If You Got Boom Roster Concentrate It's All the Best Components of European Stencils about That That Study Is Telling about So Here's How the Little so Even Though the They Publish Is Great Study Showing the Bonus Request Rate Help the Complex Factors Heal 50% Faster and All of Them Healed The Ones That Didn't Have Bone Marrow Not All of Them Healed and They Took Twice As Long Hill That Was Published 20 Years Ago Right with Bomer Asper Concentrate but Would They Knew so Little about Bomer Also Concentrate That They Spun down Whole Blood to In the Same Machine to Try to Give It More Volume Because We Thought the PRP Might Help the Bummer Work Better When in Actuality You're Already Getting PRP When You Spend Them Boomers of All We Were Doing Were Literally in a Study in Israel What They Were Just Alluding It's Just Laughing This Is Similar When You Say No You Think It's Them so You Got Blood You Are Talking about Fasting And I Had Some Friends and Maybe Overdone It on Adderall Little Bit like Three Days without Eating and Not Basing Radiology Headsets of Money I Was in Jail All Weekend and You Know There's a Great View We Talked about Intermittent Fasting There's a Great Study Published in Cell Metabolism Last Year That Showed That the That Are Correlated to Longevity in Mice And the the Mice Had the Longest Food Free Intervals Actually Increase Life Plaintiff Please Got a Hold of Our Dr. Wayne McKenna I Will Lose Track down the Same 10,000 This Is the Only 24 Hour Take Anywhere Platforms Dedicated to Food and Fun Clear Spoony Our Townhall.com, Where the Mother Report about to Be Released Atty. Gen. Bars As a Special Counsel's Russia Probe Leaves No Doubt That Moscow Did Try to Interfere in the 2016 US Electric Thanks to the Special Counsel's Thorough Investigation We Now Know That the Russian Operatives Who Perpetrated These Schemes Did Not Have the Cooperation of Pres. Trump or the Trunk Campaign or the Knowing Assistance of Any Other American for That Matter Bars As White House Cooperated Fully with the Special Counsel's Investigators And He Says the Evidence Gathered by Those Investigators Not Sufficient to Establish Obstruction of Justice White House Officials Had to Go over Each and Every Page of the 400 Page Molar Report the Trump White House Is Already Pronounced Total Exoneration for the President in the Russia Collusion Case but Attorneys and White House Staff Members Will Be Pouring over the Molar Documents Findings so They Can Craft an Official White House Response the President's Legal Team Also Is Planning to Release a Rebuttal of the Molar Report That's White House Correspondent Greg Clarkson Boeing Says It's Making Good Progress in a Software Fix for a Troubled Mac Series of 737s Following Highlights Engineers Technical Experts and Our Partners Were Comprehensively Testing the Software Make Sure That It Does the Job And They're Taking the Time To Get It Right Boeing CEO Dennis Wallenberg Max Gets Taken Out Of Service Just about Everywhere Pending the Software Update A Booming Job Market Means a Number of Americans Filing for Unemployment Now Dropped to the Lowest Level since November 1969 Retail Sales Surge Last Month up 1.6% On Wall Street Dow 53 Points Higher the S&P Is Two Points Lower the NASDAQ down 25 More of These Stories Said Townhall.com Now You Can Fly Anywhere in the World and Paid Discount Prices on Your Airline Tickets Flight to Date Alignment Harassment to Read or Anywhere Else You Want to Go and Pay A Lot Less Guarantee Quality International Travel Department Right Now Low-Cost Airlines 800 452 1075 800-452-1075 That's 800-452-1075 Got an Old Car You Can Donate It Whether It's Running or Not to the United Breast Cancer Foundation and Save the Life They'll Even Come and Pick It up for Free The United Breast Cancer Foundation Has Saved Hundreds of Women's Lives through Their Free or Low-Cost Breast Screening Exams but Now They Need Your Help The United Breast Cancer Foundation Wants to Save More Lives through Early Detection by Offering Women Free or Low-Cost Breast Screening Exams In Donating Your Old Car SUV or Truck Whether It's Running or Not Helps Pay for Them Plus You Get a Charitable Tax Deduction Help the United Breast Cancer Foundation Save Lives by Donating Your Old Car SUV or Truck Call Now for Free Pickup 800-245-0823 800-245-0823 800-245-0823 All Right Now That Number Again Is 800-245-0823 Never Forgotten Apparel Is More Than Just a Premium Women's and Men's Clothing Line It's a Movement to Remind Us to Wear American-Made and Serve Those Who Serve Us Our Heroes Never Forgotten Apparel Gives 20% of Their Total Sales to Nonprofits That Support Homeless Veterans and Off-Duty Firefighters and 50% to Individual Veterans and Firefighters in Need Nationwide Checkout Never Forgotten Apparel.com Use Promo Code Matt and ATT And Get 15% off Your Purchase Welcome Back It Is Now the Second Hour at Episode Number Seven I Got Kicked Back to Chimeric Rigor Jointly with Your Host Brown and We Also Have a Studio Today Dr. Wade McKenna Stem Cell Expert and Longtime Orthopedic Surgeon Well Everything We Start Firing up A Lot Of Brain Energy Going on on That Last Time I Started Hot and I'm Wearing My Tequila 512 Shirts Nicely Believe the Two People Sit in Front of Me Happened to Be Some Ownership in It and Big Fans of the Product and Enjoy Some Tequila Moms Will Have Some Tequila If I Want to Yes All Modifier Question but I Will Still Signal CBD Here to Regenerate My Ananda Mind into E.G. Nice Because I Think I Spent Some There with the Trying to Keep up with Dr. McKenna I Went through to Believe That I Write the Name That It It It but It Was Some System Select for I Was Looking See Who's behind Me but It But Literally We Been over 10,000 Cases in US Now so so Let Me Let Me Throw One out for You We Got a Message from Victoria That Says Can You Fix Bone on Bone Degenerative Discs so Here's the Short Answer in the You Know Me Enough Already by Now You Notice the In a Good Prospective Randomized Study of This Started with Just Pure P Because Again Bo Mastro Concentrate the Only Reason PRP Exists Is a Product Is Because We Couldn't Get Approval for Bone Marrow so We Were Spinning down Horseplay on the Equine Market You Could Sell PRP into People Pay More to Have Their Horse Injected My World Are Kid and That Horse Has Four Extremities and so Were Spinning on Whole Blood Created PRP While We're Waiting on the FDA to Approve Bone Marrow Whole Blood Is Exempt In Bone Marrow Was an and so Did Get the Validations on Bone Marrow and A Lot Of Pure P Was Created so There You Go so the Short Answer Is Enter Disco Injections Hemorrhoids Are Several Great Papers Are Republished and the Most Recent One at Two Years 92% of the Patients Had Inner Disco Injections on Degenerative Disc Disease And and I Would I Would Take It Even Step Farther and That in Our in Our Clinical Practice with over 300 Discs Now In That If You Have an Annular Tear Bring a Tear in the Covering of the Desk That Is a Primary Pain Generator It's a Bright Spot on the MRI Called Heisey Lesion Are High in Tinsel Science Is a Signal A Lot Of Radiologist Don't Now Failed to Mention That I Guess but If There's an Annular Tear High Intensity Signal within That within the Disc That Alone Is a Dramatic Pain Generator It Can Even Cause This Exact Same Symptoms As a Herniated Disc Would As Far As Lower Extremity Pain and Weakness Because the Nerve Crosses the Annular Tear in Your Tear Generate Substance P Generates the Cytokines Generates Exact Same Pain Response so Those Two Times of Radicular Pain Coming from the Back Leg and Hip Pain Radicular so That's the Old Folks Called Sciatica If It Can Be Pressure Because My Big Herniated Disc Neural Foraminal Stenosis Meeting the Canal Gets to Tie the Facets Your Baguette Hypertrophic Get Extra-Large They Were out so There's There's You Can Have Pressure Stenotic Pressure on Her Nerve Were Still Think It's Just Been Crushed That Causes Leg and Hip Pain and Back Pain Or You Can Have a Chemical Radiculopathy Created by a Tear in the Desk and It Feels the Same Patient until Difference One of Almost Everybody Else Is My Bulging Disc yet I Could Be There but but but Bulging to Start the Problem Because Here's What We Know Here's What's Already Published If Either MRIs and A Lot Of People Have No Back and Leg Pain A Lot Of Them Have Bulging This So How's It Happen How How Can Some People Smoking Just Hurts a Bit Was Bulging the Stone Right Now Is That If You Experience If You Have a Bulging Disc the Nonoperative Follow-Up At Two Years Is the Same As Opera Follow If You Don't Have Weakness If You Have Pain That You Can Tolerate and You Have Weakness in the Lower Extremity at Two Years Your Doom the Same As a People How Discectomy If You Don't Have Surgery What That's Published For Long Time so Were Not so If You Intrude a Free Fragment That Free Fragment Will Absorb Your Body Is Really Adept at Getting Rid of Items Extruded into the Canal and It Will Actually Absorb and and Get Rid of a Free Fragment As Long As Every Fragment Is Important so Much Pressure on the Nerve That It Decreases the Blood Supply from the Pressure Causes Weakness in the Lower Extremity from the Pressure the Nurse Stops Working You Have Leg Pain Your Little Foot Drop below Witnessing That Surgery You Have Weakness and You Start Paying What's Publish Now Is If We Inject That Disk the Annular Tear That Doesn't Go Away on Its Own Will Heal And Most of the Time 92% in a Study If the Annular Tear Heals The Back and Leg Pain Go Away 92 Persons 92% of the Playhouse That Had an Injection Didn't Go on to a Primary Fusion While Now Now Here's the Other Side of The Patients Had a Fusion at Five Years 30% of Them Had Two Surgeries So If You Have Fusion at One Level 30% Time of the Next Five Years Your Risk of Having a Second Surgery Either Refused at the Level above or below or Hardware Removal or Revision or You Get Extra Bone from the Fusion at the Re-Open up the Nerve Roots Have To Do in Reframing Autonomy Every Time You Do Surgery The Muscles of the Back Diablo So the Paravertebral Muscles the Muscle Mass so It Would When You Look at Some of Been for You It Will Look like Dinosaur Right That Little Thing the Bridge the Sticks up Familiar Back Check Is Called the Spinous Process The Muscles That Lay on Each Side of the Spinous Process Have To Be Moved Out Of the Way For You to Do a Back Surgery Weeks Make Fun of Spine Surgeons When I Was a Fellow in Trauma Because They Don't Have One Incision Is Midline Low Back Tonight We Had Learn All These Other Incisions and Sponsors in Certain Were More Online But What They Don't Learn That Trauma Surgeons Learn As We Make an Incision The Is Designed to Not Limit the Structure Function Blood Spire Nerve to the Muscle Were Moving In the Spine The Multiply the Small Muscle Each Side of That Bridge The Nerve and Vessel Come from Midland in the Back but Were Moving Away Is It When You Do an MRI of Someone's Back Is Set Back Surgery Initially The Muscles on Each Side of That Look like Filet Mignon That's Tenderloin Right That's the Backstrap Vassar Hunters Were Not Talking Tenderly Farewell Real Hungry so When You Move That Muscle out a Way And You Go Back and You Do an MRI That Back That Had Surgery and Other Fibers out And Having Pain Again And the Neurosurgeon Looks at the Films Goes While the Disc Looks Great the Nerve Roots Wide Open Usual Payments and That There Is No Reason for Your Backs on Well If You Look at the MRI Used To Look like Tenderloin Now Looks like Prime Bootleg like Strip Steak or or or Worse Just We Call White Muscle Syndrome Is Just Whole Thing Just Looks like Fatting a Filtration like Arby's Like Times like More like Crime-Ridden Right Is a Little Bit of Muscle Aches and Their Diagnosis Are You You Are Fasting for What You Are Armies Roy Rogers for My Generation Has To Follow-Up Question That She Was Asking How Many Injections Did That Take to Achieve the Most of Time Is One One Engine One Treatment One Injection We Do What We What We Do Is We Will You Be More Specific Rest of My My Problems Stem Cell Science As a Whole Is A Lot Of Times Patients Get in the Mix Were There Never Really Diagnosed and I Am a Firm Believer That You Can't Treat Something If You Didn't Diagnose It If I Don't Know What I'm Treating My Chances of Making It Go Away Are Pretty Slim and Entered and Unfortunately A Lot Of Stem Cell Injectors out There Don't Make the Effort to Actually Diagnose a Problem but to Get There Won't Inject Anything Well That's That That's Very Nonspecific Treatment in It, It Kinda Puts Your Your Results Risk If You Don't Know What You're Treating Because You Know What You Can Heal What You Can't Heal When You Do What We so What We Do We Were Doing Disc Injections I Want to Know That Was the Painter So There's an Old Test That Used To Be the Standard of Care before You Had a Fusion Now Because Fusions Are so Rampant in Our Country We've Gotten Away from More Specific Diagnosis on Making Sure T

By definition, Brain Drain is the emigration of highly trained or intelligent people from a particular country. India and other surrounding countries experience the Brain Drain epidemic with their society’s top talent seeking prosperous opportunities abroad in countries such as USA or Canada. Why does this happen? What do countries “abroad” offer that makes the move across the globe worth it? What is the impact? Join the Carolina Desis as they pretend to be geo-politicians. Thank you, Nick Joseph for this month’s episode topic! And as always, thank you Yash Mistry for editing this month’s episode!

Dr. Carolyn Lam:               Welcome to Circulation on the Run your weekly podcast summary and backstage pass to the journal and it's editors. I'm Dr. Carolyn Lam, associate editor from the National Heart Center and Duke National University of Singapore.                                                 Is it time to end our debates on short versus long duration of dual anti-platelet therapy? Well I will be discussing this with two very special guests in just a moment. But first here is your summary of this week's journal.                                                 The first paper tells us that HDL particle number may serve as a biomarker of residual risk when assessed on statin therapy. First author Dr. Khera, corresponding author Dr. Mora from Brigham and Women's Hospital and colleagues of the JUPITER trial assessed HDL cholesterol levels, apolipoprotein A-1, cholesterol efflux capacity and HDL particle number at baseline and 12 month in a nested case control study of the JUPITER trial. That was a randomized primary prevention trial that compared rosuvastatin to placebo in individuals with normal LDL but increased CRP levels.                                                 In the current study the authors found that cholesterol efflux capacity was moderately correlated with HDL cholesterol, apoA-I, and HDL particle number. Baseline HDL particle number was inversely associated with incident cardiovascular disease, while there was no significant association for baseline cholesterol efflux capacity, HDL or apoA-I levels. On-statin cholesterol efflux capacity was inversely associated with incident cardiovascular disease but HDL particle number again emerged as the strongest predictor.                                                 Thus for both baseline and on-statin analyses, HDL particle number was the strongest of four HDL-related biomarkers as an inverse predictor of incident events and biomarker of residual risk.  Whether therapies designed to enhance cholesterol efflux capacity or an increased HDL particle number can also reduce cardiovascular risk however remains uncertain.                                                 The next study sheds light on mechanisms underlying the de-differentiation and lineage conversion of adult human fibroblast into functional endothelial cells. First author Dr. Zhang, corresponding authors Dr. Rehman and Malik from University of Illinois College of Medicine first generated CD34+ progenitors by de-differentiating adult human skin fibroblasts and showed that these intermediate progenitors could give rise to endothelial cells as well as erythrocytes. They then showed that lineage conversion of fibroblasts via partial de-differentiation recapitulated in part the embryonic development of the vasculature as evidenced by up regulation of anti-aging enzyme telomerase and the bi-lineage potential of the generated progenitors.                                                 Importantly they showed that transcription factor SOX17 functioned as a switch which regulated the cell fate of CD34+ progenitors towards an endothelial versus erythroid lineage. Finally implanted fibroblast derived CD34+ progenitors stably engrafted to form functional human blood cells in mice that improved cardiac function after myocardial infarction. Thus the molecular switch SOX17 provides a means to optimize the generation of endothelial cells for vascular tissue regeneration or disease modeling.                                                 What do drones have to do with out of hospital cardiac arrest? Well in this next study by first author Dr. Boutilier corresponding author Dr. Chan and colleagues from University of Toronto, the authors hypothesized that a drone network designed with the aid of a mathematical model combining both optimization and queuing could reduce the time to AED arrival.                                                 Using data from over 50,000 historical out of hospital cardiac arrests covering over 26,000 square kilometers in Ontario, Canada, they found that a drone network designed to reduce the median AED arrival time by three minutes relative to the historical 911 response could also reduce the 90th percentile of the AED arrival time by between 6 minutes and 43 seconds in most urban regions and 10 minutes and 34 seconds in most rural regions.                                                 Thus this study tells us that drone delivered AEDs have the potential to be a transformative innovation in the provision of emergency care to cardiac arrest patients especially those who arrest in a private or rural setting.                                                 The next study provides thresholds for ambulatory blood pressure among African Americans. Dr. Ravenall and colleagues from New York University School of Medicine analyzed data from the Jackson Heart Study, a population-based cohort comprised exclusively of African-American adults and of whom more than 1000 participants completed ambulatory blood pressure monitoring at baseline.                                                 Based on the outcome derived approach for systolic blood pressure and a regression derived approach for diastolic blood pressure, the following definitions corresponded to clinic blood pressure of 140/90 and were proposed as ambulatory blood pressure definitions for African Americans. Daytime blood pressure above 140/85, 24 hour blood pressure above 135/80 and nighttime blood pressure above 130/75 mmHg. Note that these ambulatory blood pressure thresholds identified for African Americans were higher than those from published recommendations mainly derived in European, Asian and South American populations. The use of these ambulatory blood pressure thresholds for African Americans will lead to a lower prevalence of daytime, 24 hour and nighttime hypertension compared with the current published recommendations.                                                 The next paper provides pre-clinical evidence of a novel target in plaque information in atherosclerosis. Dr. Stachon and colleagues from Heart Center Friburg University in Germany hypothesized a functional role of the signal axis ATP binding to purinogenic receptor P2X7 in inflammasone activation and chronic inflammation driving atherosclerosis.                                                 In an elegant series of experiments they showed that P2X7 receptor activation was crucial for inflammasone assembly and interleukin-1-beta secretion. The lack of P2X7 in mice abolished inflammasone activation in atherosclerotic lesions. P2X7 was expressed in murine and human atherosclerotic lesions. LDL receptor deficient mice lacking P2X7 receptor had reduced plaque inflammation and were less prone to develop atherosclerosis.                                                 Thus this study shows that P2X7 inhibition could be a treatment strategy against plaque inflammation in atherosclerosis.                                                 The next paper describes the first prospective clinical study of adenosine use in pediatric and young adult patients after heart transplantation. Now prior to this study adenosine was relatively contraindicated post-transplant due to a presumed risk of prolonged atrioventricular block in denervated hearts.                                                 In the current study first author Dr. Flyer corresponding author Dr. Silver and colleagues from Columbia University performed a single center prospective clinical study testing whether adenosine caused prolonged asystole after transplant and if it was effective in blocking AV nodal conduction in healthy heart transplant recipients aged 6 months to 25 years presenting for routine cardiac catheterization. Following catheterization, a transvenous pacing catheter was placed and adenosine was given following a dose escalation protocol until AV block was achieved.                                                 Eighty patients completed adenosine testing. And no patient required rescue ventricular pacing. AV block was observed in 77 patients with the median longest AV block of 1.9 seconds and the mean duration of adenosine effect of 4.3 seconds.                                                 Thus, this study suggests that adenosine may be safe and effective in patients post transplantation and establishes both a safe and effective starting dose of 25mcg/kg or 1.5mg for patients weighing 60kg and more. It also establishes a stepwise therapy escalation plan to avoid prolonged bradycardia. Although patients after heart transplantation may require less adenosine to achieve AV block it appears to be safe and effective as therapy for evaluation and or treatment of tachycardia in this population.                                                 Well those were your summaries, now for our feature discussion.                                                 Today for our feature discussion we are talking about a very familiar situation, dual anti-platelet therapy following coronary intervention and the decision of long versus short duration of therapy. A debate we've heard many times but according to the perspective piece in today's journal, maybe a debate we should end. And I am so pleased to have the author, Dr. Glenn Levine from Baylor College of Medicine as well Dr. Laura Mauri associate editor from Brigham and Women's Hospital.                                                 Welcome both. Dr. Laura Mauri:               Thank you Carolyn. Dr. Glenn Levine:             Thank you. Dr. Carolyn Lam:               Glenn would you like to start by presenting your case. It's time to end a dualistic short versus long duration of DAPT debate. I really like that title, tell us more. Dr. Glenn Levine:             Thank you Carolyn.                                                 The point we make in our editorial is that over the last five or six years there have been studies comparing what I term standard, which is usually about 12 months DAPT versus shorter duration DAPT and there are other studies comparing standard DAPT versus longer duration DAPT. Those generated important information in different people interpret them in different ways. What though has happened over the last several years is certainly for both educational and entertainment value at meetings as well in editorials, the idea of how long people should be treated with DAPT has been oversimplified to whether all patients should be treated with short duration or long duration. And Laura herself knows that as she has been in many of these debates.                                                 While I think that initially that was educational and entertaining, I think these days people understand those points and a greater issue is in that we should treat some people with short duration, some with what I call standard and some with long duration. And rather than debating whether everyone should treated with short or everyone should be treated with long, I think what we need to focus on now is which patients should be treated with short duration, which are probably best treated with a standard duration and which are best treated with prolonged or extended duration DAPT.                                                 And that in a nutshell is the main point that we make in this perspective editorial. Dr. Carolyn Lam:               Laura, so do you agree? Dr. Laura Mauri:               I couldn't agree more. I think clinicians really are looking for guidance and what happens at these debates is you see these polarizing opinions that debaters are asked to defend when in actuality there's such a wide spectrum of what individual patients need. And the real question I think going forward is how to end these debates and how to provide really more tailored information so clinicians and patients can make better decisions together. And I think that's really where the piece that Glenn has written really helps direct us. Dr. Carolyn Lam:               Yeah, Glenn, I mean are we talking about the usual risk versus benefits and precision metsan or individualized risk assessment here? Dr. Glenn Levine:             Yeah, what we're talking about is looking at the ischemic risks which are primarily leg stent thrombosis or spontaneous MI versus the bleeding risks which is obviously bleeding and balancing them. And there clearly are decision tools available to clinicians. Laura has pioneered the DAPT score which is an incredibly user friendly and easy tool to use to assess which patients should be continued with prolonged DAPT or not. And there are also some other tools out there including the Paris registry score perhaps a little more complex and then there's also now the precise DAPT score which one can at least assess bleeding risk and indirectly assess the ischemic and bleeding risk.                                                 But really I think that is the focus now on balancing bleeding and ischemic risks and having pools to allow clinicians to easily do that. Dr. Carolyn Lam:               That's true. Now do you think guidelines have to catch up or have they caught up? Dr. Glenn Levine:             Our DAPT duration guideline was coming out just as Laura's DAPT score was about to be published, several months after it had been presented. And we did mention the DAPT score in our paper, it was too early to formally incorporate it into the guidelines. Nevertheless, the way our guidelines are written, they clearly give practitioners the option for individualizing therapy based on ischemic and bleeding risk and Laura's DAPT score fits perfectly into what we aim to do, namely to encourage practitioners to assess patients on an individual level and assess what duration of DAPT is best. Dr. Carolyn Lam:               I do have a question for Laura here though. I see Asian patients, I'm talking to you here from Singapore. And sometimes you wonder the trial situations and what you derive there. How does it differ from real world and how is it impacting your practice for example Laura? Dr. Laura Mauri:               That's a great question. I think you have a number of points there. One is the generalized ability or results from one trial across the world where you might have many different patient populations. And while the DAPT score was an international trial it would be interesting to see more data coming out from other different countries. And as you know there are trials in Asia that have looked at randomized DAPT duration as well.                                                 I think now that we have better access to information especially in cardiology globally, we can get that information and better tailor therapy. When we look at any one randomized trial the results might seem kind of black and white and to certain extent so do guideline recommendations but we are getting better at using the results from randomized trials to really identify risk factors. I think that with time we'll be able to either validate the DAPT score in other patient populations or develop tailored scores from unique data sets. I think the challenge really is making sure that we still get good randomized evidence for our treatment decisions but then when we have treatments that have both benefit and risk that we identify which sub-populations of patients really do achieve most of the benefit. And then the other populations that might be harmed. And that's really what we try to do with this score.                                                 And I think what you'll see, you asked about precision medicine which usually we think about using genetics but I think there's so much just really basic information that we have about patient lesion characteristics and other specific factors that we record routinely in their medical records that we can use and you'll see this, I think more and more frequently across different areas of investigation and in cardiovascular medicine.                                                 One really interesting example recently was this French trial. Data was used to be able to predict, very similar to what we did, but to predict which patients would benefit from lower blood pressure without the risk of more aggressive treatment. Dr. Carolyn Lam:               Yeah, I love the way you put that. Those are really words of wisdom, I do think that that is the way cardiovascular medicine is gonna move. Glenn, how do you put all this into practice for yourself? Dr. Glenn Levine:             I think whether or not I formally calculate a DAPT score or Paris registry score, I think clearly we integrate the factors in those scores into our everyday practice. And clearly there are patients who are at high bleeding and low ischemic risk and vice versa. I would also encourage listeners to in addition to all the scores, one has to think about the consequences of a recurrent MI or stent thrombosis. Obviously someone who has stent thrombosis of a proximal LED lesion, if they already have a depressed EF or occluded RCA, those consequences are likely much more dire then someone who occludes say at a distal OM3 stent who has the normal ejection fraction. It also encourages them to think about the consequences of stent thrombosis as well as the consequences of a recurring MI. Dr. Laura Mauri:               Just to make it clear, we know that clinicians have always tried to balance these different risks of ischemia and bleeding when faced with this decisions. I think the challenge really has been the limited amount of information that we've had to be able to do that. And so we've really just used kind of our gut until recently when we've had several large randomized data sets to be able to look to. And what that's done is it's given us the ability to construct these new tools to be able to make practice more data driven. Now still individualized but based on data that's tailored to our patients.                                                 And so I think we can use that to be able to improve outcomes. That being said, we don't want to rely on a statistic or a score alone and things like the DAPT score are based on patients like the ones that were enrolled into the randomized trial. Those were patients who could take longer anti-platelet therapy. It helps to identify who can take it for longer. But there are patients who get anti-coagulation or have other serious bleeding risks who really are going to benefit from new technologies to be able to shorten anti-platelet therapy. Dr. Carolyn Lam:               Well thank you Glenn once again for a wonderful perspective piece that has really got us thinking about situations even beyond dual anti-platelet therapy. Thank you Laura for your insights and thank you listeners for joining us today. Join us again next week.

Dr. Thomas Povsic and Dr. C. Michael Gibson Discuss

Commentary by Dr. Valentin Fuster

Interview with Eugene O. Major, PhD, author of JC Virus in CD34+ and CD19+ Cells in Patients With Multiple Sclerosis Treated With Natalizumab

A study in the March issue of Gastroenterology examined whether mobilization of bone marrow–derived stem cells with granulocyte colony–stimulating factor (G-CSF) might promote hepatic regeneration for patients with acute-on-chronic liver failure (ACLF).

Background: Cardiovascular disease has been linked to endothelial progenitor cell (EPC) depletion and functional impairment in atherosclerosis and aortic stenosis. EPCs may play a pivotal role in vascular grafting. However, the EPC depletion in coronary artery bypass grafting (CABG) patients has not been compared to coronary artery disease-free valvular replacement patients with aortic stenosis. Methods: We aimed to assess the basal number of CD34(+)/KDR+ and CD34(+)/CD144(+) cells in CABG patients, compared to aortic stenosis valvular replacement patients. 100 patients (51 CABG and 49 valvular surgery ones) were included in the present study. All CABG or valvular patients had angiographic demonstration of the presence or the absence of coronary artery disease, respectively. Numbers of CD34(+)/KDR+ and CD34(+)/CD144(+) were assessed by flow cytometry of pre-surgical blood samples. Results: We found a lower number of CD34(+)/CD144(+) cells in CABG patients compared to valvular patients (0.21 +/- 0.03% vs. 0.47 +/- 0.08%), and this difference remained statistically significant after the P was adjusted for multiple comparisons (P = 0.01428). Both groups had more EPCs than healthy controls. Conclusions: Pre-surgical CD34(+)/CD144(+) numbers are decreased in CABG patients, compared to valvular patients with absence of coronary disease.

Thu, 7 Jul 2011 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/13426/ https://edoc.ub.uni-muenchen.de/13426/1/Gatti_Monika.pdf Gatti, Monika

Die allogene Knochenmarks- bzw. Stammzelltransplantation wird seit den späten 70er Jahren als kurativer Behandlungsansatz bei myeloproliferativen Syndromen wie Leukämien und Lymphomen etabliert. Eine schwere und häufige (25-45%) Komplikation dieser Transplantation ist die Wirt-gegen-Spender-Erkrankung bzw. Graft-versus-Host Disease (GvHD). Die Erkrankung ist mit einer hohen Letalität von etwa 30% unter moderner Therapie verbunden und manifestiert sich häufig zunächst an der Haut. Eine zuverlässige und rasche Diagnosesicherung ist für die Früherkennung und adäquate Therapie der GvHD entscheidend. Leider ist die akute, das heißt binnen 100 Tagen nach Transplantation auftretende GvHD (aGvHD) von akuten Arzneimittelreaktionen (AR) klinisch und histologisch schwer zu unterscheiden. Etablierte Kriterien für diese Differentialdiagnostik existieren nicht. Die Feststellung des histologischen Schweregrads der aGvHD ist bislang eher untersucherabhängig, die des klinischen Schweregrads ist dermatologisch sehr grob und zur Verlaufskontrolle eher ungeeignet. Diese Punkte zu optimieren und einen Beitrag zur Aufklärung der Immunpathologie der aGvHD zu leisten waren die Hauptziele der vorliegenden Dissertation. Zwanzig Patienten mit klinisch gesicherter aGvHD nach allogener Knochenmarks- oder Blutstammzelltransplantation und dreizehn Patienten mit klinisch verifizierter AR wurden in die Studie aufgenommen. Die klinischen Befunde wurden nach dem etablierten Glucksberg-Score sowie dem neu entwickelten klinischen GvHD-Schweregrad-Score (GvHSco) klassifiziert. Zusätzlich wurden Hautproben entnommen und histopathologisch sowie immunhistochemisch (Expression von CD1a, CD2, CD11c, CD20, CD25, CD34, CD68, CD197, CD206, CD207, CD 208, CD209, CD303 und S100) analysiert. Klinische und histologische Ergebnisse wurden einzeln analysiert und miteinander korreliert. Zur besseren Beschreibung des klinischen Schweregrades der kutanen GvHD wurde der klinische GvHSco a priori entwickelt. Er bietet durch die Standardisierung und die hundertteilige Skala im Vergleich zum Glucksberg Score Vorteile bezüglich der individuellen Verlaufskontrolle. Als histologische Schweregradkriterien korrelierten epidermotrope lymphozytäre Infiltration und Kontinuitätsverluste der Basalmembran (Epidermolyse) am deutlichsten mit dem klinischen Schweregrad. Aufgrund dieser Ergebnisse wurde auf der Basis des histologischen Scores nach Lerner durch Ergänzung des Kriteriums Epidermolyse und durch besondere Gewichtung des Kriteriums Lymphozyteninfiltration der Modifizierte Histologische Score zur Abschätzung des Schweregrads akuter GvHD (GvHiScore) entwickelt. Die Vorteile dieser modifizierten Klassifikation sind die genaue, Untersucher-unabhängige Definition und die feinere Stratifizierung der Schweregrade. So wird eine bessere inter- und intraindividuelle Differenzierbarkeit erreicht. Als differentialdiagnostische Parameter sprachen hohe Zahlen reifer T-Zellen (CD2+, CD45RA+) und Makrophagen (CD68+), Epidermolyse, Basalzellballonierung, junktionales lymphozytäres Infiltrat differentialdiagnostisch für aGvHD, eosinophiles Infiltrat jedoch gegen eine aGvHD. Basierend auf diesen neuen Erkenntnissen wurde der differentialdiagnostische Test DSHIG („Differentialdiagnostischer Score mittels Histopathologie und Immunhistochemie für akute Graft versus Host Disease“) entwickelt. Der Test errechnet sich aus der Addition sieben dichotomer Kriterien. Die retrospektive Analyse des DSHIG ergibt eine Testspezifität und -sensitivität von 95% für die Differentialdiagnose „Akute GvHD“ versus „Akutes Arzneiexanthem“. Der differentialdiagnostisch vielversprechende DSHIG sollte prospektiv validiert werden. Bei der Lupusband-positiven akuten GvHD zeigte sich ein histologisch besonders schweres Bild mit ausgeprägter Epidermolyse. Ein Einfluss quoad vitam oder auf den klinischen Schweregrad ließ sich nicht zeigen. Die Lupusband-positiven Fälle traten bevorzugt in der späteren Phase von aGvHD auf. Für den klinischen Schweregrad und das Ein-Jahres-Überleben bei aGvHD günstig waren hohe Zellzahlen von IDEC (CD206+/CD11c+), plasmazytoiden Dendritischen Zellen (BDCA-2+) und Mastzellen. Diese Zusammenhänge wurden bislang nicht an Hautbiopsien gezeigt und könnten klinisch bedeutsam sein. Die in dieser Arbeit an Hand einer kleineren Fallzahl retrospektiv erstellten Scores sollten in zukünftigen Untersuchungen mit höherer Patientenzahl unabhängig prospektiv validiert werden. Die Dynamik der kutanen GvHD könnte darüber hinaus mit weitern Methoden wie durchflußzytometrischer Analyse und Gewinnung von sequentiellen Hautproben im zeitlichen Verlauf analysiert werden.

Objective: Liver regeneration is mainly based on cellular self-renewal including progenitor cells. Efforts have been made to harness this potential for cell transplantation, but shortage of hepatocytes and premature differentiated progenitor cells from extra-hepatic organs are limiting factors. Histological studies implied that resident cells in adult liver can proliferate, have bipotential character and may be a suitable source for cell transplantation. Methods: Particular cell populations were isolated after adequate tissue dissociation. Single cell suspensions were purified by Thy-1 positivity selection, characterised in vitro and transplanted in immunodeficient Pfp/Rag2 mice. Results: Thy-1+ cells that are mainly found in the portal tract and the surrounding parenchyma, were isolated from surgical liver tissue with high yields from specimens with histological signs of regeneration. Thy-1+ cell populations were positive for progenitor (CD34, c-kit, CK14, M2PK, OV6), biliary (CK19) and hepatic (HepPar1) markers revealing their progenitor as well as hepatic and biliary nature. The potential of Thy-1+ cells for differentiation in vitro was demonstrated by increased mRNA and protein expression for hepatic (CK18, HepPar1) and biliary (CK7) markers during culture while progenitor markers CK14, chromogranin A and nestin were reduced. After transplantation of Thy-1+ cells into livers of immunodeficient mice, engraftment was predominantly seen in the periportal portion of the liver lobule. Analysis of in situ material revealed that transplanted cells express human hepatic markers HepPar1 and albumin, indicating functional engraftment. Conclusion: Bipotential progenitor cells from human adult livers can be isolated using Thy-1 and might be a potential candidate for cell treatment in liver diseases.

Mortality due to acute myocardial infarction has been drastically reduced by optimal means of intervention, but loss of myocytes is leading to heart failure later on and with it to an impaired quality and expectancy of the patient´s life. The research of adult stem and progenitor cells gave hope that transplantation of these cells into infarcted tissue could restore its contractile function. One potentially therapeutically viable kind of cells are endothelial progenitor cells, but there are only limited numers of cells available. Also at present there is no consensus how best to obtain endothelial progenitor cells. In this study we used two different procedures of culturing endothelial progenitor cells from peripheral blood that had been applied regularly in earlier studies and compared both cell types with each other, regarding their manner of growth, their morphology, expression of surface markers and their angiogenic potential on matrigel. It turned out that cells, which were obtained from unfractioned mononuclear cells, survived only for a short period of time in culture, showed little proliferation and expressed different kinds of markers. We called these cells angiogenic cells. Cells which were obtained from CD34+ mononuclear cells on the other hand survived in culture for up to 20 weeks, were highly proliferative and showed a homogenous expression of markers. We refered to these cells as endothelial cells. Both kinds of cells were transplanted into the myocardium of athymic nude rats, after experimental infarction and the ventricular function was assessed via sonography. After 14 days a significant improvement of the ventricular function was found in both cell groups compared to the control group that had only received culture medium, but no significant effect on the size of the infarction was found. Assessment of left ventricular morphology showed a significant decrease of ventricular dilation and wall thinning in infarction area. Vessel formation was studied after 3 and 14 days, using markers for smooth muscle cells and von Willebrand factor-positive cells. No differences were found in angiogenesis. These results show that different ways of obtaining endothelial progenitor cells lead to distinctly different cell populations which however have very similar effects after transplantation into the infarcted area, which is an improvement of ventricular function and remodeling, probably caused by paracrine effects.

Bei dem Kaposi-Sarkom (KS) handelt es sich um eine eng mit dem Humanen-Herpes-Virus 8 (HHV 8) assoziierte vaskuläre Neoplasie. Die Etablierung eines kommerziell erhältlichen, stabilen Antikörpers gegen das virale „Latenz-assoziierte-nukleäre Antigen“ (LANA) von HHV 8 ermöglicht den Nachweis HHV-8-infizierter Zellen an Ex-vivo- Paraffinmaterial. In dieser Arbeit wird diese Methode an einem Kollektiv aus insgesamt 61 Patienten mit HIV-assoziiertem KS angewendet, um einen Beitrag zur Beantwortung von bislang ungeklärten Fragen nach der quantitativen Verteilung von HHV 8 im Organismus und zur formalen Pathogenese des KS zu leisten. Das Kollektiv besteht aus diagnostischen Exzisaten (20 Früh- und 20 Spätstadien des KS) sowie aus 21 Sektionsfällen mit KS wovon Lympknoten, Leber-, Milz- und Lungengewebe untersucht wurden. Durch die Etablierung und Auswertung technisch aufwendiger immunhistochemischer Doppelfärbeexperimente mit dem viralen Protein LANA und verschiedenen strukturellen und regulatorischen Humanproteinen (CD31, CD34, Ki67 und p21) sowie durch die Anpassung des LANA-Nachweises an Autolyse- bzw. Sektionsmaterial kommen wir in der vorliegenden Arbeit zu folgenden Ergebnissen: Die KS-Progression geht von der Proliferation nicht-infizierter Zellen aus. Dies spricht für ein parakrin-stimuliertes KS-Wachstum und stützt Hypothesen, die das KS als viral-getriggerte, reaktive Angioproliferation einstufen. Außerdem unterscheidet dieser Befund das KS von dem ebenfalls HHV-8-assoziiertem „Primären Effusions Lymphom“ (PEL), dessen Progression ein autonomer Zellzyklus zugrunde liegt. Weitere Ergebnisse dieser Arbeit stützen die mittels In-vitro-Experimenten von Wang et al. aufgestellte Hypothese, dass HHV 8 das Expressionsmuster der infizierten Zellen in Richtung der endothelialen Vorläuferzelle verändert. Durch Koexpressionsexperimente mit Endothelzellmarkern kann außerdem gezeigt werden, dass die HHV-8-infizierten Zellen an der Gefäßbildung im KS direkt beteiligt sind. Schließlich lassen sich im Sektionsmaterial HHV-8-infizierte mononukleäre Zellen in mehreren Organen als disseminierte Einzelzellen außerhalb der klinisch/makroskopisch sichtbaren KS-Läsionen nachweisen, und es finden sich mikroskopisch kleine KS-Frühstadien. Der bisher nur in vitro durch den Nachweis von HHV-8-infizierten Zellen im Blut vermutete formalpathogenetische Weg, wonach HHV-8-infizierte mononukleäre Zellen zur Infektion der endothelialen KS- „Zielzellen“ beitragen, wird somit ex vivo an dem Untersuchungskollektiv histomorphologisch belegt.

The hallmark of hematopoietic stem cells (HSC) is their ability of self-renewal and differentiation into multiple hematopoietic cell lineages. Although the molecular network controlling stem cell fate decisions is largely unknown, multiple studies have attributed a key role to transcription factors in this developmental process. In this context the family of homeobox genes was characterized as ‘master genes’ of this early hematopoietic development. The identification of new genes involved in normal and leukemic hematopoiesis and the development of therapies against deregulated processes in hematopoiesis are the major goals in experimental and clinical hematology. . The identification of new genes involved in normal and leukemic hematopoiesis and the development of therapies against deregulated processes in hematopoiesis are the major goals in experimental and clinical hematology. Therefore, the focus of this thesis was the characterization of two novel putative regulatory proteins of early human hematopoiesis, the hematopoietic PBX-interacting protein (HPIP) and the human Vent-like Homeobox gene (VENTX2) and to investigate to the activity of the FLT3 protein kinase inhibitor SU5614 on leukemic blast from AML patient samples. Using complex in vitro assays we analyzed the impact of constitutive expression of HPIP and VENTX2 on stem cell and early human hematopoietic development. To detect clonal progenitor cells primary and secondary colony-forming-unit (CFC) assays were performed. In addition the in vitro equivalent of HSC long-term culture initiating cells were detected with the (LTC-IC) assay. We were able to show that the constitutive expression of HPIP can rapidly lead to increased numbers of cells detected on the level of committed clonogenic progenitor cells and LTC-ICs. In addition, the production of CFC per LTC-IC is markedly enhanced when cord blood (CB) cells are transduced with HPIP as compared to the control. Notably, besides its effect on maintenance of primitive hematopoietic progenitor cells, constitutive expression of HPIP did not block terminal hematopoietic differentiation. Additional we could show that the constitutive expression of HPIP leads to an increase of myeloid cells in transplanted NOD/SCID mice. These data characterize HPIP as a novel regulator of the early human hematopoietic stem cell, demonstrating that its constitutive expression has a notable impact on self renewal and differentiation of human hematopoietic stem cells. In vitro and in vivo analyses shed light on the understanding to the function of the homeobox gene VENTX2. On the level of the most primitive hematopoietic progenitors we could not observe a significant increase in the frequency of HSCs. Furthermore, the number of colonies generated per LTC-IC did not significantly differ between the VENTX2 arm and the control arm. A strong effect was obtained on the level of clonogenic progenitor cells. VENTX2 increased the production of myeloid cells 1.7-fold in comparison to the control. Secondary replating assay confirmed the amplificatory effect of VENTX2 transduced cells in the number of secondary G-CFU indicating that VENTX2 promote myeloid lineage differentiation. Interestingly, on the level of clonogenic progenitors VENTX2 expression resulted in a significantly decreased growth of erythroid colonies by 4.2-fold compared to the control suggesting that constitutive expression of VENTX2 may inhibit early erythroid differentiation. This inhibition did not occur on the level of primitive hematopoietic cells detected by Limiting Dilution LTC-IC assay where VENTX2 increased within a 2.2 fold compared to the control. The observation that VENTX2 overexpression drives CD34+ to differentiate into myeloid lineage was additional proved by in vivo experiments. In NOD/SCID mice VENTX2 induced a 3-fold increase in the proportion of CD15+ mature myeloid cells within the GFP-positive compartment compared to the control. A 7-fold increase was observed in the total of CD38+ GFP+ cells in comparison to the MIG mice control (p

Leukämische Blasten bei AML können ex vivo in DC umgewandelt werden, wodurch Anti-gen-präsentierende Zellen entstehen, welche leukämische Antigene präsentieren. Im kleineren Rahmen sollten zunächst Daten zu methodischen Vorversuchen ausgewertet werden, um bei AML und MDS unter serumfreien Bedingungen DC zu generieren. Diese me-thodischen Vorversuche an 50 AML-, 24 MDS-Patienten und 23 gesunden Probanden erga-ben, dass adhärente Zellfraktionen die DC-Ausbeute im Vergleich zu totalen MNC-Fraktionen nicht verbessern, dass bei MACS-depletierten `MNC(-)`- und aufgetauten MNC-Fraktionen niedrigere DC-Zahlen erreicht werden, und dass die DC-Ernte bei AML- und MDS-Patienten nach 10-14tägiger Kulturzeit, bei gesunden Probanden jedoch nach 7tägiger Kulturzeit höher ist. Außerdem zeigte sich, dass die Zugabe von FL die DC-Ernte erhöht, der Einsatz von autologem Plasma dagegen in vielen Fällen einen inhibitorischen Effekt auf die DC-Generierung hat. Die Kulturmedien CellGro und Xvivo erzielten vergleichbare DC-Ausbeuten. Nach Ermittlung der optimalen Zellfraktion, Kulturdauer und –zusätze, wurden serumfreie DC von 100 AML-, 55 MDS- und 38 gesunden Proben in einem 10-14-tägigem serumfreien Xvivo-Kultursystem mit GM-CSF, IL-4, FL und TNFα angezüchtet und charak-terisiert. Bei der DC-Generierung unter standardisierten Bedingungen betrug die Ausbeute der MNC-Fraktionen durchschnittlich 20% bei MDS, 34% bei AML und 25% bei gesunden Probanden. Zwischen 53-58% der DC waren reife CD83+DC. Die DC-Ernten waren in den monozytären FAB-Klassen (AML-M4/5, MDS-CMML) am höchsten, dagegen unabhängig von den zyto-genetischen Risikogruppen. Das Oberflächenmarkerprofil der DC von den AML- und MDS-Proben (einschließlich 1 MDS- und 3 AML-Zelllinien) war mit dem der gesunden DC ver-gleichbar. In parallelen Kulturansätzen konnte außerdem gezeigt werden, dass in einem `MCM-Mimic`-Medium mit PGE2 der Anteil reifer, CCR7+DC besonders hoch war. Der leu-kämische Ursprung der AML- und MDS-DC wurde bei 5 AML- und 4 MDS-Fällen mittels FISH durch die Persistenz klonaler, zytogenetischer Aberrationen in den DC oder in übrigen Fällen durch die Coexpression leukämischer Antigene auf den DC bewiesen. Durch eine kombinierte FISH/Immunophänotyp-Analyse (FISH-IPA) konnte zudem nachgewiesen wer-den, dass obige klonale, numerische Aberrationen konstant in Kombination mit DC-Markern detektierbar, jedoch nicht alle klonalen Zellen zu leukämischen DC umwandelbar waren (durchschnittlich 53% der AML- und MDS-Blasten). Umgekehrt trugen auch nicht alle gene-rierten DC die klonale Aberration (im Durchschnitt 51% der DC). In 41 AML-Fällen mit ei-ner Leukämie-spezifischen bzw. aberranten Antigenexpression oder in AML-Fällen mit ei-nem CD33+ Blastenphänotyp und gleichzeitig

Adult mesenchymal stem cells with multilineage differentiation potentially exist in the bone marrow, but have also been isolated from the peripheral blood. The differentiation of stem cells after leaving their niches depends predominately on the local milieu and its new microenvironment, and is facilitated by soluble factors but also by the close cell-cell interaction in a three-dimensional tissue or organ system. We have isolated CD34-negative, mesenchymal stem cell lines from human bone marrow and peripheral blood and generated monoclonal cell populations after immortalization with the SV40 large T-antigen. The cultivation of those adult stem cell clones in an especially designed in vitro environment, including self-constructed glass capillaries with defined growth conditions, leads to the spontaneous establishment of pleomorphic three-dimensional cell aggregates ( spheroids) from the monoclonal cell population, which consist of cells with an osteoblast phenotype and areas of mineralization along with well-vascularized tissue areas. Modifications of the culture conditions favored areas of bone-like calcifications. After the transplantation of the at least partly mineralized human spheroids into different murine soft tissue sites but also a dorsal skinfold chamber, no further bone formation could be observed, but angiogenesis and neovessel formation prevailed instead, enabling the transplanted cells and cell aggregates to survive. This study provides evidence that even monoclonal adult human CD34-negative stem cells from the bone marrow as well as peripheral blood can potentially differentiate into different mesenchymal tissues depending on the local milieu and responding to the needs within the microenvironment. Copyright (C) 2005 S. Karger AG, Basel.

Die vorliegende Arbeit beschäftigt sich mit der funktionellen und molekularen Charakterisierung von humanen CD34- Zelllinien aus dem peripheren Blut (V54/1, V54/2) im Vergleich zu den aus dem Knochenmark etablierten Zelllinien (L87/4, L88/5). Die Klone V54/1 und V54/2 wurden aus dem peripheren Blut nach Stammzellmobilisierung und CD6 Depletion durch Zugabe eines Faktorengemisches aus IL-1b, IL-3, IL-6, IL-7, IL-8 und IL-11 erzeugt. L87/4 und L88/5 hingegen sind adhärente und wachstumsarretierte Stromazellen, die die Erhaltung und Differenzierung von hämatopoetischen Vorläuferzellen durch Mediatoren ermöglichen (Thalmeier et al. 2000). Das Ziel dieser Arbeit war die Untersuchung von Stammzelleigenschaften bei den Zelllinien L87/4, L88/5, V54/1 und V54/2. Dazu soll die Färbung mit den Farbstoffen Rhodamin 123 (Rh123) und Hoechst 33342 zeigen, ob Subpopulationen innerhalb der Klone mit unterschiedlichen Färbeeigenschaften, bestehen. Die biologische Bedeutung der beiden Farbstoffe liegt darin, dass Sie dazu geeignet sind frühe Stammzellen zu identifizieren. Als Substrat der P-Glykoproteinpumpe, die u.a. auf frühen Vorläuferzellen mit stark erhöhter Repopulationskapazität gefunden wird, werden diese Farbstoffe aus der Zelle gepumpt. Der Farbstoff-Efflux kommt durch die mdr-Gen-kodierte (multi-drug-resistance) und Kalzium-abhängige P-Glykoproteinpumpe zustande. Das P-Glykoprotein hat neben der Bedeutung in der Stammzellbiologie in der angewandten Medizin eine wichtige Funktion in der Resistenzentwicklung von Tumoren. Des weiteren wurden bei den Zelllinien stammzellrelevante Oberflächenantigene (CD10, CD34, CD14, CD105, SH3 und CD117) untersucht, um Unterschiede zwischen L87/4, L88/5 und den Klonen V54/1, V54/2 zu erkennen. Versuche zur Induktion der Differenzierung sollten Hinweise auf die Plastizität der Zelllinien geben. Experimente an den durch den Rh123-Efflux unterscheidbaren Subpopulationen der Zelllinie V54/2 dienen der Aufklärung von Unterschieden in Morphe, zellulären Transportfunktionen und Funktionseinheiten von Transkriptionsfaktor Netzwerken. Methodisch wurde für die Analyse der Epitope und der Färbungen mit Rh123 und Hoechst 33342 ein Durchflußzytometer verwendet. Die Analyse der Funktionseinheiten von Transkriptionsfaktor Netzwerken wurde mittels Reverse Transkriptase Polymerase Ketten Reaktion durchgeführt. Die Ergebnisse der Färbeexperimente zeigten, dass bei allen untersuchten Zelllinien durch eine unterschiedliche Anfärbbarkeit der Zellen mit dem Farbstoff Rh123 zwei Subpopulationen unterschieden werden können. Die jeweils größere Subpopulation der Zelllinien färbt sich mit Rh123 an und bleibt auch nach einer definierten Inkubationszeit, die den Rh123-Efflux ermöglichen soll, gefärbt. Sie wird Rh123high genannt. Die übrigen Zellen, die bei allen Zelllinien unter 10% der Gesamtpopulation betragen, sind in der Lage den Farbstoff aus der Zelle zu pumpen. Diese Subpopulation wird Rh123low genannt und ist mit Stammzelleigenschaften wie tausendfach erhöhter Repopulationsfähigkeit in NOD/SCID-Mäusen assoziiert. Es konnte also innerhalb der untersuchten monoklonalen Linien eine Rh123low Subpopulation identifiziert werden, die sich durch zahlreiche biologische Eigenschaften von der Gesamtpopulation unterscheidet. Da der Rh123 Efflux durch eine Kalzium-abhängige Pumpe zustande kommt, lässt sie sich durch den Kalziumantagonisten Verapamil hemmen. Eine Hemmung der Pumpe bewirkt, dass die Rh123low Zellen nicht mehr in der Lage sind Rh123 aus der Zelle zu pumpen, so dass sie nach einer definierten Inkubationszeit mit Rh123 gefärbt bleiben. Neben diesem funktionellen Beweis für die P-Glykoproteinpumpe konnte durch den strukturellen Nachweis der Pumpe mittels eines Antikörpers gegen P-Glykoprotein ein definitiver Beweis für das Vorhandensein der aktiven P-Glykoproteinpumpe bei der Rh123low Population erbracht werden. Mit dem anderen Farbstoff Hoechst 33342 können die jeweiligen Anteile der Zelllinien in den einzelnen Stadien des Zellzyklus nachgewiesen und zudem ein kleiner Anteil an Zellen bestimmt werden, der als „Side Population“ (SP-Zellen) definiert wird. Diesen SP-Zellen werden Eigenschaften von aktiven Stammzellen zugeschrieben. Hierbei besteht ein Unterschied zwischen den aus dem Knochenmark und den aus dem peripheren Blut etablierten Linien, da die Zellen aus dem peripheren Blut nicht nur ein anderes Zellzyklusmuster aufweisen, sondern auch einen höheren Anteil an SP-Zellen besitzen. Es wurden vergleichende Untersuchungen zwischen den Zelllinien und zwischen den Rh123high und Rh123low Subpopulationen innerhalb einer Zelllinie mit Antikörpern gegen die Epitope CD14, CD45, HLA-DR, CD10, CD117, CD105 und SH3 durchgeführt. Dabei waren CD14 und CD45 auf allen Zelllinien negativ, wobei alle Zelllinien eine positive Expression für den mesenchymalen Marker Endoglin (CD105) und für SH3 (CD73) zeigten. CD117 konnte nur auf den aus dem Knochenmark etablierten Zelllinien L87/4 und L88/5 nachgewiesen werden. CD34, ein charakteristischer Marker für hämatopoetische Vorläuferzellen, aber auch für Endothelzellen, konnte nur auf den Zellen der Rh123low Subpopulation nachgewiesen werden. Im Gegensatz dazu exprimieren die Rh123high Zellen kein CD34. Da es sich bei den Zelllinien um Klone handelt, ist der Unterschied in der Expression von CD34 zwischen der Rh123low und der Rh123high Population ein deutlicher Hinweis auf die Plastizität der Zelllinien und das Fließgleichgewicht zwischen Rh123low und Rh123high. Durch eine Zellsortierung der Zelllinie V54/2 wurde die Rh123low von der Rh123high Subpopulation getrennt, um sie dann bezüglich ihrer Morphologie, dem Wachstum in Methylzellulose und der Expression ausgewählter Funktionseinheiten von Transkriptionsfaktor Netzwerken zu untersuchen. Dabei erhärtete sich die Hypothese, dass es sich bei der Rh123low Subpopulation um aktivere Zellen mit einer gesteigerten Expression von erythroid/myeloischen und mesodermalen Eingaben (z.B. VEGF, BMP-4), Rezeptoren (z.B. tie-1), vernetzter Transkriptionsfaktoren (z.B. GATA, ETS) und letztendlich Ausgaben (z.B. PECAM) handelt. Diese fungieren in Netzwerken mit dem Ziel, stammzellrelevante Funktionen zu ermöglichen. Die Morphologie zeigte in den Zytozentrifugationspräparaten deutliche Unterschiede zwischen Zellen der Rh123low und der Rh123high Subpopulation. Die Rh123low Subpopulation besteht aus lymphoid-ähnlichen Zellen, was für Zellen mit Stammzellfunktion charakteristisch ist. Die Rh123high Subpopulation dagegen hat ein insgesamt größeres Zellvolumen und einen gebuchteten Kern mit perinukleärer Aufhellung. Untersuchungen des klonalen Wachstums in der Methylzellulose ergaben bei keiner der Subpopulationen eine wesentliche Koloniebildung. Durch die Inkubation der Zelllinie V54/2 mit dem Neurotropen Wachstumsfaktor (NGF) konnte eine morphologische Änderung in Richtung einer neuronalen/glialen Differenzierung nach 8-12 Stunden induziert werden. Der immunhistochemische Nachweis von Glial Fibrillary Acidic Protein (GFAP) bestätigte die mesenchymale Potenz zumindest in Richtung einer glialen Differenzierung. Das unterschiedliche Expressionsmuster ausgewählter, für die Differenzierung notwendiger Zusammenspieler innerhalb von Transkriptionsfaktor Netzwerken innerhalb der Rh123high und der Rh123low Population bei V54/2 war ein weiterer Hinweis, dass es sich bei der Rh123low Subpopulation um aktive Vorläuferzellen mit möglicher Stammzellpotenz handelt. In der Rh123low Subpopulation wurde im Gegensatz zur Rh123high Population eine Expression von BMP4, GATA1, GATA3 nachgewiesen, die essentiell für die Hämatopoese und für eine mesenchymale Differenzierung ist. Die Faktoren für GATA2, GATA3, beta globin, Elf-1 und PECAM1 wurden in einem stärkeren Maß in der Rh123low als in der Rh123high Population exprimiert. BMP-Rez., Myb, sowie die Endothel-assoziierten Faktoren Tie-1 und VEGF waren in beiden Subpopulationen gleich stark vorhanden. Bei den wenigen Funktionseinheiten der größeren und Rh123high Population handelt es sich vor allem um angiogenetische Faktoren, was auf eine limitierte Differenzierungseigenschaft der Rh123high Subpopulation und die enge Beziehung zwischen Blut- und Endothelzellen („Hämangioblast“) hinweist. Ein Nachweis für die Plastizität der Stammzellen innerhalb der von uns etablierten Zelllinien wurde dadurch erbracht, dass die zellsortierten Subpopulationen Rh123low und Rh123high nach dem Sortierexperiment getrennt rekultiviert wurden, wobei das Wachstum der Rh123low Subpopulation deutlich langsamer war als das der Rh123high Subpopulation. Nach zwei Wochen wurden die zellsortierten Subpopulationen erneut einer Rh123 Färbung unterzogen, wobei sich wiederum das ursprüngliche Verhältnis zwischen den Rh123low und Rh123high Subpopulationen einstellte. So kann man aus der Transdifferenzierung der Zelllinien von Rh123low in Rh123high und umgekehrt die Plastizität der hier untersuchten adulten Stammzelllinien ableiten. Die Ergebnisse sollen zum grundlegenden Verständnis der Biologie adulter (nicht embryonaler) Stammzellen beitragen und damit die Möglichkeit schaffen, adulte Stammzellen bzw. deren Subpopulationen gezielt für einen reparativen Gewebe- und Organersatz zu verwenden. Dabei liefern sie die Basis für weitergehende Untersuchungen zum besseren Verständnis der physiologischen und regenerativen Vorgänge, z.B. auch bei Alterung oder bei gesteigerter Funktion. Darüber hinaus kann aufgrund der vorliegenden Ergebnisse durch weitere Untersuchungen möglicherweise besser verstanden werden, ob es gelingen kann das Potential adulter Stammzellen zur therapeutischen Gewebereparation, z.B. zur Verhinderung oder Verringerung einer Narbenbildung, zu nutzen.

The human colon cancer cellline HT29 was investigated in various models regarding different criteria. The in-vivo-experiments were carried out with female SCID mice and consisted of subcutaneous cell injection, orthotopic cell injection into the cecal wall and orthotopic fixation of a tumor fragment onto the cecum. The subcutaneous experiment took 41 days; the orthotopic animal experiments were equally divided into three points of necropsy each two weeks apart. On these days tumor, liver and lung were withdrawn, fixed in 3,8% formaldehyde and analyzed histologically. In addition blood samples of all animals of the orthotopic experiments were taken on days of autopsy and the CEA (carcinoembryonic antigen) content was determined using an ELISA. The vascularization of the orthotopic primary tumors was examined by staining of CD34, i.e. number and area of the tagged vessels were ascertained. Additionally the green fluorescent protein (GFP) was studied in view of its suitability as quantifiable reporter gene in these models. Therefore not only HT29-wildtype but also HT29 cells transfected with GFP were used in vitro and in the first two in vivo assays. Advantages and disadvantages summarized: - The subcutaneous model was realized easily, measurement of the primary tumor was simple, the tumor take rate was 100 % and the laboratory animals appeared to suffer only from a relative slight amount of stress. Besides these advantages this setting cannot be used for investigations regarding metastasis because of the low number of metastases. - The orthotopic cell injection generated small, hardly measurable primary tumors, but this approach is a suitable model of metastasis because of the number of metastases detected. The technical effort and the burden for the animals exceeded that found in the subcutaneous setting but was below the effort of orthotopic fixation of a tumor fragment. - Of all investigated models the orthotopic fixation of a tumor fragment represents the model with the greatest effort. The primary tumors were big enough to be measured, but the number of metastases was too low to make statistical valuable evaluations. - The CD34 staining successfully marked the vessels of the primary tumor and facilitated a computer-assisted quantitative analysis of the vascularization of orthotopic colon tumors. It was assessed that a broad neoangiogenesis occurred at the beginning of tumor growth prior to the development of metastases. The number of metastases increased with proceeding length of time, whereas the number of vessels decreased. The continuous extension in tumor volume resulted in a necrotic tumor center so that vessels were detectable in the border area only. - The used HT29-GFP clone was not stable enough to generate sufficient fluorescence. The cells both in vitro an in vivo grew more slowly, the subcutaneous tumors showed necrotic areas and there were less metastases after orthotopic injection of GFP cells than after injection of wildtype cells. Because of the insufficient fluorescence it was not possible to execute a quantifiable analysis of metastasis. The application of GFP was not advantageous within these models. - CEA suits to be a valuable tumor marker for colon cancer in both investigated models. The CEA content in the blood samples of tumor bearing animals increased dependent on tumor burden and tumor invasiveness. A direct correlation between tumor size and CEA might be established with an improved measurement system or rather an advanced measuring of tumor volume. Due to the comparative characterization of injection and implantation techniques as well as other detailed examinations carried out for this thesis, it is possible to select suitable models for preclinical trials depending on the individual purpose.

Verschiedene Transkriptionsfaktoren spielen eine Rolle in der Entwicklung myeloischer Zellen. PU.1, ein Transkriptionsfaktor aus der ETS-Familie, ist sowohl für die Entwicklung lymphatischer als auch für die Entwicklung myeloischer Zellen von Bedeutung. Der Transkriptions faktor C/EBPalpha, ein an den CCAAT-Enhancer bindendes Protein, ist hingegen wesentlich verantwortlich für die Entwicklung von Granulozyten. Wir stellen hier den ersten Nachweis dafür vor, dass C/EBPalpha die Funktion von PU.1 blockiert. PU.1 und C/EBPalpha können einander binden und sind in myeloischen Zellen kolokalisiert. Wenn C/EBPalpha PU.1 bindet, kann PU.1 einen minimalen Promotor mit Bindungsstelle für PU.1 nicht mehr aktivieren. Wir zeigen, dass der Leuzin-Zipper in der DNA-bindenden Domäne von C/EBPalpha mit der beta3/beta4-Region in der DNA-bindenden Domäne von PU.1 interagieren kann. Dadurch wird der Koaktivator von PU.1, c-jun, aus seiner Bindung mit PU.1 verdrängt. C/EBPalpha hemmt PU.1 nicht, indem es Korepressoren rekrutiert. Vielmehr vermindert C/EBPalpha die Expression von PU.1 in U-937-Zellen mit induzierbarem C/EBPalpha, indem es den autoregulatorischen Effekt PU.1 auf den PU.1-Promotor hemmt. Ausserdem blockiert C/EBPalpha die durch PU.1 bedingte Entwicklung dendritischer Zellen aus CD34+ menschlichen Nabel blutzellen. Diese funktionelle Blockade von PU.1 durch C/EBPalpha könnte einer der Mechanismen sein, mit denen C/EBPalpha den durch PU.1 determinierten Weg der Zelldifferenzierung hemmt und sich Zellen unter dem Einfluss von C/EBPalpha zu Granulozyten entwickeln.

High-dose chemotherapy with autologous peripheral blood stem cell transplantation is the standard treatment of patients with multiple myeloma today. In this study we used a combination mobilizing chemotherapy containing ifosfamide with G-CSF before stem cell collection. The chemotherapy regimen consisted of ifosfamide (2,500 mg/m(2) days 1-3), epirubicin (100 mg/m(2) day 1) and etoposide (150 mg/m2 days 1-3) followed by G-CSF (5 mug/kg from day 5). In 30 younger patients (median age 51 years; range 41-60 years) who received the IEV regimen in 100% dosage, a median of 11.15 x 10(6) CD34(+) cells/kg (range 0-44.60 x 10(6) CD34(+) cells/kg) was collected. In 22 elder patients (median age 64 years; range 59-72 years) similar collection results were obtained with a median of 10.82 x 10(6) CD34(+) cells/kg (range 0.99-42.22 x 10(6) CD34(+) cells/kg) after the IEV regimen in 75% dosage. The pretreatment chemotherapy cycles before mobilization were fewer in elder patients with a median of 0 cycles (range 0-7 cycles) compared with younger patients with a median of 4 cycles (range 0-7 cycles). These collection results were favorable and allowed to support a tandem transplantation procedure in younger and elder patients in 97 and 95%, respectively. In the majority of patients, the hematological toxicity of IEV was of WHO grade 3/4. The extramedullary toxicity was mild to moderate and there were only few cases (5-10%) of relevant nephrotoxicity or neurotoxicity associated with the application of ifosfamide. Copyright (C) 2003 S. Karger AG, Basel.

In meiner Arbeit sollte untersucht werden, ob eine Veränderung des Zelltropismus von Epstein-Barr Virus mit genetischen Methoden möglich ist. Ziel war es, durch Verwendung hybrider Glykoproteine oder Glykoproteine anderer Viren die Bindung bzw. die Fusion von EBV Partikeln zu vermitteln und dadurch eine sogenannte Pseudotypisierung zu erreichen. Zu diesem Zweck wurden im ersten Teil EBV Glykoproteinmutanten des viralen Liganden gp350 und des für die Membranfusion wichtigen gp85 Proteins hergestellt. Die Charakterisierung der gp350-negativen EBV Mutante zeigte im Gegensatz zu früheren Beobachtungen, daß gp350 sowohl für die Immortalisierung und Infektion von primären B-Lymphozyten als auch für die Infektion von Burkitt-Lymphom Zellinien wie Raji Zellen nicht absolut notwendig ist. Die Infektionseffizienz war jedoch reduziert. HLA-Klasse-II-negative Raji 2.2.5 Zellen konnten ebenfalls, wenn auch mit einer noch niedrigeren Infektionsrate, infiziert werden. Diese Ergebnisse weisen darauf hin, daß gp350 zwar der Hauptligand für die Infektion von B-Zellen sein dürfte, daß es aber einen gp350-unabhängigen Infektionsmechanismus gibt, der durch einen zusätzlichen viralen Liganden vermittelt wird. Die Identität eines solchen Liganden konnte im Rahmen dieser Arbeit nicht untersucht werden. Die Charakterisierung der gp85-negativen Mutante sowie der Doppel-KO-Mutante bestätigte, daß das gp85 Glykoprotein für die Infektion der Zielzellen von EBV essentiell ist. Durch die Infektion verschiedener, z. T. CD21-positiver Epithelzellinien mit der gp350-negativen Mutante konnte gezeigt werden, daß auch im Fall von Epithelzellen ein weiterer Ligand existieren muß, der die Funktion von gp350 bis zu einem gewissen Grad ersetzen kann. Die Analyse der CD21 Expression von 293 Zellen zeigte, daß diese Zellen geringe Mengen dieses EBV Rezeptors exprimieren und die Infektion durch Wildtyp-EBV in diesem Fall durch die Interaktion zwischen gp350 und CD21 vermittelt wird. Zusätzlich deuten die Ergebnisse darauf hin, daß der Viruseintritt der gp350-negativen Mutante in Epithelzellen über einen anderen Rezeptor als CD21 erfolgt. Dies scheint auch auf die Infektion von B-Zellen zuzutreffen. Im zweiten Teil meiner Arbeit konnte zunächst mit Hilfe von Fusionsproteinen zwischen GFP und gp350 gezeigt werden, daß große N-terminale Bereiche des gp350 Proteins deletiert werden können, ohne dadurch die Expression sowie den Transport dieser Chimären zur Plasmamembran zu beeinträchtigen. Darauf aufbauend wurde ein gp350 Fusionsprotein mit dem humanen Stammzellfaktor konstruiert. Es wurde gezeigt, daß dieses hybride Glykoprotein in die Virushülle von EBV eingebaut wird. Die mit dem Fusionsprotein pseudotypisierten Viren waren in der Lage, c-kit exprimierende Zellen wie TF-1 Zellen und CD34-positive, hämatopoetische Vorläuferzellen, wenn auch mit einer relativ niedrigen Infektionsrate, zu infizieren. Entsprechende Blockierungsexperimente bestätigten, daß die beobachtete Infektion durch die spezifische Interaktion des hSCF Anteils mit dem c-kit Rezeptor erfolgt. Die Retargetierungsversuche mit der nicht infektiösen gp350/gp85-negativen EBV Mutante mit Hilfe des Glykoproteins des "Lymphocytic Choriomeningitis Virus" zeigten, daß ein einziges heterologes, virales Glykoprotein sowohl die Bindung an die Zelloberfläche wie auch den Viruseintritt in HeLa Zellen vermitteln kann.

Two human stromal cell lines were established previously from bone marrow-derived primary long-term cultures by immortalization using the SV40 large T antigen and cellular cloning. After irradiation, the fibroblast-like cell lines L87/4 and L88/5 support hematopoietic differentiation of allogeneic cord blood cells in vitro. The stromal cells do not express CD34 and CD50, but some adhesion molecules and integrins, such as CD44, CD54 and CD58. Their expression profiles on RNA and protein levels are suggestive of their osteogenic potency. The quality and quantity of osteocalcin and osteopontin protein expression depended on the culture conditions. Expression of the osteogenic markers increased over time in culture, especially in cells growing in clusters. The stromal cells also expressed collagens I and V, but did not show any expression of collagens II and III. The potentially osteoblastic stromal cells were transplanted into NOD/SCID recipient mice by intravenous injection and were found in various mesenchymal organs up to 10 weeks after transplantation. Osteocalcin-positive human stromal cells could be detected in the bone marrow, thymus, liver, brain and gut of the recipient animals. In summary, there is evidence that human bone-marrow-derived stromal cells have to be considered mesenchymal progenitors, persistently expressing osteogenic markers in vitro and in vivo. Copyright (C) 2001 S, Karger AG, Basel.

We have already shown that cytokine cocktails (IL-1 beta, IL-3, IL-6, SCF, GM-CSF) and/or lymphokine-activated killer (LAK) cells can reduce the amounts of clonal, CD34-positive mononuclear bone marrow cells (BM-MNC) in acute myeloid leukemia (AML). In addition, the influence of those cocktails and/or LAK cells on the clonogenic potential of AML BM-MNC was investigated. BM colonies cultured in agar during different stages of the disease were immunophenotyped in situ: 17 patients at diagnosis, 14 patients in complete remission, 8 patients at relapse, 8 healthy donors. A significant reduction in leukemic cells and colonies positive for CD34 after in vitro culture of BM-MNC with cytokine cocktails was achieved with all samples obtained at diagnosis (n = 8, p < 0.01), in 6 of 8 cases in complete remission but only in 2 of 6 cases at relapse. Cytokine cocktails stimulated granulopoiesis as well as B and T lymphopoiesis. Colonies with leukemic phenotype could never be detected in healthy BM. A significant reduction in leukemic colonies was achieved by coculture of BM-MNC (uncultured or cytokine precultured) with autologous LAK cells in all 4 cases at diagnosis and in 1 case at relapse. An additive effect of in vitro cytokine preincubation of BM samples on the leukemia-reducing effect of LAK cells could be demonstrated in all samples studied (p