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Background & Aim: The pro-angiogenic, immunoregulatory and anti- inflammatory properties of MSCs are being exploited for the development of cellular therapies, including the treatment of graft versus host disease (GvHD), inflammatory bowel disease and COVID-19. SNBTS have developed a GMP process to bank umbilical cord MSCs (UC-MSCs) whereby we can reliably bank 100 vials of 10 million P2 UC-MSCs per cord. Each of these vials can be extensively expanded and stored for specific applications. The ultimate aim of the bank is for off-the-shelf clinical use, e.g., in GvHD or as an adjuvant therapy in Islet transplantations. Methods, Results & Conclusion(s): During process development, different basal media and supplements were screened for proliferation and MSC marker expression. Cells grown in promising media combinations were then tested for tri-lineage differentiation (identity), their chemokine/cytokine expression and T-cell inhibition (function) assessed. Medium selected for further GMP development and scale up was ultimately determined by all round performance and regulatory compliance. GMP-like UC-MSCs were shown to have immune-modulatory activity in T-cell proliferation assays at 4:1 or 16:1 ratios. Co-culture of UC-MSCs and freshly isolated leukocytes, +/- the immune activating agent LPS, show a dose dependent survival effect on leukocytes. In particular, neutrophils, which are normally very short lived in vitro demonstrated increased viability when co-cultured with UCMSCs. The survival effect was partially reproduced when UC-MSC were replaced with conditioned medium or cell lysate indicating the involvement of soluble factors. This improved neutrophil survival also correlates with results from leukocyte migration studies that demonstrate neutrophils to be the main cell type attracted to MSCs in in vitro and in vivo. Genetic modification of UC-MSC may improve their therapeutic potential. We have tested gene editing by CRISPR/Cas9 technology in primary UC-MSCS. The CXCL8 gene, highly expressed in UC-MSC, was targeted in isolates from several different donors with editing efficiencies of 78-96% observed. This translated to significant knockdown of CXCL8 protein levels in resting cells, however after stimulation levels of CXCL8 were found to be very similar in edited and non-edited UC-MSCs. This observation requires further study, but overall the results show the potential to generate future banks of primary UC-MSCS with genetically enhanced pro-angiogenic, immunoregulatory and/or anti-inflammatory activities.Copyright © 2023 International Society for Cell & Gene Therapy
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Background & Aim: The long-term effects of human mesenchymal stem cell (MSC) treatment on COVID-19 patients have not been fully characterized. The aim of this study was to evaluate the safety and efficacy of a MSC treatment administered to severe COVID-19 patients enrolled in a randomized, double-blind, placebo-controlled clinical trial (NCT 04288102). Methods, Results & Conclusion(s): A total of 100 patients experiencing severe COVID-19 received either MSC treatment (n = 65, 4x107 cells per infusion) or a placebo (n = 35) combined with standard of care on days 0, 3, and 6. Patients were subsequently evaluated 18 and 24 months after treatment to evaluate the long-term safety and efficacy of the MSC treatment. The outcomes measured included: 6-minute walking distance (6-MWD), lung imaging, quality of life according to the Short Form 36 questionnaire, COVID-19-related symptoms, titers of SARS-CoV-2 neutralizing antibodies, MSC-related adverse events (AEs), and tumor markers. Two years after treatment, a marginally smaller proportion of patients had a 6-MWD below the lower limit of the normal range in the MSC group than in the placebo group (OR = 0.19, 95% CI: 0.04-0.80, Fisher's exact test, p = 0.015). On the SF-36 questionnaire, a marginally higher general health score was received by the MSC group at month 18 compared with the placebo group (50.00 vs. 35.00;95% CI: 0.00-20.00, Wilcoxon rank sum test, p = 0.016). In contrast, there were no differences in the total severity score of lung imaging or the titer of neutralizing antibodies between the two groups. Meanwhile, there were no MSC-related AEs reported at the 18- or 24-month follow-ups. The serum levels of most of the tumor markers examined remained within normal ranges and were similar between the MSC and placebo groups. Long-term safety was observed for the COVID-19 patients who received MSC treatment. Yet few sustained efficacy of MSC treatment was observed at the end of the 2-year follow-up period. Funding(s): The National Key Research and Development Program of China (2022YFA1105604, 2020YFC0860900), the specific research fund of The Innovation Platform for Academicians of Hainan Province (YSPTZX202216) and the Fund of National Clinical Center for Infectious Diseases, PLA General Hospital (NCRCID202105,413FZT6). [Figure presented]Copyright © 2023 International Society for Cell & Gene Therapy
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Background & Aim: Ricin is one of the most lethal toxins, particularly if inhaled, and is considered a biological threat agent due to its wide availability and ease of production. Pulmonary ricin intoxication manifests in ARDS, cytokine storm, immune infiltration, and severe edema. Passive immunization is the preferred measure against pulmonary ricinosis, but only if administered shortly after exposure. Despite their potential to remedy pulmonary injury and inflammation, mesenchymal cell (MSC) therapies were never investigated in ricinosis. Here, we report the potential for treating pulmonary ricinosis with MesenCure, a professionalized allogeneic MSC therapy shown to reduce the mortality of patients suffering from severe pulmonary manifestations of COVID by 68%. Methods, Results & Conclusion(s): Preliminary studies demonstrated positive MesenCure effects in a sub-lethal pulmonary ricinosis model in CD1 mice. This model is regarded as highly translational due to the broad heterogeneity of these outbred mice. Positive effects included a reduction in excess protein content of the bronchoalveolar lavage fluid (BALF) by 45% when MesenCure was injected intravenously (IV) at 125k cells/animal, 48h post-exposure (PE) and evaluated one day later (p<0.05, Fig. 1A). Moreover, we found up to 52% reduction in the excess BALF leukocytes, when MesenCure was injected IV, 24h PE using the same dose (p<0.05, Fig. 1B) or 6h PE using a double dose (p<0.01, Fig. 1C), and evaluated two days PE. Optimizing the dose and administration route further improved the therapeutic outcome of MesenCure applied 6h PE as assessed by weight loss. As shown in Fig. 1D-E, IV injection of 250k-500k MesenCure cells/animal slightly protected the intoxicated animals against weight loss (p for treatment x time interaction <0.01 or <0.05 for 250k and 500k cells/animal, respectively). Interestingly, one million cells IV resulted in a lesser effect (not shown), however when injected subcutaneously (SC), 1M cells were very effective (p<0.001, Fig. 1F), seemingly even more effective than 2M cells/animal SC (Fig. 1G). Surprisingly, 2M thawed cells/animal injected SC protected the animals against weight loss almost completely (p<0.0001, Fig. H). In conclusion, we provide evidence for the potential of SC MSCs, specifically MesenCure, for treating pulmonary ricinosis and possibly other forms of ARDS. In agreement with Giri and Galipeau (2020), we provide further evidence for the dependency of MSC outcomes on their specific state and administration route. [Figure presented]Copyright © 2023 International Society for Cell & Gene Therapy
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Introduction: Hepatic inflammatory pseudotumor (HIP), albeit rare, is an important pathology to be included in differentials for hepatic masses. The benign nature and treatment of this disease process should be considered especially in comparison to malignant hepatic processes. Case Description/Methods: A 66-year-old male with pre-existing history of compensated Hepatitis C cirrhosis status post direct-acting antivirals with sustained virologic response presented in shock after a syncopal episode. Initial work up revealed leukocytosis, thrombocytopenia, acute renal injury, elevated liver enzymes, and COVID-19 positive test. Patient underwent initial liver ultrasound revealing intrahepatic and extrahepatic biliary ductal dilation. Subsequent MRCP demonstrated diffuse thickening of intra and extra hepatic bile ducts suggestive of cholangitis and several hepatic masses concerning for abscesses versus possible metastatic cholangiocarcinoma. Patient improved symptomatically with antibiotics and supportive care. A liver biopsy was performed with pathology showing lymphoplasmacytic inflammation and fibroblastic infiltration suggestive of hepatic inflammatory pseudotumor. A repeat MRCP one week later showed interval decrease in size of liver lesions and repeat liver function tests also showed improvement. Patient was discharged on a course of ciprofloxacin and metronidazole. Patient had repeat MRCP 3 months after discharge, with further significant improvement in size of liver lesions. After multi-disciplinary discussion the plan was for further surveillance with imaging and labs in 2 months. Discussion(s): Inflammatory pseudotumors are benign and non-neoplastic lesions that can occur in any organ. They can appear as a malignant lesion when they arise in the liver and an accurate identification can allow for conservative management and prevent unnecessary invasive procedures. Hepatic inflammatory pseudotumors are often seen with concomitant infection or inflammatory processes. Liver biopsies distinguish these tumors from other malignant processes as they demonstrate a characteristic dense inflammatory infiltrate interspersed in stroma of interlacing bundles of myofibroblasts. This case highlights the importance of maintaining HIP on the differential diagnosis. (Figure Presented).
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Introduction: The coronavirus infections ( disease) (COVID-19) pandemic 2019 caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) began in December 2019 and has affected millions of lives worldwide, even though many aspects of the disease are still unknown.Current data shows that many hospitalized patients with COVID-19 suffer from kidney damage manifesting as proteinuria, hematuria, or acute kidney injury (AKI). AKI is especially common among severely and critically ill patients with COVID-19 and may be a predictor of mortality.The pathophysiology of AKI associated with COVID-19 may be due to non-specific mechanisms, but also to mechanisms specific to COVID. Evaluation of morphological changes in the kidneys and their mechanisms could help determine their spectrum and immediate or long-term effects. The aim of our study was to compare the initial clinical and laboratory data of patients with COVID 19 who developed AKI and to analyze the morphological changes in the kidneys that underwent autopsy when patients died of COVID 19 with AKI. Method(s): We conducted a preliminary retrospective analysis of cured patients with COVID 19 and AKI during the pandemic period of July 2021 - December 2021. We analyzed the clinical and laboratory parameters of 2 groups of patients: thise who survived and thise who died of AKI caused by COVID 19. We also studied he main frequently detected morphological changes in the renal biopsy: the subject ground was a dead group. Statistical processing of the obtained data was performed using the STATA program. The two groups were compared, and the statistical significance of the scores of the two groups was calculated using the Mann Whitney analysis and the chi-quadrant Results: According to the results of analyzes of clinical and laboratory parameters, it can be summarized that in the group of the dead in 80% of patients, the volume of lung damage is more than the deceased than the survivors. There is a deep lymphopenia and thrombinemia in the group of the dead. The level of azotemia also prevails in the group of the deceasedAccording to our studies, out of 10 lethal cases, 90% of the histology of samples taken from the kidneys of those who died from COVID 19 revealed the following changes: edema and plethora of renal glomeruli, atrophy and sclerosis from 10-50% of the glomeruli. In the tubules, total necrosis of the epithelium. In the stroma, vascular thrombosis, focal sclerosis with lymphoid infiltration, small focal hemorrhages [Formula presented] Conclusion(s): Acute kidney injury remains a fairly common and serious manifestation of the severe course of COVID 19. Regardless of the type of development of the mechanism, it is often associated with high mortality. Our results of the study can serve as a subject for further research. No conflict of interestCopyright © 2023
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The coronavirus infection (COVID-19), an acute viral disease with predominant affection of the upper respiratory tract, is a challenge for modern medicine. Considering the fact that in the patho-genesis of coronavirus pneumonia there is violation of the im-mune response (hyper-response, cytokine storm) the drugs that locally regulate it may be promising in the pneumonia treatment. Biological activity of exosomes is widely investigated in the world. Small extracellular vesicles of mesenchymal cells have the following effects: anti-apoptotic, proliferation stimulation, anti-inflammatory and immunomodulatory. Objective(s): to evaluate the safety and efficacy of the method of inhalation administration of small extracellular vesicles in bilateral pneumonia caused by a new SARS-CoV-2 coronavirus infection. To study these effects an interventional, prospective, random-ized, double-blind, placebo-controlled study has been conducted to evaluate the safety and efficacy of inhaled small extracellular vesicles administration to the patients with bilateral pneumonia caused by the new coronavirus infection SARS-CoV-2. Altogether 36 patients with confirmed new coronavirus infection COVID-19, complicated by bilateral pneumonia of moderate severity (12 patients each in study groups 1 and 2, depending on the type of given small extracellular vesicles, and the control group) participated in the study. Small extracellular vesicles were inhaled twice a day in the dose of 2-10x1010 particles. The efficacy and safety of the method were assessed judging by the patients' general state, as-sessment of the disease severity, general and biochemical blood tests, coagulogram, saturation, CT scan of the lungs before and after 10 days of treatment. The observation period was 30 days after hospitalization. During the study the safety of the method was proved, all the patients recovered. Reliable differences of the blood CRP index, which normalized by day 10 of treatment in groups 1 and 2, but remained elevated in the control group. No significant differences were found in other assessed parameters.Copyright © 2022, Human Stem Cell Institute. All rights reserved.
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The cellular and molecular hallmarks of aging include genomic instability, telomere attrition, epigenetic alterations, changes in intracellular signaling, cellular senescence, and mitochondrial dysfunction. These lead to complex remodeling and changes involving both the innate and adaptive immune systems. Besides age related changes in immune cells, the microenvironment in the lymphoid and non-lymphoid organs, as well as circulating factors interacting with the immune system also contribute to immunosenescence. Overall, immunosenescence is characterized by reduction of immune response, an increase in inflammatory and oxidation background (inflamm-aging), and production of autoantibodies. One of the most prominent age-related changes in the adaptive immune system is the decline in regenerative thymic capacity. Similar aging related defects have also been observed in stroma of the bone marrow. While lymphocytes in infants show a naive phenotype, memory phenotype predominates after midlife. Though immune responses against recall antigens may still be conserved, the ability to mount primary response against novel antigens declines with age. As a result, increased susceptibility to infections, and suboptimal vaccine response is observed in the elderly. Apart from functional alternation in immune cells, there is a low-grade persistent elevation in inflammatory molecules. Inflamm-aging may result from the accumulation of misfold proteins, compromised gut barrier function, chronic infection, obesity, etc. Furthermore, aging is associated with immune dysregulation, with defective resolution of immune response after activation, and impaired clearance of dead cells with sustained inflammation. Excessive inflammation not only impairs antigen specific immunity, but also leads to tissue damage. In fact, this may partly account for the increased mortality of COVID infection in the elderly. Apart from vulnerability to infection and weakened vaccine response, immunosenescence also plays an important role in cancer and autoimmunity in the elderly. Because of increased tissue damage and apoptosis, coupled with inflamm-aging, increased autoantibodies production is observed in the elderly. Nevertheless, there is an age-related increase in peripheral regulatory T cells. While there is an increase in autoimmunity with aging, this does not always translate into an increase in autoimmune disease. On the other hand, the increase in regulatory T cells, along with other immunosuppressive cells and the senescence associated proinflammatory environment, promotes tumor development and progression in the elderly. As immunosenescence has a significant impact on health and disease, better understanding on this process is crucial for research and development in the future geriatric health care.
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The coronavirus infection (COVID-19), an acute viral disease with predominant affection of the upper respiratory tract, is a challenge for modern medicine. Considering the fact that in the patho-genesis of coronavirus pneumonia there is violation of the im-mune response (hyper-response, cytokine storm) the drugs that locally regulate it may be promising in the pneumonia treatment. Biological activity of exosomes is widely investigated in the world. Small extracellular vesicles of mesenchymal cells have the following effects: anti-apoptotic, proliferation stimulation, anti-inflammatory and immunomodulatory. Objective(s): to evaluate the safety and efficacy of the method of inhalation administration of small extracellular vesicles in bilateral pneumonia caused by a new SARS-CoV-2 coronavirus infection. To study these effects an interventional, prospective, random-ized, double-blind, placebo-controlled study has been conducted to evaluate the safety and efficacy of inhaled small extracellular vesicles administration to the patients with bilateral pneumonia caused by the new coronavirus infection SARS-CoV-2. Altogether 36 patients with confirmed new coronavirus infection COVID-19, complicated by bilateral pneumonia of moderate severity (12 patients each in study groups 1 and 2, depending on the type of given small extracellular vesicles, and the control group) participated in the study. Small extracellular vesicles were inhaled twice a day in the dose of 2-10x1010 particles. The efficacy and safety of the method were assessed judging by the patients' general state, as-sessment of the disease severity, general and biochemical blood tests, coagulogram, saturation, CT scan of the lungs before and after 10 days of treatment. The observation period was 30 days after hospitalization. During the study the safety of the method was proved, all the patients recovered. Reliable differences of the blood CRP index, which normalized by day 10 of treatment in groups 1 and 2, but remained elevated in the control group. No significant differences were found in other assessed parameters. Copyright © 2022, Human Stem Cell Institute. All rights reserved.
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Background & Aim: Multilineage-differentiating stress enduring (Muse) cells, collectable as pluripotent surface marker SSEA-3-positive, are naturally existing non-tumorigenic pluripotent stem cells that distribute in the bone marrow, peripheral blood, connective tissue of every organ, and exhibit triploblastic differentiation and self-renewability at a single cell level. They are also contained in cultured MSCs and fibroblasts as several percent, and are expandable to a clinical scale. Circulating Muse cells, both endogenous and intravenously injected exogenous cells, selectively home to damaged tissue by sensing sphingosine-1-phosphate (S1P), one of the general alert signals produced by the damaged tissue, and then spontaneously differentiate into multiple tissue-constituent cells to replace damaged/ apoptotic cells. In this manner, they repair tissues. In addition, they have a specific immunomodulatory system, represented by HLA-G expression, allowing allogenic-Muse cells to directly administrate to patients without HLA-matching or long-term immunosuppressant (Figure Presented) treatment, and to remain in the host tissue as differentiated functional cells for more than half a year, as shown by animal models. Methods, Results & Conclusion: For these characteristics, intravenous drip is the main route of treatment and do not require surgery for their administration, nor do they require gene introduction or cytokine treatment to be rendered pluripotency and/or differentiation. Currently, clinical trials using intravenously administered donor- Muse cells have been conducted for myocardial infarction, stroke, epidermolysis bullosa, spinal cord injury, perinatal hypoxic ischemic encephalopathy, amyotrophic lateral sclerosis and COVID19-ARDS, all by intravenous drip of donor-derived Muse cell formula, CL2020, without HLA-matching or immunosuppressant treatment. The result of randomized, double-blind, placebo-controlled clinical trial in stroke patients confirmed safety and efficacy of Muse cell-based product for up to 52 weeks (1 year). Muse cells may safely provide beneficial effects compatible with the ‘body’s natural repair systems’ by a simple cost- effective strategy;collection, expansion and intravenous drip.
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Background & Aim: The wide gap in severe Covid-19 management is increasingly addressed by mesenchymal cell (MSC) therapies, despite studies that failed to show significant efficacy in ARDS. To improve the therapeutic utility of MSCs in ARDS, Bonus BioGroup developed MesenCure: An allogeneic adipose-derived MSC product professionalized by a combination of culture conditions enhancing the cells’ potency and stability, producing unique transcriptomic, proteomic, and morphological signatures. Up to 100k fresh MesenCure doses with a shelf life sufficient for global supply can be produced from a single donor under 20 PDLs, further preventing potency loss due to cryopreservation and culture aging. Based on preclinical data presented during ISCT2021, demonstrating MesenCure’s advantages over non-professionalized MSCs, and its safety in a Phase I study, Bonus BioGroup initiated a multi-center Phase II trial in severe Covid-19 patients that was recently concluded. Methods, Results & Conclusion: The Phase II trial included 50 severe Covid-19 patients suffering from diffuse pneumonia and oxygen desaturation treated with up to 3 MesenCure doses (1.5x106 cells/kg on days 1, 3, and 5), on top of the Standard of Care (SoC), and 150 similar severe control patients treated by the SoC only and stratified according to gender, age, and comorbidities. A substantial 68% reduction in the mortality rate of the test patients was measured (Fig. 1A, p<0.05), along with a 57% drop in their risk of intubation relative to the control (Fig. 2A, p<0.05). Over 50% of the patients treated with MesenCure were released from the hospital within two days after treatment, and a 38% reduction was measured in the hospital length of stay (LoS) of patients having LoS>7 days (Fig. 1C, p<0.01). Starting from a similar baseline as the control, the median CRP and CK levels of the test patients, after MesenCure treatment, ended 52% (p<0.0001) and 33% (p<0.01) lower than their respective control levels. As shown in Fig. 2 [Figure Presented] Fig. 1 ( 25). (A) Mortality rates among test and control patients at Visit 8 (one month after the first MesenCure dose or the equivalent time points for the control). (B) Test and control patients’ risk of deteriorating to mechanical ventilation. (C) Average hospital length of stay (LoS) of patients having LoS > 7 days. Two-sided p values were calculated using the Fisher Exact test (A and B) or t-test (C). [Figure Presented Fig. 2 ( 25). (A) CRP and (B) CK levels measured at Visit 6, the earliest of two weeks after the first MesenCure dose (Visit 2) or upon hospital release, or the equivalent time points for the control. The test and control groups started from similar median CRP and CK levels. (C) Changes in control and test patients’ LDH levels from Visit 1 (screening) to Visit 6. (D) Area of test patients’ diffuse pneumonia during Visits 1, 6, and 8 (one month after Visit 2). (E) Blood oxygen saturation measured during test patients visits 1, 2-4 (upon or before receiving the first to third MesenCure dose), Visit 5 (the earliest of one week after Visit 2 or upon hospital release), and Visit 6. (F) Test patients’ blood lymphocytes levels (absolute) across Visits 1 and 6. Charts are presented as box-and-whiskers (according to the Tukey method). p values were calculated using the Mann-Whitney test (A, B, and C), Dunn’s multiple comparisons (D and E), or the Wilcoxon test (F). the more profound improvements in inflammatory and tissue damage markers observed in test patients were accompanied by a rapid recovery in pneumonia, respiratory functions, and lymphopenia, emphasizing MesenCure’s powerful effect. In conclusion, we show that MesenCure saves patients’ lives and accelerates their healing, possibly reducing the risk of long-term damages while freeing ICU beds allowing better care for other patients, and reducing the burden associated with hospitalization and additional long-term healthcare costs.
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Background & Aim: Cytokine Release Syndrome (CRS) and Immune effector Cell-Associated Neurotoxicity Syndrome (ICANS) are related side effects of immunotherapies seen in up to 76% of patients treated with CAR-T and 48% of those treated with BiTEs. In up to 27% of the patients, these syndromes may lead to severe consequences. Current treatments for severe CRS are ineffective in >30% of the cases and can worsen ICANS prognosis, calling for novel treatments, especially in light of the expanding use of immunotherapies. Despite their obvious potential, mesenchymal cell (MSC) therapies were seldom investigated in this context. In the present study, Bonus BioGroup has set to assess the potential for treating CRS with MesenCure™, our allogeneic MSC platform, professionalized to enhance the cells’ potency and shown safe and effective in severe COVID patients. Methods, Results & Conclusion: A highly translational and validated CRS model was established in humanized NSG mice bearing human PBMCs, B-cell lymphoma, and CAR-T cells. CAR-T introduction significantly increased the serum levels of proinflammatory cytokines in model animals, indicative of CRS (Fig. 1A). Two IV MesenCure injections were well-tolerated in this model (Fig. 1B) and did not obstruct the CAR-Ts’ ability to inhibit tumor growth by 89% (Fig. 1C, p<0.0001). Remarkably, significant reductions in all proinflammatory cytokines tested (excluding IL-6) were measured in model animals treated with MesenCure, substantiating its potential to treat CRS (Fig. 1A). Interestingly, the magnitudes of these reductions resembled those observed in 50 severe COVID patients treated with MesenCure. MesenCure’s robust immunomodulatory capacity was further demonstrated in vitro by its ability to inhibit the proliferation of activated CD4 T cells with an IC50 of 6k MSC/200k PBMCs, twice more effectively than non-professionalized MSCs. Comparable results were also obtained with CD8 T cells. Similarly, MesenCure inhibited neutrophils’ ROS production by up to 80% within an hour following activation (IC50 19k MSC/200k neutrophils). These effects are likely mediated, in part, by IDO, whose RNA levels were found to be 6.8-fold higher in MesenCure cells than in non-professionalized MSCs (p<0.05), two hours after activation with IFNγ. Moreover, IDO inhibition by 1-MT (1 mM) reduced MesenCure’s (Figure Presented) Fig. 1 (A) The levels of serum proinflammatory cytokines measured in tumor-bearing NSG mice after CRS induction by injection of human PBMCs/CAR-Ts (or saline control) and MesenCure treatment (or saline control). Experimental groups’ designation: Control – not injected with PBMCs/CAR-Ts and not treated by MesenCure;CAR-T – CRS model animals, injected with PBMCs/CAR-Ts but not treated with MesenCure;MesenCure – treated with MesenCure but not injected with PBMCs/CARTs;and CAR-T + MesenCure – CRS model animals treated with MesenCure. (B) Relative change in body weight from the day of tumor induction (Day 0) and (C) IVIS analysis of tumor burden (dorsal aspect) in the above four experimental groups. Statistical significance indicators: ns – not significant, * p<0.05, *** p<0.001, **** p<0.0001. Statistical tests: Holm-Šídák’s multiple comparisons test (A) and two- sided t-test (C). ability to inhibit T cells’ proliferation by 73%. In conclusion, we provide the first evidence for the potential of MSCs and MesenCure, in particular, for treating immunotherapy-related CRS.
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Recent evidence indicates that targeting IL-6 provides broad therapeutic approaches to several diseases. In patients with cancer, autoimmune diseases, severe respiratory infections [e.g. coronavirus disease 2019 (COVID-19)] and wound healing, IL-6 plays a critical role in modulating the systemic and local microenvironment. Elevated serum levels of IL-6 interfere with the systemic immune response and are associated with disease progression and prognosis. As already noted, monoclonal antibodies blocking either IL-6 or binding of IL-6 to receptors have been used/tested successfully in the treatment of rheumatoid arthritis, many cancer types, and COVID-19. Therefore, in the present review, we compare the impact of IL-6 and anti-IL-6 therapy to demonstrate common (pathological) features of the studied diseases such as formation of granulation tissue with the presence of myofibroblasts and deposition of new extracellular matrix. We also discuss abnormal activation of other wound-healing-related pathways that have been implicated in autoimmune disorders, cancer or COVID-19.
Subject(s)
Autoimmune Diseases , COVID-19 , Neoplasms , Humans , Autoimmunity , Inflammation , Autoimmune Diseases/drug therapy , Neoplasms/drug therapy , Wound Healing , Tumor MicroenvironmentABSTRACT
Background: Placental pathology in pregnant women infected by SARS-CoV-2 has generated many reports with non-specific or contradictory results. The objective is to identify histological and molecular findings of infected placentas and its impact on the course of pregnancy. Design: Placentas from 279 women infected with SARS-CoV-2 during pregnancy were reviewed, 137 of them with active infection within 10 days prior to delivery and 142 with past infection beyond this period. Four fetal autopsies were submitted for pathological study. Placental and fetal infection was investigated by immunohistochemistry (IHC) in 279 cases, RT-PCR in 27 cases and in situ hybridization (ISH) in 5 cases. Significance of association between qualitative variables was tested by Fisher's exact test. Results: SARS-CoV-2 infection of placental tissues was demonstrated in 16/279 (5,7%) by IHQ (granular cytoplasmic villous trophoblast staining) and confirmed by ISH and RT-PCR (carried on 5 IHQ positive cases). All these 16 infected placentas showed diffuse (6/16) or focal (10/16) Trophoblast Damage (DTD/FTD) characterized by trophoblast necrosis with preserved villous stroma, collapse of the intervillous space, intervillous fibrinoid deposits, and variable polymorphous inflammatory infiltrates. Cases with DTD (>80% of the parenchyma affected) resulted in 5 stillbirth and 1 premature alive baby (27w) born after induction of labor for pathologic fetal non-stress test. In 4/5 deaths an autopsy was performed and infection of fetal tissues was not demonstrated by IHQ. The cases with FTD (<35% of the parenchyma affected) hadn't negative impact on the fetus. Correlation between placental infection and DTD/FTD had statistical significance (p<0,001). 15/16 placentas belonged to the group of pregnant women with active infection within 10 days prior delivery. Correlation between DTD/FTD and maternal active infection had statistical significance (p<0,002). Conclusions: Placentas infected by SARS-CoV2 has a characteristic histological pattern that we have defined as DTD or FTD, characterized by a predominant villous trophoblast necrosis, intervillous space collapse and variable degrees of mixed inflammation and intervillous fibrinoid deposition. DTD/FTD is an infrequent lesion observed in 5,7% of pregnant women affected by COVID-19 in our series. DTD can be the cause of fetal death due to placental insufficiency. In this study, vertical transmission of the virus to the fetus has not been demonstrated.
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During the ongoing Covid-19 pandemic, collection and donation of human cadaveric corneas are cumbersome. Decellularized corneas (DC) have gained intense popularity as a possible scaffold for corneal remodeling and as an alternative tissue source for corneal replacement. However, DC elicits immune response inspite of elimination of the cellular contents/antigens due to distortion of the collagen fibrils that exposes certain antigenic sites, which often lead to graft rejection. Therefore, here, we tested the hypothesis that cross-linking DC with chondroitin sulfate (CS) may help in restoring distorted conformational changes of the fibrous matrix and would reduce graft rejection. An in vitro immune response study confirmed that the cross-linked DC elicited the least immune response than DC. We implanted three sets of corneal scaffolds obtained from goat, i.e., native, decellularized, and DC conjugated with CS into rabbit stroma. Histology analysis, three months post-implantation confirmed seamless graft integration, cell migration, and no sign of inflammation in the crosslinked cornea. However, so far we have checked the immunogenic potential of decellularized and crosslinked cornea among cross-species(goat to rabbit). Now, before moving to a human clinical trial (patients with infectious keratitis), we are validating the decellularization of the human stromal layer using discarded human corneas not suitable for implantation, for the regeneration of the corneal endothelial layer. The decellularized, chemically decorated cornea will be tectonically strong, offer less immunogenicity, can be sterilized, and will have a longer shelf life. Through this novel study, we can meet the demand for alternative bioengineered human cornea for keratitis patients.
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MicroRNAs have been projected as promising tools for diagnostic and prognostic purposes in cancer. More recently, they have been highlighted as RNA therapeutic targets for cancer therapy. Though miRs perform a generic function of post-transcriptional gene regulation, their utility in RNA therapeutics mostly relies on their biochemical nature and their assembly with other macromolecules. Release of extracellular miRs is broadly categorized into two different compositions, namely exosomal (extracellular vesicles) and non-exosomal. This nature of miRs not only affects the uptake into target cells but also poses a challenge and opportunity for RNA therapeutics in cancer. By virtue of their ability to act as mediators of intercellular communication in the tumor microenvironment, extracellular miRs perform both, depending upon the target cell and target landscape, pro- and anti-tumor functions. Tumor-derived miRs mostly perform pro-tumor functions, whereas host cell- or stroma-derived miRs are involved in anti-tumor activities. This review deals with the recent understanding of exosomal and non-exosomal miRs in the tumor microenvironment, as a tool for pro- and anti-tumor activity and prospective exploit options for cancer therapy.
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The Annual Meeting of the International Society for Stem Cell Research (ISSCR) brought together experts from across the world in the field of stem cell therapy research and regenerative medicine. The sessions were organized around 5 main themes: clinical applications, cellular identity, modeling development & disease, new technologies, and tissue stem cell & regeneration. This report covers some of the highlights of the virtual meeting.
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Introduction: Obesity is a major risk factor for severe coronavirus disease, and clinical evidence now supports the GI tract, in addition to the respiratory system, as a potential route for SARS-CoV-2 infection. Expression of viral entry factors ACE2, TMPRSS2, and CTSL have been detected along the human GI tract including gastric, ileal and colonic mucosa. It is unclear whether obesity confers increased susceptibility to initial SARS-CoV-2 infection, or what gut mechanisms in obesity predispose to vulnerability to SARS-CoV-2. Thus, we aimed to investigate, by single cell RNA-sequencing (scRNA-Seq) of human colonic mucosa, whether patients with obesity may be more susceptible to SARS-CoV-2 infection, by virtue of enhanced expression of SARS-CoV2 entry cofactors followed protein assessment in colon biopsies. Methods: We studied 19 patients: 10 lean (age 33±3y, BMI 23±1kg/m2, 90% female), and 9 with obesity (age 43±3y, BMI 36±1kg/m2, 89% female). Human colonic biopsies from lean (n=4 scRNA-Seq;n=6 validation) and obesity (n=6 scRNASeq;n=3 validation) participants were obtained by sigmoidoscopy. Biopsies were dissociated, and viable cells were FACS-isolated. Chromium-10X Genomics was used for scRNA-Seq library prep, followed by Illumina HiSeq4000 sequencing. COVID-19 entry factors displaying significant differential expression between lean and obesity were then validated for gene, and protein expression in the validation cohort using Illumina TruSeq, and quantitative immunofluorescence confocal microscopy, respectively. Results: The initial dataset analysis revealed sequencing of 59,653 cells, 705 million reads, at 127,000 reads per cell. The human colonic mucosa partitioned into 20 cell subsets (Fig1A,B), and 15 of the 20 clusters displayed detectable expression of at least one of the COVID-19 entry factors: TMPRSS2, CTSL, or ACE2 (Fig1C,D,E). Goblet cell expression of TMPRSS2 was increased 4.6-fold (p<0.05), stromal cell expression of CTSL was increased 1.2-fold (p<0.0001), and ACE2 expression was increased 1.27-fold (p<0.001) in crypt-top (CT) colonocytes of obesity compared to lean controls (Fig2A). Colonic overexpression of TMPRSS2 mRNA (p<0.05) and protein (p<0.05), and CTSL (p<0.05) mRNA, but not ACE2 mRNA, in obesity was further validated in a second validation cohort (Fig2B-F). Conclusions: scRNA-Seq analysis of human colonic epithelium in obesity compared to healthy controls revealed multiple epithelial cell subsets (goblet cell, stromal, and colonocytes) with overexpression of COVID-19 entry factors TMPRSS2, CTSL, and ACE2, confirming the digestive system as a portal for infection by SARS-CoV-2.Furthermore goblet, stromal, and colonocyte-specific overexpression of TMPRSS2, CTSL, and ACE2 in obesity may play a significant role in increased initial susceptibility to COVID-19, and worse disease outcomes in human obesity.(Figure Presented) Single-Cell RNA-Seq Profiling of Human Colonic Epithelium in Obesity. A) t-SNE plot of single-cell RNA-seq profiles of native human colonic epithelial cells, colored by cluster number and identity, listed by largest to smallest population, and annotated by cluster identity, determined by highest ranking gene marker for the colonic cells clustered and profiled between lean and obesity, where dotted blue, red, and green circles represents goblet cells, stromal cells, and CT colonocytes, respectively. B) Proposed cluster identities based on conserved expression of known markers for annotated cell types. Clusters identified displayed expression of at least one of the COVID-19 entry factors: TMPRSS2, CTSL, or ACE2. The average proportion of cells in annotated clusters expressing, C) TMPRSS2, D) CTSL, and E) ACE2 among all studied participants.
ABSTRACT
Mesenchymal stromal cells (MSC) are widely investigated for treating ARDS in Covid-19. Nonetheless, these efforts are overshadowed by studies predating the pandemic that mostly failed to show MSC efficacy in ARDS and recent disappointments with repurposed MSC products. Relying on years of MSC-related experience, Bonus BioGroup developed MesenCure: An enhanced allogeneic MSC therapy for Covid-19, professionalized by a unique combination of culture conditions and optimized in ARDS-relevant models. MesenCure is currently evaluated in a Phase II study in severe Covid-19 patients and administered (IV) in three doses (1.5M cells/kg, d1, d3, d5). A Phase I/II study on ten severe patients demonstrated a significant improvement in ARDS-related parameters following MesenCure treatment. Patients were discharged within one day (median) following treatment, requiring no respiratory support. Speedy recovery from local inflammation was observed in these patients, demonstrated by a rapid reduction in diffuse lung pneumonia, from 55% of the lung area to 15% within 5-6 days from the first dose (p<0.01, Fig. A-C). A corresponding drop in CRP was detected (p<0.01), which returned to normal. A multivariate regression analysis revealed that the reduction in CRP was mainly associated with the number of doses administered and not and the time elapsed since the first dose. MesenCure efficacy may be attributable to the cells' de novo expression of the gene encoding for the IL-6 receptor, making them more responsive to inflammation than non-professionalized naïve MSC (NA-MSC);as well as >8-fold upregulation of the EDIL3 gene, encoding for an endogenous inhibitor of immune infiltration. A corresponding immunosuppressive effect of MesenCure MSCs was demonstrated in vitro, showing their ability to suppress T cells activation twice more effectively than NA-MSC. In this study, MesenCure inhibited the proliferation of primary CD4 T cells in a concentration-depended manner following non-specific activation. Over 98% inhibition was achieved in co-culture of 1:10 MSC-to-PBMC with an IC 50 of 6k MSC/200k PBMCs (r 2=1.00) compared to 12k NA-MSC/200k PBMCs (r 2=0.95). Comparable results were also obtained for CD8 T cells. Similarly, MesenCure inhibited ROS production by primary neutrophils remarkably fast and by up to 80% within less than 40 minutes following their activation (IC 50 = 19k MSC/200k neutrophils, r 2=1.00). In addition to local immunosuppressive outcomes, a significant increase in blood leukocytes was observed in patients treated with MesenCure (p<0.05, Fig. D-F). Further analysis suggested that the increase in total WBCs and neutrophils was associated with the number of MesenCure doses administered (p<0.05, Fig. G-H). In contrast, the increase in lymphocytes was time-dependent (R=0.72, Fig. I). The seemingly exclusively localized anti-inflammatory effects seen in severe patients treated with MesenCure were also observed in animal (murine) studies. An in vivo study in an acute lung injury model demonstrated a dose-dependent localized reduction in leukocyte counts in the lung fluids of animals treated with MesenCure (IV) using two dose levels. Relative to untreated animals, MesenCure reduced lung leukocyte counts by 35%-43% in animals treated with the low dose and by 62%-67% following high-dose MesenCure treatment (p<0.05). The leukocytes' clearance from the lungs was accompanied by a 41%-57% reduction in lung edema (p<0.05) following MesenCure treatment. Notably, NA-MSC did not achieve the same effect. Similar to our clinical findings, a significant increase was measured in neutrophil counts in animals treated with low-dose MesenCure (p<0.05), which decreased dramatically (p< 0.01) in animals treated with a four-times higher dose. MesenCure is administered at a much lower dose compared to other MSC products administered at up to 10M cells/kg. Considering the increase in blood leukocytes measured in patients treated with low-dose MesenCure and comparable preclinical findings, our data suggest that low-dose MesenCure could elicit a potent ocal anti-inflammatory effect without suppressing, and even enhancing, peripheral immunity that is needed to fight the virus. Further research is inevitably required into the mechanism behind this phenomenon. However, our results indicate MesenCure's potential in relieving local inflammation while giving the patient a fighting chance against viremia. [Formula presented] Disclosures: Bronshtein: Bonus BioGroup: Current Employment. Ben David: Bonus BioGroup: Current Employment. Novak: Bonus BioGroup: Current Employment. Kivity: Bonus BioGroup: Current Employment. Meretzki: Bonus BioGroup: Current Employment. Rozen: Bonus BioGroup: Consultancy.
ABSTRACT
TER94 is a multifunctional AAA+ ATPase crucial for diverse cellular processes, especially protein quality control and chromatin dynamics in eukaryotic organisms. Many viruses, including coronavirus, herpesvirus, and retrovirus, coopt host cellular TER94 for optimal viral invasion and replication. Previous proteomics analysis identified the association of TER94 with the budded virions (BVs) of baculovirus, an enveloped insect large DNA virus. Here, the role of TER94 in the prototypic baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) life cycle was investigated. In virus-infected cells, TER94 accumulated in virogenic stroma (VS) at the early stage of infection and subsequently partially rearranged in the ring zone region. In the virions, TER94 was associated with the nucleocapsids of both BV and occlusion-derived virus (ODV). Inhibition of TER94 ATPase activity significantly reduced viral DNA replication and BV production. Electron/immunoelectron microscopy revealed that inhibition of TER94 resulted in the trapping of nucleocapsids within cytoplasmic vacuoles at the nuclear periphery for BV formation and blockage of ODV envelopment at a premature stage within infected nuclei, which appeared highly consistent with its pivotal function in membrane biogenesis. Further analyses showed that TER94 was recruited to the VS or subnuclear structures through interaction with viral early proteins LEF3 and helicase, whereas inhibition of TER94 activity blocked the proper localization of replication-related viral proteins and morphogenesis of VS, providing an explanation for its role in viral DNA replication. Taken together, these data indicated the crucial functions of TER94 at multiple steps of the baculovirus life cycle, including genome replication, BV formation, and ODV morphogenesis.IMPORTANCE TER94 constitutes an important AAA+ ATPase that associates with diverse cellular processes, including protein quality control, membrane fusion of the Golgi apparatus and endoplasmic reticulum network, nuclear envelope reformation, and DNA replication. To date, little is known regarding the role(s) of TER94 in the baculovirus life cycle. In this study, TER94 was found to play a crucial role in multiple steps of baculovirus infection, including viral DNA replication and BV and ODV formation. Further evidence showed that the membrane fission/fusion function of TER94 is likely to be exploited by baculovirus for virion morphogenesis. Moreover, TER94 could interact with the viral early proteins LEF3 and helicase to transport and further recruit viral replication-related proteins to establish viral replication factories. This study highlights the critical roles of TER94 as an energy-supplying chaperon in the baculovirus life cycle and enriches our knowledge regarding the biological function of this important host factor.