Multiple Sclerosis and MEN2 Neoplasia in a Female Patient: A Unique Co-Existence with Expanded Immunological Interest and Therapeutical Challenges, before and after Patient's COVID-19 Infection.

Multiple sclerosis (MS) and its various comorbidities that may be observed are of great interest due to the complexity of MS pathophysiology and all of the immunological changes that follow. The incidence of cancer in MS has been investigated for several years, as not only does it affect ongoing therapeutical decisions, but also, certain disease-modifying treatments (DMTs) may increase the risk of tumorigenesis. For the first time, we present a case of a female patient with pediatric-onset MS (POMS) and multiple endocrine neoplasia 2B (MEN2B) and analyze the immunological impact of these diseases on the therapeutical choice, under the umbrella of her COVID-19 infection and the SARS-CoV-2 pandemic as a whole. We also review the existing literature regarding the immunogenetic and immunological correlations between these two extremely rare diseases and discuss the most suitable treatment for our case, which seems to be an anti-CD20 agent due to a better outcome in putative MS worsening and tumor progression, when killer immunoglobulin-like receptors' (KIR) expression is reduced in natural killer (NK) cells. We also broaden our concerns on this comorbidity issue, at the same time focusing on the future research needed in this unexplored field of the comorbidity of MS and cancers.

HLA-genotyping by next-generation-sequencing reveals shared and unique HLA alleles in two patients with coexisting neuromyelitis optica spectrum disorder and thymectomized myasthenia gravis: Immunological implications for mutual aetiopathogenesis?

The exact immunopathogenesis, genetic mechanisms and triggering factors underlying myasthenia gravis (MG) and neuromyelitis optica (NMO) remain unknown and the coexistence may underline an aetiopathogenetic link be- tween these two diseases. We report the cases of two thymectomized patients with acetylcholine receptor (AChR) antibody (Ab)-positive MG who eventually developed AQP4-NMO. Next-Generation Sequencing (NGS) analysis showed that patient-1 had two HLA alleles previously associated with MG, mainly HLA-A*01:01:01 and HLA-DRB1*03:01, present in a haplotype in Caucasian MG patients (HLA-A1-B8-DR3-DQ2). Patient-2, expressed HLA-C*07:01:01, a well characterized MG risk factor and HLA-DQB1*05:02:01, previously described both in MG and NMO patients. Finally, we observed two common alleles in patient 1 and 2, HLA-DQA1*05:01:01 and HLA-DPB1*04:02:01. We believe that this study provides clinical evidence of the role of specific HLA alleles in rare forms of combined human peripheral and CNS autoimmunity, a fact that enhances the aim towards tailor-made therapeutic decision making.

Mesenchymal stromal cell delivery as a potential therapeutic strategy against COVID-19: Promising evidence from in vitro results.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 2019 (COVID-19) pandemic, which was initiated in December 2019. COVID-19 is characterized by a low mortality rate (< 6%); however, this percentage is higher in elderly people and patients with underlying disorders. COVID-19 is characterized by mild to severe outcomes. Currently, several therapeutic strategies are evaluated, such as the use of anti-viral drugs, prophylactic treatment, monoclonal antibodies, and vaccination. Advanced cellular therapies are also investigated, thus representing an additional therapeutic tool for clinicians. Mesenchymal stromal cells (MSCs), which are known for their immunoregulatory properties, may halt the induced cytokine release syndrome mediated by SARS-CoV-2, and can be considered as a potential stem cell therapy. AIM: To evaluate the immunoregulatory properties of MSCs, upon stimulation with COVID-19 patient serum. METHODS: MSCs derived from the human Wharton's Jelly (WJ) tissue and bone marrow (BM) were isolated, cryopreserved, expanded, and defined according to the criteria outlined by the International Society for Cellular Therapies. Then, WJ and BM-MSCs were stimulated with a culture medium containing 15% COVID-19 patient serum, 1% penicillin-streptomycin, and 1% L-glutamine for 48 h. The quantification of interleukin (IL)-1 receptor a (Ra), IL-6, IL-10, IL-13, transforming growth factor (TGF)-ß1, vascular endothelial growth factor (VEGF)-a, fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), and indoleamine-2,3-dioxygenase (IDO) was performed using commercial ELISA kits. The expression of HLA-G1, G5, and G7 was evaluated in unstimulated and stimulated WJ and BM-MSCs. Finally, the interactions between MSCs and patients' macrophages were established using co-culture experiments. RESULTS: Thawed WJ and BM-MSCs exhibited a spindle-shaped morphology, successfully differentiated to "osteocytes", "adipocytes", and "chondrocytes", and in flow cytometric analysis were characterized by positivity for CD73, CD90, and CD105 (> 95%) and negativity for CD34, CD45, and HLA-DR (< 2%). Moreover, stimulated WJ and BM-MSCs were characterized by increased cytoplasmic granulation, in comparison to unstimulated cells. The HLA-G isoforms (G1, G5, and G7) were successfully expressed by the unstimulated and stimulated WJ-MSCs. On the other hand, only weak expression of HLA-G1 was identified in BM-MSCs. Stimulated MSCs secreted high levels of IL-1Ra, IL-6, IL-10, IL-13, TGF-ß1, FGF, VEGF, PDGF, and IDO in comparison to unstimulated cells (P < 0.05) after 12 and 24 h. Finally, macrophages derived from COVID-19 patients successfully adapted the M2 phenotype after co-culturing with stimulated WJ and BM-MSCs. CONCLUSION: WJ and BM-MSCs successfully produced high levels of immunoregulatory agents, which may efficiently modulate the over-activated immune responses of critically ill COVID-19 patients.

Mesenchymal stromal cells as potential immunomodulatory players in severe acute respiratory distress syndrome induced by SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus-2 and the related coronavirus disease-19 (COVID-19) is a worldwide emerging situation, which was initially reported in December 2019 in Wuhan, China. Currently, more than 7258842 new cases, and more than 411879 deaths have been reported globally. This new highly transmitted coronavirus is responsible for the development of severe acute respiratory distress syndrome. Due to this disorder, a great number of patients are hospitalized in the intensive care unit followed by connection to extracorporeal membrane oxygenation for breath supporting and survival. Severe acute respiratory distress syndrome is mostly accompanied by the secretion of proinflammatory cytokines, including interleukin (IL)-2, IL-6, IL-7, granulocyte colony-stimulating factor (GSCF), interferon-inducible protein 10 (IP10), monocyte chemotactic protein-1 (MCP1), macrophage inflammatory protein 1A (MIP1A), and tumor necrosis factor alpha (TNF-α), an event which is known as "cytokine storm". Further disease pathology involves a generalized modulation of immune responses, leading to fatal multiorgan failure. Currently, no specific treatment or vaccination against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been developed. Mesenchymal stromal cells (MSCs), which are known for their immunosuppressive actions, could be applied as an alternative co-therapy in critically-ill COVID-19 patients. Specifically, MSCs can regulate the immune responses through the conversion of Th1 to Th2, activation of M2 macrophages, and modulation of dendritic cells maturation. These key immunoregulatory properties of MSCs may be exerted either by produced soluble factors or by cell-cell contact interactions. To date, several clinical trials have been registered to assess the safety, efficacy, and therapeutic potential of MSCs in COVID-19. Moreover, MSC treatment may be effective for the reversion of ground-glass opacity of damaged lungs and reduce the tissue fibrosis. Taking into account the multifunctional properties of MSCs, the proposed stem-cell-based therapy may be proven significantly effective in critically-ill COVID-19 patients. The current therapeutic strategy may improve the patient's overall condition and in parallel may decrease the mortality rate of the current disease.

Evaluation of Peripheral Blood and Cord Blood Platelet Lysates in Isolation and Expansion of Multipotent Mesenchymal Stromal Cells.

Abstract: Background: Multipotent Mesenchymal Stromal Cells (MSCs) are used in tissue engineering and regenerative medicine. The in vitro isolation and expansion of MSCs involve the use of foetal bovine serum (FBS). However, many concerns have been raised regarding the safety of this product. In this study, alternative additives derived either from peripheral or cord blood were tested as an FBS replacement. Methods: Platelet lysates (PL) from peripheral and cord blood were used for the expansion of MSCs. The levels of growth factors in peripheral blood (PB) and cord blood (CB) PLs were determined using the Multiple Reaction Monitoring (MRM). Finally, the cell doubling time (CDT), tri-lineage differentiation and phenotypic characterization of the MSCs expanded with FBS and PLs were determined. Results: MSCs treated with culture media containing FBS and PB-PL, were successfully isolated and expanded, whereas MSCs treated with CB-PL could not be maintained in culture. Furthermore, the MRM analysis yielded differences in growth factor levels between PB-PL and CB-PL. In addition, the MSCs were successfully expanded with FBS and PB-PL and exhibited tri-lineage differentiation and stable phenotypic characteristics. Conclusion: PB-PL could be used as an alternative additive for the production of MSCs culture medium applied to xenogeneic-free expansion and maintenance of MSCs in large scale clinical studies.

Optimizing isolation culture and freezing methods to preserve Wharton's jelly's mesenchymal stem cell (MSC) properties: an MSC banking protocol validation for the Hellenic Cord Blood Bank.

BACKGROUND: Mesenchymal stem or stromal cells (MSCs) are a heterogeneous population that can be isolated from many tissues including umbilical cord Wharton's jelly (UC-WJ). Although initially limited in studies such as a hematopoietic stem cell transplantation adjuvant, an increasing number of clinical trials consider MSCs as a potential anti-inflammatory or a regenerative medicine agent. It has been proposed that creating a repository of MSCs would increase their availability for clinical applications. The aim of this study was to assess the optimal isolation and cryopreservation procedures to facilitate WJ MSC banking. STUDY DESIGN AND METHODS: Cells were isolated from UC-WJ using enzymatic digestion or plastic adhesion methods. Their isolation efficacy, growth kinetics, immunophenotype, and differentiation potential were studied, as well as the effects of freezing. Flow cytometry for common MSC markers was performed on all cases and differentiation was shown with histocytochemical staining. Finally, the isolation efficacy on cryopreserved WJ tissue fragments was tested. RESULTS: MSC isolation was successful using both isolation methods on fresh UC-WJ tissue. However, UC-WJ MSC isolation from frozen tissue fragments was impossible. Flow cytometry analysis revealed that only MSC markers were expressed on the surface of the isolated cells while differentiation assays showed that they were capable of trilinear differentiation. All the above characteristics were also preserved in isolated UC-WJ MSCs over the cryopreservation study period. CONCLUSION: These data showed that viable MSCs can only be isolated from fresh UC-WJ tissue, setting the foundation for clinical-grade banking.

Successful short-term cryopreservation of volume-reduced cord blood units in a cryogenic mechanical freezer: effects on cell recovery, viability, and clonogenic potential.

BACKGROUND: Cord blood (CB) units are stored from weeks to years in liquid- or vapor-phase nitrogen until they are used for transplantation. We examined the effects of cryostorage in a mechanical freezer at -150°C on critical quality control variables of CB collections to investigate the possible use of mechanical freezers at -150°C as an alternative to storage in liquid- (or vapor-) phase nitrogen. STUDY DESIGN AND METHODS: A total of 105 CB units were thawed and washed at different time intervals (6, 12, 24, and 36 months). For every thawed CB unit, samples were removed and cell enumeration (total nucleated cells [TNCs], mononuclear cells [MNCs], CD34+, CD133+) was performed. In addition, viability was obtained with the use of flow cytometry, and recoveries were calculated. Also, total absolute colony-forming unit counts were performed and progenitor cell recoveries were studied by clonogenic assays. RESULTS: Significant differences (p < 0.05) were observed in certain variables (TNCs, MNC numbers, viability) when they were examined in relation with time intervals, while others (CD34+, CD133+) were relatively insensitive (p = NS) to the duration of time interval the CB units were kept in cryostorage condition. CONCLUSIONS: The data presented suggest that cryopreservation of CB units in a mechanical freezer at -150°C may represent an alternative cryostorage condition for CB cryopreservation.

Autologous transplantation of adipose-derived stem cells enhances skin graft survival and wound healing in diabetic rats.

BACKGROUND: Diabetes can lead to impaired wound healing and skin grafts used surgically for diabetic wounds are often complicated with necrosis, although different therapies have been proposed. Adipose-derived stem cells (ASCs) participate in tissue repair processes and may have a role during impaired wound healing. In this study, autologous transplantation of ASCs was used to determine if it increases angiogenesis and skin graft survival and enhances wound healing in diabetic rats. METHODS: Adipose-derived stem cells were successfully isolated and cultured. A full-thickness skin graft model was used to determine the effects of locally administered ASCs in 10 rats rendered diabetic (group 1), whereas 10 others served as controls (group 2). Histological examination of skin grafts followed after 1 week. Additionally, immunohistochemical staining intensity of vascular endothelial growth factor (VEGF) and transforming growth factor ß3 (TGF-ß3) was assessed in all grafts. RESULTS: The gross and histological results showed significantly increased survival, angiogenesis, and epithelialization. Mean area of graft necrosis was significantly less in group 1 than in group 2 (7.49% vs 39.67%, P < 0.001). Statistically significant increase of capillary density, collagen intensity, VEGF, and TGF-ß3 expression was noted in group 1 compared with group 2. CONCLUSIONS: These findings suggest that autologous ASC transplantation can enhance skin graft survival in diabetic rats through differentiation, vasculogenesis, and secretion of growth factors such as VEGF and TGF-ß3. This might represent a novel therapeutic approach in skin graft surgery for diabetic wounds.

Stable expression of a neuronal dopaminergic progenitor phenotype in cell lines derived from human amniotic fluid cells.

Cells from human amniotic fluid derived from the fetus are considered a source of multipotent cells. Their properties have not been fully exploited, partially because unlike other embryonic sources such as embryonic stem (ES) cells, cell lines from amniocentesis samples have not been generated. We have established and characterized the properties of eight individual cell lines. Flow cytometry using several cell surface markers showed that all cell lines generated consisted of homogeneous populations that lack HLAII antigenicity. Using a combination of immunocytochemistry, Western blotting, and RT-PCR, we found weak expression of Oct4 and nestin and strong expression of tubulin-betaIII, MAP2, and tau. Specific markers for cholinergic, (nor)adrenergic, and GABAergic neurons or glia were weakly expressed or absent, whereas expression of factors implicated in early induction of dopaminergic neurons, TGF-beta3 and beta-catenin were present. Further analysis showed strong expression of EN-1, c-RET, PTX3, and NURR1 essential for induction and survival of midbrain dopaminergic neurons, TH, AADC, and VMAT2 components of dopamine synthesis and secretion, and syntaxin1A and SNAP-25 necessary for neurotransmitter exocytosis. This phenotype was retained throughout passages and up to the current passage 36. Expression of neuronal and dopaminergic markers in individual AF cell lines was comparable to expression in neurons induced from ES cells and in IMR-32 and SH-SY5Y neuroblastomas. Our data show that cell lines can be derived from subcultures of amniocentesis, and are primarily composed of a population of progenitors with a phenotype similar to that of committed mesencephalic dopaminergic neurons.