Albumin-based solution is the ideal post-thawing suspension medium for cord blood hematopoietic stem cells: A stability and proliferative evaluation.

BACKGROUND: Cryopreservation and thawing protocols represent key factors for the efficacy of cellular therapy products, such as hematopoietic stem cells (HSCs). While the HSC cryopreservation has already been standardized, the thawing procedures have been poorly studied. This study aimed to evaluate the thawing and washing protocol of cord blood (CB) derived HSCs or the HPC(CB), by selecting the optimal thawing solution and determining CD34+ cells' stability over time. STUDY DESIGN AND METHODS: Seven cryopreserved CB products were thawed, washed, and resuspended in three different solutions (10% Dextran40 in NaCl equally prepared with 5% human albumin; 5% human albumin in PBS/EDTA; and normal saline) and stored at 4°C (±2°C). Mononuclear cell (MNC) count, CD45+/CD34+ cell enumeration, and cell viability were tested at 0, 1, 2, 4, 6, 8, 12, 24, 36, and 48 h. The protocol with the selected solution was further validated on additional 10 CB samples. The above parameters and the colony-forming unit (CFU) assay were analyzed at time points 0, 2, 4, 6, and 8 h. RESULTS AND DISCUSSION: The results showed that the 5% human albumin was the most suitable thawing solution. MNCs were stable up to 4 h (p = 0.009), viable CD45+ cells were unstable even at 2 h (p = 0.013), and viable CD34+ cells were stable until 6 h (p = 0.019). The CFU assay proved the proliferative potential up to 8 h, although significantly decreased after 4 h (p = 0.013), and correlated with the viable CD34+ cell counts. We demonstrated that the post-thawed and washed HPC(CB) using 5% human albumin is stable for up to 4 h.

Clofarabine and Treosulfan as Conditioning for Matched Related and Unrelated Hematopoietic Stem Cell Transplantation: Results from the Clo3o Phase II Trial.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) can be curative for patients with hematologic malignancies. The ideal conditioning regimen before allo-HSCT has not been established. We conducted a Phase II study to evaluate the tolerability and efficacy of clofarabine and treosulfan as conditioning regimen before allo-HSCT. The primary objective was to evaluate the cumulative incidence of nonrelapse mortality (NRM) on day +100. Forty-four patients (36 with acute myelogenous leukemia, 5 with acute lymphoblastic leukemia, 3 with myelodysplastic syndromes) were enrolled. The median patient age was 47 years, and the median duration of follow-up was 27 months. The conditioning regimen was based on clofarabine 40 mg/m2 (days -6 to -2) and treosulfan 14 g/m2 (days -6 to -4). Allogeneic hematopoietic stem cells were derived from a sibling (n = 22) or a well-matched unrelated donor (n = 22). Graft-versus-host disease (GVHD) prophylaxis consisted of antithymocyte globulin, rituximab, cyclosporine, and a short-course of methotrexate. The regimen allowed for rapid engraftment and a 100-day NRM of 18%, due mainly to bacterial infections. The incidences of grade II-IV acute GVHD and chronic GVHD were 16% and 19%, respectively. The rates of overall survival (OS), progression-free survival, and relapse at 2 years were 51%, 31%, and 50%, respectively. Significantly different outcomes were observed between patients with low-intermediate and patients with high-very high Disease Risk Index (DRI) scores (1-year OS, 78% and 24%, respectively). Our findings show that the use of treosulfan and clofarabine as a conditioning regimen for allo-HSCT is feasible, with a 78% 1-year OS in patients with a low-intermediate DRI score. However, 1-year NRM was 18%, and despite the intensified conditioning regimen, relapse incidence remains a major issue in patients with poor prognostic risk factors.

A modular framework for the development of targeted Covid-19 blood transcript profiling panels.

BACKGROUND: Covid-19 morbidity and mortality are associated with a dysregulated immune response. Tools are needed to enhance existing immune profiling capabilities in affected patients. Here we aimed to develop an approach to support the design of targeted blood transcriptome panels for profiling the immune response to SARS-CoV-2 infection. METHODS: We designed a pool of candidates based on a pre-existing and well-characterized repertoire of blood transcriptional modules. Available Covid-19 blood transcriptome data was also used to guide this process. Further selection steps relied on expert curation. Additionally, we developed several custom web applications to support the evaluation of candidates. RESULTS: As a proof of principle, we designed three targeted blood transcript panels, each with a different translational connotation: immunological relevance, therapeutic development relevance and SARS biology relevance. CONCLUSION: Altogether the work presented here may contribute to the future expansion of immune profiling capabilities via targeted profiling of blood transcript abundance in Covid-19 patients.

Tailoring cells for clinical needs: Meeting report from the Advanced Therapy in Healthcare symposium (October 28-29 2017, Doha, Qatar).

New technologies and therapies designed to facilitate development of personalized treatments are rapidly emerging in the field of biomedicine. Strikingly, the goal of personalized medicine refined the concept of therapy by developing cell-based therapies, the so-called "living drugs". Breakthrough advancements were achieved in this regard in the fields of gene therapy, cell therapy, tissue-engineered products and advanced therapeutic techniques. The Advanced Therapies in Healthcare symposium, organized by the Clinical Research Center Department of Sidra Medicine, in Doha, Qatar (October 2017), brought together world-renowned experts from the fields of oncology, hematology, immunology, inflammation, autoimmune disorders, and stem cells to offer a comprehensive picture of the status of worldwide advanced therapies in both pre-clinical and clinical development, providing insights to the research phase, clinical data and regulatory aspects of these therapies. Highlights of the meeting are provided in this meeting report.

Phase II Study of Sequential Infusion of Donor Lymphocyte Infusion and Cytokine-Induced Killer Cells for Patients Relapsed after Allogeneic Hematopoietic Stem Cell Transplantation.

Seventy-four patients who relapsed after allogeneic stem cell transplantation were enrolled in a phase IIA study and treated with the sequential infusion of donor lymphocyte infusion (DLI) followed by cytokine-induced killer (CIK) cells. Seventy-three patients were available for the intention to treat analysis. At least 1 infusion of CIK cells was given to 59 patients, whereas 43 patients received the complete cell therapy planned (58%). Overall, 12 patients (16%) developed acute graft-versus-host disease (aGVHD) of grades I to II in 7 cases and grades III to IV in 5). In 8 of 12 cases, aGVHD developed during DLI treatment, leading to interruption of the cellular program in 3 patients, whereas in the remaining 5 cases aGVHD was controlled by steroids treatment, thus allowing the subsequent planned administration of CIK cells. Chronic GVHD (cGVHD) was observed in 11 patients (15%). A complete response was observed in 19 (26%), partial response in 3 (4%), stable disease in 8 (11%), early death in 2 (3%), and disease progression in 41 (56%). At 1 and 3 years, rates of progression-free survival were 31% and 29%, whereas rates of overall survival were 51% and 40%, respectively. By multivariate analysis, the type of relapse, the presence of cGVHD, and a short (<6 months) time from allogeneic hematopoietic stem cell transplantation to relapse were the significant predictors of survival. In conclusion, a low incidence of GVHD is observed after the sequential administration of DLI and CIK cells, and disease control can be achieved mostly after a cytogenetic or molecular relapse.

Two hits in one: whole genome sequencing unveils LIG4 syndrome and urofacial syndrome in a case report of a child with complex phenotype.

BACKGROUND: Ligase IV syndrome, a hereditary disease associated with compromised DNA damage response mechanisms, and Urofacial syndrome, caused by an impairment of neural cell signaling, are both rare genetic disorders, whose reports in literature are limited. We describe the first case combining both disorders in a specific phenotype. CASE PRESENTATION: We report a case of a 7-year old girl presenting with a complex phenotype characterized by multiple congenital abnormalities and dysmorphic features, microcephaly, short stature, combined immunodeficiency and severe vesicoureteral reflux. Whole Genome Sequencing was performed and a novel ligase IV homozygous missense c.T1312C/p.Y438H mutation was detected, and is believed to be responsible for most of the clinical features of the child, except vesicoureteral reflux which has not been previously described for ligase IV deficiency. However, we observed a second rare damaging (nonsense) homozygous mutation (c.C2125T/p.R709X) in the leucine-rich repeats and immunoglobulin-like domains 2 gene that encodes a protein implicated in neural cell signaling and oncogenesis. Interestingly, this mutation has recently been reported as pathogenic and causing urofacial syndrome, typically displaying vesicoureteral reflux. Thus, this second mutation completes the missing genetic explanation for this intriguing clinical puzzle. We verified that both mutations fit an autosomal recessive inheritance model due to extensive consanguinity. CONCLUSIONS: We successfully identified a novel ligase IV mutation, causing ligase IV syndrome, and an additional rare leucine-rich repeats and immunoglobulin-like domains 2 gene nonsense mutation, in the context of multiple autosomal recessive conditions due to extensive consanguinity. This work demonstrates the utility of Whole Genome Sequencing data in clinical diagnosis in such cases where the combination of multiple rare phenotypes results in very intricate clinical pictures. It also reports a novel causative mutation and a clinical phenotype, which will help in better defining the essential features of both ligase IV and leucine-rich repeats and immunoglobulin-like domains 2 deficiency syndromes.

Granulocyte transfusions in children and adults with hematological malignancies: benefits and controversies.

Bacterial and fungal infections continue to pose a major clinical challenge in patients with prolonged severe neutropenia after chemotherapy or hematopoietic stem cell transplantation (HSCT). With the advent of granulocyte colony-stimulating factor (G-CSF) to mobilize neutrophils in healthy donors, granulocyte transfusions have been broadly used to prevent and/or treat life-threatening infections in patients with severe febrile neutropenia and/or neutrophil dysfunction. Although the results of randomized controlled trials are inconclusive, there are suggestions from pilot and retrospective studies that granulocyte transfusions may benefit selected categories of patients. We will critically appraise the evidence related to the use of therapeutic granulocyte transfusions in children and adults, highlighting current controversies in the field and discussing complementary approaches to modulate phagocyte function in the host.

Treatment of graft versus host disease with mesenchymal stromal cells: a phase I study on 40 adult and pediatric patients.

This phase I multicenter study was aimed at assessing the feasibility and safety of intravenous administration of third party bone marrow-derived mesenchymal stromal cells (MSC) expanded in platelet lysate in 40 patients (15 children and 25 adults), experiencing steroid-resistant grade II to IV graft-versus-host disease (GVHD). Patients received a median of 3 MSC infusions after having failed conventional immunosuppressive therapy. A median cell dose of 1.5 × 10(6)/kg per infusion was administered. No acute toxicity was reported. Overall, 86 adverse events and serious adverse events were reported in the study, most of which (72.1%) were of infectious nature. Overall response rate, measured at 28 days after the last MSC injection, was 67.5%, with 27.5% complete response. The latter was significantly more frequent in patients exhibiting grade II GVHD as compared with higher grades (61.5% versus 11.1%, P = .002) and was borderline significant in children as compared with adults (46.7 versus 16.0%, P = .065). Overall survival at 1 and 2 years from the first MSC administration was 50.0% and 38.6%, with a median survival time of 1.1 years. In conclusion, MSC can be safely administered on top of conventional immunosuppression for steroid resistant GVHD treatment. Eudract Number 2008-007869-23, NCT01764100.

The Promise of Precision Nutrition for Modulation of the Gut Microbiota as a Novel Therapeutic Approach to Acute Graft-versus-host Disease.

Acute graft-versus-host disease (aGVHD) is a severe side effect of allogeneic hematopoietic stem cell transplantation (aHSCT) that has complex phenotypes and often unpredictable outcomes. The current management is not always able to prevent aGVHD. A neglected actor in the management of aGVHD is the gut microbiota. Gut microbiota dysbiosis after aHSCT is caused by many factors and may contribute to the development of aGVHD. Diet and nutritional status modify the gut microbiota and a wide range of products are now available to manipulate the gut microbiota (pro-, pre-, and postbiotics). New investigations are testing the effect of probiotics and nutritional supplements in both animal models and human studies, with encouraging results. In this review, we summarize the most recent literature about the probiotics and nutritional factors able to modulate the gut microbiota and we discuss the future perspective in developing new integrative therapeutic approaches to reducing the risk of graft-versus-host disease in patients undergoing aHSCT.

CD14+/CD31+ monocytes expanded by UM171 correct hemophilia A in zebrafish upon lentiviral gene transfer of factor VIII.

Emerging gene therapy clinical trials test the correction of hemophilia A (HA) by replacing factor VIII (FVIII) in autologous hematopoietic stem cells (HSCs). Although it is known that platelets, monocyte/macrophages, and mesenchymal stromal cells can secrete transgenic FVIII, a systematic examination of blood lineages as extrahepatic sources of FVIII, to our knowledge, has not yet been performed. In this study, we sought to provide a comprehensive map of native and lentivirus-based transgenic FVIII production from HSC stage to mature blood cells, through a flow cytometry analysis. In addition, we generated a model of transient HA in zebrafish based on antisense RNA, to assess the corrective potential of the FVIII-transduced HSCs. We discovered that FVIII production begins at the CD34+ progenitor stage after cytokine stimulation in culture. Among all mature white blood cells, monocytes are the largest producers of native FVIII and can maintain protein overexpression during differentiation from HSCs when transduced by a FVIII lentiviral vector. Moreover, the addition of the HSC self-renewal agonist UM171 to CD34+ cells during transduction expanded a subpopulation of CD14+/CD31+ monocytes with excellent ability to carry the FVIII transgene, allowing the correction of HA phenotype in zebrafish. Finally, the HA zebrafish model showed that f8 RNA is predominantly localized in the hematopoietic system at the larval stage, which indicates a potential contributory role of FVIII in hematopoiesis that warrants further investigation. We believe that this study may be of broad interest to hematologists and researchers striving to advance knowledge and permanent treatments for patients with HA.

Decreased Plasma Level of Cytokeratin 20 (KRT20) Is Indicative of the Emergence and Severity of Acute GvHD Irrespective to the Type of Organ Involvement.

Accurate risk prediction of acute graft versus host disease (aGvHD) is currently an unmet clinical need. This study sought to analyze whether three plasma proteins expressed in a largely skin- and gut-restricted manner would be affected by the development of acute cutaneous and gastrointestinal aGvHD. The diagnostic sensitivity, specificity, and prognostic value of plasma cytokeratin-15 (KRT15) cytokeratin-20 (KRT20), and occludin (OCLN) were evaluated in a discovery and a validation cohort using ELISA in comparison with elafin (PI3) and regenerating family member 3 alpha (REG3A), two established markers of skin- and gut aGvHD. The discovery cohort (n = 39) revealed that at the time of diagnosis, plasma KRT20 showed a progressive decrease from unaffected individuals to patients with single-, and patients with multi-organ aGvHD. KRT20 was affected by cutaneous (p = 0.0263) and gastrointestinal aGvHD (p = 0.0242) independently and in an additive manner. Sensitivity and specificity of KRT20 for aGvHD involving both target organs (AUC = 0.852) were comparable to that of PI3 for skin-aGvHD (AUC = 0.708) or that of REG3A for gut-aGvHD (AUC = 0.855). Patient follow-up in the validation cohort (n = 67) corroborated these observations (p < 0.001), and linked low KRT20 to grade 2+ disease (p < 0.001), but failed to confirm low KRT20 as an independent risk factor. These data established a link between low plasma KRT20 levels and moderate to severe aGvHD involving multiple target organs.

High-temporal resolution profiling reveals distinct immune trajectories following the first and second doses of COVID-19 mRNA vaccines.

Knowledge of the mechanisms underpinning the development of protective immunity conferred by mRNA vaccines is fragmentary. Here, we investigated responses to coronavirus disease 2019 (COVID-19) mRNA vaccination via high-temporal resolution blood transcriptome profiling. The first vaccine dose elicited modest interferon and adaptive immune responses, which peaked on days 2 and 5, respectively. The second vaccine dose, in contrast, elicited sharp day 1 interferon, inflammation, and erythroid cell responses, followed by a day 5 plasmablast response. Both post-first and post-second dose interferon signatures were associated with the subsequent development of antibody responses. Yet, we observed distinct interferon response patterns after each of the doses that may reflect quantitative or qualitative differences in interferon induction. Distinct interferon response phenotypes were also observed in patients with COVID-19 and were associated with severity and differences in duration of intensive care. Together, this study also highlights the benefits of adopting high-frequency sampling protocols in profiling vaccine-elicited immune responses.

Flow-Cytometry Platform for Intracellular Detection of FVIII in Blood Cells: A New Tool to Assess Gene Therapy Efficiency for Hemophilia A.

Detection of factor VIII (FVIII) in cells by flow cytometry is controversial, and no monoclonal fluorescent antibody is commercially available. In this study, we optimized such an assay and successfully used it as a platform to study the functional properties of phosphoglycerate kinase (PGK)-FVIII lentiviral vector-transduced cells by directly visualizing FVIII in cells after different gene transfer conditions. We could measure cellular stress parameters after transduction by correlating gene expression and protein accumulation data. Flow cytometry performed on transduced cell lines showed that increasing MOI rates resulted in increased protein levels, plateauing after an MOI of 30. We speculated that, at higher MOI, FVIII production could be impaired by a limiting factor required for proper folding. To test this hypothesis, we interfered with the unfolded protein response by blocking proteasomal degradation and measured the accumulation of intracellular misfolded protein. Interestingly, at higher MOIs the cells displayed signs of toxicity with reactive oxygen species accumulation. This suggests the need for identifying a safe window of transduction dose to avoid consequent cell toxicity. Herein, we show that our flow cytometry platform for intracytoplasmic FVIII protein detection is a reliable method for optimizing gene therapy protocols in hemophilia A by shedding light on the functional status of cells after gene transfer.

Influence of storage conditions of small volumes of blood on immune transcriptomic profiles.

OBJECTIVE: Transcriptome analysis of human whole blood is used to discover biomarkers of diseases and to assess phenotypic traits. Here we have collected small volumes of blood in Tempus solution and tested whether different storage conditions have an impact on transcriptomic profiling. Fifty µl of blood were collected in 100µl of Tempus solutions, freezed at - 20 °C for 1 day and eventually thawed, stored and processed under five different conditions: (i) - 20 °C for 1 week; (ii) +4 °C for 1 week; (iii) room temperature for 1 week; (iv) room temperature for 1 day, - 20 °C for 1 day, room temperature until testing at day 7, (v) - 20 °C for 1 week, RNA was isolated and stored in GenTegra solution. We used 272 immune transcript specific assays to test the expression profiling using qPCR based Fluidigm BioMark HD dynamic array. RESULTS: RNA yield ranged between 0.17 and 1.39µg. Except for one sample, RIN values were > 7. Using Principal Component Analysis, we saw that the storage conditions did not drive sample distribution. The condition that showed larger variability was the RT-FR-RT (room temperature-freezing-room temperature), suggesting that freezing-thawing cycles may have a worse effect on data reproducibility than keeping the samples at room temperature.

Induction of pro-inflammatory programs in enteroendocrine cells by the Toll-like receptor agonists flagellin and bacterial LPS.

Enteroendocrine cells are hormone-secreting cells spread along the intestinal epithelium. Their principal function is to promote the digestion of food. However, little is known about other functions that these cells may play, since they are difficult to study as a whole endocrine organ due to their diffuse localization. It is known that the intestinal epithelial barrier is actively involved in the host defense against pathogen invasion. Here we applied gene expression profiling to characterize the response of the human LCC-18 enteroendocrine cell line to physiological and pathological stimuli mimicked by fatty acids (FAs), flagellin and LPS exposure. We observed that these cells participate in an innate immune reaction to pathogens through the expression of pro-inflammatory factors (i.e. CXCL1 and 3 and IL-32) that we could validate by molecular and proteomic approach. Interestingly, IL-32 has been recently found over-expressed in the inflamed mucosa of patients affected by inflammatory bowel disease. This is very important because modifications of enteroendocrine cells during intestinal inflammation have been so far considered as secondary effects of the inflammatory status rather than due to direct pathogen/enteroendocrine cell interaction. As expected, FAs exposure up-regulates pro-differentiative genes and the production of cholecystokinin but it does not enhance the expression of pro-inflammatory genes. The present observations enlighten a new aspect of the cross talk between immune and endocrine system and suggest enteroendocrine cells as important contributors of inflammatory processes occurring in the gut in response to pathogen exposure and direct enhancers of the inflammatory status associated with human inflammatory bowel disease.

GM-CSF/IL-3/IL-5 receptor common beta chain (CD131) expression as a biomarker of antigen-stimulated CD8+ T cells.

BACKGROUND: Upon Ag-activation cytotoxic T cells (CTLs) produce IFN-gamma GM-CSF and TNF-alpha, which deliver simultaneously pro-apoptotic and pro-inflammatory signals to the surrounding microenvironment. Whether this secretion affects in an autocrine loop the CTLs themselves is unknown. METHODS: Here, we compared the transcriptional profile of Ag-activated, Flu-specific CTL stimulated with the FLU M1:58-66 peptide to that of convivial CTLs expanded in vitro in the same culture. PBMCs from 6 HLA-A*0201 expressing donors were expanded for 7 days in culture following Flu M1:58-66 stimulation in the presence of 300 IU/ml of interleukin-2 and than sorted by high speed sorting to high purity CD8+ expressing T cells gated according to FluM1:58-66 tetrameric human leukocyte antigen complexes expression. RESULTS: Ag-activated CTLs displayed higher levels of IFN-gamma, GM-CSF (CSF2) and GM-CSF/IL-3/IL-5 receptor common beta- chain (CD131) but lacked completely expression of IFN-gamma receptor-II and IFN-stimulated genes (ISGs). This observation suggested that Ag-activated CTLs in preparation for the release of IFN-gamma and GM-CSF shield themselves from the potentially apoptotic effects of the former entrusting their survival to GM-SCF. In vitro phenotyping confirmed the selective surface expression of CD131 by Ag-activated CTLs and their increased proliferation upon exogenous administration of GM-CSF. CONCLUSION: The selective responsiveness of Ag-activated CTLs to GM-CSF may provide an alternative explanation to the usefulness of this chemokine as an adjuvant for T cell aimed vaccines. Moreover, the selective expression of CD131 by Ag-activated CTLs proposes CD131 as a novel biomarker of Ag-dependent CTL activation.

Molecular purging of multiple myeloma cells by ex-vivo culture and retroviral transduction of mobilized-blood CD34+ cells.

BACKGROUND: Tumor cell contamination of the apheresis in multiple myeloma is likely to affect disease-free and overall survival after autografting. OBJECTIVE: To purge myeloma aphereses from tumor contaminants with a novel culture-based purging method. METHODS: We cultured myeloma-positive CD34+ PB samples in conditions that retained multipotency of hematopoietic stem cells, but were unfavourable to survival of plasma cells. Moreover, we exploited the resistance of myeloma plasma cells to retroviral transduction by targeting the hematopoietic CD34+ cell population with a retroviral vector carrying a selectable marker (the truncated form of the human receptor for nerve growth factor, DeltaNGFR). We performed therefore a further myeloma purging step by selecting the transduced cells at the end of the culture. RESULTS: Overall recovery of CD34+ cells after culture was 128.5%; DeltaNGFR transduction rate was 28.8% for CD34+ cells and 0% for CD138-selected primary myeloma cells, respectively. Recovery of CD34+ cells after DeltaNGFR selection was 22.3%. By patient-specific Ig-gene rearrangements, we assessed a decrease of 0.7-1.4 logs in tumor load after the CD34+ cell selection, and up to 2.3 logs after culture and DeltaNGFR selection. CONCLUSION: We conclude that ex-vivo culture and retroviral-mediated transduction of myeloma leukaphereses provide an efficient tumor cell purging.

Molecular signatures induced by interleukin-2 on peripheral blood mononuclear cells and T cell subsets.

Experimentally, interleukin-2 (IL-2) exerts complex immunological functions promoting the proliferation, survival and activation of T cells on one hand and inducing immune regulatory mechanisms on the other. This complexity results from a cross talk among immune cells which sways the effects of IL-2 according to the experimental or clinical condition tested. Recombinant IL-2 (rIL-2) stimulation of peripheral blood mononuclear cells (PBMC) from 47 donors of different genetic background induced generalized T cell activation and anti-apoptotic effects. Most effects were dependent upon interactions among immune cells. Specialized functions of CD4 and CD8 T cells were less dependent upon and often dampened by the presence of other PBMC populations. In particular, cytotoxic T cell effector function was variably affected with a component strictly dependent upon the direct stimulation of CD8 T cells in the absence of other PBMC. This observation may provide a roadmap for the interpretation of the discrepant biological activities of rIL-2 observed in distinct pathological conditions or treatment modalities.