Risk of SARS-CoV-2 infection and severe COVID-19 in hematological patients who received or not pre-exposure prophylaxis with tixagevimab/cilgavimab: a target trial emulation.

We emulated a hypothetical target trial in which hematological subjects cared at the University Hospital of Pisa (Italy) received or not SARS-CoV-2 prophylaxis with tixagevimab/cilgavimab. Subjects who received prophylaxis (cases) were compared to those who did not (controls). The main outcome was SARS-CoV-2 infection in the subsequent 6 months. Inverse probability weighting (IPW) was used to adjust for confounders. A multivariable analysis was performed to identify variables associated with SARS-CoV-2 infection. We recruited 462 patients: 228 received prophylaxis, 234 were controls. COVID-19 was lower in cases compared to controls (16.7% vs 24.8%, p = 0.03, after IPW 14.3% vs 24.6%, p = 0.01). On multivariable analysis, B-cell depleting therapies (HR 2.09, 95%CI 1.05-4.18, p = 0.037) were associated with increased risk of COVID-19, while tixagevimab/cilgavimab prophylaxis (HR 0.45, 95%CI 0.27-0.73, p = 0.001) and previous SARS-CoV-2 infection (HR 0.27, 95%CI 0.14-0.51, p < 0.001) were protective. In conclusion, prophylaxis with monoclonal antibodies may reduce the risk of COVID-19 in hematological patients.

Multicenter Observational Retrospective Study on Febrile Events in Patients with Acute Myeloid Leukemia Treated with Cpx-351 in "Real-Life": The SEIFEM Experience.

In the present study, we aimed to evaluate the absolute risk of infection in the real-life setting of AML patients treated with CPX-351. The study included all patients with AML from 30 Italian hematology centers of the SEIFEM group who received CPX-351 from July 2018 to June 2021. There were 200 patients included. Overall, 336 CPX-351 courses were counted: all 200 patients received the first induction cycle, 18 patients (5%) received a second CPX-351 induction, while 86 patients (26%) proceeded with the first CPX-351 consolidation cycle, and 32 patients (10%) received a second CPX-351 consolidation. A total of 249 febrile events were recorded: 193 during the first or second induction, and 56 after the first or second consolidation. After the diagnostic work-up, 92 events (37%) were classified as febrile neutropenia of unknown origin (FUO), 118 (47%) were classifiable as microbiologically documented infections, and 39 (17%) were classifiable as clinically documented infections. The overall 30-day mortality rate was 14% (28/200). The attributable mortality-infection rate was 6% (15/249). A lack of response to the CPX-351 treatment was the only factor significantly associated with mortality in the multivariate analysis [p-value: 0.004, OR 0.05, 95% CI 0.01-0.39]. Our study confirms the good safety profile of CPX-351 in a real-life setting, with an incidence of infectious complications comparable to that of the pivotal studies; despite prolonged neutropenia, the incidence of fungal infections was low, as was infection-related mortality.

The CoV-2 outbreak: how hematologists could help to fight Covid-19.

COVID-19 is a medical emergency, with 20 % of patients presenting with severe clinical manifestations. From the pathogenetic point of view, COVID-19 mimics two other well-known diseases characterized by cytokine storm and hyper-activation of the immune response, with consequent organ damage: acute graft-versus-host disease (aGVHD) and macrophage activation syndrome (MAS). Hematologists are confident with these situations requiring a prompt therapeutic approach for switching off the uncontrolled cytokine release; here, we discuss pros and cons of drugs that are already employed in hematology in the light of their possible application in COVID-19. The most promising drugs might be: Ruxolitinib, a JAK1/2 inhibitor, with a rapid and powerful anti-cytokine effect, tyrosine kinase inhibitors (TKIs), with their good anti-inflammatory properties, and perhaps the anti-Cd26 antibody Begelomab. We also present immunological data from gene expression experiments where TKIs resulted effective anti-inflammatory and pro-immune drugs. A possible combined treatment algorithm for COVID-19 is here proposed.

Phase II Trial of Maintenance Treatment With IL2 and Zoledronate in Multiple Myeloma After Bone Marrow Transplantation: Biological and Clinical Results.

Background: Maintenance treatment after autologous bone marrow transplantation in multiple myeloma improves the outcome of patients. We designed a phase II clinical trial to evaluate the treatment with IL2 and zoledronate after autologous bone marrow transplantation in myeloma patients. Methods: Patients with a histologically proven diagnosis of multiple myeloma become eligible if achieved a very good partial remission in bone marrow samples after 3 months from autologous bone marrow transplantation. IL2 was administered from day 1 to 7. In the first cycle, the daily dose was 2 × 106 IU, whereas, in subsequent ones the IL2 dose was progressively escalated, with +25% increases at each cycle, until evidence of toxicity or up to 8 × 106 IU. Four mg of zoledronic acid were infused on day 2. Flow cytometry analysis of γδ-lymphocytes was performed at days 1 and 8 of treatment cycles. Results: Forty-four patients have been enrolled between 2013 and 2016. The median time to progression was 22.5 months (95% CI 9.7-35.2). A complete remission with a negative immunofixation was obtained in 18% of patients and correlated with a significantly longer time to progression (p = 0.015). Treatment was well tolerated without G3 or 4 toxicities. After a week of treatment with IL2 and zoledronate, γδ lymphocytes, Vγ9δ2, CD57+, effector, late effector, and memory γδ increased but in subsequent cycles, there was a progressive reduction of this expansion. Conclusions: The maintenance treatment with IL2 and Zoledronate has a modest activity in myeloma patients after autologous bone marrow transplantation. EudraCT Number: 2013-001188-22.

Treatment with Allogenic Mesenchymal Stromal Cells in a Murine Model of Systemic Lupus Erythematosus.

OBJECTIVE: Pre-clinical and uncontrolled studies in patients with systemic lupus erythematosus (SLE) showed that mesenchymal stromal cells (MSCs) have a potential therapeutic role in refractory cases. The optimal therapeutic strategy in these patients remain to be elucidated. Our aim was to test the hypothesis that repeated administrations of 1×106/kg body weight of allogenic MSCs, that is a significantly lower dosage with respect to the fixed 1×106 MSC used in animal models, can be effective in improving the clinical course of a murine SLE model. METHODS: Bone marrow derived MSCs were obtained from 12-week-old C57BL/6J mice. Seventy-five 8 weeks old female NZ mice were randomly assigned to receive via caudal vein the following alternative treatments: 1) single infusion of 106 MSCs/kg body weight at 18 weeks of age (NZs18) or at at 22 weeks of age (NZs22); 2) multiple monthly infusions of 106 MSCs/kg body weight starting at 18 weeks of age (NZM18) or at 22 weeks of age (NZM22); 3) saline infusions (NZc) Fifteen 8 weeks old C57BL/6J mice (Envigo, Huntingdon, UK) were used as untreated controls (C). Weekly, body weight was recorded and twenty-four hour urines were collected by metabolic cages for each animal; proteinuria was detected by dipstick analysis. At sacrifice, peripheral blood samples were collected from mice and anti-dsDNA antibodies were detected by enzyme immunoassorbent assay (ELISA) method using commercial kits. At sacrifice, kidneys were analyzed for histopathology and immunohistochemical analysis for B220, CD4, MPO, CD4＋Foxp3, F40/80 infiltration was performed. RESULTS: Proteinuria occurrence was delayed NZS and NZM mice, no differences were observed in anti-dsDNA autoantibody titer among the groups at the different time-points; at 36 weeks, no significant differences were observed in term of nephritis scores. Inflammatory cells deposition (MPO and F4/80 positive cells) in NZM was significantly higher than in NZ and NZS. An overexpression of B lymphocytes (B220) was found in NZM while T regulatory cells (CD4＋ Foxp3＋ cells) were reduced in both NZS and NZM with respect to NZc. CONCLUSIONS: Overall, our study failed to show a positive effect of a treatment with murine MSCs in this model and, for some aspects, even deleterious results seem to be observed.

Mesangiogenic Progenitor Cells Derived from One Novel CD64(bright)CD31(bright)CD14(neg) Population in Human Adult Bone Marrow.

Mesenchymal stromal cells (MSCs) have been the object of extensive research for decades, due to their intrinsic clinical value. Nonetheless, the unambiguous identification of a unique in vivo MSC progenitor is still lacking, and the hypothesis that these multipotent cells could possibly arise from different in vivo precursors has been gaining consensus in the last years. We identified a novel multipotent cell population in human adult bone marrow that we first named Mesodermal Progenitor Cells (MPCs) for the ability to differentiate toward the mesenchymal lineage, while still retaining angiogenic potential. Despite extensive characterization, MPCs positioning within the differentiation pathway and whether they can be ascribed as possible distinctive progenitor of the MSC lineage is still unclear. In this study, we describe the ex vivo isolation of one novel bone marrow subpopulation (Pop#8) with the ability to generate MPCs. Multicolor flow cytometry in combination with either fluorescence-activated cell sorting or magnetic-activated cell sorting were applied to characterize Pop#8 as CD64(bright)CD31(bright)CD14(neg). We defined Pop#8 properties in culture, including the potential of Pop#8-derived MPCs to differentiate into MSCs. Gene expression data were suggestive of Pop#8 in vivo involvement in hematopoietic stem cell niche constitution/maintenance. Pop#8 resulted over three logs more frequent than other putative MSC progenitors, corroborating the idea that most of the controversies regarding culture-expanded MSCs could be the consequence of different culture conditions that select or promote particular subpopulations of precursors.

Specific integrin expression is associated with podosome-like structures on mesodermal progenitor cells.

Mesenchymal stromal cells (MSCs) are a heterogeneous cell population capable of differentiating toward several cell lines in vitro and, possibly, in vivo. Within cultured MSCs, we identified and purified a precursor cell population [mesodermal progenitor cells (MPCs)] retaining robust proliferation potential and ability to differentiate into endothelial or mesenchymal cells. MPC-derived MSCs retain the ability to further differentiate into osteoblasts, cartilage, or fat cells. Here we further characterized MPCs and MSCs by evaluating expression of integrins and adhesion molecules showing their ability to assemble the molecular machinery involved in endothelium adhesion. MPCs were shown to interact with activated and nonactivated endothelium, whereas MSCs exhibited activation of focal adhesion complexes, higher cell motility, and reduced or absent adhesiveness onto endothelial cells, suggesting a matrix remodeling vocation. We also reported a consistent expression of CXCR4 on the MPC cell surface, suggesting that the different phenotypic behavior could be related to specific functions of the cell in each differentiation stage.

Mesodermal progenitor cells (MPCs) differentiate into mesenchymal stromal cells (MSCs) by activation of Wnt5/calmodulin signalling pathway.

BACKGROUND: Mesenchymal Stromal Cells (MSCs) remain poorly characterized because of the absence of manifest physical, phenotypic, and functional properties in cultured cell populations. Despite considerable research on MSCs and their clinical application, the biology of these cells is not fully clarified and data on signalling activation during mesenchymal differentiation and proliferation are controversial. The role of Wnt pathways is still debated, partly due to culture heterogeneity and methodological inconsistencies. Recently, we described a new bone marrow cell population isolated from MSC cultures that we named Mesodermal Progenitor Cells (MPCs) for their mesenchymal and endothelial differentiation potential. An optimized culture method allowed the isolation from human adult bone marrow of a highly pure population of MPCs (more than 97%), that showed the distinctive SSEA-4+CD105+CD90(neg) phenotype and not expressing MSCA-1 antigen. Under these selective culture conditions the percentage of MSCs (SSEA-4(neg)CD105+CD90(bright) and MSCA-1+), in the primary cultures, resulted lower than 2%. METHODOLOGY/PRINCIPAL FINDING: We demonstrate that MPCs differentiate to MSCs through an SSEA-4+CD105+CD90(bright) early intermediate precursor. Differentiation paralleled the activation of Wnt5/Calmodulin signalling by autocrine/paracrine intense secretion of Wnt5a and Wnt5b (p<0.05 vs uncondictioned media), which was later silenced in late MSCs (SSEA-4(neg)). We found the inhibition of this pathway by calmidazolium chloride specifically blocked mesenchymal induction (ID50 =  0.5 µM, p<0.01), while endothelial differentiation was unaffected. CONCLUSION: The present study describes two different putative progenitors (early and late MSCs) that, together with already described MPCs, could be co-isolated and expanded in different percentages depending on the culture conditions. These results suggest that some modifications to the widely accepted MSC nomenclature are required.

High-dose (40,000 IU twice/week) alpha recombinant human erythropoietin as single agent in low/intermediate risk myelodysplastic syndromes: a retrospective investigation on 133 patients treated in a single institution.

We investigated the efficacy of alpha recombinant human erythropoietin (α-rHuEPO) administered as single agent to 133 patients affected by myelodysplastic syndromes referring to our Institution in the last 10 years. WPSS score was "very low" in 67%, "low" in 19%, "intermediate" in 14%. The starting schedule was: 40,000 IU bi-weekly, with reduction or suspension, when necessary, in responsive patients. According to new IWG criteria, response rate (RR) was 75%, 66%, 59% after 8, 16, 24 weeks, respectively. Comparing "very low" and "low/intermediate" risk, RR was 81% vs. 43% (P < 0.001); 70% vs. 45% (P = 0.040); 63% vs. 42% (P = NS) after 8, 16, 24 weeks. RR was significantly influenced by transfusion dependence (P = 0.039) and basal serum EPO level (P < 0.001). Mean Hb value was 94 ± 11 g/l before therapy; 114 ± 19 after 8 weeks (P < 0.001); 116 ± 18 after 16 weeks (P < 0.001); 114 ± 17 after 24 weeks (P < 0.001). Reduction or suspension of therapy significantly affected Hb level after 4 (P < 0.001) and 8 weeks (P < 0.01). Conversely, restart of full dosage significantly enhanced again Hb level after 4 (P < 0.01) and 8 weeks (P < 0.001). 65% patients are alive (mean survival: 74 weeks). Seventy percent are alive in the "very low risk" group and 38% in "low/intermediate risk" group (P < 0.001). Overall mean follow-up was 69 weeks (range, 8-376): it was 80 weeks in responsive patients (max 376) and 38 weeks in patients who progressively became unresponsive (max 168) (P < 0.01). Median response was 36 weeks, with 33% of patients still responding after one year. Treatment was well tolerated.

Bendamustine with or without rituximab in the treatment of relapsed chronic lymphocytic leukaemia: an Italian retrospective study.

To retrospectively assess the efficacy of bendamustine alone and with rituximab (R-B), 109 patients with relapsed chronic lymphocytic leukaemia (CLL) were enrolled in 24 Italian centres. The median age was 66 years (range 39-85). Forty-three percent of patients had relapsed and 57% were resistant (median previous therapies = 3; range 1-8). Twenty-two patients received bendamustine alone and 87 patients received R-B (median B dosage: 100 mg/m(2) per day, range 90-130 mg/m(2) per day). The overall response rate was 69·6% (complete response 28·6%; partial response 41%), and was significantly higher in patients treated with R-B (P = 0·014) and in those responsive to the previous treatment (P=0·04). After a median follow-up of 7·9 months (range 1-148), the median progression-free survival was 16 months and the median duration of response was 13 months. Median overall survival (OS) was 16·8 months for the whole cohort; patients not responding to the treatment had a significantly worse outcome than those who attained a response (P = 0·0001). In multivariate analysis, only resistant disease status at start of bendamustine treatment (HR 3·2, 95% CI 1·4-7·3, P = 0·006) had an independent prognostic value for OS. Toxicity was manageable and mostly haematological. In conclusion, in our experience R-B was an effective and well-tolerated treatment for relapsed/refractory CLL patients, producing a remarkable high CR rate and mild toxicity.

Constitutive expression of pluripotency-associated genes in mesodermal progenitor cells (MPCs).

BACKGROUND: We recently characterized a progenitor of mesodermal lineage (MPCs) from the human bone marrow of adults or umbilical cord blood. These cells are progenitors able to differentiate toward mesenchymal, endothelial and cardiomyogenic lineages. Here we present an extensive molecular characterization of MPCs, from bone marrow samples, including 39 genes involved in stem cell machinery, differentiation and cell cycle regulation. METHODOLOGY/PRINCIPAL FINDINGS: MPCs are cytofluorimetrically characterized and quantitative RT-PCR was performed to evaluate the gene expression profile, comparing it with MSCs and hESCs lines. Immunofluorescence and dot-blot analysis confirm qRT-PCR data. MPCs exhibit an increased expression of OCT4, NANOG, SALL4, FBX15, SPP1 and to a lesser extent c-MYC and KLF4, but lack LIN28 and SOX2. MPCs highly express SOX15. CONCLUSIONS/SIGNIFICANCE: MPCs express many pluripotency-associated genes and show a peculiar Oct-4 molecular circuit. Understanding this unique molecular mechanism could lead to identifying MPCs as feasible, long telomeres, target cells for reprogramming with no up-regulation of the p53 pathway. Furthermore MPCs are easily and inexpensively harvested from human bone marrow.

Selective culture of mesodermal progenitor cells.

We have recently identified mesodermal progenitor cells (MPCs) isolated from adult human bone marrow. These cells show unusual phenotypes, having putative embryonic markers and aldehyde dehydrogenase (ALDH) activity. Interestingly, these resting cells, which have been selected by culturing them in the presence of adult human serum, can easily be induced to differentiate into mature mesenchymal stromal cells (MSCs) after substituting the adult human serum for fetal bovine serum (FBS) or human cord serum. MPC-derived MSCs are, in turn, able to differentiate toward osteoblasts, chondrocytes, and adipocytes. Furthermore, MPCs are able to differentiate into endothelial cells. MPCs have been proven to be strongly adherent to plastic culture bottles and to be trypsin-resistant. In the present article, we show a simple and inexpensive method to isolate highly selected mesodermal progenitors from bone marrow or cord blood. The optimization of standard culture conditions (using commercial human AB sera and appropriate concentrations for cell seeding in plastics) allows a pure population of MPCs to be obtained even after a short culture period. We believe that this simple, repeatable, and standardized method will facilitate studies on MPCs.

Identification and purification of mesodermal progenitor cells from human adult bone marrow.

Bone marrow-derived mesodermal stem cells may differentiate toward several lines and are easily cultured in vitro. Some putative progenitors of these cells have been described in both humans and mice. Here, we describe a new mesodermal progenitor population [mesodermal progenitors cells (MPCs)] able to differentiate into mesenchymal cells upon appropriate culture conditions. When cultured in presence of autologous serum, these cells are strongly adherent to plastic, resistant to trypsin detachment, and resting. Mesodermal progenitor cells may be pulsed to proliferate and differentiate by substituting autologous serum for human cord blood serum or fetal calf serum. By these methods cells proliferate and differentiate toward mesenchymal cells and thus may further differentiate into osteoblats, chondrocytes, or adipocytes. Moreover MPCs are capable to differentiate in endothelial cells (ECs) showing characteristics similar to microvessel endothelium cells. Mesodermal progenitors cells have a defined phenotype and carry embryonic markers not present in mesenchymal cells. Moreover MPCs strongly express aldehyde dehydrogenase activity, usually present in hematopoietic precursors but absent in mesenchymal cells. When these progenitors are pulsed to differentiate, they lose these markers and acquire the mesenchymal ones. Interestingly, mesenchymal cells may not be induced to back differentiate into MPCs. Our results demonstrate the adult serum role in maintaining pluripotent mesodermal precursors and allow isolation of these cells. After purification, MPCs may be pulsed to proliferate in a very large scale and then induced to differentiate, thus possibly allowing their use in regenerative medicine.

Good manufacturing practice-grade fibrin gel is useful as a scaffold for human mesenchymal stromal cells and supports in vitro osteogenic differentiation.

BACKGROUND: Recently, there has been an increased interest in using mesenchymal stromal cells (MSCs) in bone tissue engineering coupled with a suitable scaffold of both biological and synthetic origin. The cells and these constructs can be combined in vitro or directly in vivo to enhance tissue repair. MSCs are spindle-shaped cells capable of self-renewal and can be induced to differentiate mainly into osteo-, chondro-, and adipogenic-progeny types. Several biomaterials are currently available and, among them, fibrin-based constructs seem to be suitable for guiding the cells during tissue repair or regeneration due to their biocompatibility and biodegradability. STUDY DESIGN AND METHODS: Here, this study describes a simple in vitro system using human mesenchymal stromal cells (hMSCs) and fibrin scaffold prepared at different concentrations in fibrinogen (1.5%-3% and 6%) to evaluate cell proliferation and viability inside these constructs. RESULTS: The data demonstrate that the constructs with 3 percent in fibrinogen resulted in the best scaffolds, because within them the cells were able to proliferate and were uniformly distributed. Finally, analyzing the capability of the clots to support osteogenic differentiation of MSCs, we observed that they differentiated into osteoblasts. CONCLUSION: These results suggest that fibrin gel could be useful as a delivery system for hMSCs.

The small peptide OGP(10-14) acts through Src kinases and RhoA pathways in Mo-7e cells: morphologic and immunologic evaluation.

BACKGROUND: Osteogenic growth peptide (OGP) is an endogenous tetradecapeptide present in micromolar concentrations in mammalian serum; its carboxy-terminal pentapeptide, OGP(10-14), represents its physiologically active fragment. OGP(10-14) induces proliferation and differentiation in fibroblast and osteoblast cell lines, and it enhances hematopoiesis in vitro and in vivo. The signaling pathways triggered by OGP(10-14) are not yet fully known. In the present report, we evaluated the effect of OGP(10-14) on differentiation of a cancer megakaryoblast cell line and its involvement on RhoA and Src family kinases signaling pathway. MATERIAL/METHODS: Cell proliferation of the Mo-7e line was evaluated using the MTT test. Mo-7e differentiation was evaluated by microscopic observation of cell morphology and by expression of the factor VIII-related antigen. Involvement of RhoA and Src kinases on signaling pathways triggered by OGP(10-14) was analyzed using RhoA and Src family kinase (SFK) inhibitors (C3 and PP2) and an immunoperoxidase technique. RESULTS: OGP(10-14) induces expression of the factor VIII-related antigen, morphologic changes indicative of megakaryocytic differentiation, and a down-regulation of the Fyn Src kinase. These OGP(10-14) effects were prevented by C3 and enhanced by PP2. CONCLUSIONS: The anti-proliferative and pro-differentiating activities of OGP(10-14) on thrombopoietin (TPO)-primed Mo-7e cells are mediated by RhoA and Src kinase pathways as demonstrated by the use of C3 and PP2.