Cellular and transcriptional dynamics of human neutrophils at steady state and upon stress.

Traditionally viewed as poorly plastic, neutrophils are now recognized as functionally diverse; however, the extent and determinants of neutrophil heterogeneity in humans remain unclear. We performed a comprehensive immunophenotypic and transcriptome analysis, at a bulk and single-cell level, of neutrophils from healthy donors and patients undergoing stress myelopoiesis upon exposure to growth factors, transplantation of hematopoietic stem cells (HSC-T), development of pancreatic cancer and viral infection. We uncover an extreme diversity of human neutrophils in vivo, reflecting the rates of cell mobilization, differentiation and exposure to environmental signals. Integrated control of developmental and inducible transcriptional programs linked flexible granulopoietic outputs with elicitation of stimulus-specific functional responses. In this context, we detected an acute interferon (IFN) response in the blood of patients receiving HSC-T that was mirrored by marked upregulation of IFN-stimulated genes in neutrophils but not in monocytes. Systematic characterization of human neutrophil plasticity may uncover clinically relevant biomarkers and support the development of diagnostic and therapeutic tools.

Early bone marrow alterations in patients with adenosine deaminase 2 deficiency across disease phenotypes and severities.

BACKGROUND: Deficiency of adenosine deaminase 2 (DADA2) is a complex monogenic disease caused by recessive mutations in the ADA2 gene. DADA2 exhibits a broad clinical spectrum encompassing vasculitis, immunodeficiency, and hematologic abnormalities. Yet, the impact of DADA2 on the bone marrow (BM) microenvironment is largely unexplored. OBJECTIVE: This study comprehensively examined the BM and peripheral blood of pediatric and adult patients with DADA2 presenting with rheumatologic/immunologic symptoms or severe hematologic manifestations. METHODS: Immunophenotyping of hematopoietic stem cells (HSCs), progenitor cells, and mature cell populations was performed for 18 patients with DADA2. We also conducted a characterization of mesenchymal stromal cells. RESULTS: Our study revealed a significant decrease in primitive HSCs and progenitor cells, alongside their reduced clonogenic capacity and multilineage differentiation potential. These BM defects were evident in patients with both severe and nonsevere hematologic manifestations, including pediatric patients, demonstrating that BM disruption can emerge silently and early on, even in patients who do not show obvious hematologic symptoms. Beyond stem cells, there was a reduction in mature cell populations in the BM and peripheral blood, affecting myeloid, erythroid, and lymphoid populations. Furthermore, BM mesenchymal stromal cells in patients with DADA2 exhibited reduced clonogenic and proliferation capabilities and were more prone to undergo cellular senescence marked by elevated DNA damage. CONCLUSIONS: Our exploration into the BM landscape of patients with DADA2 sheds light on the critical hematologic dimension of the disease and emphasizes the importance of vigilant monitoring, even in the case of subclinical presentation.

B-cells depletion after treatment with rituximab predicts relapse of IgG4-related disease.

OBJECTIVES: B cell depletion therapy with rituximab is effective in most patients with IgG4-related disease (IgG4-RD) but requires repeated cycles to prevent disease flares. We here aimed to assess B cells after rituximab to predict relapse of IgG4-RD and guide retreatment. METHODS: Patients with active IgG4-RD included in this retrospective study fulfilled the ACR/EULAR Classification Criteria. Total CD19+ B cells, plasmablasts, naïve and memory B cells were measured on peripheral blood by flow-cytometry at baseline and six months after rituximab. All patients were treated with two 1 g infusions of rituximab 15 days apart and monitored for 48 months. Disease response was assessed using the IgG4-RD Responder Index. RESULTS: Thirty-three patients were included. Six months after rituximab, disease response was observed in all patients. Complete depletion of CD19+ B cells, plasmablasts, naïve and memory B cell depletion was achieved in 30%, 55%, 39%, and 42% of cases, respectively. Twenty-three relapses (70%) were observed at a median time of 24 months after rituximab. Relapse rate was significantly higher in patients who failed to achieve complete depletion of CD19+ cells (60% vs 17%, p= 0.02), naïve B cells (54% vs 15%, p= 0.01), or memory B cells (50% vs 16%, p= 0.03) six months after rituximab. The median relapse free survival time was shorter in patients who failed to achieve complete depletion of CD19+ cells (19 vs 38 months, p= 0.02), naïve B cells (16 vs 38 months, p= 0.01), or memory B cells (19 vs 38 months, p= 0.03) six months after rituximab. CONCLUSIONS: The degree of B cell depletion six months after rituximab may predict disease flare and may instruct on the pacing of B cell depletion therapy in IgG4-RD.

A Novel Assay in Whole Blood Demonstrates Restoration of Mitochondrial Activity in Phagocytes After Successful HSCT in Hyperinflamed X-Linked Chronic Granulomatous Disease.

X-linked chronic granulomatous disease is a rare disease caused by mutations in the CYBB gene. While more extensive knowledge is available on genetics, pathogenesis, and possible therapeutic options, mitochondrial activity and its implications on patient monitoring are still not well-characterized. We have developed a novel protocol to study mitochondrial activity on whole blood of XCGD patients before and after transplantation, as well as on XCGD carriers. Here we present results of these analyses and of the restoration of mitochondrial activity in hyperinflamed X-linked Chronic Granulomatous Disease after hematopoietic stem cell transplantation. Moreover, we show a strong direct correlation between mitochondrial activity, chimerism, and DHR monitored before and after transplantation and in XCGD carriers. In conclusion, based on these findings, we suggest testing this new ready-to-use marker to better characterize patients before and after treatment and to investigate disease expression in carriers.

Emapalumab treatment in an ADA-SCID patient with refractory hemophagocytic lymphohistiocytosis-related graft failure and disseminated bacillus Calmette-Guérin infection.

Emapalumab, a fully human anti-IFNγ monoclonal antibody, has been approved in the US as second-line treatment of primary hemophagocytic lymphohistiocytosis (HLH) patients and has shown promise in patients with graft failure (GF) requiring a second allogeneic hematopoietic stem cell transplantation (HSCT). The blockade of IFNγ activity may increase the risk of severe infections, including fatal mycobacteriosis. We report a case of secondary HLH-related GF in the context of HLA-haploidentical HSCT successfully treated with emapalumab in the presence of concomitant life-threatening infections, including disseminated tuberculosis (TB). A 4 years old girl with Adenosine Deaminase-Severe Combined Immunodeficiency complicated by disseminated TB came to our attention for ex-vivo hematopoietic stem cell-gene therapy. After engraftment failure of gene corrected cells, she received two HLA-haploidentical T-cell depleted HSCT from the father, both failed due to GF related to concomitant multiple infections and secondary HLH. Emapalumab administration allowed to control HLH, as well as to prevent GF after a third haplo-HSCT from the mother. Remarkably, all infections improved with antimicrobial medications and disseminated TB did not show any reactivation. This seminal case supports emapalumab use for treatment of secondary HLH and prevention of GF in patients undergoing haplo-HSCT even in the presence of multiple infections, including TB.

B lymphocytes directly contribute to tissue fibrosis in patients with IgG4-related disease.

BACKGROUND: IgG4-related disease (IgG4-RD) is a fibroinflammatory condition marked by rapid clinical improvement after selective depletion of B lymphocytes with rituximab. This feature suggests that B cells might participate in fibrogenesis and wound healing. OBJECTIVE: In the present work we aimed to demonstrate that B lymphocytes contribute directly to tissue fibrosis in patients with IgG4-RD. METHODS: Total circulating CD19+ B lymphocytes, naive B cells, memory B cells, or plasmablasts from patients with IgG4-RD were cultivated with human fibroblasts. Profibrotic soluble factors and collagen production in cocultures were assessed by using ELISAs and Luminex assays. RNA sequencing and quantitative RT-PCR were used to assess fibroblast activation in the presence of B cells, as well as induction of profibrotic pathways in B-cell subsets. Relevant profibrotic and inflammatory molecules were confirmed in vitro by using functional experiments and on IgG4-RD tissue sections by using multicolor immunofluorescence studies. RESULTS: B cells from patients with IgG4-RD (1) produced the profibrotic molecule platelet-derived growth factor B and stimulated collagen production by fibroblasts; (2) expressed enzymes implicated in extracellular matrix remodeling, such as lysyl oxidase homolog 2; (3) produced the chemotactic factors CCL4, CCL5, and CCL11; and (4) induced production of these same chemokines by activated fibroblasts. Plasmablasts expressed sets of genes implicated in fibroblast activation and proliferation and therefore represent cells with intrinsic profibrotic properties. CONCLUSION: We have demonstrated that B cells contribute directly to tissue fibrosis in patients with IgG4-RD. These unanticipated profibrotic properties of B lymphocytes, particularly plasmablasts, might be relevant for fibrogenesis in patients with other fibroinflammatory disorders and for wound-healing processes in physiologic conditions.

Effects of glucocorticoids on B-cell subpopulations in patients with IgG4-related disease.

OBJECTIVES: Glucocorticoids induce prompt clinical improvement in patients with IgG4-related disease (IgG4-RD) but their mechanisms of action in this specific condition are not fully understood. B lymphocytes appear central to IgG4-RD pathogenesis because B-cell depletion with rituximab leads to swift clinical responses. In the present work we aim to assess the effects of glucocorticoids on B-cell subpopulations in patients with IgG4-RD. METHODS: Fifty patients with active untreated IgG4-RD and 20 healthy controls were enrolled in the present study. Flow cytometry analysis for total circulating CD19+ and CD20+ cells, naïve B cells, memory B cells, plasmablasts, and plasma cells was performed at baseline in all patients, and after 6 months of glucocorticoid treatment in 30 patients. Correlation studies with biomarkers of disease activity were also performed. RESULTS: At baseline, patients with IgG4-RD showed reduced CD19+ and CD20+ B cells compared to healthy controls, but increased circulating plasmablasts and plasma cells. Circulating plasmablasts and plasma cells correlated with clinical and serological biomarkers of IgG4-RD activity. Glucocorticoid-induced disease remission was accompanied by a reduction of naïve B cell count, an increase of memory B cells, and by a depletion of circulating plasmablasts and plasma cells. CD19+ and CD20+ B cells, were not affected by glucocorticoids. CONCLUSIONS: The efficacy of glucocorticoids in IgG4-RD is associated with selective effects on different B-cell subpopulations. Further studies are warranted to fully understand possible perturbations of the naïve and memory B-cell compartments in patients with IgG4-RD.

Autologous conditioned serum (ACS) for intra-articular treatment in Osteoarthritis: Retrospective report of 28 cases.

INTRODUCTION: Autologous conditioned serum (ACS) is a novel blood product developed for intra-articular injection as a novel therapy for Osteoarthritis (OA). This study is a retrospective evaluation of 28 cases (25 Knee-OA and 3 hip-OA) treated with ACS between November 2013 and February 2016. MATERIALS AND METHODS: ACS was prepared according to standards in an accredited Cell Manipulation Lab, and applied by an expert clinician (2 ml injection once weekly over 4 weeks). At any injection visit (Timepoints 1-4), and after a follow-up of 1 (Timepoint 5) and 6 months (Timepoint 6), patients were asked to describe the intensity of their pain with the VAS (visual analog scale) psychometric scale, and the objective parameter ROM (Range Of Motion) was recorded in case of injection in the knee. RESULTS: Pain (VAS) reduced in all cases since the first injection up to Timepoint 5. A significant improvement was observed in VAS between Timepoint 1 and 6 (primary objective), with a median VAS decrease of 60 mm (range 20-100, p < 0.01). A significant difference was also recorded in ROM between Timepoint 1 and 6 (secondary objective), with a median increase of 25° (range 5-40, p < 0.01). Ten out of 14 patients (71%) who were undergoing a chronic therapy to relieve pain were able to interrupt it. No serious adverse events were recorded. CONCLUSIONS: Treatment with ACS produced a rapid decline in pain, accompanied by a large improvement in ROM. These results suggest that ACS is a valid option for the treatment of OA.

Multiparametric Whole Blood Dissection: A one-shot comprehensive picture of the human hematopoietic system.

Human hematopoiesis is a complex and dynamic system where morphologically and functionally diverse mature cell types are generated and maintained throughout life by bone marrow (BM) Hematopoietic Stem/Progenitor Cells (HSPC). Congenital and acquired hematopoietic disorders are often diagnosed through the detection of aberrant frequency or composition of hematopoietic cell populations. We here describe a novel protocol, called "Whole Blood Dissection" (WBD), capable of analyzing in a single test-tube, hematopoietic progenitors and all major mature cell lineages composing either BM or peripheral blood (PB) through a multiparametric flow-cytometry analysis. WBD allows unambiguously identifying in the same tube up to 23 different blood cell types including HSPC subtypes and all the major myeloid and lymphoid lineage compartments at different stages of maturation, through a combination of 17 surface and 1 viability cell markers. We assessed the efficacy of WBD by analyzing BM and PB samples from adult (n = 8) and pediatric (n = 9) healthy donors highlighting age-related shift in cell composition. We also tested the capability of WBD on detecting aberrant hematopoietic cell composition in clinical samples of patients with primary immunodeficiency or leukemia unveiling expected and novel hematopoietic unbalances. Overall, WBD allows unambiguously identifying >99% of the cell subpopulations composing a blood sample in a reproducible, standardized, cost-, and time-efficient manner. This tool has a wide range of potential pre-clinical and clinical applications going from the characterization of hematopoietic disorders to the monitoring of hematopoietic reconstitution in patients after transplant or gene therapy. © 2017 The Authors. Cytometry Part A Published by Wiley Periodicals, Inc. on behalf of ISAC.

Quantitative measurement of 18F-FDG PET/CT uptake reflects the expansion of circulating plasmablasts in IgG4-related disease.

Objective: [18F]Fluorodeoxyglucose (18F-FDG) PET/CT is increasingly used to assess organ involvement and response to treatment in IgG4-related disease (IgG4-RD), but clear correlations between 18F-FDG uptake and disease activity have not been established yet. We aimed to correlate the intensity and distribution of 18F-FDG uptake with validated clinical, serological and immunological parameters of IgG4-RD activity. Methods: Twenty patients with active IgG4-RD underwent a baseline 18F-FDG PET/CT. Ten patients repeated 18F-FDG PET/CT after immunosuppressive treatments. 18F-FDG tissue uptake was measured using the standardized uptake value corrected for the partial volume effect (PVC-SUV) and the total lesion glycolysis (TLG) with (TLGtot+ln) and without (TLGtot-ln) lymph nodes. Disease activity was assessed by means of clinical parameters [IgG4-RD Responder Index (RI)], serological (ESR and CRP) and immunological (serum IgG4 and circulating plasmablasts) biomarkers. The enhanced liver fibrosis score was exploited as a biomarker for fibroblast activation. Results: Thirteen (65%) patients had two or more organs affected by IgG4-RD. All patients had active IgG4-RD as defined by a median IgG4-RD RI value of 9 (range 6-15; normal < 3). Serum IgG4 and plasmablasts were elevated in 85% of patients. Circulating plasmablasts positively correlated with PVC-SUV (P = 0.027), inversely correlated with TLGtot-ln (P = 0.023) and did not correlate with TLGtot+ln (P > 0.05). No statistically significant correlation was found between PVC-SUV or TLG and IgG4-RD RI, ESR, CRP, serum IgG4 or enhanced liver fibrosis score (P > 0.05). Clinical response to immunosuppressive therapies was associated with a consensual reduction of circulating plasmablasts, PVC-SUV, TLGtot+ln and TLGtot-ln values (P < 0.05 for all comparisons). Conclusions: 18F-FDG uptake of IgG4-RD lesions reflects immunological perturbations of the B cell compartment rather than fibroblast activation and extracellular matrix deposition. Conventional biomarkers of disease activity, namely IgG4-RD RI, ESR, CRP and serum IgG4 levels, do not appear to correlate with the radiometabolic activity of IgG4-RD lesions. In light of our results PET/CT represents a reliable instrument for assessing IgG4-RD activity, although lymph-node uptake deserves careful interpretation.

Deconstructing IgG4-related disease involvement of midline structures: Comparison to common mimickers.

OBJECTIVE: A series of destructive and tumefactive lesions of the midline structures have been recently added to the spectrum of IgG4-related disease (IgG4-RD). We examined the clinical, serological, endoscopic, radiological, and histological features that might be of utility in distinguishing IgG4-RD from other forms of inflammatory conditions with the potential to involve the sinonasal area and the oral cavity. METHODS: We studied 11 consecutive patients with erosive and/or tumefactive lesions of the midline structures referred to our tertiary care center. All patients underwent serum IgG4 measurement, flow cytometry for circulating plasmablast counts, nasal endoscopy, radiological studies, and histological evaluation of tissue specimens. The histological studies included immunostaining studies to assess the number of IgG4 + plasma cells/HPF for calculation of the IgG4+/IgG + plasma cell ratio. RESULTS: Five patients with granulomatosis with polyangiitis (GPA), three with cocaine-induced midline destructive lesions (CIMDL), and three with IgG4-RD were studied. We found no clinical, endoscopic, or radiological findings specific for IgG4-RD. Increased serum IgG4 and plasmablasts levels were not specific for IgG4-RD. Rather, all 11 patients had elevated blood plasmablast concentrations, and several patients with GPA and CIMDL had elevated serum IgG4 levels. Storiform fibrosis and an IgG4+/IgG + plasma cell ratio >20% on histological examination, however, were observed only in patients with IgG4-RD. CONCLUSIONS: Histological examination of bioptic samples from the sinonasal area and oral cavity represents the mainstay for the diagnosis of IgG4-RD involvement of the midline structures.

Post-transplantation Cyclophosphamide and Sirolimus after Haploidentical Hematopoietic Stem Cell Transplantation Using a Treosulfan-based Myeloablative Conditioning and Peripheral Blood Stem Cells.

Haploidentical hematopoietic stem cell transplantation (HSCT) performed using bone marrow (BM) grafts and post-transplantation cyclophosphamide (PTCy) has gained much interest for the excellent toxicity profile after both reduced-intensity and myeloablative conditioning. We investigated, in a cohort of 40 high-risk hematological patients, the feasibility of peripheral blood stem cells grafts after a treosulfan-melphalan myeloablative conditioning, followed by a PTCy and sirolimus-based graft-versus-host disease (GVHD) prophylaxis (Sir-PTCy). Donor engraftment occurred in all patients, with full donor chimerism achieved by day 30. Post-HSCT recovery of lymphocyte subsets was broad and fast, with a median time to CD4 > 200/µL of 41 days. Cumulative incidences of grade II to IV and III-IV acute GVHD were 15% and 7.5%, respectively, and were associated with a significant early increase in circulating regulatory T cells at day 15 after HSCT, with values < 5% being predictive of subsequent GVHD occurrence. The 1-year cumulative incidence of chronic GVHD was 20%. Nonrelapse mortality (NRM) at 100 days and 1 year were 12% and 17%, respectively. With a median follow-up for living patients of 15 months, the estimated 1-year overall and disease-free survival (DFS) was 56% and 48%, respectively. Outcomes were more favorable in patients who underwent transplantation in complete remission (1-year DFS 71%) versus patients who underwent transplantation with active disease (DFS, 34%; P = .01). Overall, myeloablative haploidentical HSCT with peripheral blood stem cells (PBSC) and Sir-PTCy is a feasible treatment option: the low rates of GVHD and NRM as well as the favorable immune reconstitution profile pave the way for a prospective comparative trial comparing BM and PBSC in this specific transplantation setting.

Plerixafor on demand in ten healthy family donors as a rescue strategy to achieve an adequate graft for stem cell transplantation.

BACKGROUND: In allogeneic hematopoietic stem cell (HSC) transplantation, the collection of an appropriate number of HSCs while maintaining a high level of safety for healthy donors is fundamental. Inadequate HSC mobilization can be seen with the standard use of granulocyte-colony-stimulating (G-CSF). Plerixafor (PL) is a chemokine receptor CXC Type 4-stromal-derived factor 1 inhibitor; its HSC-mobilizing properties are synergistic with G-CSF in poor mobilizing patients. The use of PL as adjuvant or alternative to G-CSF in healthy donors has shown a good safety profile but is so far off-label. STUDY DESIGN AND METHODS: We report 10 healthy HSC donors treated with PL because of insufficient response to G-CSF alone or contraindication to G-CSF. Eight donors did not mobilize enough CD34+ cells with G-CSF alone because poor mobilizers or because insufficient HSCs were harvested according to the clinical need of the patient; in two cases G-CSF administration and marrow harvest were unfeasible or contraindicated in the donor. RESULTS: The use of PL for mobilization increased the number of circulating CD34+ cells by 2.8-fold and the CD34+/kg collection by 3.0-fold. Only mild adverse events were reported (bone pain or discomfort) and not univocally attributable to PL. Rate of engraftment and graft-versus-host disease were similar to those seen in recipients of grafts from G-CSF only-mobilized donors. CONCLUSION: We exposed 10 allogeneic donors to mobilization with PL. PL was well tolerated in all cases and ensured procurement of an adequate graft for transplantation resulting in a normal hematopoietic engraftment.

Binge drinking, reflection impulsivity, and unplanned sexual behavior: impaired decision-making in young social drinkers.

BACKGROUND: The repeated pattern of heavy intoxication followed by withdrawal from alcohol (i.e., "binge drinking") has been found to have substantial adverse effects on prefrontal neural systems associated with decision-making and impulse control. Repeated binge drinking has been linked to risky and unplanned sexual behavior; however few studies have examined the role of impulsivity and related cognitive processes in understanding this association. The aim of this study was to examine the relationship between binge drinking, "reflection impulsivity" (deficits in gathering and evaluating information during decision-making), alcohol-related expectancies, and unplanned sexual behavior in a sample of young social drinkers. METHODS: Ninety-two university students completed the alcohol use questionnaire (AUQ) to measure alcohol intake and binge drinking. Two groups (low-binge and high-binge) were generated from the AUQ data. The Information Sampling Task (IST) was used to measure reflection impulsivity; the Alcohol Expectancy Questionnaire (AEQ) for alcohol outcome expectancies; and an unplanned sexual behavior questionnaire, which asked about the number of unplanned sexual events. RESULTS: When compared to the low-binge drinking group, the high-binge drinkers had significantly more unplanned sexual encounters and were impaired on the IST, reflection-impulsivity task. They scored higher on the alcohol expectancy factors of sociability, risk and aggression, negative self-perception, and in particular liquid courage. In a regression analysis, number of unplanned sexual encounters, binge drinking score, and liquid courage were all significantly related. CONCLUSIONS: These results support the role of binge drinking in reduced impulse control and decision-making deficits. The findings indicate that high-binge drinkers demonstrate impairments on an impulse control task similar to that observed in dependent samples and this may be a factor in understanding the negative behavioral consequences associated with excessive alcohol use.

Alcohol Misuse post Metabolic and Bariatric Surgery: A Systematic Review of Longer-term Studies with Focus on new Onset Alcohol use Disorder and Differences Between Surgery Types.

BACKGROUND: Evidence suggests an increased risk of alcohol problems post-surgery where no problematic alcohol use was present prior to surgery which may be different across types of surgery. OBJECTIVE: To characterise the risk of new onset alcohol misuse post bariatric surgery, differences between surgeries and the impact over time. METHODS: All published studies on new and relapsing alcohol use were reviewed. Data were classed as 'subjective' (clinical interview, self-report questionnaires) and 'objective' (hospital admissions, substance misuse programmes) and further categorised by follow up time - 'shorter-term' (one year), 'medium-term' (one year to two years) and 'long-term' (> two years). RESULTS: Twenty-three of the forty-two studies included in the review reported new onset data. Nine studies reported on differences between surgery types. In those reporting objective measures, all of which were long term, RYGB carried a higher risk than SG, followed by LAGB. All but one study using subjective measures reported a small but significant number of new onset concerning alcohol use, and comparisons between surgery types had more varied results than the objective measures. Studies of substance abuse programmes found high rates of new onset cases (17-60%). CONCLUSION: This systematic review provides support for the consensus guidance suggesting patients should be informed of a small but significant risk of new onset alcohol use following bariatric surgery, with the strongest evidence in the medium- to long-term and in those who have had RYGB followed by SG.

Managing leukapheresis in adult and pediatric patients eligible for chimeric antigen receptor T-cell therapy: suggestions from an Italian Expert Panel.

Chimeric antigen receptor (CAR) T-cell therapy relies on T cells engineered to target specific tumor antigens such as CD-19 in B-cell malignancies. In this setting, the commercially available products have offered a potential long-term cure for both pediatric and adult patients. Yet manufacturing CAR T cells is a cumbersome, multistep process, the success of which strictly depends on the characteristics of the starting material, i.e., lymphocyte collection yield and composition. These, in turn, might be affected by patient factors such as age, performance status, comorbidities, and previous therapies. Ideally, CAR T-cell therapies are a one-off treatment; therefore, optimization and the possible standardization of the leukapheresis procedure is critical, also in view of the novel CAR T cells currently under investigation for hematological malignancies and solid tumors. The most recent Best Practice recommendations for the management of children and adults undergoing CAR T-cell therapy provide a comprehensive guide to their use. However, their application in local practice is not straightforward and some grey areas remain. An Italian Expert Panel of apheresis specialists and hematologists from the centers authorized to administer CAR T-cell therapy took part in a detailed discussion on the following: 1) pre-apheresis patient evaluation; 2) management of the leukapheresis procedure, also in special situations represented by low lymphocyte count, peripheral blastosis, pediatric population <25 kg, and the COVID-19 outbreak; and 3) release and cryopreservation of the apheresis unit. This article presents some of the important challenges that must be faced to optimize the leukapheresis procedure and offers suggestions as to how to improve it, some of which are specific to the Italian setting.

Dynamics of polyclonal immuno-reconstitution after allogeneic transplant with post-transplant cyclophosphamide and letermovir.

Cytomegalovirus (CMV) reactivations are strong stimulators of immune-reconstitution (IR) in hematopoietic stem cell transplantation (HSCT) recipients. Herein, we analyzed 317 CMV-seropositive consecutive patients (n = 109 letermovir, LTV; n = 208 no-LTV), undergoing HSCT with post-transplant cyclophosphamide (PTCy) and calcineurin inhibitor- (CNI) free graft-versus-host-disease (GvHD) prophylaxis. At day+90, median CD19+/mm3 was higher in LTV-cohort: 5.5 [0;439] versus 2 [0;294], p = 0.008; median CD3+/mm3 counts were lower in LTV-cohort, with no differences in CD4+, CD8+ and NK-cells. At day+180 median CD3+, CD4+ and CD8+/mm3 values were comparable between groups. Higher CD19+/mm3 counts were observed in LTV-cohort: 62 [0; 2983] versus 42 [0; 863]. Significantly higher median NK/mm3 values were seen in LTV-cohort: 225.5 [0;763] versus 163.5 [0;1181], p = 0.0003. The impact of LTV on B-cell IR at 3 months and NK-cell levels at 6 months was retained in multivariate analysis (p < 0.01), whereas the effect on T-cells was not confirmed. Moreover, we confirmed a significant reduction of clinically-relevant CMV, and moderate-to- severe chronic GvHD in LTV-cohort. Overall, in our study the use of LTV was associated with a slight improvement of B-cell and NK-cells reconstitution, with only minor impact on T-cell subsets, giving new insights on polyclonal IR for HSCT recipients in the LTV era.

Intrabone Transplantation of a Single Unwashed Umbilical Cord Blood Unit with Antithymocyte Globulin-Free and Sirolimus-Based Graft-versus-Host Disease Prophylaxis: Fast Immune Reconstitution and Long-Term Disease Control in Patients with High-Risk Diseases.

Several strategies have been explored with the attempt of improving the safety and feasibility of umbilical cord blood transplantation (UCBT) in adults. The aim of this retrospective analysis was to examine the safety and efficacy of intrabone transplantation of a single unwashed cord blood unit in an antithymocyte globulin-free, sirolimus-based graft-versus-host disease prophylaxis platform. We collected data for all consecutive UCBTs infused intrabone (IB) and unwashed at San Raffaele Hospital in Milan between 2012 and 2021. Thirty-one consecutive UCBTs were identified. All but 3 UCB units had a high-resolution HLA typing on 8 loci at the time of selection. At the time of cryopreservation, the median CD34+ cell count was 1 × 105/kg (range, .6 to 12.0 × 105/kg) and the median total nucleated cell (TNC) count was 2.8 × 107/kg (range, 1.48 to 5.6 × 107/kg). Eighty-seven percent of patients received myeloablative conditioning, and 77% underwent transplantation for acute myeloid leukemia. The median duration of follow-up among survivors was 38.2 months (range, 10.4 to 123.6 months). No adverse events were related to the IB infusion at bedside under short-conscious periprocedural sedation or to the no wash technique. After thawing, median CD34+ cell and TNC counts were .8 × 105/kg (range, .1 to 2.3 × 105/kg) and 1.42 × 107/kg (range, .69 to 3.2 × 107/kg). The median time to engraftment was 27 days for neutrophils and 53 days for platelets. One patient experienced graft rejection and was subsequently rescued with a salvage transplantation. The median time to a CD3+ cell count >100/µL was 30 days. The 100-day cumulative incidence of grade III-IV acute graft-versus-host disease (GVHD) was 12.9% (95% confidence interval [CI], 4% to 27.3%), and the 2-year cumulative incidence of moderate-to-severe chronic GVHD (cGVHD) was 11.8% (95% CI, 2.7% to 28.3%). At 2 years, overall survival (OS) was 52.7% (95% CI, 33% to 69%), relapse incidence was 30.7% (95% CI, 13.7% to 49.6%), and transplantation-related mortality was 29% (95% CI, 14.3% to 45.6%). In univariate analysis, infused CD34+ cell count did not impact transplantation outcomes. In patients who underwent transplantation in first complete remission, relapse rate was 13%, with a 2-year OS >90%. In our cohort, IB infusion of a single cord blood unit was feasible, with no adverse reactions related to the no wash/IB infusion, low rates of cGVHD and disease relapse, and rapid immune reconstitution.

2020 COVID-19-Related Lockdown: the Relationships Between Coping Strategies, Psychological Adjustment and Resilience Among a Non-clinical Sample of British Adults.

To curb COVID-19 infections, the British government enforced a series of lockdowns resulting in restrictions on movement and socialisation. This study assessed which groups may have been at higher risk of emotional distress among a non-clinical sample of British adults. It also examined which coping strategies, if any, related to more positive psychological adjustment and higher resilience scores. A cross-sectional, correlational study was carried out. Using a convenience sample, an online survey was conducted in April-June 2020. One hundred ninety-four participants completed the Brief COPE (coping), the GAD-7 (anxiety), the PHQ-9 (depression), the CD-RISC (resilience), and provided demographic information. Participants used mainly coping strategies considered to be adaptive. They exhibited mild/moderate anxiety and depression symptoms, and moderate resilience scores. However, some individuals displayed significantly higher distress symptoms and lower resilience scores than others, especially those aged under 35 (particularly 18-24), those not working, those who were single and/or childless. Results also show that coping strategies including substance use, behavioural disengagement and self-blame were associated with anxiety and/or depression symptoms, conversely, positive reframing related to lower anxiety symptomatology. Interventions promoting positive reframing may be helpful. Similarly, interventions promoting connection to others, a factor known to enhance resilience, may be beneficial. This is particularly relevant to groups who may be more at risk of psychological distress, such as young individuals.