A surge of late-occurring meiotic double-strand breaks rescues synapsis abnormalities in spermatocytes of mice with hypomorphic expression of SPO11

Meiosis is the biological process that, after a cycle of DNA replication, halves the cellular chromosome complement, leading to the formation of haploid gametes. Haploidization is achieved via two successive rounds of chromosome segregation, meiosis I and II. In mammals, during prophase of meiosis I, homologous chromosomes align and synapse through a recombination-mediated mechanism initiated by the introduction of DNA double-strand breaks (DSBs) by the SPO11 protein. In male mice, if SPO11 expression and DSB number are reduced below heterozygosity levels, chromosome synapsis is delayed, chromosome tangles form at pachynema, and defective cells are eliminated by apoptosis at epithelial stage IV at a spermatogenesis-specific endpoint. Whether DSB levels produced in Spo11 +/− spermatocytes represent, or approximate, the threshold level required to guarantee successful homologous chromosome pairing is unknown. Using a mouse model that expresses Spo11 from a bacterial artificial chromosome, within a Spo11 −/− background, we demonstrate that when SPO11 expression is reduced and DSBs at zygonema are decreased (approximately 40 % below wild-type level), meiotic chromosome pairing is normal. Conversely, DMC1 foci number is increased at pachynema, suggesting that under these experimental conditions, DSBs are likely made with delayed kinetics at zygonema. In addition, we provide evidences that when zygotene-like cells receive enough DSBs before chromosome tangles develop, chromosome synapsis can be completed in most cells, preventing their apoptotic elimination

SARS-CoV-2 Vaccine Induced Atypical Immune Responses in Antibody Defects: Everybody Does their Best

Background: Data on immune responses to SARS-CoV-2 in patients with Primary Antibody Deficiencies (PAD) are limited to infected patients and to heterogeneous cohorts after immunization. Methods: Forty-one patients with Common Variable Immune Deficiencies (CVID), six patients with X-linked Agammaglobulinemia (XLA), and 28 healthy age-matched controls (HD) were analyzed for anti-Spike and anti-receptor binding domain (RBD) antibody production, generation of Spike-specific memory B-cells, and Spike-specific T-cells before vaccination and one week after the second dose of BNT162b2 vaccine. Results: The vaccine induced Spike-specific IgG and IgA antibody responses in all HD and in 20% of SARS-CoV-2 naive CVID patients. Anti-Spike IgG were detectable before vaccination in 4 out 7 CVID previously infected with SARS-CoV-2 and were boosted in six out of seven patients by the subsequent immunization raising higher levels than patients naïve to infection. While HD generated Spike-specific memory B-cells, and RBD-specific B-cells, CVID generated Spike-specific atypical B-cells, while RBD-specific B-cells were undetectable in all patients, indicating the incapability to generate this new specificity. Specific T-cell responses were evident in all HD and defective in 30% of CVID. All but one patient with XLA responded by specific T-cell only. Conclusion: In PAD patients, early atypical immune responses after BNT162b2 immunization occurred, possibly by extra-follicular or incomplete germinal center reactions. If these responses to vaccination might result in a partial protection from infection or reinfection is now unknown. Our data suggests that SARS-CoV-2 infection more effectively primes the immune response than the immunization alone, possibly suggesting the need for a third vaccine dose for patients not previously infected

Point-of-care fresh CAR T cells for pediatric or young adult BCP-ALL that is relapsed/refractory or in very-high-risk first relapse

: The point-of-care, automated manufacturing of CAR T cells can reduce the waiting window and increase the therapy accessibility to patients with relapsed/refractory (r/r) BCP-ALL as compared to commercially available drug products (DP). We conducted a phase I/II trial (NCT04787263) on the use of 2nd generation (4.1BB), CD19-CAR T cells, for the treatment of BCP-ALL patients with either 1st relapse and very high-risk (VHR) characteristics or >2nd relapse and rapidly progressing disease preventing access to the commercial DP. CAR-T cells were manufactured with a 12-day process using CliniMACS Prodigy®, from a fresh apheresis and infused fresh. Three dose levels were tested in the phase I, namely 1.0, 2.0 and 3.0×106 CAR+T cells/kg. Nineteen pts were enrolled, 13/19 (68%) in first VHR relapse. The designed dose was always successfully produced. No dose-limiting toxicities were observed in the phase I. Grade 1-2 cytokine release syndrome was observed in 13/19 (68%) patients. Grade 1-2 immune effector cell-associated neurotoxicity syndrome occurred in 2 patients and resolved spontaneously. All patients achieved bone marrow complete remission with negative minimal residual disease. Eight pts (42%) received hematopoietic stem cell transplantation (HSCT) consolidation. Overall, CR was maintained in 13/19 patients (68%), 7 of them having received HSCT. Importantly, all patients but one treated with 3.0x106 CAR+T cells/kg maintain CR. The 3y-EFS and OS of the whole cohort are 68% and 83%, respectively. Our data suggest that the point-of-care, manufacturing of autologous, anti-CD19 CAR T cells can successfully treat patients with BCP-ALL including those with 1st relapse and VHR characteristics. (NCT04787263)

Donor-derived GD2-specific CAR T cells in relapsed or refractory neuroblastoma

Allogeneic chimeric antigen receptor (CAR) T cells targeting disialoganglioside-GD2 (ALLO_GD2-CART01) could be a therapeutic option for patients with relapsed or refractory, high-risk neuroblastoma (r/r HR-NB) whose tumors did not respond to autologous GD2-CART01 or who have profound lymphopenia. We present a case series of five children with HR-NB refractory to more than three different lines of therapy who received ALLO_GD2-CART01 in a hospital exemption setting. Four of them had previously received allogeneic hematopoietic stem cell transplantation. All patients experienced grade 2 or 3 cytokine release syndrome and one grade 2 neurotoxicity. Moderate acute graft-versus-host-disease occurred in four patients. ALLO_GD2-CART01 persisted for >6 weeks. Post-treatment, two complete responses were achieved and one maintained; in addition, one partial response and one stable disease were observed. Comparing the transcriptomic profiles obtained by RNA sequencing analyses of drug products with patient-matched, peripheral blood ALLO_GD2-CART01 collected at expansion, we found upregulation of genes associated with T cell activation and migration. In addition, after infusion, transcriptomic signaling analysis showed enrichment of genes involved in response to decreased oxygen levels, humoral immune response, cell polarization and immune-synapse formation. In comparison to autologous CAR T cells, ALLO_GD2-CAR T cells were characterized by pathways associated with T cell proliferation, immune-synapse formation and cell chemotaxis. The safety and efficacy of ALLO_GD2-CART01 in children with r/r HR-NB deserve further investigation in a prospective trial

Bone marrow megakaryocytic activation predicts fibrotic evolution of Philadelphia-negative myeloproliferative neoplasms

Philadelphia-negative chronic myeloproliferative neoplasms (MPNs) have been traditionally considered as indistinctly slowly progressing conditions; recent evidence proves that a subset of cases have a rapid evolution, so that MPNs' prognosis needs to be personalized. We identified a new morphological parameter, defined as Megakaryocytic Activation (M-ACT) based on the coexistence of megakaryocytic emperipolesis, megakaryocytes (MK) clusters formation and evidence of arrangement of collagen fibers around the perimeter of MK. We retrospectively analyzed the bone marrow biopsy of two MPNs cohorts of patients with polycythemia (PV) (n=64) and non-PV patients [including essential thrombocythemia (ET), and early/prefibrotic primary myelofibrosis (PMF)] (n=222). M-ACT showed a significant correlation with splenomegaly, white blood cell (WBC) count, and LDH serum levels in both groups, with JAK2 V617F allele burden in PV patients, and with CALR mutations, and platelet count in non-PV patients. Progression-free survival, defined as PV-to-secondary MF progression and non-PV-to-overt PMF, was worse in both PV and early/prefibrotic PMF patients with M-ACT in comparison to those without M-ACT (P<.0001). Interestingly, M-ACT was not found in the subgroup of ET patients. In conclusion, M-ACT can be helpful in the differential diagnosis of MPNs and can represent a new morphologic parameter with a predictive value for progression of MPNs

The new Friuli Earthquake Damage (Fr.E.D.) database

After the May 6, 1976 earthquake in Friuli Venezia Giulia (NE of Italy) the buildings in the shaken area were investigated and 84780 forms were filled in. After a first organization of these data, we provided a re-designed database in order to make the data more usable. Thanks to the new technologies that have emerged in the last years, the original data were enlarged, adding, where possible, the geographic coordinates for each form acquired. We are now able to exploit new information for deeper studies and analyses to a level of detail unthinkable until a few years ago. All this information is now available on-line upon request