Experimental Results and Mechanistic Insights on the Reactions of Indolylmethyl Acetates with Soft Carbon Pronucleophiles.

The palladium-catalyzed reaction of N-protected 2-indolylmethyl acetates with soft carbon pronucleophiles is described. Besides the formation of the expected coupling reaction at the C1' position, unprecedented attack at the C3 position of the plausible η3-indolyl-palladium intermediate has been observed, and the selectivity control C1'/C3 seems to depend on the nature of the protecting group and ligand. The reactivity of 3-indolylmethyl acetates has also been also investigated. Quantum chemical calculations support the experimental results.

Palladium-Catalyzed Tsuji-Trost-Type Reaction of 3-Indolylmethylacetates with O, and S Soft Nucleophiles.

The chemical valorization of widespread molecules in renewable sources is a field of research widely investigated in the last decades. In this context, we envisaged that indole-3-carbinol, present in different Cruciferae plants, could be a readily available building block for the synthesis of various classes of indoles through a palladium-catalyzed Tsuji-Trost-type reaction with O and S soft nucleophiles. The regiochemical outcome of this high-yielding functionalization shows that the nucleophilic substitution occurs only at the benzylic position. Interestingly, with this protocol, the sulfonyl unit could be appended to the indole nucleus, providing convenient access to new classes of molecules with potential bioactivity.

Sustained post-rituximab B-cell depletion is common in ANCA-associated vasculitis and is affected by sex and renal function.

OBJECTIVE: Despite the increasing use of rituximab in anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), it remains unclear what the optimal dosing is, especially for maintenance of remission. A deeper understanding of post-rituximab B-cell repopulation patterns may aid better-tailored treatment. METHODS: This is a monocentric, retrospective study including ANCA-positive AAV patients receiving a single course of rituximab induction. CD19+ B cells were longitudinally monitored with flow cytometry. B-cell repopulation was defined as CD19+ >10 cells/µL. RESULTS: Seventy-one patients were included, the majority with microscopic polyangiitis (75%), myeloperoxidase-ANCA positivity (75%) and with renal involvement (79%). During a median follow-up of 54 months since the first rituximab infusion, 44 patients (62%) repopulated B cells, with a median time to repopulation of 39 months (range 7-102). Patients experiencing B-cell depletion lasting longer than the overall median time to repopulation (39 months) exhibited a lower risk of flare and higher risk of serious infection. In multivariate Cox regression, higher estimated glomerular filtration rate (eGFR) [hazard ratio (HR) 1.84, 95% confidence interval (CI) 1.13-2.98 per 30 mL/min/1.73 m2 eGFR] and female sex (HR 2.70, 95% CI 1.37-5.31) were independent predictors of increased rate of B-cell repopulation. CONCLUSION: A subset of AAV patients develop sustained post-rituximab B-cell depletion, which associates with reduced risk of flare and increased risk of serious infection in the long term. Preserved renal function and female sex are associated with faster B-cell repopulation. These observations further highlight the need to personalize immunosuppression to improve clinical outcomes.

Immunoglobulin light chain transcript detection by ultrasensitive RNA in situ hybridization for B-cell lymphoma diagnosis.

Evaluation of B-cell clonality can be challenging in the interpretation of lymphoid infiltrates on tissue sections. Clonality testing based on IG gene rearrangements analysis by PCR (IG-PCR) is the gold standard. Alternatively, B-cell clonality can be assessed by the recognition of immunoglobulin light chain (IgLC) restriction, by immunohistochemistry (IHC), chromogenic in situ hybridization (ISH) or flow cytometry (FC). IG-PCR requires molecular facilities, and FC requires cell suspensions, both not widely available in routine pathology units. This study evaluates the performance of B-cell clonality detection by IgLC-RNAscope® (RNAsc) in a group of 216 formalin-fixed, paraffin-embedded samples including 185 non-Hodgkin B-cell lymphomas, 11 Hodgkin lymphomas (HL) and 20 reactive samples. IgLC-RNAsc, performed in parallel with FC in 53 cases, demonstrated better performances (93% vs 83%), particularly in diffuse large B-cell lymphoma (98% vs 71%) and follicular lymphoma (93% vs 83%) diagnosis. IgLC-RNAsc was also superior to IHC and ISH especially in samples with limited tumor cell content, where IG-PCR was not informative. Performed for the first time on mediastinal lymphomas, IgLC-RNAsc identified monotypic IgLC transcripts in 69% of primary mediastinal large B-cell lymphoma (PMBCL) and 67% of mediastinal gray zone lymphomas (MGZL). IGK/L double-negative cells were detected in 1 PMBCL, 2 MGZL, and all classical HL, while monotypic IgLC expression appeared to be a hallmark in nodular lymphocyte-predominant HL. IgLC-RNAsc demonstrates to be a powerful tool in B-cell lymphoma diagnosis, above all in challenging cases with limited tumor cell content, ensuring in situ investigations on mechanisms of Ig regulation across lymphoma entities.

Clinical and transcriptomic characteristics of a novel SMARCD2 mutation that disrupts neutrophil maturation and function.

We report a novel case of SMARCD2 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily D, member 2) mutation successfully treated with hematopoietic stem cell transplantation. The female patient presented delayed cord separation, chronic diarrhea, skin abscesses, skeletal dysmorphisms, and neutropenia with specific granule deficiency. Analysis of the transcriptomic profile of peripheral blood sorted mature and immature SMARCD2 neutrophils showed defective maturation process that associated with altered expression of genes related to specific, azurophilic, and gelatinase granules, such as LTF, CRISP3, PTX3, and CHI3L1. These abnormalities account for the prevalence of immature neutrophils in the peripheral blood, impaired function, and deregulated inflammatory responses.

Rituximab Monotherapy Is Effective as First-Line Treatment for Granulomatous Lymphocytic Interstitial Lung Disease (GLILD) in CVID Patients.

Granulomatous lymphocytic interstitial lung disease (GLILD) represents a fatal immune dysregulatory complication in common variable immunodeficiency (CVID). Evidence-based diagnostic guidelines are lacking, and GLILD treatment consists in immunosuppressive drugs; nonetheless, therapeutical strategies are heterogeneous and essentially based on experts' opinions and data from small case series or case reports.We aimed to evaluate the efficacy and safety of first-line Rituximab monotherapy for CVID-related GLILD, by assessing symptoms and quality of life alterations, immunological parameters, pulmonary function tests, and lung computed tomography.All six GLILD patients received Rituximab infusions as a first-line treatment. Rituximab was administered at 375 mg/m2 monthly for six infusions followed by maintenance every 3 months; none of the patients experienced severe adverse events. Symptom burden and quality of life significantly improved in treated patients compared to a control group of CVID patients without GLILD. Rituximab treatment indirectly caused a trend toward reduced T-cell activation and exhaustion markers sCD25 and sTIM-3. Lung function improved in treated patients, with statistically significant increases in TLC and DLCO. Lung CT scan findings expressed by means of Baumann scoring system displayed a reduction in the entire cohort.In conclusion, first-line monotherapy with Rituximab displayed high efficacy in disease remission in all treated patients, with improvement of symptoms and amelioration of quality of life, as well as restoration of PFTs and lung CT scan findings.

Antibody Deficiency in Patients with Biallelic KARS1 Mutations.

Biallelic KARS1 mutations cause KARS-related diseases, a rare syndromic condition encompassing central and peripheral nervous system impairment, heart and liver disease, and deafness. KARS1 encodes the t-RNA synthase of lysine, an aminoacyl-tRNA synthetase, involved in different physiological mechanisms (such as angiogenesis, post-translational modifications, translation initiation, autophagy and mitochondrial function). Although patients with immune-hematological abnormalities have been individually described, results have not been collectively discussed and functional studies investigating how KARS1 mutations affect B cells have not been performed. Here, we describe one patient with severe developmental delay, sensoneurinal deafness, acute disseminated encephalomyelitis, hypogammaglobulinemia and recurrent infections. Pathogenic biallelic KARS1 variants (Phe291Val/ Pro499Leu) were associated with impaired B cell metabolism (decreased mitochondrial numbers and activity). All published cases of KARS-related diseases were identified. The corresponding authors and researchers involved in the diagnosis of inborn errors of immunity or genetic syndromes were contacted to obtain up-to-date clinical and immunological information. Seventeen patients with KARS-related diseases were identified. Recurrent/severe infections (9/17) and B cell abnormalities (either B cell lymphopenia [3/9], hypogammaglobulinemia [either IgG, IgA or IgM; 6/15] or impaired vaccine responses [4/7]) were frequently reported. Immunoglobulin replacement therapy was given in five patients. Full immunological assessment is warranted in these patients, who may require detailed investigation and specific supportive treatment.

Longitudinal Characterization of Immune Response in a Cohort of Children Hospitalized with Multisystem Inflammatory Syndrome.

BACKGROUND: Multisystem Inflammatory Syndrome in Children (MIS-C) is a severe complication of SARS-CoV-2 infection caused by hyperactivation of the immune system. METHODS: this is a retrospective analysis of clinical data, biochemical parameters, and immune cell subsets in 40 MIS-C patients from hospital admission to outpatient long-term follow-up. RESULTS: MIS-C patients had elevated inflammatory markers, associated with T- and NK-cell lymphopenia, a profound depletion of dendritic cells, and altered monocyte phenotype at disease onset, while the subacute phase of the disease was characterized by a significant increase in T- and B-cell counts and a rapid decline in activated T cells and terminally differentiated B cells. Most of the immunological parameters returned to values close to the normal range during the remission phase (20-60 days after hospital admission). Nevertheless, we observed a significantly reduced ratio between recently generated and more differentiated CD8+ T- and B-cell subsets, which partially settled at longer-term follow-up determinations. CONCLUSIONS: The characterization of lymphocyte distribution in different phases of MIS-C may help to understand the course of diseases that are associated with dysregulated immune responses and to calibrate prompt and targeted treatments.

Direct Regioselective Hydro(hetero)arylation/Cyclocondensation Reactions of ß-(2-Aminophenyl)-α,ß-ynones by Means of Transition-Metal Catalysis/Brønsted Acid Synergism: Experimental Results and Computational Insights.

Experimental results and computational insights explain the key role of transition-metal catalysis/Brønsted acid synergism in the achievement of the sequential regioselective direct heteroarylation/cyclocondensation reactions of ß-(2-aminophenyl)-α,ß-ynones with a variety of electron-rich aromatic heterocyclic/arenes to afford quinoline-(hetero)aromatic hybrids. The first approach to the synthesis of 4-(1H-pyrrol-2-yl)quinolines is described. The effectiveness of various transition metals is compared.

CARD11 dominant negative mutation leads to altered human Natural Killer cell homeostasis.

Dominant negative mutations in CARD11 have been reported in patients with immune dysregulation, severe atopic features, and variable T cell alterations. Data on Natural killer (NK) cells from affected patients are lacking. We report on a 12-year-old boy with severe atopic dermatitis, food induced anaphylaxis and hypogammaglobulinemia harbouring a novel de novo heterozygous variant c.169G > A; p.Glu57Lys in CARD11. The dominant negative effect of this mutation was confirmed on both CD4+ and CD8+. CTLA4+Foxp3+CD4+ Tregs were severely reduced. Patient's NK cells showed reduced expression of NKp46, NKG2D and CD69. Patient's CD56bright NK cells showed in vitro impaired production of IFN-γ. Steady state pS6 levels on patient's NK cells were increased and remained elevated upon IL2 + IL12 + IL18 overnight stimulation. Overall, the effect of CARD11 mutation on mTORC1 differs between T and NK cells. These findings may explain the increased susceptibility to viral infections and the reduced immune surveillance in affected patients.

Targeting the immune microenvironment in Waldenström macroglobulinemia via halting the CD40/CD40-ligand axis.

Recent investigations have improved our understanding of the molecular aberrations supporting Waldenström macroglobulinemia (WM) biology; however, whether the immune microenvironment contributes to WM pathogenesis remains unanswered. First, we showed how a transgenic murine model of human-like lymphoplasmacytic lymphoma/WM exhibits an increased number of regulatory T cells (Tregs) relative to control mice. These findings were translated into the WM clinical setting, in which the transcriptomic profiling of Tregs derived from patients with WM unveiled a peculiar WM-devoted messenger RNA signature, with significant enrichment for genes related to nuclear factor κB-mediated tumor necrosis factor α signaling, MAPK, and PI3K/AKT, which was paralleled by a different Treg functional phenotype. We demonstrated significantly higher Treg induction, expansion, and proliferation triggered by WM cells, compared with their normal cellular counterpart; with a more profound effect within the context of CXCR4C1013G-mutated WM cells. By investigating the B-cell-to-T-cell cross talk at single-cell level, we identified the CD40/CD40-ligand as a potentially relevant axis that supports WM cell-Tregs interaction. Our findings demonstrate the existence of a Treg-mediated immunosuppressive phenotype in WM, which can be therapeutically reversed by blocking the CD40L/CD40 axis to inhibit WM cell growth.

COVID-19 does not impact on outcome of myeloma patients infected while undergoing autologous stem cell transplantation.

Here we report two cases of myeloma patients who became positive for SARS-CoV-2 infection during the acute phase of autologous stem cell transplant. Both patients were promptly treated with monoclonal antibodies and remdesivir, and, despite the profound neutropenia and lymphopenia, they did not develop respiratory failure and they remained paucisymptomatic during the entire period of aplasia. Neutrophil engraftment took place as expected and the patients were discharged quickly and did not experience adverse effects after discharge.

Lack of a prompt normalization of immunological parameters is associated with long-term care and poor prognosis in COVID-19 affected patients receiving convalescent plasma: a single center experience.

OBJECTIVES: Being COVID-19 convalescent plasma (CCP) a therapeutic option that can have a potential impact on the normalization of immunological parameters of COVID-19 affected patients, a detailed analysis of post-infusion immunological changes was conducted in CCP treated patients, aiming to identify possible predictive hallmarks of disease prognosis. METHODS: This prospective observational study describes a cohort of 28 patients who received CCP shortly after being hospitalized for COVID-19 and diagnosed for Acute Respiratory Distress Syndrome. All patients were subjected to a detailed flow cytometry based evaluation of immunological markers at baseline and on days +3 and +7 after transfusion. RESULTS: At baseline almost all patients suffered from lymphopenia (25/28 on T-cells and 16/28 on B-cells) coupled with neutrophil-lymphocyte ratio exceeding normal values (26/28). Lymphocyte subsets were generally characterized by increased percentages of CD19+CD20-CD38hiCD27+ plasmablasts and reduction of CD4+CD45RA+CCR7+CD31+ recent thymic emigrants, while monocytes presented a limited expression of CD4 and HLA-DR molecules. Amelioration of immunological parameters began to be evident from day +3 and became more significant at day +7 post-CCP transfusion in 18 patients who recovered within 30 days from hospitalization. Conversely, baseline immunological characteristics generally persisted in ten critical patients who eventually progressed to death (6) or long-term care (4). CONCLUSIONS: This study demonstrates that proper immunophenotyping panels can be potentially useful for monitoring CCP treated patients from the first days after infusion in order to presume higher risk of medical complications.

Successful CAR-T cell therapy in a refractory MCL patient with bacterial, fungal and COVID-19 infection: a case report.

Background: The COVID-19 pandemic has had a significant impact on the management and care of onco-hematological patients, particularly those with lymphoproliferative disorders who are at higher risk for COVID-19 associated bacterial and fungal superinfections. Case presentation: We present the successful treatment of a 44-year-old male patient with refractory mantle cell lymphoma treated with chimeric antigen receptor T (CAR-T) cell therapy, despite concurrent COVID-19 infection. The patient developed grade II cytokine release syndrome, requiring admission to the intensive care unit. The CAR-T cells expanded effectively, and the patient achieved complete metabolic remission. During the treatment course, the patient experienced complications including COVID-19-associated pulmonary aspergillosis and a co-infection with Stenotrophomonas maltophilia and the SARS-CoV-2 omicron variant. Prompt antifungal and antibacterial therapy, along with appropriate COVID-19 treatment, led to the resolution of these infections. Dexamethasone was also administered to reduce inflammation and aid hematologic recovery. Despite the presence of multiple infections, the patient achieved complete remission of lymphoma, highlighting the effectiveness of CAR-T cell therapy in this high-risk patient. Conclusion: Despite the challenges posed by concurrent infections, the decision to proceed with CAR-T cell therapy in this patient proved to be successful, resulting in complete remission of lymphoma. Early initiation of supportive therapies and the use of dexamethasone contributed to the resolution of complications. This case underscores the importance of individualized decision-making and the potential benefits of CAR-T cell therapy in similar high-risk patients.

Electrochemical-Induced Cascade Reaction of 2-Formyl Benzonitrile with Anilines: Synthesis of N-Aryl Isoindolinones.

An electrochemical initiated tandem reaction of anilines with 2-formyl benzonitrile has been developed. Thus, unprecedented 3-N-aryl substituted isoindolinones have been conveniently achieved by constant current electrolysis in a divided cell using catalytic amount of electricity and supporting electrolyte and a Pt-cathode as working electrode. The origin of the electrochemical activation as well as the mechanism of the subsequent chemical cascade reactions have been investigated by DFT calculations.

Timely Leukapheresis May Interfere with the "Fitness" of Lymphocytes Collected for CAR-T Treatment in High Risk DLBCL Patients.

The development of chimeric antigen receptor (CAR)-T cell therapy has revolutionized the treatment of hematological diseases. However, approximately 60% of patients relapse after CAR-T cell therapy, and no clear cause for this failure has been identified. The objective of the Bio-CAR-T BS study (ClinicalTrials.gov: NCT05366569) is to improve our understanding of the lymphocyte harvest to maximize the quality of the CAR-T cell product. Of the 14 patients enrolled, 11 were diagnosed with DLBCL, 2 with PMBCL, and 1 with ALL. Five of 11 DLBCL patients met the criteria for "pre-emptive" Lymphocytes-apheresis (being at high risk of second relapse), and 6 were included in the standard-of-care Lymphocytes-apheresis group. Previous autologous stem cell transplantation (ASCT) and age were significantly different between the two groups. At the time of Lymphocyte-apheresis, patients in the "pre-emptive" group had more "fit" lymphocytes (higher CD4+/CD8+ ratio; higher naïve T cells levels) compared with standard group, probably due to the impact of ASCT. At the same time, also being older than 60 years results in a more "exhausted" lymphocyte profile. Overall, "pre-emptive" Ly-apheresis in DLBCL patients at high risk of relapse appears to be feasible and may allow the timely collection of "fit" lymphocytes for CAR-T cell manufacturing.