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Introduction: In the present study, the impact of BromAc®, a specific combination of bromelain and acetylcysteine, on the SARS-CoV-2-specific inflammatory response was evaluated., Methods: An in vitro stimulation system was standardized using blood samples from 9 healthy donors, luminex assays and flow cytometry were performed., Results and Discussion: BromAc® demonstrated robust anti-inflammatory activity in human peripheral blood cells upon SARS-CoV-2 viral stimuli, reducing the cytokine storm, composed of chemokines, growth factors, and proinflammatory and regulatory cytokines produced after short-term in vitro culture with the inactivated virus (iSARS-CoV-2). A combined reduction in vascular endothelial growth factor (VEGF) induced by SARS-CoV-2, in addition to steady-state levels of platelet recruitment-associated growth factor-PDGFbb, was observed, indicating that BromAc® may be important to reduce thromboembolism in COVID-19. The immunophenotypic analysis of the impact of BromAc® on leukocytes upon viral stimuli showed that BromAc® was able to downmodulate the populations of CD16+ neutrophils and CD14+ monocytes observed after stimulation with iSARS-CoV-2. Conversely, BromAc® treatment increased steady-state HLA-DR expression in CD14+ monocytes and preserved this activation marker in this subset upon iSARS-CoV-2 stimuli, indicating improved monocyte activation upon BromAc® treatment. Additionally, BromAc® downmodulated the iSARS-CoV-2-induced production of TNF-a by the CD19+ B-cells. System biology approaches, utilizing comprehensive correlation matrices and networks, showed distinct patterns of connectivity in groups treated with BromAc®, suggesting loss of connections promoted by the compound and by iSARS-CoV-2 stimuli. Negative correlations amongst proinflammatory axis and other soluble and cellular factors were observed in the iSARS-CoV-2 group treated with BromAc® as compared to the untreated group, demonstrating that BromAc® disengages proinflammatory responses and their interactions with other soluble factors and the axis orchestrated by SARS-CoV-2., Conclusion: These results give new insights into the mechanisms for the robust anti-inflammatory effect of BromAc® in the steady state and SARS-CoV-2-specific immune leukocyte responses, indicating its potential as a therapeutic strategy for COVID-19., Competing Interests: SV and DM are shareholders of Mucpharm Pty Ltd. and provided scientific input on the protocol and design of the study. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declare that this study received funding from Mucpharm Pty Ltd. The funder had the following involvement in the study: provided only scientific input on the protocol and design of the study., (Copyright © 2023 Ferreira, Clarindo, Ribeiro, Gomes-de-Pontes, de Carvalho, Martins-Filho, da Fonseca, Teixeira, Sabino, Eapen, Morris, Valle and Coelho-dos-Reis.)

Post-transplantation cyclophosphamide (PTCy) reduces the incidence and severity of graft-versus-host disease (GVHD), thereby improving the safety and accessibility of allogeneic hematopoietic cell transplantation (HCT). We have shown that PTCy works by inducing functional impairment and suppression of alloreactive T cells. We also have identified that reduced proliferation of alloreactive CD4 + T cells at day +7 and preferential recovery of CD4 + CD25 + Foxp3 + regulatory T cells (T regs ) at day +21 are potential biomarkers associated with optimal PTCy dosing and timing in our B6C3F1→B6D2F1 MHC-haploidentical murine HCT model. To understand whether the effects of PTCy are unique and also to understand better the biology of GVHD prevention by PTCy, here we tested the relative impact of cyclophosphamide compared with five other optimally dosed chemotherapeutics (methotrexate, bendamustine, paclitaxel, vincristine, and cytarabine) that vary in mechanisms of action and drug resistance. Only cyclophosphamide, methotrexate, and cytarabine were effective in preventing fatal GVHD, but cyclophosphamide was superior in ameliorating both clinical and histopathological GVHD. Flow cytometric analyses of blood and spleens revealed that these three chemotherapeutics were distinct in constraining conventional T-cell numerical recovery and facilitating preferential T reg recovery at day +21. However, cyclophosphamide was unique in consistently reducing proliferation and expression of the activation marker CD25 by alloreactive CD4 + Foxp3 - conventional T cells at day +7. Furthermore, cyclophosphamide restrained the differentiation of alloreactive CD4 + Foxp3 - conventional T cells at both days +7 and +21, whereas methotrexate and cytarabine only restrained differentiation at day +7. No chemotherapeutic selectively eliminated alloreactive T cells. These data suggest that constrained alloreactive CD4 + Foxp3 - conventional T-cell numerical recovery and associated preferential CD4 + CD25 + Foxp3 + T reg reconstitution at day +21 may be potential biomarkers of effective GVHD prevention. Additionally, these results reveal that PTCy uniquely restrains alloreactive CD4 + Foxp3 - conventional T-cell proliferation and differentiation, which may explain the superior effects of PTCy in preventing GVHD. Further study is needed to determine whether these findings also hold true in clinical HCT., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hadjis, Nunes, Khan, Fletcher, Pohl, Venzon, Eckhaus and Kanakry.)

Anti-drug antibody formation poses tremendous obstacles for optimal treatment of hemophilia A (HA). In this study, we sought to utilize chimeric receptor-modified natural regulatory T cells (Tregs) to target FVIII-specific memory B cells, which are responsible for persistent anti-FVIII neutralizing antibodies (inhibitors) in HA patients. Thus, CD4 + CD25 hi CD304 + natural Tregs were FACS sorted from naïve C57BL/6 mice and retrovirally transduced to express a chimeric B-cell antibody receptor (BAR) containing the immunodominant A2 domain of FVIII. Plasmablast-depleted (CD138 neg ) splenocytes from FVIII immunized FVIII-knockout HA mice served as the source for FVIII-specific memory B cells, which were specifically stimulated in vitro with FVIII and enumerated in a B-cell ELISPOT assays. Adding A2-BAR Tregs (1 per 150 splenocytes), but not conventional T cells, to the CD138 - splenocytes significantly suppressed the formation of anti-FVIII antibody secreting cells (ASC), compared to the non-relevant OVA-BAR Tregs control group. The observation that A2-BAR Tregs can suppress the response to FVIII suggests that bystander suppression can occur in the local milieu in this system. Transwell experiments confirmed that the suppression was contact-dependent. Moreover, even in the presence of antibodies to FVIII (so-called inhibitors), similarly prepared CD4 + CD25 hi CD127 low A2-BAR human natural Tregs completely suppressed polyclonal anti-FVIII ASC formation. In conclusion, we demonstrated in vitro that FVIII domain-expressing BAR Tregs could efficiently target and suppress FVIII-specific memory B cells., (Copyright © 2020 Pohl, Venkatesha, Zhang and Scott.)