Successful high flow nasal cannula therapy for severe COVID-19 pneumonia is associated with tocilizumab use.

INTRODUCTION: Our aim was to determine the rate of success of HFNO and its relationship with current treatments for severe COVID-19. METHOD: This was a cohort study including patients admitted for HFNO because of respiratory failure despite oxygen therapy through a facial mask. Care was standardized, with systematic use of steroids and prevention or treatment of thromboembolic complications, and tocilizumab when deemed useful. HFNO failure was defined by the requirement for mechanical ventilation and/or death. RESULTS: In August 2021, among 1397 patients with COVID-19 admitted in the emergency department, 110 (7.8%) received HFNO (mean age 55 years, sex-ratio M/F 1.4). Thirteen patients (12%) had received a steroid treatment before hospital admission. At least one comorbid condition was observed in 57% of the patients. Mean duration of the disease at admission was 8.8 days and mean respiratory rate was 34/min. A CT scan was performed for 101 patients (92%), among whom 13 had a pulmonary embolism. All patients received a steroid treatment, and tocilizumab was prescribed in 79 cases (72%). Failure of HFNO was observed in 54 cases (49%); the only risk factor was the absence of tocilizumab administration: AOR [IC95%] 3.50 [1.40-8.69]. We observed a trend toward failure with steroid use before hospital admission: AOR 3.83 [0.96-16.66]. CONCLUSION: Success of HFNO, when all therapeutic means of treatment for severe COVID-19 pneumonia were applied, was associated with tocilizumab administration. Our data suggest the interest of a randomized study to determine whether HFNO is the right signal for prescription of anti-IL6 drugs.

FOXO1 is a TXN- and p300-dependent sensor and effector of oxidative stress in diffuse large B-cell lymphomas characterized by increased oxidative metabolism.

Molecular profiling has led to identification of subtypes of diffuse large B-cell lymphomas (DLBCLs) differing in terms of oncogenic signaling and metabolic programs. The OxPhos-DLBCL subtype is characterized by enhanced mitochondrial oxidative phosphorylation. As increased oxidative metabolism leads to overproduction of potentially toxic reactive oxygen species (ROS), we sought to identify mechanisms responsible for adaptation of OxPhos cells to these conditions. Herein, we describe a mechanism involving the FOXO1-TXN-p300 redox-dependent circuit protecting OxPhos-DLBCL cells from ROS toxicity. We identify a BCL6-dependent transcriptional mechanism leading to relative TXN overexpression in OxPhos cells. We found that OxPhos cells lacking TXN were uniformly more sensitive to ROS and doxorubicin than control cells. Consistent with this, the overall survival of patients with high TXN mRNA expression, treated with doxorubicin-containing regimens, is significantly shorter than of those with low TXN mRNA expression. TXN overexpression curtails p300-mediated FOXO1 acetylation and its nuclear translocation in response to oxidative stress, thus attenuating FOXO1 transcriptional activity toward genes involved in apoptosis and cell cycle inhibition. We also demonstrate that FOXO1 knockdown in cells with silenced TXN expression markedly reduces ROS-induced apoptosis, indicating that FOXO1 is the major sensor and effector of oxidative stress in OxPhos-DLBCLs. These data highlight dynamic, context-dependent modulation of FOXO1 tumor-suppressor functions via acetylation and reveal potentially targetable vulnerabilities in these DLBCLs.

Synthesis and cytostatic properties of monoterpene derivatives of cyanuric and isocyanuric acids.

Symmetric, trisubstituted derivatives of cyanuric and isocyanuric acid 3a-e and 4a-e, respectively have been prepared by treatment of cyanuric chloride 1 with appropriate terpenic alcohols 2a-e. Under less basic conditions involving treatment of appropriate alcohols 2a-e with metallic sodium, less polar cyanuric acid derivatives 3a-e were obtained in 74-85 yield. Under more basic conditions, in the presence of sodium hydride, isocyanuric acid derivatives 4a-e were prepared in 71-88 yield. Cytostatic activity of (IR,2S,5R)-menthol derivative (3a) has been evaluated on 9 cancer subtypes including 62 tumor cell lines. The studies have shown that contrary to expectations, 2,4,6-trimentoloxy-1,3,5-triazine revealed a weak or moderate activity against most of the cell lines.

Recovery of dendritic cell counts and function in peripheral blood of cancer patients after chemotherapy.

Dendritic cell (DC) counts and function were assayed in peripheral blood of lymphoma and solid tumor patients before and after chemotherapy. The DC counts declined significantly within the first week from the start of chemotherapy, recovered in the second week, and exceeded the baseline values in the third week. DC recovery was usually similar after the first and after the last cycle of chemotherapy. DC1 and DC2 subsets followed the pattern of reconstitution found for the DC population as a whole. Monocytes and granulocytes recovered 1-2 weeks later than DC. The primary proliferative response to keyhole lympet hemocyanin (KLH), totally DC-dependent, declined within the first week from the start of chemotherapy, and in the majority of patients (including those initially unresponsive) recovered along with DC counts. The recovered responsiveness to KLH, but not to anti-CD3 antibody, disappeared at the end of chemotherapy in lymphoma and some solid tumor patients. Prolonged depletion of CD4+ T cells could contribute to the loss of responsiveness in lymphoma patients receiving multiple cycles of chemotherapy. However, in some solid tumor patients, the reactivity to KLH was absent, despite the reconstitution of both DC and CD4+ T-cell counts. Our data show that numerical reconstitution of DC is not necessarily accompanied by functional recovery. The early recovery of DC should be considered while designing protocols for DC collection for immunotherapy.

A method for directly determining the number of dendritic cells and for evaluation of their function in small amounts of human peripheral blood.

Bone marrow-derived dendritic cells (DC) are highly potent antigen-presenting cells (APC) capable of initiating primary responses of naive T lymphocytes to antigen. Studies on DC in disease have been impeded by the lack of a defined method for accurate DC counting and for evaluation of their function in a small amount of blood. In order to detect and enumerate DC in whole peripheral blood preparations, we applied a direct two-color immunofluorescence method. Blood from healthy donors was stained with a mixture of fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies (mAbs) recognizing lineage-associated molecules (CD3, CD14, CD16, CD20, CD57) and phycoerythrin (PE)-conjugated anti-HLA-DR mAb. DC were identified as lineage marker negative (lin-), HLA-DR highly positive cells. The mean percentage of these cells present in peripheral blood leukocytes (PBL) was 0.54%, and the mean absolute DC count was 31.4 x 10(6)/l of blood. DC stained directly in whole blood were heterogeneous with regard to their expression of CD2 and CD4 molecules, and did not express CD80 and CD83 molecules. Expression of CD80 and CD83 on DC was induced following a multistep isolation procedure, including overnight culture. We demonstrated a significant primary proliferative response to keyhole limpet hemocyanin (KLH) in cultures of peripheral blood mononuclear cells (PBMNC). Since primary proliferative response to neoantigens is entirely dependent on DC as APC, the cultures of unseparated PBMNC stimulated with KLH can be used to evaluate DC function in a relatively simple test. This test does not require previous isolation of DC and T lymphocytes and, therefore, can be performed on a small amount of blood. The elaborated flow cytometric method of DC counting in blood and the proliferative test of DC-dependent primary response to neoantigen are currently being applied in an ongoing study on the effect of chemotherapy on DC number and function in cancer patients.

Generation of antigen-specific CD4+ T cell lines from naive precursors.

The conditions required for sensitizing naive T cells to nominal antigen are poorly understood. In this report we describe an in vitro system for generating antigen-specific CD4+ T cells from previously unprimed individuals. Freshly isolated CD4+ T cells were cultured with keyhole limpet hemocyanin (KLH), sperm whale myoglobin (SWM), or human immunodeficiency virus (HIV) gp160, antigens to which most persons have not been sensitized, in the presence of either dendritic cells (DC) or macrophages (M phi). In short-term (< 8 days) cultures, CD4+ T cells or their CD4+, CD45RA (naive) subpopulation mounted significant proliferative responses to KLH, SWM, and HIV gp160, but only if the antigens were presented by DC. In contrast, CD4+, CD45RO (memory) T cells responded poorly to these antigens, although they responded vigorously to tetanus toxoid, a recall antigen, presented by either DC or M phi. KLH- and SWM-specific CD4+ T cell lines were established from the starting population that had been sensitized in vitro, following repeated stimulation with antigen and M phi in medium supplemented with interleukin-2 and interleukin-4. Despite the continued presence of these cytokines during T cell expansion, the expanded lines retained their ability to respond to the priming antigen in the absence of exogenous cytokines. When the CD45RA and CD45RO subpopulations were sensitized and expanded separately, the CD45RA cells alone gave rise to antigen-specific T cell lines, while the CD45RO cells proliferated nonspecifically. These results demonstrate that human naive CD4+ T cells can be sensitized in vitro to nominal antigens presented by DC and that the sensitized cells can be expanded into long-term lines that retain their antigen specificity.

Generation of antigen-specific CD8+ CTLs from naive precursors.

Class I MHC-restricted CTLs are an important component of the host immune response against viral infections, and CTL effectors can often be isolated from infected individuals. However, the mechanism responsible for the induction of CTLs is incompletely understood because, in part, of the difficulty in generating such cells in vitro from naive precursors. In the present study we have used human peripheral blood dendritic cells (DCs), devoid of CD4+ T cells, to sensitize naive CD8+ T cells to exogenous Ags, resulting in the generation of Ag specific CTL effectors. With this system, Ag-specific CTL lines were generated to a complex glycoprotein, keyhole limpet hemocyanin, and to multiple small (9-15 amino acids) synthetic peptides derived from conserved regions of the HIV-1 gag and envelope proteins. The HIV-1-specific CTLs demonstrated potent HLA class I restricted killing of both Ag pulsed and virally infected target cells. In contrast to Ag-pulsed DCs, Ag-pulsed monocytes failed to sensitize CTL precursors although they could be used as feeders for purposes of CTL expansion and as target cells in cytolytic assays. With the use of the system described herein, a detailed analysis of the primary human T cell response to foreign Ags is now feasible, and CTL of desired specificity can be generated for potential clinical use in adoptive immunotherapy protocols.

Granulocyte-macrophage colony-stimulating factor promotes differentiation and survival of human peripheral blood dendritic cells in vitro.

Interest in human dendritic cells (DC) has been heightened recently by the discovery that this cell type is a primary target of the human immunodeficiency virus, the causative agent of AIDS. DC are bone marrow-derived cells with an extraordinarily potent ability to promote the immunological activity of T lymphocytes. Unfortunately, since DC constitute less than 0.5% of peripheral blood mononuclear cells and die within a few days of their isolation, they are not readily accessible to study. We report here that granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine with well-recognized effects on granulocyte and macrophage maturation, profoundly affects the morphology and viability of DC isolated from peripheral blood. GM-CSF not only promotes DC survival but also induces DC differentiation to mobile, reversibly adherent cells with long-branched projections. DC cultured in GM-CSF survive for up to 6 wk and retain their ability to stimulate the proliferation of T cells in allogeneic and autologous mixed leukocyte reactions.

TG and TnonG lymphocyte involvement in the proliferative response to PHA. I. Reactivity and regulatory functions of TG and TnonG subsets in the proliferative response of T lymphocytes.

Both TG and TnonG subpopulations from human peripheral blood were found to secrete helper and suppressor factors for the PHA-induced T cell proliferative response. The TnonG subpopulation contained cells which were both able to secrete helper factor and to respond by proliferation, whereas in the TG subpopulation cells with a proliferative potential could not be conclusively demonstrated. Suppressor and helper activities in TG and TnonG culture supernatants displayed a different time kinetics and were influenced by the dose of mitogen in test cultures.

TG and TnonG lymphocyte involvement in the proliferative response to PHA. II. The role of TG and TnonG lymphocytes in the regulation of the PHA-induced proliferative response of non-T cells.

The TnonG subpopulation from human peripheral blood provides help for PHA induced proliferation of non-T cells, while the TG subpopulation does not. Thus the inducer cells in the proliferative response of T and non-T cells to PHA are at least in part different.