Neuronal apoptosis plays an essential role in early brain development and contributes to secondary neuronal loss after acute brain injury. Recent studies have provided evidence that neuronal susceptibility to apoptosis induced by traumatic or ischemic injury decreases during brain development. However, the molecular mechanisms responsible for this age-dependent phenomenon remain unclear. Here we demonstrate that, during brain maturation, the potential of the intrinsic apoptotic pathway is progressively reduced and that such repression is associated with downregulation of apoptotic protease-activating factor-1 (Apaf-1) and caspase-3 gene expression. A similar decline in apoptotic susceptibility associated with downregulation of Apaf-1 expression as a function of developmental age was also found in cultured primary rat cortical neurons. Injury-induced cytochrome c-specific cleavage of caspase-9 followed by activation of caspase-3 in mature brain correlated with marked increases in Apaf-1 and caspase-3 mRNA and protein expression. These results suggest that differential expression of Apaf-1 and caspase-3 genes may underlie regulation of apoptotic susceptibility during brain development, as well as after acute injury to mature brain, through the intrinsic pathway of caspase activation.

Immune thrombocytopenia (ITP) is an autoantibody-mediated bleeding disorder with both accelerated platelet destruction and impaired platelet production. We and others have described impaired regulatory CD4 ؉ CD25 hi T cells (Treg) numbers and/or suppressive function in ITP patients. Clinical trials using thrombopoietic agents to stimulate platelet production have shown favorable outcomes in ITP patients, but information on the immunologic responses of treated patients are lacking. We studied the immunologic profile of chronic ITP patients before (n ‫؍‬ 10) and during treatment with thrombopoietin receptor (TPO-R) agonists (n ‫؍‬ 9). Treg activity, as measured by suppression of proliferation of autologous CD4 ؉ CD25 ؊ cells, was improved in patients on treatment (P < .05), and the improvement correlated with reduction in interleukin-2-producing CD4 ؉ cells, consistent with dampening of immune responses. There was a concomitant increase in total circulating transforming growth factor-␤1 (TGF-␤1) levels (P ‫؍‬ .002) in patients on treatment, and the levels of TGF-␤1 correlated with the degree of improvement in platelet counts (r ‫؍‬ .8, P ‫؍‬ .0002). This suggests that platelets in patients on TPO-R treatment may play a role in improving Treg function, either directly or indirectly by enhanced release of TGF-␤1 as a result of greater platelet turnover. In conclusion, our findings suggest that thrombopoietic agents in patients with ITP have profound effects to restore immune tolerance. IntroductionImmune thrombocytopenia (ITP) is a bleeding disorder resulting from low platelet counts with an incidence of 2 and 12 per 100 000 adults and children, respectively, per year and a mortality rate of 1% to 3% per year in severely affected cases. 1,2 Autoreactive antibodies to platelet antigens, mainly the platelet glycoprotein IIb/IIIa complex, are considered responsible for accelerated destruction of platelets by the reticuloendothelial system and also reduced platelet production. 3 Whereas healthy persons harbor platelet-specific autoreactive T cells that are tolerized in the periphery, 4 patients with ITP possess activated platelet-autoreactive T cells with increasing cytokine imbalance toward interleukin-2 (IL-2) and interferon-␥, 5-9 especially in patients with chronic ITP with some also reporting higher levels of circulating proinflammatory cytokines tumor necrosis factor-␣ 10 and soluble CD40 ligand (sCD40L). 11 These data are consistent with loss of peripheral tolerance and an inflammatory phenotype in chronic ITP patients.CD4 ϩ regulatory T cells (Tregs) play a critical role in maintenance of peripheral tolerance by both directly and indirectly suppressing the activation and proliferation of many cell types, including T cells, B cells, dendritic cells, natural killer cells, and natural killer T cells in vivo and/or in vitro. 12 Because of their ability to control homeostasis and immunopathology, 13 the level of Tregs and their function are among the most informative criteria of a patient's immune status. Tregs are ...

No abstract

The development of neutralizing antibodies (nAb) against SARS-CoV-2, following infection or vaccination, is likely to be critical for the development of sufficient population immunity to drive cessation of the COVID19 pandemic. A large number of serologic tests, platforms and methodologies are being employed to determine seroprevalence in populations to select convalescent plasmas for therapeutic trials, and to guide policies about reopening. However, tests have substantial variability in sensitivity and specificity, and their ability to quantitatively predict levels of nAb is unknown. We collected 370 unique donors enrolled in the New York Blood Center Convalescent Plasma Program between April and May of 2020. We measured levels of antibodies in convalescent plasma using commercially available SARS-CoV-2 detection tests and in-house ELISA assays and correlated serological measurements with nAb activity measured using pseudotyped virus particles, which offer the most informative assessment of antiviral activity of patient sera against viral infection. Our data show that a large proportion of convalescent plasma samples have modest antibody levels and that commercially available tests have varying degrees of accuracy in predicting nAb activity. We found the Ortho Anti-SARS-CoV-2 Total Ig and IgG high throughput serological assays (HTSAs), as well as the Abbott SARS-CoV-2 IgG assay, quantify levels of antibodies that strongly correlate with nAb assays and are consistent with gold-standard ELISA assay results. These findings provide immediate clinical relevance to serology results that can be equated to nAb activity and could serve as a valuable ‘roadmap’ to guide the choice and interpretation of serological tests for SARS-CoV-2.

IntroductionClassic differentiation of naive CD4 ϩ T cells into different T helper (Th) subsets, including Th1, Th2, and Th17, occurs in lymphoid tissues after contact with antigen-presenting cells that produce polarizing cytokines. These Th subsets in turn orchestrate diverse immune responses also mediated by production of distinct cytokines. Because aberrant Th1 or Th17 activities have the potential to trigger chronic inflammatory and autoimmune diseases, 1,2 effector Th responses in healthy persons are under tight regulation mediated in part by CD4 ϩ regulatory T cells (Tregs) that are thymic-derived or naive T cell-inducible. 3 Understanding how Th1/Th17/Treg differentiation and expansion are controlled is likely to provide an explanation of how inflammation may be sustained in pathologic environments.More recently, human monocytes were shown to trigger and polarize Th responses 4,5 as well as to both stimulate and suppress T-cell responses during infection and in autoimmune diseases. 5,6 Monocytes, which are generally regarded as precursors of tissue macrophages and dendritic cells, 7 can be phenotypically divided based on surface expression of CD14 (lipopolysaccharide receptor) and CD16 (low affinity Fc␥ receptor III) expression into subsets, each with distinct functional activities. The major monocyte subpopulation characterized by high CD14 but no CD16 expression (CD14 hi CD16 Ϫ ), also referred to as classic monocytes, have higher phagocytic activity. 8 The minor CD16 ϩ cells produce higher TNF after stimulation and expand under infectious or inflammatory conditions. 9,10 With regards to the control of Th differentiation and reactivation, the specific role of the monocyte subsets has not been fully characterized.Immune thrombocytopenia (ITP) is an autoimmune bleeding disease resulting from decreased platelet production as well as accelerated platelet destruction mediated in part by autoantibody-based destruction mechanisms. 11 ITP patients harbor activated platelet-autoreactive T cells with increasing cytokine imbalance toward IL-2 and IFN-␥ 12-14 as well as altered Treg numbers and function. [15][16][17][18][19][20] A shift toward stimulatory monocytes with enhanced Fc␥R-mediated phagocytic capacity further supports a generalized immune dysregulation in ITP. 21 More recently, studies reported increased Th17 cells or IL-17 cytokine in ITP patients, 22-24 implicating a possible role for Th17 cells in ITP immunopathology, although 2 reports did not detect any difference. 25,26 Among the treatment options available to ITP patients, the recently licensed thrombopoietic agents, by increasing platelet production, have yielded overall durable responses in patients with persistent, chronic, and/or refractory ITP while on treatment. 27 Interestingly, improved Treg function in ITP patients was associated with increased platelet counts after the use of these agents, 28 despite apparent lack of immunomodulatory activity associated with such agents. Similarly, improved Treg compartment was reported in ITP patients w...

Red blood cell alloimmunization is a major complication of transfusion therapy. Host immune markers that can predict antibody responders remain poorly described. As regulatory T cells (Tregs) play a role in alloimmunization in mouse models, we analyzed the Treg compartment of a cohort of chronically transfused patients with sickle cell disease (SCD, n = 22) and β-thalassemia major (n = 8) with and without alloantibodies. We found reduced Treg activity in alloantibody responders compared with nonresponders as seen in mice. Higher circulating anti-inflammatory IL-10 levels and lower IFN-γ levels were detected in non-alloimmunized SCD patients. Stimulated sorted CD4+ cells from half of the alloimmunized patients had increased frequency of IL-4 expression compared with nonresponders, indicating a skewed T helper (Th) 2 humoral immune response in a subgroup of antibody responders. All patients had increased Th17 responses, suggesting an underlying inflammatory state. Although small, our study indicates an altered immunoregulatory state in alloantibody responders which may help future identification of potential molecular risk factors for alloimmunization.

Patients with sickle cell disease (SCD) often require transfusions to treat and prevent worsening anemia and other SCD complications. However, transfusions can trigger alloimmunization against transfused red blood cells (RBCs) with serious clinical sequelae. Risk factors for alloimmunization in SCD remain poorly understood. We recently reported altered regulatory T cell (Treg) and T helper (Th) responses with higher circulating Th1 (IFN-γ+) cytokines in chronically transfused SCD patients with alloantibodies as compared to those without alloantibodies. Since monocytes play a critical role in polarization of T cell subsets and participate in clearance of transfused RBCs, we tested the hypothesis that in response to RBC breakdown product, hemin, monocyte control of T cell polarization will differ between alloimmunized and non-alloimmunized SCD patients. Exogenous hemin induced Treg polarization in purified T-cell-monocyte cocultures from healthy volunteers through monocyte anti-inflammatory heme degrading enzyme HO-1. Importantly, hemin primarily through its effect on CD16+ monocytes induced an anti-inflammatory (higher Treg/lower Th1) polarization state in non-alloimmunized SCD group, whereas it had little effect in the alloimmunized group. Non-alloimmunized SCD CD16+ monocytes expressed higher basal levels of HO-1. Furthermore, IL-12, which contributed to a pro-inflammatory polarization state (low Treg/high Th1) in SCD, was dampened in hemin-treated stimulated monocytes from non-alloimmunized SCD patients, but not in alloimmunized group. These data suggest that unlike alloimmunized patients, non-alloimmunized SCD CD16+ monocytes in response to transfused RBC breakdown products promote an anti-inflammatory state that is less conductive to alloimmunization.

Background: The development of neutralizing antibodies (NAbs) against SARS-CoV-2, following infection or vaccination, is likely to be critical for the development of sufficient population immunity to drive cessation of the COVID19 pandemic. A large number of serologic tests, platforms and methodologies are being employed to determine seroprevalence in populations to select convalescent plasmas for therapeutic trials, and to guide policies about reopening. However, these tests have substantially variable sensitivity and specificity, and their ability to quantitatively predict levels of NAbs is unknown. Methods: We determined levels of antibodies in convalescent plasma using commercially available SARS-CoV-2 detection tests and in-house ELISA assays and correlated those measurements with neutralization activity measured using pseudotyped virus particles, which offer the most informative assessment of antiviral activity of patient sera against viral infection. Findings: Our data show that a large proportion of convalescent plasma samples have modest antibody levels and that commercially available tests have varying degrees of accuracy in predicting neutralizing activity. Nevertheless, we found particular commercially available tests are capable of accurately measuring levels of antibodies that strongly correlate with neutralization assays. Interpretation: Our findings imply that SARS-CoV-2 convalescent plasma donors have a wide range of antibody concentrations. At present it is unclear how antibody acquisition, particularly for low titer individuals, might afford future immunity to SARS-CoV-2. Further research will be required to determine the minimum threshold of antibody and neutralization activity necessary to accurately predict immunity. Correlation of clinical antibody tests with neutralization activity in this study could serve as a valuable roadmap to guide the choice and interpretation of serological tests for SARS-CoV-2.