Summ~aryDuring human immunodeficiency virus (HIV) infection there is a profound and selective decrease in the CD4 + population of T lymphocytes. The mechanism of this depletion is not understood, as only a small fraction of all CD4 + cells appear to be productively infected with HIV-1 in seropositive individuals. In the present study, crosslinking of bound gp120 on human CD4 + T cells followed by signaling through the T cell receptor for antigen was found to result in activation-dependent cell death by a form of cell suicide termed apoptosis, or programmed cell death. The data indicate that even picomolar concentrations of gp120 prime T cells for activationinduced cell death, suggesting a mechanism for CD4 + T cell depletion in acquired immune deficiency syndrome (AIDS), particularly in the face of concurrent infection and antigenic challenge with other organisms. These results also provide an explanation for the enhancement of infection by certain antibodies against HIV, and for the paradox that HIV appears to cause AIDS after the onset of antiviral immunity.T he immunodeficiency that defines AIDS is due primarily to a progressive decline in the number and function of CD4 + T cells. The mechanism of this decline is debated, though lyric infection of cells targeted by interaction of CD4 with the envelope glycoprotein of the HIV virion, gp120, is an obvious model (1-4), and recent data suggest an apoptotic mechanism of cell death after HIV infection (5). However, previous studies have found that only 1 in 1-10 x 104 PBMC actively express HIV-1 in patients with AIDS (6-10), and immune dysfunction is seen early in infection, before a significant proportion of CD4 + cells has been eliminated (11-15). Thus, it is likely that mechanisms other than direct viral destruction contribute to CD4 + T cell loss and to the anergy associated with CD4 + T cell-dependent immune responses.Mouse splenic T cells pretreated with anti-CD4 antibodies die by apoptosis when stimulated through the oL/~ TCR (16). Apoptosis is an active form of physiologic cell death, requiring RNA and protein synthesis, which is characterized by the activation of endogenous endonucleases that cleave chromatin DNA between nucleosomes (17, 18). Here we report that crosslinking of gp120 on human CD4 + T cells followed by signaling through the TCR results in activation-induced cell death. This cell death has the characteristic features of apoptosis, including the histologic changes of nuclear and cytoplasmic condensation and DNA fragmentation into nucleosome-sized multimers of 200 bp. Our data provide a mechanism for the recent observation that CD4 + T cells from HIV-infected individuals are primed in vivo for suicide by apoptosis, upon TCR activation by both superantigen and MHC class II-restricted antigens (19). Materials and Methodslsola~'on ofCD4 + T Cells. Human T ceils were separated from Ficoll-Hypaque-isolated PBMC by rosetting with 2-aminoethylisothio-uronium bromide hydrobromide (AET)-treated SRBC, as described (20). CD4 + calls were isolated by incu...

BackgroundInsulin glargine (Lantus®) is a long-acting basal insulin analog that demonstrates effective day-long glycemic control and a lower incidence of hypoglycemia than NPH insulin. After subcutaneous injection insulin glargine is partly converted into the two main metabolites M1 ([GlyA21]insulin) and M2 ([GlyA21,des-ThrB30]insulin). The aim of this study was to characterize the glargine metabolites in vitro with regard to their insulin receptor (IR) and IGF-1 receptor (IGF1R) binding and signaling properties as well as their metabolic and mitogenic activities.MethodsThe affinity of human insulin, insulin glargine and its metabolites to the IR isoforms A and B or IGF1R was analyzed in a competitive binding assay using SPA technology. Receptor autophosphorylation activities were studied via In-Cell Western in CHO and MEF cells overexpressing human IR-A and IR-B or IGF1R, respectively. The metabolic response of the insulins was studied as stimulation of lipid synthesis using primary rat adipocytes. Thymidine incorporation in Saos-2 cells was used to characterize the mitogenic activity.ConclusionsThe binding of insulin glargine and its metabolites M1 and M2 to the IR were similar and correlated well with their corresponding autophosphorylation and metabolic activities in vitro. No differences were found towards the two IR isoforms A or B. Insulin glargine showed a higher affinity for IGF1R than insulin, resulting in a lower EC50 value for autophosphorylation of the receptor and a more potent stimulation of thymidine incorporation in Saos-2 cells. In contrast, the metabolites M1 and M2 were significantly less active in binding to and activation of the IGF1R and their mitogenicity in Saos-2 cells was equal to human insulin. These findings strongly support the idea that insulin glargine metabolites contribute with the same potency as insulin glargine to blood glucose control but lead to significantly reduced growth-promoting activity.

In this study, the effect of soluble IL-4 receptors (sIL-4R) on murine allergen-induced IgE and IgG1 production was examined. Lymphocytes from mice sensitized to the allergens ragweed (RW) or ovalbumin (OVA) in vivo were restimulated in vitro with the sensitizing allergen in the presence of either a soluble murine sIL-4R, a dimeric sIL-4R Ig fusion protein (sIL-4R/Fc), or anti-IL-4 antibody in 14-day cultures. Both monomeric and dimeric SIL-4R inhibited polyclonal IgE (∼70%) and IgG1 (∼35%) production in a dose-dependent fashion, similar to that observed in the presence of the anti-IL-4 antibody. Allergen-specific IgE and IgG1 were inhibited to a greater degree. Addition of sIL-4R was most effective when present in the culture during the first 3 days and added not later than day 6. In kinetic experiments, we distinguished ongoing IgE production from precommitted B cells versus newly induced IgE synthesis and found that newly induced IgE production was the major target of the sIL-4Rs. These data demonstrate the efficacy of sIL-4R in inhibiting the early stages of the IgE B-cell maturation pathway and indicate the potential of sIL-4R for the inhibition of IgE production in vivo.

In recent years, several strategies that selectively inhibit pro-inflammatory cytokines, have yielded effective protein-based therapies for inflammatory disorders, validating the therapeutic hypothesis that intervention in cytokine signalling can provide clinical benefit. However, these protein-based products must be administered by injection, a constraint associated with inconvenience, adverse effects and expense for patients, caregivers and insurers. Besides interfering with the effects of cytokines such as TNF-alpha or IL-1beta that have already been produced, inhibition of pro-inflammatory cytokine production or signalling with low-molecular weight orally-active drugs would combine the convenience of conventional pharmaceuticals with the focused efficacy of the protein therapies. Reducing IL-1beta and IL-18 production by inhibition of IL-1beta converting enzyme (ICE, caspase-1) is one promising strategy because of the key roles of these cytokines in many inflammatory diseases. Pralnacasan, the first orally available, potent and selective ICE inhibitor to enter clinical trials, is currently under investigation in rheumatoid arthritis.

We have evaluated the effects of a treatment with soluble interleukin-1 receptor (sIL-1R) in the accelerated model of autoimmune diabetes induced by cyclophosphamide (CY) in the non-obese diabetic (NOD) mouse. Prior to the CY challenge (350 mgkg body weight), female euglycemic NOD mice were randomly divided into three groups (A-C). Groups B and C were treated daily from 1 day before to 13 days after the CY challenge with sIL-1R at doses of 0.2 and 2 mg/kg body weight. Group A was treated with PBS. By 2 weeks after CY administration, an acute form of autoimmune diabetes with glycosuria, hyperglycemia and severe insulitis occurred in the majority (13/20, 65%) of the control mice (group A). In contrast, repeated injections with sIL-1R protected NOD mice from insulin-dependent diabetes mellitus (IDDM) development in a dose-dependent fashion; the incidence of IDDM was 53.3% (8/15) in the mice treated with 0.2 mg/kg and only 6.7% (1/15) in those treated with 2 mg/kg. However, none of the doses of the sIL-1R reduced the extent of insulitis in NOD mice. Importantly, the anti-diabetogenic property of sIL-1R may not involve major T cell function impairment; accordingly, in parallel experiments, splenic lymphoid cells from NOD mice not challenged with CY, but treated with 2 mg/kg sIL-1R for 5 consecutive days showed a normal distribution of mononuclear cell subsets and maintained their capacity to secrete interferon-gamma and IL-2 and to proliferate in response to polyclonal mitogenic stimulation with concanavalin A.