vWf inhibitor detection by competitive ELISA

An inhibitor to von Willebrand factor (vWf) was detected in the plasma from two patients with histories of mild bleeding and one patient with a severe deficiency in the Factor VIII complex using a competitive enzyme-linked immunosorbent assay (ELISA) procedure. IgG antibodies from the patients' plasmas were shown to bind to vWf immobilised on polystyrene beads by flow cytometry. The inhibitor also potentiated a recently described platelet function assay based on stirring vWf immobilised on polystyrene beads with platelet rich plasma (PRP). Upon addition of mAb IV.3, potentiation of vWf bead-induced platelet activation was lost indicating that the enhancement of platelet activation was Fc receptor-dependent. Since the ELISA described can be used to quantitate vWf and to detect inhibitors to vWf in plasma samples, the method should prove useful in differentiating acquired vWd from congenital vWd.     

Simultaneous determination of the HIV protease inhibitors indinavir, amprenavir, saquinavir, ritonavir and nelfinavir in human plasma by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method for the simultaneous quantitative determination of five HIV protease inhibitors (i.e. indinavir, amprenavir, saquinavir, ritonavir and nelfinavir) in human plasma is described. An aliquot of 500 microl plasma was extracted with 0.5 ml of 0.1 M NH4OH and 5 ml of methyl tert.-butyl ether. After evaporating, the residue was dissolved in eluent mixture of acetonitrile and 50 mM KH2PO4 adjusted to pH 5.6 with 50 mM Na2HPO4 (43:57, v/v). Subsequently, the eluent was washed with hexane. Chromatography was performed using a C18 reversed-phase column. Ultraviolet detection at 215 nm was used. Linearity of the method was obtained in the concentration range of 0.05-20 microg ml(-1) for all five protease inhibitors. Our method is now in use to analyse plasma samples from patients treated with co-administration of HIV protease inhibitors.     

Improved metabolic profile after switch to darunavir/ritonavir in HIV positive patients previously on protease inhibitor therapy

Metabolic abnormalities associated with cumulative exposure to antiretroviral therapy have been linked to an increased risk of myocardial infarction in HIV positive individuals. The aim of this study was to evaluate whether the switch from lopinavir/ritonavir (LPV/r) or fosamprenavir/ritonavir (FPV/r) to darunavir/ritonavir (DRV/r) is able to improve the lipid profile. A total of 13 Caucasian subjects (7 from LPV/r and 6 from FPV/r) were enrolled in the study and received DRV/r at the dose of 800/100 mg, without change in their NRTI backbone. Viro‐immunological parameters, triglycerides (TGs), total cholesterol (TCh), high‐density lipoprotein (HDL) and low‐density lipoprotein (LDL) cholesterol, fasting glucose, HOMA‐IR, indexes of hepatic and renal functionality, microalbuminuria and cystatin C were measured at baseline (T0), 3 months (T3), 6 months (T6), and 12 months (T12). The switch to DRV/r reduced levels of TCh, LDL, and TGs at T3. Similar improvements were confirmed further at T6 and at T12. A 14% increase in CD4+ count cells (P < 0.05) was observed. Serum cystatin C values showed a statistically significant decrease. After 12 months of switching to DRV/r from LPV/r or FPV/r, patients infected with HIV with TGs above 200 mg/dl, showed a 49% decrease in TGs, along with a 16% reduction of LDL and 19% reduction of TCh. Switching to DRV/r also improved immunological parameters, such as CD4+ cells count and cystatin C plasmatic levels, which may translate into a reduction of the cardiovascular risk. In conclusion, a switch to DRV/r should be considered in those HIV positive patients undergoing antiretroviral therapy, who also present abnormal lipid profiles. J. Med. Virol. 85:755–759, 2013. © 2013 Wiley Periodicals, Inc.     

Quantification of total IgM and IgG levels in rat sera by a sandwich ELISA technique

Routine determination of IgG and IgM levels is likely to be included in future international immunotoxicity assessment guidelines with the rat as the required animal species. Only a few historical data are available on these two parameters in the rat. We describe a quantitative ELISA sandwich method using commercially available purified rat immunoglobulin as standards and provide the normal range for IgG and IgM values in healthy untreated 10 ± 2 to 30 ± 3 weekold Sprague-Dawley rats. Our data indicate that both IgM and IgG levels increase gradually with age. In addition, we determined IgG values in rats hyperimmunised with protamine, lysozyme and casein.     

HIV-1 protease inhibitor ritonavir modulates susceptibility to apoptosis of uninfected T cells.

OBJECTIVE: Clinical experience with HIV-1 protease inhibitors (PIs) in the treatment of AIDS frequently has shown that increases in CD4+ T-cell counts can be independent of HIV-1 inhibition by these drugs. This disconnection between viral load and CD4 counts led us to investigate how the PI ritonavir directly affects leukocyte activation in vitro, using peripheral blood mononuclear cell (PBMC) fractions derived from normal donors. METHODS AND RESULTS: When uninfected PBMC cultures were treated for 72 hours with ritonavir at concentrations similar to or lower than that shown to be effective in vivo, an increase in cell viability was observed. The susceptibility of PBMCs to apoptosis was markedly decreased after ritonavir treatment and correlated with lower levels of caspase-1 expression, decreases in annexin V staining, and reduced caspase-3 activity. Induction in vitro of tumor necrosis factor (TNF) production by PBMCs and monocytes was inhibited by ritonavir in a time- and dose-dependent manner at nontoxic concentrations. CONCLUSION: Based on our data, we conclude that the HIV-1 PI ritonavir is an immune modulator that may affect leukocyte activation and apoptosis as an important part of its therapeutic benefit.     

Cerebrospinal fluid HIV RNA and drug levels with combination ritonavir and saquinavir.

Combination antiretroviral therapy with ritonavir and saquinavir has established potent and durable activity on plasma viremia. CNS HIV infection may be sequestered from drug therapy that does not penetrate the blood-brain barrier. Penetration of these protease inhibitors into the cerebrospinal fluid (CSF) and CSF HIV RNA levels on such therapy has not been well described. DESIGN/METHODS: In a cross-sectional study, 28 HIV1-infected study subjects were evaluated either before initiation of or before maximal response to ritonavir-saquinavir therapy, during maximal plasma virologic response, and after virologic failure. Simultaneous samples of plasma and cerebrospinal fluid were obtained from 24 study subjects to measure HIV RNA and protease inhibitor levels. RESULTS: Across the treatment groups, a strong correlation was found between plasma and CSF HIV RNA levels (r = 0.870; p < .001). In each study subject with plasma HIV RNA levels below assay limit (80 copies/ml), the CSF HIV RNA level was also below the limit of quantitation. Low levels of saquinavir (<2 ng/ml) and ritonavir (<25 ng/ml) in the CSF were observed, with a CSF:plasma drug concentration ratio of < or = 0.005 (0.5%) in all study subjects evaluated (n = 11). The plasma:CSF HIV RNA ratio was high before or early in treatment (median, 38; interquartile range [IQR], 13,97), but low (median, 0.29; IQR, 0.17, 7.5) in those failing therapy (group C, p < .001). CONCLUSIONS: CSF ritonavir and saquinavir levels are consistent with the estimated known fraction of unbound drug in plasma (<2%). Across these treatment response groups, suppression of plasma viremia can predict low CSF HIV RNA levels. This correlation may represent HIV RNA transport and equilibrium between CSF and plasma, or it may represent CNS anti-HIV activity of protease inhibitors. The low drug levels and inverted ratio of HIV RNA in the CSF compared with plasma early in plasma virologic breakthrough suggests CSF virologic failure may contribute to failure of plasma virologic response.     

An evaluation of competitive and second generation ELISA screening tests for antibody to HIV

Two competitive anti-HIV ELISA screening assays (Behring and Wellcozyme) and two second generation assays using antigens generated by recombinant DNA technology (Abbott) and synthetic peptides (Biochrom) were evaluated against common panels of anti-HIV positive sera and sera known or thought likely to give false positive reactions. The assays were also tested on fresh sequential blood donations. Conventional estimates of sensitivity and specificity did not reveal a significant difference between the assays. Statistical analyses using log10 transformed data to determine delta values (the distance of the mean optical density (OD) ratio from the cut-off measured in standard deviation units) showed the Abbott assays to have the highest probability (greater than 99.99%) of detecting anti-HIV positive samples and the Behring assay as having the highest probability (greater than 99.99%) of correctly identifying anti-HIV negative specimens. The combined data from conventional estimates of sensitivity and specificity and delta values suggests that the Abbott assay is the test of choice for screening purposes.     

Preparation and validation of monoclonal antibody-based indirect competitive ELISA for detecting testosterone levels

This paper presents the generation of monoclonal antibodies (mAbs) against testosterone (TES) through cell fusion technology, and the development of a mAb-based indirect competitive enzyme-linked immunosorbent assay (icELISA) method to detect TES residue in bovine edible tissues. Under optimal conditions, this assay exhibited a working range of 0.04–19 ng/mL, with half-maximum inhibition (IC₅₀) and limit of detection values of 0.27 and 0.02 ng/mL, respectively. After three sample pretreatment procedures were checked, a simple dilution method was adopted for further use. The stabilisation studies demonstrated that the icELISA kits can be stored for at least 180 to 240 days, at 4°C and ?20°C, respectively. When applied to bovine samples, the data from icELISA and gas chromatography coupled to mass spectrometry showed excellent correlation (r²=0.9923 in muscle, 0.9884 in liver and 0.9957 in kidney). Therefore, this assay has the potential to be incorporated into a quantitative monitoring programme for the rapid screening of TES residue in foods.     

Evaluation of simplified protease inhibitor dosing regimens for the treatment of HIV infection

Complicated dosing regimens can unfavorably affect patient adherence and drug efficacy. In an effort to increase adherence while maximizing the benefits of the protease inhibitor (PI) component of HAART, the efficacy and safety of less frequent PI dosing regimens have recently been under scrutiny. Limiting the number of drugs required in antiretroviral therapy regimens may also minimize toxicity and drug-drug interactions. This article reviews the current movement toward twice-daily regimens and examines the efficacy and safety data available on twice-daily dosing of amprenavir, indinavir, nelfinavir, saquinavir soft-gel capsules, and ritonavir. Future trends in dosing are also discussed.     

HIV血清型分析及其对HIV抗体酶联免疫诊断试剂敏感性的影响

目的 分析HIV的血清型并比较不同HIV抗体酶联免疫诊断试剂检测不同血清型HIV抗体的敏感性。方法 利用血清型特异性寡肽竞争抑制EIA法对基因型明确的92份HIV样品进行血清型分析。选择血清型与基因型一致的样品以及血清型和基因型不一致的样品各4份进行系列稀释，并用7家HIV抗体酶联免疫诊断试剂进行检测。结果 对92份样品中的77份样品成功地完成了血清型分析，其中血清型A、B、C、D和E分别为3份、17份、56份、0份和1份，与基因型的一致率为93．51％，对其余15份样品无法完成血清型分析。7家HIV抗体酶联免疫诊断试剂检测不同血清型样品的灵敏度虽有差异，但无规律性。结论 HIV血清型对HIV抗体酶联免疫诊断试剂的敏感性无规律性影响。     

HIV gp41-induced apoptosis is mediated by caspase-3-dependent mitochondrial depolarization, which is inhibited by HIV protease inhibitor nelfinavir

Apoptotic loss of CD4+ T cells has been proposed as a mechanism of T cell depletion in human immunodeficiency virus (HIV) infections resulting in immunodeficiency. The Env glycoprotein has been implicated in apoptosis of uninfected bystander cells via gp120 binding to CD4/CXC chemokine receptor 4 as well as the fusion/hemifusion process mediated by gp41. Using an in vitro model of coculture of Env-expressing cells as effectors and CD4+ T cells as targets, we find that apoptosis mediated by Env glycoprotein in bystander cells in fact correlates with gp41-induced hemifusion. Further, the apoptotic pathway initiated by this interaction involves caspase-3-dependent mitochondrial depolarization and reactive oxygen species production. HIV gp41-induced mitochondrial depolarization is inhibited by protease inhibitor nelfinavir but not by other HIV protease inhibitors or inhibitors of calpain and cathepsin. This "kiss of death" (hemifusion) signaling pathway is independent of p38 mitogen-activated protein kinase and p53, making it distinct from the apoptosis seen in syncytia. We also show that virion-induced apoptosis is gp41-dependent. Our findings provide new insights into the mechanism via which HIV gp41 mediates apoptosis in bystander cells.