Anti-phospholipid human monoclonal antibodies inhibit CCR5-tropic HIV-1 and induce β-chemokines

Traditional antibody-mediated neutralization of HIV-1 infection is thought to result from the binding of antibodies to virions, thus preventing virus entry. However, antibodies that broadly neutralize HIV-1 are rare and are not induced by current vaccines. We report that four human anti-phospholipid monoclonal antibodies (mAbs) (PGN632, P1, IS4, and CL1) inhibit HIV-1 CCR5-tropic (R5) primary isolate infection of peripheral blood mononuclear cells (PBMCs) with 80% inhibitory concentrations of <0.02 to ∼10 µg/ml. Anti-phospholipid mAbs inhibited PBMC HIV-1 infection in vitro by mechanisms involving binding to monocytes and triggering the release of MIP-1α and MIP-1β. The release of these β-chemokines explains both the specificity for R5 HIV-1 and the activity of these mAbs in PBMC cultures containing both primary lymphocytes and monocytes

Virus burden in long-term survivors of human immunodeficiency virus (HIV) infection is a determinant of anti-HIV CD8+ lymphocyte activity

　　Persons infected with human immunodeficincy virus (HIV) for > 8 years were studied to delinate virologic and immunologic attributes of long- term survival. Whereas those　with 300-700 CD4+ cell/mL often had circulating cytotoxic T lymphocytes (CTL) agaist HIV antigens, those with > 1000 CD4+ cells/mL did not. The subjects with > 1000 CD4+ cells/mL had low virus burden, low levels of Gag-specific CTL precursors, and minimal CD8+ cell activation. Overall,　elevated levels of CD8+ cells , CD38 antigen expression on CD8+ cells, and anti-HIV functions were correlated with increased virus burden, provirus load, and HIV plasma RNA levels. A factor that supressed HIV replication was spontaneously secrected from Cd8+ cells of most subjects but not from those with high CD 4+ cell counts. CD8+ cell activities, therefore, may reflect chronic viral stimulation of the immune system. Long-term survivors with　high level of CD4+ cells maintained control of viral replication but lacked the CD8 + cell activation

Feasibility of a Multiplex Flow Cytometric Bead Immunoassay for Detection of Anti-Epoetin Alfa Antibodies▿

Immunogenicity profiles of recombinant therapeutic proteins are important to understand because antibodies raised against these molecules may have important clinical sequelae. The purpose of the present study was to demonstrate that a flow cytometric bead array could be used to detect clinically relevant antibodies with specificity to such therapeutics. We chose to evaluate well-characterized specimens from persons treated with epoetin alfa that developed antibody-mediated pure red blood cell aplasia as a means to demonstrate the utility of this platform. Our data show that this assay is capable of detecting anti-epoetin alfa antibodies with a relative antibody concentration of 50 ng/ml, where 25 of 25 sera spiked with antibodies at this concentration scored positive. Moreover, the assay was designed to include positive and negative control beads for each specimen that is processed to ensure the specificity of the signal when detected. Measurement of interassay precision supports quantitative estimates of relative antibody concentrations in the range of 313 to 5,000 ng/ml, where the percent coefficient of variation did not exceed 20%. With respect to clinical specimens, antibodies with specificity for epoetin alfa could be easily detected in a set of specimens from persons with pure red blood cell aplasia that had prior exposure to the EPREX brand of recombinant epoetin alfa. Further development and validation of this approach may facilitate successful widespread application of the method for detection of anti-epoetin alfa antibodies, as well as antibodies directed against other recombinant therapeutic proteins

Endotoxin Neutralization as a Biomonitor for Inflammatory Bowel Disease

<div><p>Gram-negative bacterial endotoxin is a potent immunostimulant implicated in the development and/or progression of a variety of diseases. The mammalian immune system has both innate and adaptive immune responses to neutralize endotoxin. In this study, a system was developed to monitor bacterial exposure by measuring the extent and nature of endotoxin neutralization in plasma. In control patients, females had higher levels of endotoxin neutralization than males, mirroring clinical outcomes from bacterial infection and sepsis. In addition to the total amount of neutralization, we used inactivation techniques to elucidate the nature of this activity and develop a system to compare early and late immune responses. Using this method to monitor patients with inflammatory bowel disease, we found a more robust total response that relies more on long-term, adaptive components of the immune system and less on early, innate components. Our results indicate that endotoxin neutralization is a valuable method to discern inflammatory bowel disease patients from a control population. Additionally, the nature of neutralization may be valuable in monitoring disease severity and/or the role of medication.</p> </div

Effect of age on endotoxin neutralization.

<p>The average of 3 replicates was determined for each neutralization value and the result was plotted against patient age. A linear regression line was fit to the data and the r value was determined. For each sample set the male graph is on the left and the female is on the right. (A–B) TEN increases with age in males (<i>r</i> = 0.8107) but stays relatively constant in females (<i>r</i> = -0.1497). (C–D) HSEN decreases with age in males (<i>r</i> = -0.8670) but stays constant in females (<i>r</i> = -0.0964). (E–F) ASEN increases with age in males (<i>r</i> = 0.4210) but stays constant in females (<i>r</i> = -0.0055). (G–H) HAREN increases with age in males (<i>r</i> = 0.4534) but stays constant in females (<i>r</i> = 0.0265).</p

Acid-sensitive endotoxin neutralization (ASEN).

<p>Each data point with error bar represents the average and standard deviation of 3 replicates. The mean and standard error of the mean are indicated for each sample group. The <i>p</i> value indicates the statistical difference compared with the control group as determined using Student’s t-test. (A) In males, ASEN is increased from 46.4 ± 4.3% in controls to 56.7 ± 3.9% (<i>p</i> = 0.09183) in UC and 57.1 ± 2.9% (<i>p</i> = 0.04859) in CD. (B) In females, ASEN is increased from 45.1 ± 1.9% in controls to 57.8 ± 2.0% (<i>p</i> = 0.00005) in UC and 57.1 ± 2.9% (<i>p</i> = 0.00041) in CD.</p