Alexander V. Panyutich

Mechanism of extracellular release of human neutrophil calprotectin complex

Calprotectin is an abundant cytosolic protein complex of humanneutrophils with in vitro extracellular antimicrobial activity. Studiessuggest that calprotectin may be actively secreted from intact HL‐60cells and that it can be translocated to polymorphonuclear neutrophil(PMN) cell membranes. To examine whether calprotectin is secretedextracellularly, we incubated soluble and particulate stimuli,including live and heat‐inactivated Candida albicans, withwhole blood and measured calprotectin levels in the plasma. We comparedthe release of calprotectin to that of lactoferrin, a protein known tobe secreted by PMNs. Extracellular lactoferrin was detected afterincubation with any of the particulate stimuli. In contrast, asignificant increase in extracellular calprotectin was found only afterincubation with live C. albicans. Specifically, theincrease in extracellular calprotectin correlated directly with aproportional decrease in PMN viability. Our results indicate that humanPMN calprotectin is not secreted extracellularly except as a result ofcell disruption or death.

A sensitive enzyme-linked immunosorbent assay for human interleukin-8

In order to quantify human interleukin-8 (IL-8), which is chemotactic for T cells and basophils as well as neutrophils, we developed an enzyme-linked immunosorbent assay (ELISA). Since binding inhibition tests indicated that three monoclonal antibodies (mAbs; BS-1, WS-4, WS-6) blocked the binding of 125I-labelled IL-8 to neutrophils, we tested an ELISA using these mAbs as primary antibodies, rabbit anti-IL-8 Ab as the secondary antibody, and alkaline phosphatase-labelled goat anti-rabbit Ab as the conjugate. Among the three mAbs tested, WS-4 was the most sensitive with a detection limit of 16 pg/ml. Several other cytokines, including monocyte chemotactic and activating factor (MCAF), which is structurally related to IL-8, showed no cross-reactivity in this system, indicating that this ELISA is specific for IL-8. The coefficients of variation for the intra- and interassays were below 10%. Furthermore, this ELISA also detected natural IL-8 (including both 72 and 77 amino acid forms) produced by cultured human cells and cell lines stimulated with IL-1, suggesting that this system will be useful in the detection of natural IL-8 in various body fluids.

An enzyme immunoassay for human defensins

We developed and optimized an enzyme immunoassay for human neutrophil defensins, cationic cysteine-rich peptides that participate in host defense and inflammation. The assay utilizes a sandwich design with a monoclonal capture antibody and a biotinylated monoclonal detecting antibody. Cetrimonium bromide is employed to obviate non-specific binding of defensins to surfaces. The assay has a sensitivity of 0.04-0.05 ng/ml and a working range of 0.05-10 ng/ml.

Identification of defensin binding to C1 complement

In human serum we found strong defensin binding to the complexes of activated C1 complement (C ) and C1 inhibitor (C1i). Purified C1q, activated C1 tetramer ( 2 2) and C1i did not bind defensin. When ( 2 2) was dissociated by EDTA, only the activated C1s (C s) bound defensin. Binding of defensins to C complement represents a newly recognized bridge between the complement‐ and phagocyte‐mediated host defenses, and a potential mechanism for protecting infected tissue from cytotoxic injury by defensin.

Induction of tumor necrosis factor synthesis in human monocytes treated by transcriptional inhibitors

Human blood monocytes and lymphocytes were separated by Percoll gradient fractionation. The synthesis of RNA was inhibited by actinomycin D (AcD) or alpha-amanitin (Amn). Monocytes were stimulated with LPS, lymphocytes were stimulated with phytohaemagglutinin (PHA). The activity of tumor necrosis factor (TNF-alpha) and lymphotoxin (LT) was measured by L-929 cell assay. It was shown that induction of TNF-alpha synthesis by LPS was not blocked by AcD and Amn. In contrast, the production of LT was blocked in cultures of lymphocytes treated by the inhibitors. Moreover, TNF-alpha synthesis was induced in resting monocyte cultures by AcD. Cycloheximide (Cy) inhibited the production of TNF-alpha. The data imply that TNF-alpha synthesis by human blood monocytes can be induced by posttranscriptional regulation of TNF-alpha mRNA presynthesized in vivo.

The effect of biotinylation on the antigenic specificity of anti-defensin monoclonal antibodies

We studied the effects of biotinylation on three monoclonal antibodies (Mabs) that were raised against carrier protein conjugates of human defensin HNP-1, and of rabbit defensins NP-2 and NP-5 respectively. Before biotinylation, each Mab specifically bound to its peptide hapten. Biotinylation of these Mabs by the N-hydroxysuccinimide-biotin (NHS-biotin) resulted in crossreactivity of each Mab with the two irrelevant defensin peptides. In contrast, Mab specificity was preserved after biotinylation with biotin hydrazide, which links biotin to the glycan moiety of antibodies. The effects of NHS-biotinylation were in part mimicked by 2,4-dinitrofluorobenzene, another agent that modified primary amine groups of proteins, suggesting that this modification contributed to the change in antibody specificity. When a high degree of antigenic specificity against peptide immunogens is required, biotinylation on the glycan moiety of Mabs may be preferable.

Tumor necrosis factor α induction in human monocytes

Abstract The present study was undertaken to assess the presence of tumor necrosis factor (TNF)-α mRNA and protein in circulating human blood monocytes and to study the TNF-α gene expression in human monocytes isolated by continuous Percoll gradient fractionation. The technique of RNA isolation directly from the blood samples was used to study TNF-α mRNA expression in circulating human blood leukocytes. It was shown that human blood leukocytes of healthy donors contained no presynthesized pool of TNF-α mRNA as well as no TNF-α protein. It was found that early pretreatment with cycloheximide interfers with TNF-α mRNA induction by Staphylococcus aureus .