Effect of nerve growth factor on endothelial cell biology: proliferation and adherence molecule expression on human dermal microvascular endothelial cells.

In addition to its effect on the central nervous system, nerve growth factor (NGF) appears to play a key role in the initiation and maintenance of inflammation in many organs. NGF degranulates mast cells, recruits inflammatory cellular infiltrates and activates T cells. Extravascular migration of leukocytes is initially controlled by the interaction of cell surface adhesion molecules of leukocytes and endothelial cells. A marked upregulation of NGF in keratinocytes is also observed in conditions characterized by angiogenesis such as psoriasis and wound healing. In this study we investigated the role of NGF in inflammation by studying its effects on endothelial cell proliferation and intracellular adhesion molecule expression by endothelial cells. The effect of NGF on human dermal microvascular endothelial cell (HDMEC) proliferation was measured using the hexosaminidase assay. ICAM-1 expression on HDMEC was measured by ELISA. The function of ICAM-1 was assessed by adherence of peripheral blood mononuclear cells (PBMC) to HDMEC using 51Cr-labeled PBMC. There was a significant increase in proliferation of HDMEC stimulated with NGF as compared to unstimulated HDMEC (P < 0.001). NGF-neutralizing antibody decreased the mitogenic effect of NGF significantly (P < 0.05). NGF also increased ICAM expression on HDMEC as compared to unstimulated HDMEC (P < 0.05). NGF-neutralizing antibody decreased ICAM expression on NGF-stimulated HDMEC (P < 0.05). The percentage of PBMC adherence was higher in NGF-stimulated HDMEC (P < 0.001). Anti-ICAM antibody decreased PBMC adherence. In the study reported here, the role of NGF in two important aspects of inflammation, i.e. angiogenesis and inflammatory cell recruitment at the site of inflammation, was investigated.

Monocyte adhesion to fibronectin in psoriasis.

BACKGROUND: After vascular extravasation, mononuclear cells (MNC) undergo chemotaxis and adhesion to extracellular matrix proteins, resulting in their differentiation into macrophages. Although endothelial adhesion and chemotaxis are altered in psoriasis, MNC adhesion to extracellular matrix proteins has not been previously studied in the disease. Since MNC adhesion to endothelial cells is abnormally regulated in psoriasis by TGF-beta, we tested they hypothesis that in psoriasis substance P also regulates the adhesion of monocytes to the extracellular matrix protein fibronectin. METHODS: Monocytes from 16 normal controls and 11 psoriatic individuals were isolated and purified using a two-step gradient centrifugation procedure. Adhesion to fibronectin was studied by plating monocyte suspensions onto fibronectin-precoated microtiter plates. The number of adherent cells was quantified by measuring their hexosaminidase activity. RESULTS: Although statistically significant differences in the basal (unstimulated) adhesion or in the substance P-stimulated adhesion between normal control monocytes and those obtained from psoriatic individuals were not observed, a subpopulation of psoriatics was identified who responded to substance P. Furthermore, this in vitro response to substance P was correlated with the clinical status of the subpopulation which was characterized by unstable psoriasis triggered by stressful life events. CONCLUSIONS: The results of this study indicate that priming of monocytes by the extracellular matrix protein fibronectin or by elevated levels of substance P are not critical steps in the pathogenesis of stable, chronic psoriasis. Substance P may contribute to the appearance of new lesions in some individuals with unstable psoriasis.

Carrier bound templates for single tube reverse transcriptase assays and for combined purification and activity analyses, with special reference to HIV.

Polyriboadenosine (prA) was coupled to polycarbonate macrobeads or magnetic beads. The efficiency of the beads and of prA-Sepharose, after priming with odT, as templates in activity assays of purified AMV- and HIV-reverse transcriptase (RT), using [125I]iododeoxyuridine-triphosphate as substrate, was studied. Although the use of immobilized templates, compared with soluble template, resulted in a decreased total molar turnover, it did not affect the sensitivity of the assay for detecting RT. The utility of the new assay was analyzed by mixing purified AMV- or HIV-Rt with different dilutions of the untreated clinical specimen. This showed that RT activity was unaffected by 100 microliters of an extract of whole blood cells resuspended to their original blood volume and diluted 1/64, and also by 100 microliters of serum diluted 1/64. To improve the utility of the assay at the inhibitory concentrations of clinical specimens, the following procedure was adopted: the sample to be analyzed was incubated with the carrier bound template in order to allow the RT to bind, the carrier was washed to remove inhibitory factors, and the reaction components were then added to determine the amount of bound RT. This procedure greatly enhanced the recovery of RT activity from crude specimens and made the direct detection of HIV-RT possible. The assay is easily automated and useful for RT determination in multiple samples and for determining RT-inhibiting substances such as substrate analogs and antibodies.