Peter Borgs

Hemostatic factors in rabbit limb lymph: relationship to mechanisms regulating extravascular coagulation

Mechanisms regulating extravascular coagulation in interstitial fluids of peripheral tissues are poorly understood, since measurements of hemostatic factors in these fluids are unavailable. Because lymph from a body region reflects the composition of its interstitial fluid, we measured hemostatic factors in limb lymph of rabbits both as activity and as antigen. Mean lymph-to-plasma activity ratios were the following: fibrinogen, 0.28; prothrombin, 0.26; factor X, 0.27; factor VII, 0.17; and factors V and VIII, 0.08. All lymph fibrinogen was clottable; fibrin degradation products were absent. Lymph von Willebrand factor antigen was <10% of plasma antigen and consisted primarily of lower molecular weight multimers. Mean lymph-to-plasma activity ratio for antithrombin was 0.38 and for tissue factor pathway inhibitor the ratio was 0.40. Low levels of antithrombin-factor Xa were measurable in lymph. The data are compatible with a basal factor VIIa-tissue factor-catalyzed extravascular activation of factor X that is prevented from progressing to generation of fibrin in limb interstitial fluid and lymph by low levels of factor VIII and factor V and by the inhibitory activity of antithrombin and tissue factor pathway inhibitor.

AIDS, alcohol, endothelium, and immunity

Abstract Analogies are drawn between important unknowns in AIDS and alcohol research, related to underlying common pathogenetic mechanisms, immunodysregulation, cofactors, and prominent vascular manifestations. The central role of the blood and lymphatic vasculatures and specifically their endothelial lining in many facets of the immune response is reviewed. Evidence is presented that both alcohol and HIV (as well as other coinfecting viruses in AIDS) target and alter endothelial cells and the angiogenic process. These concepts are further illustrated by a serendipitous viral epidemic among rats on continuous long-term alcoholic and control nonalcoholic diets, where synergism between alcohol and virus appeared to underlie multiple vascular proliferative lesions in the liver. In AIDS and alcoholism/alcoholic liver disease (ALD), the prominent features of dysregulated angiogenesis point to the endothelium as a key player in pathogenesis of these seemingly disparate disorders and potentially in immunomodulation.

Effective stabilization of ethanol levels in multiple-well tissue culture plates.

Underestimation of ethanol (EtOH) volatility in vitro is a potential source of experimental error. EtOH (0-5% in culture medium) was added to 24- or 96-well tissue culture plates with standard low evaporation lids and incubated at 37 degrees C in humidified 7.5% CO2 and 92.5% air. After 72 hours, approximately 70% of the initial EtOH had disappeared from the aqueous phase of the plate (EtOH volatilization). EtOH concentrations gradually decreased in high-concentration wells (1-5%) and increased in low-concentration wells (0-0.1%) over time. This temporal redistribution of EtOH (EtOH diffusion) was detected after only 1 hour of incubation. Parafilm, Blenderm surgical tape, and ELISA plate-sealing tape barriers inconsistently or inadequately prevented EtOH volatilization and diffusion, but a newly designed plate-sealing clamp (PSC) apparatus inhibited this phenomenon. Rat hepatic sinusoidal endothelial cells cultured with the PSC apparatus maintained intact cell membranes for 72 hours and stable levels of monolayer permeability for at least 48 hours. By stabilizing in vitro EtOH concentrations, the PSC apparatus eliminates a potential source of major experimental error.