The association of adult Onchocerca volvulus with lymphatic vessels.

Immunocytochemical examination of onchocercal nodule tissues containing adult Onchocerca volvulus using immuno-markers for blood and lymphatic vessels (vWF, D2-40, podoplanin, Prox-1, and Lyve1) shows a distinct pattern of distribution of these vessels within nodules. Blood vessels were commonly seen associated with organized lymphoid cellular aggregates in the both the outer and inner areas of the nodules. In contrast, the majority of the lymphatic vessel positivity was seen in the central zone in close apposition to the adult parasites, and the remainder usually associated with microfilariae in the outer areas of the nodule. These findings suggest an intimate relationship between adult O. volvulus and lymphatic vessels, including the likely proliferation of lymphatic endothelial cells (lymphangectasia) akin to that seen with other filariae. These findings indicate that adult O. volvulus may migrate via the lymphatic system, and that clinical manifestations of this disease that involve tissue edema may be the result of the location of these worms in the lymphatic system.

An ultrastructural evaluation of acute 1-nitronaphthalene induced hepatic and pulmonary toxicity in the rat.

1-Nitronaphthalene is a mutagenic particulate of diesel exhaust which causes acute liver and lung toxicity in rodents. The studies presented here describe morphological changes in the lung and liver at several time intervals following a single injection of 1-nitronaphthalene (100 mg/kg, i.p.) in male Sprague-Dawley rats using transmission and scanning electron microscopy. Although both the lungs and liver are injured by 1-nitronaphthalene, the lungs appear to be the primary target organ. Within 4 h of treatment, all 1-nitronaphthalene treated animals exhibited respiratory distress characterized by labored breathing, severe gasping and chromodacryorrhea. The primary ultrastructural alteration were hydropic changes in the non-ciliated bronchiolar (Clara) cells of the distal-most bronchioles of the lung. These were apparent as early as 1 h after 1-nitronaphthalene injection, while adjacent ciliated cells showed no alterations. Over a 24 h period, the bronchioles showed progressive ultrastructural changes leading to necrosis and exfoliation of both ciliated and Clara cells. Interstitial pneumonitis and edema were observed in all animals treated with 1-nitronaphthalene, and was usually associated with bronchioles containing necrotic epithelium. In the liver, ultrastructural changes were observed in the centrilobular hepatocytes at 8 h and consisted of cytomegaly, loss of continuous inner membrane and reduced matrix density of the mitochondria. At 48 h, cellular damage to centrilobular hepatocytes was severe and nearly all mitochondria were damaged. Elevated levels of alanine aminotransferase, aspartate aminotransferase and bilirubin were detected in the serum of animals treated with 1-nitronaphthalene at 8-48 h. In conclusion, 1-nitronaphthalene is a pulmonary toxicant with a unique progression of injury, which primarily damages Clara cells followed by ciliated cells. This disparity is likely due to a difference in the bioactivation of 1-nitronaphthalene. Furthermore, this systemic toxicant also has injurious effects on the centrilobular region of the liver which precedes lung injury.

Leukotriene C(4) biosynthesis in isolated August rat peritoneal leukocytes.

The mixed leukocyte population obtained from the peritoneum of the August rat is a potentially important experimental model of inherent eosinophilia that has not been well characterized. In the present study, isolated cell preparations generated a concentration-dependent release of leukotriene (LT) C(4) when exposed to the Ca(2+) ionophore A23187, reaching maximal stimulation at 5.0 muM. This response was inhibited by the 5-lipoxygenase activating protein antagonist MK-886 (0.1 muM), nominally Ca(2+) and Mg(2+)-free incubation media and by activation of protein kinase C via phorbol 12-myristate 13-acetate (50 nM). These findings establish a model system for investigating LTC(4) profiles contingent with innate peritoneal eosinophilia and are consistent with the hypothesis that cellular LTC(4) biosynthesis is phosphoregulated.

Efficient lipid-mediated transfection of DNA into primary rat hepatocytes.

Cationic lipids are an effective means for transfecting nucleic acids into a variety of cell types. Very few of these lipids, however, have been reported to be effective with primary cells. We report on the efficacy of several commercially available cationic lipid reagents to transfect plasmid DNA into primary rat hepatocytes in culture. The reagents tested in this study include TransfectAce, LipofectAmine, Lipofectin, N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammoniumchloride (DOTMA), (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate (DOTAP), and cetyltrimethyl-ammonium bromide/dioleoylphosphatidylethanol-amine (CTAB/DOPE). Electron micrographic (EM) studies indicate that similar size Lipofectin and DOTAP vesicles contain DNA-like material internally and that these vesicles attach to the cell membrane. DOTAP vesicles are multilamellar, appear as clusters, and have a high DNA-to-lipid ratio. Lipofectin vesicles appear to attach to the cell surface as individual vesicles. The EM observations are consistent with current theories on the mechanism of transfection by cationic lipids. While Lipofectin has proven to be effective in transfection studies of primary cells in culture, we have found DOTAP to be a viable alternative. DOTAP yields transfection rates in hepatocytes comparable to DOTMA and Lipofectin, however, at lower concentrations of reagent and at considerably less cost. Optimal conditions for transfecting 5 micrograms of plasmid DNA with DOTAP were achieved by utilizing multilamellar (vortexed) vesicles at a concentration of 15 micrograms DOTAP per 2 ml media in 60-mm plates for 2 h transfection time. In this study, DOTAP has proven to be economical, easy to prepare, and very effective in transfecting DNA into primary rat hepatocytes.

Protective effect of the 21-aminosteroid lipid peroxidation inhibitor tirilazad mesylate (U74006F) on hepatic endothelium in experimental hemorrhagic shock.

The effects of the 21-aminosteroid lipid peroxidation inhibitor tirilazad mesylate (U74006F) on ultrastructural damage to the hepatic endothelium in a rat model of hemorrhagic shock were examined. Male Sprague-Dawley rats were anesthetized with urethane and subjected to a 2 hr period of hemorrhagic hypotension (mean arterial pressure clamped at 43-45 mm Hg), followed by reinfusion and follow-up for 2 hr. At the end of the experiment, light microscopic analysis of the livers of animals that received an i.v. injection of vehicle (citrate buffer) just prior to reinfusion showed substantial sinusoidal neutrophil influx. Electron microscopic morphometry revealed significant sinusoidal endothelial degeneration. In contrast, rats that received a 10 mg/kg i.v. bolus of U74006F just prior to posthemorrhage reinfusion displayed a significant preservation of endothelial structural integrity. However, this occurred despite the fact that there was the same degree of hepatic neutrophil influx as in the vehicle-treated rats. These results show that U74006F is capable of protecting endothelial structure, even in the face of significant neutrophil invasion, probably via protection from endothelial cell membrane free radical-induced lipid peroxidation.