Application of laser scanning confocal microscopy in the analysis of particle-induced pulmonary fibrosis.

Laser scanning confocal microscopy (LSCM) allows us to simultaneously quantitate the degree of lung fibrosis and distinguish various pathological lesions of intact lung tissue. Lucifer Yellow has been shown an ideal fluorescent stain to examine the connective tissue matrix components of embedded lung tissue with LSCM. We evaluated the use of LSCM in quantitating lung fibrosis and compared this procedure with the more traditional method of assessing fibrosis by measuring hydroxyproline, a biochemical assay of collagen. CD/VAF rats were intratracheally dosed with silica (highly fibrogenic), Fe2O3 (non-fibrogenic), and saline (vehicle control) at a high dose of 10-mg/100 g body weight. At 60 days post-instillation, the left lung was dissolved in 6 M HCl and assayed for hydroxyproline. Silica induced increases of 58% and 94% in hydroxyproline content over the Fe2O3 and control groups, respectively. The right lung lobes were fixed, sectioned into blocks, dehydrated, stained with Lucifer Yellow (0.1 mg/ml), and embedded in Spurr plastic. Using LSCM and ImageSpace software, the tissue areas of ten random scans from ten blocks of tissue for each of the three groups were measured, and three-dimensional reconstructions of random areas of lung were generated. The silica group showed increases of 57% and 60% in the lung areas stained by Lucifer Yellow over the Fe2O3 and control groups, respectively. Regression analysis of hydroxyproline vs. lung tissue area demonstrated a significant positive correlation (p < 0.05) with a correlation coefficient of 0.91. Histological analysis of right lung tissue revealed a marked degree of granulomatous interstitial pneumonitis for the silica group, which was absent in the Fe2O3 and control groups. No significant differences (p < 0.05) in hydroxyproline content and measured tissue area were observed between the Fe2O3 and control groups. LSCM, and its associated advanced image analysis and three-dimensional capabilities, is an alternative method to both quickly quantitate and examine fibrotic lung disease without physical disruption of the tissue specimen.

Occludin is a functional component of the tight junction.

Occludin's role in mammalian tight junction activity was examined by 'labeling' the occludin pool with immunologically detectable chick occludin. This was accomplished by first transfecting MDCK cell with the Lac repressor gene. HygR clones were then transfected with chick occludin cDNA inserted into a Lac operator construct. The resulting HygR/NeoR clones were plated on porous inserts and allowed to form tight junctions. Once steady state transepithelial electrical resistance was achieved, isopropyl- beta-D-thiogalactoside was added to induce chick occludin expression. Confocal laser scanning microscopy of monolayers immunolabeled with Oc-2 monoclonal antibody revealed that chick occludin localized precisely to the preformed tight junctions. When sparse cultures were maintained in low Ca2+ medium, chick occludin and canine ZO-1 co-localized to punctate sites in the cytoplasm suggesting their association within the same vesicular structures. In low calcium medium both proteins also co-localized to contact sites between occasional cell pairs, where a prominent bar was formed at the plasma membrane. Chick occludin was detectable by western blot within two hours of adding isopropyl- beta-D-thiogalactoside to monolayers that had previously achieved steady state transepithelial electrical resistance; this coincided with focal immunofluorescence staining for chick occludin at the cell membrane of some cells. A gradual rise in transepithelial electrical resistance, above control steady state values, began five hours after addition of the inducing agent reaching new steady state values, which were 30-40% above baseline, 31 hours later. Upon removal of isopropyl- beta-D-thiogalactoside chick occludin expression declined slowly until it was no longer detected in western blots 72 hours later; transepithelial electrical resistance also returned to baseline values during this time. While densitometric analysis of western blots indicated that the presence of chick occludin had no detectable effect on E-cadherin or ZO-1 expression, the possibility cannot be excluded that ZO-1 might be a limiting factor in the expression of chick occludin at the cell surface. To test whether expression of chick occludin affected the process of tight junction assembly, monolayers in low Ca2+ medium were treated with isopropyl- beta-D-thiogalactoside for 24 or 48 hours, before Ca2+ was added to stimulate tight junction assembly. Chick occludin did not alter the rate at which transepithelial electrical resistance developed, however, steady state values were 30-40% above control monolayers not supplemented with the inducing agent. By freeze fracture analysis, the number of parallel tight junction strands shifted from a mode of three in controls to four strands in cells expressing chick occludin and the mean width of the tight junction network increased from 175 +/- 11 nm to 248 +/- 16 nm. Two days after plating confluent monolayers that were induced to express chick occludin, mannitol flux was reduced to a variable degree relative to control monolayers. With continued incubation with the inducing agent, mannitol flux increased on day 11 to 50%, and TER rose to 45% above controls. Both of these changes were reversible upon removal of isopropyl- beta-D-thiogalactoside. These data are consistent with the notion that occludin contributes to the electrical barrier function of the tight junction and possibly to the formation of aqueous pores within tight junction strands.

Characterization of RAFTK, a novel focal adhesion kinase, and its integrin-dependent phosphorylation and activation in megakaryocytes.

We have recently isolated a cDNA encoding a novel human intracellular tyrosine kinase, termed RAFTK (for a related adhesion focal tyrosine kinase). The RAFTK cDNA, which encodes a polypeptide of 1,009 amino acids, shares 65% homology to the focal adhesion kinase (FAK), including several consensus motifs. In this report, we describe the biochemical characterization and functional analysis of the RAFTK protein. Coexpression of RAFTK and FAK proteins in megakaryocytic cells and blood platelets was observed. Using a specific antibody to RAFTK and the monoclonal antibody 2A7 to FAK, FAK and RAFTK could be distinguished antigenically. RAFTK had intrinsic tyrosine kinase and autokinase activities. It was phosphorylated on tyrosine in growing cultures of COS cells transfected with the pCDNAIII/flag-RAFTK expression vector containing the RAFTK cDNA ligated with the 8 amino acid flag peptide sequence. Similar to FAK, dephosphorylation of RAFTK was observed when adherent transfected COS cells were detached. Phosphorylation was regained upon replating of these cells on the fibronectincoated dishes. Analysis of tyrosine-phosphorylated RAFTK from adherent transfected COS cells showed that the Src homology 2 (SH2) domains of the Src and Fyn protein kinases as well as the Grb2 adaptor protein were able to specifically associate with RAFTK. Tyrosine phosphorylation of endogenous RAFTK was observed upon fibronectin-induced activation of human megakaryocytic cells. Furthermore, colocalization of RAFTK protein with vinculin, a focal adhesion protein, was observed by confocal microscopy in focal adhesion-like structures in adherent CMK cells and in transfected pCDNAIII/flag-RAFTK COS cells upon fibronectin activation. These data suggest that RAFTK is a novel member of the FAK family, that it localizes to focal adhesion-like structures in CMK megakaryocytic cells, that it participates in integrinmediated signaling pathways in megakaryocytes, and that it is able to associate with the tyrosine kinases Src and Fyn as well as the adaptor protein Grb2 via SH2-phosphotyrosine interactions.

Pharmacologic modulation of the cutaneous vasculature in the isolated perfused porcine skin flap.

The isolated perfused porcine skin flap (IPPSF), an ex vivo model system used in cutaneous toxicology and pharmacology, is capable of assessing the percutaneous absorption of vasoactive compounds. However, the vascular responses of the IPPSF to classical pharmacologic agents have not been calibrated. The ability of acetylcholine, nitroglycerin, tolazoline, and norepinephrine to affect vasculature resistance and glucose utilization was investigated in the IPPSF. Norepinephrine infusions between 10(-7) and 10(-5) M increased vascular resistance in a dose-dependent manner; half-maximal (EC50) and maximal responses occurred at 3.18 x 10(-6) and 10(-5) M, respectively. In non-preconstricted flaps, neither acetylcholine, nitroglycerin, nor tolazoline vasodilated the IPPSF; however, acetylcholine, nitroglycerin, and tolazoline each lowered vascular resistance in a dose-dependent manner in norepinephrine-preconstricted flaps. Maximal relaxation was induced at 10(-4), 10(-6), and 5 x 10(-5) M, by tolazoline, acetylcholine, and nitroglycerin, respectively, whereas the EC50 values were 2.88 x 10(-7), 1.35 x 10(-8), and 1.72 x 10(-7) M, respectively. In flaps pretreated with norepinephrine and methylene blue (a potential blocker of edothelium-derived relaxing factor), no concentration of acetylcholine, and only the highest concentration of nitroglycerin, lowered vascular resistance. In non-preconstricted flaps, glucose utilization decreased in norepinephrine-infused flaps, increased in nitroglycerin- and tolazoline-infused flaps, and was biphasic in acetylcholine-infused flaps. These results indicate that the IPPSF responds to pharmacologic agents in a manner similar to classic in vitro and in vivo models. Thus, the IPPSF would be a relevant model for investigating the delivery and/or toxicity of pharmacologically active compounds.

Nonuniform alteration of cis-diamminedichloroplatinum(II) tissue distribution in dogs with whole body hyperthermia.

The purpose of this study was to investigate the pharmacokinetics and tissue disposition of cisplatin (CDDP) in euthermic and hyperthermic dogs to determine if hyperthermic alteration of tissue CDDP concentration is uniform. Eighteen female beagle dogs received 20, 50, or 80 mg/m2 CDDP by constant infusion for 60 min under normothermic or hyperthermic conditions (n = 3/subgroup). Blood, plasma, and ultrafiltered plasma samples were collected during the infusion. At termination of infusion, animals were immediately sacrificed, all major tissues were collected, and platinum levels were determined by atomic absorption spectroscopy. Platinum concentrations in all blood fractions of hyperthermic dogs tended to be lower than those of normothermic dogs. The correlation between dose and blood area under the concentration-time curve was linear at both temperatures. Each tissue concentration was normalized for that individual dog's blood area under the curve. The ratio of relative extraction at 42 degrees C to that at 37 degrees C were compared for each tissue. Values of 1.0 were interpreted as indicating uniform relative tissue extraction at each temperature. Values of greater than 2.0 were obtained in lung and ileum, while values of greater than 1.5 were obtained in liver, adrenal, stomach, colon, duodenum, spleen, and pancreas. Values of less than 1.0 were obtained in skin and superficial lymph nodes. These results indicate that hyperthermia significantly alters the pattern of CDDP tissue disposition in a nonuniform manner and that pharmacokinetic data obtained at one temperature, e.g., areas under the curve, cannot be used to directly predict tissue concentrations at another temperature.