Micropapillary transitional cell carcinoma is a rare and highly aggressive variant. Paradoxically, our study demonstrated no significant p53 abnormalities. The lacunar histological pattern did not appear to represent invasion of vascular spaces. Rather, these tumors seemed to have the ability to disrupt and replace the normal stromal matrix to achieve rapid nonendothelial extension. Thus, micropapillary histology may predict a lesser likelihood of surgical cure.

SUMMARY Tissue kallikrein releases kinins by specific proteolysis, an activity inhibited by kallistatin. In this study, kallikrein and kallistatin were localized to endothelial and smooth muscle cells of large, medium, and small normal blood vessels by immunohistochemical techniques. Immunostaining for both proteins was strong in the endothelium of all sizes of blood vessels and was more intense in medial smooth muscle cells of small and mediumsized blood vessels than in elastic arteries. The sites of synthesis by endothelial and smooth muscle cells were demonstrated in normal blood vessels of all sizes by in situ hybridization histochemistry. Kallikrein and kallistatin levels were measured by immunoassays in homogenates of human aorta, vena cava, and iliac artery and vein. Tissue kallikrein and kallistatin transcripts were identified in human blood vessels by RT-PCR followed by Southern blot analysis with specific oligonucleotide probes. The results demonstrated the expression and co-localization of tissue kallikrein and kallistatin in human vessels and suggest a potential role of kallistatin in regulating tissue kallikrein in blood vessels.

These findings indicated that kallikrein gene delivery attenuates hypertension and protects against renal injury and cardiac remodeling in the rat remnant kidney model of chronic renal failure.

Production of superoxide radicals by osteoclasts is necessary for normal bone degradation. White blood cell superoxide, needed for bacterial killing, is produced by activated NADPH oxidase. Since osteoclasts and white blood cells share a common hematopoietic origin, we initiated experiments to test the hypothesis that superoxide radicals at the osteoclast-bone interface are produced by NADPH oxidase. Diphenyl iodonium (IDP), an inhibitor of NADPH oxidase, blocked superoxide generation and decreased osteoclastic bone resorption in cultures of calvarial explants from normal mice. Interferon (IFN) gamma, a stimulant of NADPH oxidase activity, increased superoxide production and bone resorption in cultures of calvarial explants from osteopetrotic (microphthalmic) mice. IDP blocked the stimulatory effects of IFN in this bone resorption model. These data suggest that osteoclastic superoxide is produced by NADPH oxidase.

Serine proteinases modulate the interaction of tumor cells with extracellular matrix components during extravasation and metastasis. The serine proteinase tissue kallikrein has been previously demonstrated in several human adenocarcinomas, and we presently report the localization of immunoreactive kallikrein and its mRNA in pancreatic adenocarcinoma. In addition, a synthetic peptide-based inhibitor specific for tissue kallikrein (FE999024) was used in our studies to explore a possible role for kallikrein in cancer cell invasiveness. Matrigel invasion assays were performed with a human breast-cancer cell line, MDA-MB-231, which expresses tissue kallikrein in culture. In the presence of FE999024 invasion through Matrigel was inhibited in a dose-dependent manner to a maximum of 39%. We also developed a novel ex vivo assay in which breast cancer cells are infused into the pulmonary circulation of artificially ventilated explanted rat lungs. At intervals up to 6 hours after infusion pulmonary invasion was quantified by bronchial alveolar lavage to recover human cancer cells from the airspace. Invading cells in the lung interstitium were also quantified after immunohistochemistry with a monoclonal antibody specific for human cytokeratin 18. The synthetic kallikrein inhibitor attenuates breast cancer cell invasion into the airspace by 33% when quantified by lavage recovery and up to 34% as quantified in the lung interstitium by cytokeratin 18 immunostaining. Cancer cells exploit serine proteinases to influence the local blood supply, extravasate into and out of vessels, and to migrate through tissue matrix during metastasis. The serine proteinase tissue kallikrein has been localized in human adenocarcinomas and related cell lines from a number of organs including prostate, breast, pituitary, colon, ovary, endometrium, kidney, and esophagus.

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The tissue kallikrein-kinin system contributes to the regulation of local blood flow, vascular permeability, inflammatory responses, and ion transport. Tissue kallikrein is a serine proteinase which produces vasoactive kinin peptides. Kallistatin specifically binds to tissue kallikrein and inhibits its proteolytic activity. To investigate their anatomical relationship in the human pancreas and salivary glands, the expression and localization of kallistatin and tissue kallikrein were identified by immunoassays, immunohistochemistry, and in situ hybridization histochemistry. Human kallistatin and tissue kallikrein levels were measured by ELISA and radioimmunoassay, respectively, in pancreatic and salivary tissue extracts, and in pancreatic fluid and saliva. Immunoreactive kallistatin and kallikrein were colocalized in acinar cells of the human pancreas by immunohistochemistry. In situ hybridization histochemistry confirmed the presence of both mRNAs in pancreatic acini. In salivary glands, kallistatin and kallikrein mRNAs were also colocalized in serous acinar cells, and the kallikrein transcript was further localized to striated and interlobular ducts. Immunoreactive kallistatin was localized in serous acinar and demilune cells of salivary glands and kallikrein was localized to the epithelium of striated and interlobular ducts. The colocalization and/or coexpression of human tissue kallikrein and kallistatin in the pancreas and salivary glands suggest a role for kallistatin in the regulation of tissue kallikrein in these organs.

The purpose of this study was to explore the changes in mRNA expression levels for metallothionein subtype 2 (MT-2) and heat-shock protein 70 (HSP70) in fathead minnows in response to environmental exposure in a mercury (Hg)-contaminated freshwater ecosystem. It was hypothesized that expression levels of both genes may rise concurrent with the bioaccumulation of Hg and possibly other heavy metals during exposure to the Ouachita River. The experimental design incorporated three distinct populations of fathead minnows: (1) a negative control population of laboratory-raised fathead minnows unexposed to heavy metals or other contaminants, (2) laboratory-raised fatheads placed in cages and exposed to a contaminated ecosystem for 2 wk, and (3) wild-caught (native) fathead minnows captured at the same site where caged fatheads tested positive for Hg bioaccumulation. Study endpoints included growth rates and gross pathology at necropsy. Total Hg levels of the water at the exposure sites as well as in whole fish homogenates were determined by cold vapor atomic absorption spectroscopy (AAS). AAS was also used to assay levels of lead (Pb) and copper (Cu), though these were below detectable limits. Hepatic expression levels of MT and HSP70 mRNA were determined by quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). As hypothesized, levels of both transcripts were significantly increased in the caged exposure group and native fish group compared to unexposed control fish. In addition, the native fish group had significantly higher levels of expression for both genes when compared to caged exposed fish.