Roche, Patrick C.

Abstract The neutral protease tryptase has been isolated from a human mast cell line, HMC-1. The HMC-1 line was established from the peripheral blood of a patient with mast cell leukemia and maintained as continuously proliferating clones in vitro and as solid mast cell tumors in nude mice. HMC-1-derived tryptase was purified by sequential chromatography on Dowex 1, DEAE 5 PW, and heparin-agarose. Purified tryptase has an apparent molecular weight of 150,000, as determined by molecular sieve HPLC, but migrates as a doublet of bands of 32/35,000 on SDS-PAGE gels. Maximal enzymatic activity was observed at pH 8.5. Cleavage of tosyl-L-arginine methyl ester by purified tryptase was inhibited by dansyl-L-glutamyl-glycyl-L-arginine chloromethyl ketone 2 HCI, HgCI2, tosyl-L-lysine chloromethyl ketone, leupeptin, and PMSF but not by benzamidine, aprotinin, tosyl-L-phenylalanine chloromethyl ketone, soybean trypsin inhibitor, human plasma, ovomucoid inhibitor, or lima bean trypsin inhibitor. Microsequencing of purified tryptase yielded an amino terminal sequence that was identical to that previously reported for human pituitary-derived tryptase.

The precise localization of the calcitriol (1 alpha,25-dihydroxyvitamin D3) receptor (VDR) and the 25-hydroxyvitamin D3 [25(OH)D3] 24-hydroxylase cytochrome P-450 in the human kidney is unknown. Using newly developed polyclonal antibodies against the human VDR, we demonstrate that the receptor is present in cells of the distal tubule, the collecting duct, the proximal tubule, and in the parietal epithelial cells of the glomerulus. In the distal tubule and collecting duct not all cells contain epitopes for the receptor. The protein is not detected in glomerular capillaries, in the glomerular mesangium, in the interstitium, or in blood vessels. Specific polyclonal antibodies directed against the 25(OH)D3 24-hydroxylase cytochrome P-450 demonstrate epitopes for the cytochrome in cells of the proximal tubule, the distal tubule, glomerular parietal epithelial cells, and mesangial cells. The protein is absent from interstitial cells. Calbindin D28k is present exclusively in principal cells of the distal tubule and collecting duct. In the human kidney, the VDR is present in cells where vitamin D-inducible proteins are found; conversely it is absent from cells where vitamin D-dependent proteins are not present.

We used immunohistochemical techniques to examine the distribution of 1 alpha, 25-dihydroxyvitamin D3 receptors (VDR) in developing rat and mouse kidneys and murine metanephric organ culture. In vivo, the patterns of expression in the two species were similar despite the slight difference in gestational periods (rat, 22 days; mouse, 19 days). Starting at gestational day 15, epitopes for VDR were found in cells of branching ureteral buds and in surrounding mesenchyme and at later developmental stages in glomerular visceral and parietal epithelial cells and proximal and distal tubules (DT). Epitopes for the 28-kDa calcium-binding protein (calbindin D28k) were found exclusively in DTs starting at gestational day 19. The pattern of VDR expression during in vitro nephrogenesis in serum-free murine metanephric organ culture paralleled that seen in vivo. At the time of explantation into organ culture (gestational day 13), VDR epitopes were not detected. By 3 days of in vitro development, VDR expression was identical to that found in gestational day 15 metanephroi in vivo. VDR expression after 5 days of in vitro development mirrored the pattern of gestational day 17 metanephroi in vivo. No calbindin D28k epitopes were seen at any in vitro developmental stage studied. We demonstrate for the first time that VDR are present in specific areas of the developing rat and mouse kidney early in gestation. Calbindin D28k appears later in developing rat and mouse kidney and is distributed differently than the VDR. Metanephric organ culture may be a useful model for studying the regulation and function of VDR during early renal development.

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] is required for normal glucose-stimulated insulin release from pancreatic beta-cells. Biochemical characterization techniques have demonstrated the presence of the 1,25(OH)2D3 receptor (VDR) in homogenates of whole pancreas. Autoradiographic studies using radiolabeled 1,25(OH)2D3 suggest that the VDR is localized to beta-cells but are inconclusive. We used immunohistochemical techniques to stain serial sections from both human and rat pancreas with polyclonal antibodies to human VDR, chick calbindin D28k, insulin, glucagon, and somatostatin. VDR was present in the islet cells and also at low levels in acinar cells of the human and rat pancreas. Calbindin D28k was distributed in a manner similar to the VDR in pancreatic islets but was not present in acini. These results show for the first time that VDR and calbindin D28k are present in human pancreatic tissue. VDR and calbindin D28k are focally distributed throughout pancreatic islet cell types in humans and rats; VDR is also present in the exocrine pancreas. These findings suggest that 1,25(OH)2D3 may influence both endocrine and exocrine pancreatic function.

BACKGROUND: Several studies have reported what seem to be false-positive results using the Food and Drug Administration (FDA)–approved HercepTest (Dako Corp, Carpinteria, CA) to profile Her-2/neu amplification and overproduction in breast carcinoma. False-positive status has been based on comparisons with gene copy enumeration by fluorescence in situ hybridization (FISH) and with comparisons to immunohistochemistry (IMH) results using a monoclonal antibody. However, simple overexpression by tumor cells that have normal gene copy has not been evaluated by profiling mRNA expression, ie, such cases could simply represent true-positive, transcriptionally upregulated overexpression. MATERIALS AND METHODS: Four hundred infiltrating ductal carcinomas of breast were evaluated by IMH using monoclonal (CB11; Ventana Medical Systems, Inc, Tucson, AZ) and polyclonal (HercepTest; Dako) antibodies after antigen retrieval (AR). A polyclonal antibody sans AR (PCA/SAR) was also used. All IMH stains were evaluated and scored according to the guidelines for the FDA-approved HercepTest. A total of 145 of 400 carcinomas were subsequently evaluated by direct and digoxigenin-labeled (Dig) FISH, and 144 of 400 were evaluated by detection of mRNA overexpression via autoradiographic RNA:RNA in situ hybridization. RESULTS: Overall HercepTest/CB11 IMH discordance was 12%. Expression of mRNA was highly concordant with FISH and DigFISH amplification and with CB11 and PCA/SAR immunohistology. IMH false-positive cases (no Her-2/neu gene amplification) occurred with both HercepTest (23%) and CB11 (17%), and the majority of false-positive results (34 of 44) were scored as 2+. All 2+ false-positive cases were mRNA-negative. Combined results of HercepTest and CB11 showed that 79% (38 of 48) of 3+ cases were Her-2/neu gene amplified, but only 17% (seven of 41) of 2+ cases had increased gene copy. CONCLUSION: Discordant HercepTest/FISH results, and to a lesser extent discordance with CB11 IMH, are most commonly false-positive results with a score of 2+. The 2+ score as defined in the guidelines for the FDA-approved HercepTest should not be used as a criterion for trastuzumab therapy unless confirmed by FISH. Determination of Her-2 gene copy number by FISH may be a more accurate and reliable method for selecting patients eligible for trastuzumab therapy.

Islet amyloid derived from islet amyloid polypeptide (IAPP) is a well-recognized feature of type II diabetes. However, the mechanism of islet amyloidogenesis is unknown. In vitro studies suggest that amino acid residues 20–29 in human, but not mouse, IAPP confer amyloidogenicity consistent with the absence of spontaneous islet amyloidosis in mice. Several clinical and in vitro studies suggest that increased synthetic rates of IAPP predispose to IAPP-amyloidosis. In the present study, we sought to test the hypothesis that pharmacological induction of insulin resistance in a mouse transgenic (TG) for human IAPP would induce islet amyloid and β-cell dysfunction. TG and non-transgenic (N-TG) control mice were treated with both rat growth hormone (12 μg/day) and dexamethasone (0.24 mg/day) (dex/GH) or received no treatment for 4 weeks, after which animals were killed to examine islet morphology. Treatment with dex/GH caused hyperglycemia (7.3 ± 0.4 vs. 5.2 ± 0.1 mmol/l, TG vs. N-TG, P < 0.001) associated with a decreased plasma insulin concentration (595 ± 51 vs. 996 ± 100 pmol/1, TG vs. N-TG, P < 0.05) in TG versus control mice. Islet amyloid was induced in treated TG mice but not in control mice. Islet amyloid was identified in both intra- and extracellular deposits, the former being associated with evidence of β-cell degeneration. We conclude that dex/GH treatment in mice TG for human IAPP induces IAPP-derived islet amyloid, hyperglycemia, and islet dysfunction. The present model recapitulates the islet morphology and phenotype of type II diabetes.

Amyloid deposits that characteristically form in the pancreatic islets of patients with non-insulin-dependent diabetes mellitus (NIDDM) and in insulinomas are both derived from islet amyloid polypeptide (IAPP). Evidence from previous studies has suggested that deposition of IAPP-derived amyloid is related to inherent amyloidogenic sequences present within normal human IAPP, together with an increased production and local concentration of IAPP. However, whether the aggregation of IAPP to form amyloid fibrils is primarily an intra- or extracellular event is not clear. To address this question, we studied 20 human insulinomas by light and electron microscopy. By light microscopy, amyloid deposits were demonstrated in 13 of 20 (65%) human insulinomas. Furthermore, evaluation of Congo red-stained tumor sections showed small, globular or irregular, congophilic amyloid deposits within the cytoplasm of many tumor cells in 10 of 13 (77%) amyloid-containing insulinomas. Dense, punctate areas of IAPP immunoreactivity within tumor cells corresponded with the congophilic intracellular deposits. Ubiquitin immunoreactivity also was observed as punctate intracellular labeling and within large extracellular amyloid deposits. Among the 10 insulinomas available for electron microscopic evaluation, pathological IAPP-immunoreactive (immunogold) deposits were found in 3 of 5 insulinomas in which amyloid was demonstrated by light microscopy and in none of 5 tumors found negative for amyloid by light microscopy. Morphology of IAPP-immunoreactive deposits varied from those with the classical distinct 7- to 10-nm diameter nonbranching fibrils to those with distinct but faint fibrillarity to those without discernable fibrils. In each of the 3 tumors with ultrastructurally demonstrable amyloid deposits, intracellular aggregates of IAPP-immunoreactive amyloid fibrils were observed either free in the cytoplasmic matrix or in membrane-bound structures. Intracellular IAPP-immunoreactive deposits were up to 10 urn in the greatest dimension. Free intracellular aggregates of amyloid fibrils sometimes contained degenerating secretory vesicles and fragments of membranous organelles. These observations suggest that IAPP-derived amyloid deposits are initially formed intracellularly and are subsequently released to the extracellular space by exocytosis of the membrane-bound structures and/or after necrosis of the tumor cells. Our observations raise the possibility that IAPP-derived islet amyloid deposits (characteristically present in NIODM patients) also may be initially formed within the cytoplasm of β-cells, thus leading to β-cell necrosis and a reduction of β-cell mass as seen in NIDDM.

Abstract Sarcoidosis is a chronic inflammatory disease of unknown cause characterized by the formation of nonnecrotizing granulomas in affected tissues, most notably the lungs. Granuloma healing may result in pulmonary fibrosis and respiratory impairment in some patients. Transforming growth factor-beta 1 (TGF-beta 1) is a potent cytokine that promotes fibrosis by enhancing the synthesis of extracellular matrix components, including fibronectin and the alpha 5 beta 1 fibronectin receptor. The role of TGF-beta 1 in promoting lung fibrosis in the setting of pulmonary sarcoidosis has not yet been investigated. Accordingly, we determined the extent and distribution of TGF-beta 1 in lung tissue obtained from seven patients with clinical and histologic features of pulmonary sarcoidosis. The tissue distributions of TGF-beta 1, the TGF-beta 1 binding proteoglycan decorin, fibronectin, and the alpha 5 beta 1 fibronectin receptor were assessed immunohistochemically. In all cases, the epithelioid histiocytes comprising nonnecrotizing granulomas of pulmonary sarcoidosis contained abundant TGF-beta 1. We further demonstrated decorin, fibronectin, and the alpha 5 beta 1 fibronectin receptor within nonnecrotizing granulomas and in the fibrous tissue surrounding the lesions. TGF-beta 1 staining was also observed in bronchiolar epithelial cells, hyperplastic Type II pneumocytes, and occasional alveolar macrophages. This study demonstrates enhanced tissue localization of TGF-beta 1 and related extracellular matrix proteins associated with the nonnecrotizing granulomas of pulmonary sarcoidosis. Through its actions on matrix protein synthesis, TGF-beta 1 may modulate the fibrotic repair process accompanying granuloma healing in sarcoidosis.