Physical Deletion of the p53 Gene in Bladder Cancer: Detection by Fluorescence in Situ Hybridization

To understand better the role of physical p53 deletion in bladder cancer, 106 formalin-fixed and 45 unfixed bladder tumors were examined using fluorescence in situ hybridization. Probes for centromere 17 and the p53 locus were hybridized simultaneously to interphase tumor cells to analyze p53 and chromosome 17 copy number on a cell by cell basis. 17p deletion was found in four of 43 pTa tumors, 18 of 43 pT1 tumors and 29 of 58 pT2-4 tumors (P = 0.0001). 17p deletion was also highly correlated with grade (P = 0.0001) and with p53 immunostaining (P = 0.0005). Chromosome 17 polysomy was associated with stage, grade, 17p deletions, and p53 immunostaining (P = 0.0001). The strong difference in centromere 17 copy number and 17p deletions between pTa and pT1 tumors supports a relevant biological distinction between pTa and pT1 tumors.     

Differential Effects of Clostridium difficile Toxins on Tissue-Cultured Cells

Two immunologically distinct Clostridium difficile toxins elicited similar morphological changes on cultured cells, although there were differences in both toxin potency and cell sensitivities.     

Production of interleukin 2 and interleukin 4 by immune CD4-CD8+ and their role in the generation of antigen-specific cytotoxic T cells

In this report we investigated the production and role of interleukin (IL)2 and IL4 in the generation of antigen-specific cytotoxic T cells (CTL). We used as our model the ultraviolet-light-induced epithelial tumor 1591, a highly immunogenic regressor tumor which evokes a strong cell-mediated immune response leading to rejection. We show that IL2 and IL4 are differentially required for the development of optimal cytolytic activity to the 1591 tumor in primary and secondary in vitro splenic cultures. First, anti-IL2 receptor monoclonal antibody (mAb) significantly decreased specific cytotoxicity in both primary and secondary splenic mixed lymphocyte-tumor cell culture (MLTC) cultures, but anti-IL4 mAb inhibited the cytotoxic responses only secondary and not primary cultures. Second, when supernatants from MLTC were tested for lymphokine activity, primary cultures produced only IL2 while secondary cultures produced both IL2 and IL4. Splenic cells were then depleted of CD4+ cells by negative selection, or enriched for CD8+ cells by positive selection, and tested for lymphokine production and requirements. CD8+ cells could not generate significant CTL activity in primary cultures, but could in secondary MLTC. The addition of mAb to either IL2 or IL4 significantly inhibited the generation of CTL by CD8+ cells in these secondary MLTC.CD8+ cells were also found to produce both IL2 and IL4 in secondary MLTC by functional and Northern blot analysis. The production of IL2 and IL4 by CD8+ cells occurs during different phases of culture, with IL2 being produced early (days 1 and 2) and IL4 late (days 3-5). In addition, the requirement of CD8+ cells for both IL2 and IL4 is unique for that lymphokine. These results suggest that both IL2 and IL4 are both produced and required by CD8+ cells during secondary MLTC, and suggest an additional cellular source of IL4 production besides CD4+ T cells during antigen-specific CTL responses.     

Somatostatin analogue (octreotide) inhibits bile duct epithelial cell proliferation and fibrosis after extrahepatic biliary obstruction.

Extrahepatic biliary obstruction leads to bile duct epithelial cell proliferation. Somatostatin and its analogue, octreotide, have been shown to inhibit DNA synthesis and proliferation in hepatocytes. We investigated the effect of octreotide on the biliary epithelial cell proliferative responses to biliary obstruction. Male Sprague-Dawley rats underwent common bile duct ligation and subcutaneous injection of either saline or octreotide (6 micrograms/kg) twice daily for 7 days. Morphometric analysis of hepatocytes, bile duct epithelial cells, and periportal connective tissue was performed by computerized point counting. Hepatocyte volume was preserved with octreotide treatment, which also significantly decreased bile duct proliferation and periportal extracellular matrix deposition in response to biliary obstruction compared with saline treated, duct-ligated animals. These results indicate that octreotide prevents the morphological changes that accompany extrahepatic biliary obstruction.     

Regulated expression of the IL-31 receptor in bronchial and alveolar epithelial cells, pulmonary fibroblasts, and pulmonary macrophages.

Interleukin-31 (IL-31), an IL-6 cytokine family member, is proposed to play a role in animal models of airway hyperreactivity. It is produced by activated T cells and signals via a heterodimeric receptor complex composed of IL-31Ralpha and OSMRbeta. Only low levels of IL-31Ralpha expression have been demonstrated in pulmonary epithelial cell lines, however, and little is known about the ability to regulate its expression and signaling. Therefore, primary cultures of human bronchial and alveolar epithelial cells, pulmonary fibroblasts, pulmonary macrophages, and established lines of immortalized bronchial epithelial cells (HBE) and alveolar carcinoma cells (A549) were analyzed by RT-PCR, immunoblotting, and thymidine incorporation. Distinct, cell type-specific regulation of IL-31Ralpha expression was detected. Transforming growth factor-beta (TGF-beta) enhanced IL-31Ralpha mRNA expression in primary cultures and established lines of epithelial cells, but not in macrophages. In contrast, interferon-gamma (IFN-gamma) induced IL-31Ralpha mRNA expression in macrophages. IL-31Ralpha protein expression was below detection threshold in primary epithelial cell cultures but was detectable in A549 cells and increased with TGF-beta treatment. In HBE and A549 cells, TGF-beta pretreatment increased IL-31-mediated Stat3 and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. In A549 cells, TGF-beta magnified IL-31-dependent suppression of proliferation. The data suggest that increased IL-31Ralpha expression correlates with an enhanced response to IL-31.     

Mechanical Characterization of an In Vitro Device Designed to Quantitatively Injure Living Brain Tissue

Due to the nonlinear, viscoelastic material properties of brain, its mechanical response is dependent upon its total strain history. Therefore, a low strain rate, large strain will likely produce a tissue injury unique from that due to a high strain rate, moderate strain. Due to a lack of current understanding of specific in vivo physiological injury mechanisms, a priori assumptions cannot be made that a low strain rate injury induced by currently employed in vitro injury devices is representative of clinical, nonimpact, inertial head injuries. In the present study, an in vitro system capable of mechanically injuring cultured tissue at high strain rates was designed and characterized. The design of the device was based upon existing systems in which a clamped membrane, on which cells have been cultured, is deformed. However, the present system incorporates three substantial improvements: (1) noncontact measurement of the membrane deflection during injury; (2) precise and independent control over several characteristics of the deflection; and (3) generation of mechanical insults over a wide range of strains (up to 0.65) and strain rates (up to 15s^–1). Such a system will be valuable in the elucidation of the mechanisms of mechanical trauma and determination of injury tolerance criteria on a cellular level utilizing appropriate mechanical injury parameters.     

Declining density of intimal smooth muscle cells and age as preconditions for atheronecrosis in the basilar artery

The aging basilar artery has some differences and some similarities when compared with the aorta and coronary arteries. As the non-necrotic intimal thickness increases over time, the number of smooth muscle cells reaches a steady state around age 25–30 years in the coronaries and aorta, but continues to increase in the basilar artery, even to 90 years of age. The numbers of cells per unit of tissue (the cell density) declines with age, and the patterns of decline are quantitatively similar in all three arterial segments. All arteries so far examined behave alike in showing that atheronecrosis emerges in those specimens that have sufficiently low density of intimal smooth muscle cells. These results identify low intimal cell density as a criterion for recognizing arteries that are prone to atheronecrosis. One possible explanation is that depopulation of the fibrotically thickened and aged intima, by spreading apart the smooth muscle cells with expanding matrix materials, could be the conditioning factor that brings about the intrusion of atheronecrosis.     

Evaluation of intestinal parasite morphology in polyvinyl alcohol preservative: comparison of copper sulfate and mercuric chloride bases for use in Schaudinn fixative.

As a result of disposal problems inherent in the use of mercury compounds, many laboratories have considered using copper sulfate as a substitute for mercuric chloride in polyvinyl alcohol (PVA) preservative. The primary use for PVA-preserved specimens is the permanent stained smear, the most important technique for the identification of intestinal protozoa. A comparison of organism recovery and morphology was undertaken with PVA containing either copper sulfate or mercuric chloride base. Paired fecal specimens (417 pairs) were collected and examined with the Formalin-ether concentration and Trichrome stain techniques. Numbers of organisms recovered and helminth egg and protozoan morphology were assessed from the concentration sediment. Morphology, clarity of nuclear and cytoplasmic detail, overall color differences, and the ease or difficulty in detecting organisms in fecal debris were assessed from the permanent stained smear. No significant differences were found in the numbers and morphology of organisms seen in the concentration sediment. However, when the trichrome stain was used, the overall morphology of the intestinal protozoa preserved in PVA with copper sulfate was not equal to that seen with PVA with mercuric chloride. We do not recommend switching from mercuric chloride base to copper sulfate base unless that is the only option available for the preparation of permanent stained smears.     

Direct and indirect effects of soluble extracts of Schistosoma mansoni eggs on fibroblast proliferation in vitro.

The possibility that soluble products of Schistosoma mansoni eggs might participate in the pathogenesis of hepatic fibrosis in schistosomiasis was investigated. Both crude saline extracts of eggs (soluble egg antigen [SEA]) and a partially purified SEA fraction contained activity which stimulated guinea pig and human dermal fibroblasts to proliferate in vitro, as measured by uptake of [3H]thymidine. Maximum activity was present in fractions which eluted from Sephacryl S-200 with an apparent molecular weight of less than or equal to 12,500 and in fractions which had an estimated pI 8, as determined by preparative isoelectric focusing of partially purified SEA. Activity in crude SEA was not removed by chromatography on concanavalin A-Sepharose 4B. When concanavalin A-binding glycoproteins lacking intrinsic fibroblast-stimulating activity were incubated with spleen cells from infected or uninfected mice, fibroblasts-stimulating activity was detected in the culture supernatants. Thus, SEA contains two functionally distinct molecular species. One of these directly stimulates fibroblasts, whereas the other induces the release of a fibroblast-stimulating activity from lymphocytes or macrophages or both. Since these fibroblast-stimulating factors might be elaborated in the livers of infected individuals, these observations suggest a potential role of soluble schistome products in the pathogenesis of hepatic fibrosis in schistosomiasis.