Effects of laminin on the attachment of glioma cells to type IV collagen

The interaction of tumour cells with basement membrane components is thought to be important in influencing their invasive and metastatic properties. This paper describes the effect of laminin on the attachment of radiolabelled glioma and B16 murine melanoma cells to tissue culture plastic and type IV collagen. With the exception of the non-metastatic B16 F1 variant, laminin (and fibronectin) stimulated cell attachment to tissue culture plastic. Although laminin stimulated the attachment of the B16 BL6 metastatic variant to type IV collagen, it consistently inhibited the attachment of the glioma cells under the same conditions. Laminin appeared to exert its effect by adsorption to the collagen and was not cytotoxic to the glioma cells. In contrast, fibronectin had very little effect on cell attachment to type IV collagen. One of the most unusual features of glioma is the rarity of metastasis to extraneural sites. However, the effect of laminin observed here may not be the only factor involved in the metastatic inefficiency of this tumour type.     

Radical-mediated damage to parasites and erythrocytes in Plasmodium vinckei infected mice after injection of t-butyl hydroperoxide.

Intravenous injection of t-butyl hydroperoxide rapidly killed Plasmodium vinckei in mice, and caused haemolysis. The same dose seemed harmless to unparasitized mice. Many parasites disintegrated inside circulating erythrocytes, so parasite death was not simply a passive consequence of haemolysis. Injection of desferrioxamine, which removes the traces of free iron that promote the dissociation of t-butyl hydroperoxide into radical species, prevented both parasite death and haemolysis. Lipid peroxidation, as measured by accumulation of malonyldialdehyde over 2 h in vitro, occurred in erythrocytes exposed to t-butyl hydroperoxide, and was particularly marked in erythrocytes from parasitized mice. These erythrocytes accumulated appreciable malonyldialdehyde even without exposure to t-butyl hydroperoxide. Desferrioxamine inhibited the accumulation of malonyldialdehyde, but did not prevent depletion of reduced glutathione by t-butyl hydroperoxide. This suggests that t-butyl hydroperoxide damaged parasites and erythrocytes by dissociating into radical species, rather than by decreasing intraerythrocyte anti-oxidant capacity. In earlier experiments we suggested that intraerythrocytic parasite death and haemolysis caused by alloxan were mediated by radical species, and these experiments with t-butyl hydroperoxide add weight to this interpretation. We regard both of these systems as models for macrophage-induced parasite death and host pathology in acute malaria.     

Loss of heterozygosity mutations of tumor suppressor genes in cytologically atypical areas in chronic lymphocytic thyroiditis

The relationship between chronic lymphocytic thyroiditis (CLT) and papillary thyroid carcinoma (PTC) is a subject of controversy. Some investigators suggest a causal relationship, whereas others regard the two as only a coincidental occurrence. An additional complicating factor is the presence of atypical nuclei frequently found within lymphoid infiltrates in CLT, which resemble those in PTC. The finding of the RET-PTC translocations in CLT has been reported by two independent groups of investigators, suggesting that the areas of nuclear atypia in CLT are neoplastic rather than reactive. In the present study, we report additional molecular findings that support the hypothesis that the atypical nuclear changes in CLT may be preneoplastic or neoplastic. We microdissected small areas with atypical nuclei in glands with CLT and observed loss-of-heterozygosity mutations of tumor suppressor genes. These genetic mutations are evidence of clonal preneoplastic or neoplastic changes in the follicular cells of CLT. The clinical malignant potential of these minute foci is likely to be very small but remains to be determined.     

Molecular alterations in atypical adenomatous hyperplasia occurring in benign and cancer-bearing lungs.

Atypical adenomatous hyperplasia (AAH) is considered to be a precursor lesion of the lung adenocarcinoma. Several genetic abnormalities have been reported in AAH associated with adenocarcinoma, but little is known about AAH associated with benign lung lesions. To address this we compared the molecular characteristics of AAH present in benign conditions to those coexisting with carcinoma. Seven cases of AAH from resected non-neoplastic lungs (AAH-B) and 12 cases from lungs resected for primary lung carcinoma (AAH-M) were analyzed for loss of heterozygosity (LOH) using 21 polymorphic microsatellite markers situated in proximity to known tumor suppressor genes on chromosomes 3p, 5q, 7p, 9p, 10q, and 17p. Direct DNA sequencing for K-ras mutation was also performed. There was a broad range of LOH in both groups. No LOH was identified in 3 cases (25%) of AAH-M, but all cases of AAH-B showed LOH (P=0.26). Six cases (50%) of AAH-M and 3 cases (43%) of AAH-B showed loss at 1 marker (P=0.99). LOH at 2 or more markers was identified in 3 (25%) cases of AAH-M and 4 (57%) cases of AAH-B (P=0.32). LOH was most frequently detected on chromosomes 3p and 10q in both groups. The difference in overall fractional allelic loss between the 2 groups did not reach statistical significance. K-ras mutations were not identified in either group. Our results showed a significant overlap in LOH patterns between AAH with or without coexistent lung malignancy. Therefore, AAH may represent a smoking induced low-grade neoplastic lesion that may be a precursor lesion of only a subset of invasive lung adenocarcinoma.     

A test of the assumptions of the transtheoretical model in a post-traumatic stress disorder population

Prior reports suggest an ambivalence regarding treatment in individuals with Post-Traumatic Stress Disorder (PTSD). A model that accommodates such ambivalence is the Transtheoretical Model of Behavior Change (TTM, also known as the Stages-of-Change Model). Fifty veterans presenting for treatment completed self-report measures (94% response rate) that assessed disorder variables and constructs relating to the TTM. While the relationships between the components of each specific construct were found to be consistent with the findings of other studies and a number of predicted relationships between variables were confirmed, many results were inconsistent with the TTM. Notwithstanding questions about the suitability of the self-report measures, the unique characteristics of the veteran sample and the small sample size, the results suggest that the assumptions of the TTM were not met in veterans with PTSD. Copyright © 2005 John Wiley & Sons, Ltd.     

An evaluation of commercial radioisotope methods for the determination of folate and vitamin B12.

Five commercial kits for the determination of folate and six kits for the determination of vitamin B12 were investigated. Their performance has been compared with microbiological methods for the two vitamins and with a non-commercial radioisotopic method for B12. The results show the importance of the determination of the reference range for an individual laboratory for each method. The precision of the kits varied appreciably, as did their performance using specimens from patients with different haematological disorders. In particular, certain kits failed to detect all patients with pernicious anaemia. The relative accuracy of the kits was assessed. Various factors which should be taken into account in the final selection of a satisfactory kit are discussed.     

Localization of IFN-gamma-activated Stat1 and IFN regulatory factors 1 and 2 in breast cancer cells.

The aim of the present work was to evaluate the induction and localization of Stat1, interferon (IFN) regulatory factor-1 (IRF-1), and IRF-2 after IFN-gamma exposure of human breast cancer cell lines, SKBR3, MDA468, MCF7, and BT20. Results from growth assays, Western staining, electrophoretic mobility shift assay (EMSA), and immunohistochemical staining were collated to test our hypothesis that immunohistochemical analysis of Stat1, IRF-1, and IRF-2 would provide additional information about the functionality of the IFN-gamma signaling pathway in human tumor lines. EMSA results showed that in each of four cell lines, Stat1 expression was increased and demonstrated functional activity after IFN-gamma stimulation. Western and EMSA analysis showed upregulation of IRF-1 but not IRF-2 in each cell line. Confocal microscopy of cells stained for Stat1, IRF-1, and IRF-2 confirmed the results and also provided novel information about the intracellular localization of proteins and intercellular variations in responses. The proportion of cells with IRF-1 stimulation and translocation was positively correlated with the IFN-gamma growth suppression in vitro. In conclusion, using four independent assays, we have demonstrated that heterogeneity in IFN-gamma-mediated upregulation of signal transduction proteins can be detected in vitro and that these differences can explain distinct cellular growth effects.     

Axillary lymph node cellular immune response to HER-2/neu peptides in patients with carcinoma of the breast.

HER-2/neu peptides have recently been shown to induce a proliferative response by peripheral CD4(+) T cells in breast cancer patients. To investigate potential differences in the local cellular immune response between breast cancer patients with and without nodal metastases, lymphocytes were isolated from axillary lymph nodes from patients with breast cancer, and proliferative and cytokine responses to HER-2/neu peptides were determined. Freshly isolated lymphocytes from lymph nodes of 7 women undergoing surgery for invasive breast cancer were plated at 20 x 10(5) cells per well in triplicate. Cells were stimulated with HER-2/neu peptides at 50 microg/ml and with control antigens. Incorporation of tritium-labeled thymidine was determined 4 days later. The levels of the cytokines interferon-gamma (IFN-gamma), interleukin-4 (IL-4), and IL-10 were determined at priming and at restimulation with HER-2/neu peptides using a cytokine-specific, double-sandwich, enzyme-linked immunosorbent assay (ELISA). Lymphocytes isolated from the axillary lymph nodes of the patients mounted significant cellular immune response to HER-2/neu peptides, manifested by proliferation and specific cytokine elaboration. Proliferative responses to HER-2/neu peptides were seen in lymphocytes of patients with and without overexpression of HER-2/neu in the primary tumor. In some patients, the proliferative response to HER-2/neu peptides in lymphocytes from lymph nodes with metastases was absent or blunted compared with the response in lymphocytes from lymph nodes without metastases from the same patient (p < 0.05). HER-2/neu peptides induced a predominantly T helper type 1 (Th1) pattern of cytokine response in nodal lymphocytes isolated from breast cancer patients. A Th1-specific cytokine production pattern was maintained at priming and restimulation with HER-2/neu peptides and was amplified with IL-12 costimulation. These results indicate that HER-2/neu peptides can activate T cells in draining lymph nodes from women with invasive breast cancer. This activation is associated with a predominantly Th1 cytokine response, which suggests that conditioning with HER-2/neu peptides may be of value in the development of breast cancer vaccines.     

Spermatogenesis in the dog

The cycle of the seminiferous epithelium of the dog was divided into eight stages, using as criteria the shape of the spermatid nucleus, the location of spermatids and spermatozoa in regard to the basement membrane, the presence of meiotic figures and the release of spermatozoa from the lumen of the tubule. Based upon these criteria, a modification of the eight-stage system of classification of the cycle of the seminiferous epithelium was developed. Cell populations making up each stage are described. The relative frequencies of stages 1 through 8 were 21.9, 12.7, 2.8, 11.5, 8.3, 15.4, 13.3 and 14.0%, respectively. The duration of one cycle of the seminiferous epithelium was 13.6 days (SE ± 0.7), as determined from cells labeled by tritiated thymidine. The absolute durations of stages 1 through 8 were 3.0, 1.7, 0.4, 1.6, 1.1, 2.1, 1.8 and 1.9 days, respectively. The life span of primary spermatocytes was 20.9 days, of secondary spermatocytes 0.5 days, spermatids with round nuclei 10.5 days, spermatids with elongated nuclei up to the time they are released into the lumen, 10.6 days. Counts of the different types of spermatogenic cells in tubular cross sections revealed little or no germ cell degeneration during the two maturation divisions.