Cloning and nucleotide sequencing of Vero toxin 2 variant genes from Escherichia coli O91:H21 isolated from a patient with the hemolytic uremic syndrome

Cellular DNA extracted from Escherichia coli strain B2F1 (O91:H21) was found to contain two separate DNA sequences that hybridized with a Vero toxin 2 (VT2)-specific gene probe under stringent conditions. These two sequences were cloned and both were shown to encode a variant of Vero toxin 2 (VT2vh). The nucleotide sequences of the operons encoding VT2vh, designated as vtx2ha and vtx2hb, were determined. The two operons were nearly identical (99% overall DNA homology) and both encoded A subunits of 319 amino acid residues and B subunits of 89 amino acid residues, the A and B subunit genes being separated by a stretch of 14 bp. The A and B subunit genes of the vtx2ha operon exhibited 98.6% and 95.5% DNA homology, respectively, with those of the slt-II operon encoding Shiga-like toxin II (or VT2) cloned from a strain from a patient with hemorrhagic colitis, while the A and B subunit genes of the vtx2ha operon showed 94.5% and 82.8% DNA homology, respectively, with those of the slt-IIv operon encoding a SLT-II variant cloned from a strain isolated from a pig with edema disease. The nucleotide sequences of the presumed promoters and presumptive ribosome binding sites in the vtx2ha, vtx2hb, and slt-II, and slt-IIv operons were identical. These results indicate that nucleotide sequences encoding a family of VT2-related toxins are present in various strains of E. coli and that the sequences of the genes for A subunits are better conserved than those of the B subunit genes.     

ULTRASTRUCTURE OF AN ANAPLASTIC GIANT-CELL CARCINOMA FOUND 8 YEARS AFTER OPERATION ON A PAPILLARY CARCINOMA OF THE THYROID

Light and electron microscopic studies have been made on an anaplastic giant-cell tumor that developed in a woman 8 years after an operation on the thyroid for papillary carcinoma. Many giant cells were observed in the anaplastic tumor tissue, but no follicles. Numerous tightly-packed mitochondria and abundant ribosomes were present, but there were no desmosomes. The basement membrane was not distinct.     

Pituitary Folliculo-Stellate-Like Cells Stimulate Somatotroic Pituitary Tumor Growth in Nude Mice

A new cell line (TtT/GF) established from a murine pituitary thyrotropic tumor having characteristics similar to those of pituitary folliculo-stellate cell (FS cell) was implanted into nude mice together with cells from a rat pituitary somatotrophic tumor cell line (MtT/S) to determine whether the former enhances pituitary tumor growth. For as long as 2-3 mo after implantation, MtT/S cells implanted either alone or together with fibroblasts formed either no tumors or only very small tumors in the nude mice. In contrast all of the nude mice that had received MtT/S cells implanted together with TtT/GF cells developed large tumors. Furthermore, the mice bearing the MtT/S and TtT/GF implants showed a significantly higher body weight and serum growth hormone level than those bearing only MtT/S cells or a combination of MtT/S cells and fibroblasts. The TtT/GF cell line itself had no tumorigenicity during the experimental period. Therefore, the TtT/GF cell line as a model of FS cells enhanced pituitary endocrine cell tumor formation. Additionally, immunocytochemistry showed that TtT/GF cells positive for glial fibrillary acidic protein (GFAP) or S-100 protein were present in the parenchymatous tissue elements or connective tissue surrounding the tumor nests. In the parenchymatous tissue, the TtT/GF cells exhibited a stellate appearance and surrounded neighboring tumor cells with their long cell processes. These results suggest that TtT/GF cells can serve as a model for pituitary FS cells, and are capable of stimulating pituitary tumor growth either by modifying the microenvironment or producing growth factors.     

Cross-linking of Fc(gamma)-receptor on monocytes inhibits hepatitis C virus-specific cytotoxic T-lymphocyte induction in vitro.

In hepatitis C virus (HCV) infection, immune complex (IC)-type virus particles are frequently observed in circulation. The IC leads to cross-linking of Fcgamma receptors (FcgammaR) on monocytes and exerts immunoinhibitory function. To test the roles of IC in HCV-specific cytotoxic T lymphocyte (CTL) induction, we generated HCV CTL from peripheral blood mononuclear cells of chronic hepatitis C patients with or without HCV-IC- or immunoglobulin G (IgG)-coated culture plates and compared their lytic activities. HCV-IC or adherent IgG, which induces FcgammaR cross-linking, significantly reduced CTL activity. Expression of B7-1 on monocytes decreased on adherent IgG. In addition, tumour necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta1 (TGF-beta1) production increased from cells on adherent IgG and their mRNA expression in monocytes was enhanced. Anti-TNF-alpha antibody during induction on adherent IgG inhibited lysis; however, anti-TGF-beta completely reversed its inhibitory effect. These results demonstrated that HCV-IC or adherent IgG impaired HCV-CTL induction in vitro. The FcgammaR-mediated CTL suppression occurred via decreased expression of monocyte B7-1 and/or enhanced production of TGF-beta1.     

Augmentation of human monocyte chemiluminescence by iron-saturated lactoferrin

The effect of Fe3+ bound lactoferrin (LF) on chemiluminescence (CL) generation in human monocytes in the presence and absence of opsonized zymosan was examined. Fe3+ bound LF (10(-6) or 10(-7) M) augmented the CL response in the presence but not the absence of zymosan. In contrast, Apo LF, Fe3+ bound transferrin and Fe3+ itself had no significant effect. It appears that the effect of LF on zymosan-induced chemiluminescence is dependent on iron and is receptor specific. The augmentation of CL by LF was inhibited by superoxide dismutase (SOD) and ETYA (fatty acid cyclooxygenase and lipoxygenase inhibitor), but not indomethacin (fatty acid cyclooxygenase inhibitor). Thus the enhancement of CL by LF depends on superoxide anion production and is related to the lipoxygenase pathway. The significance of this observation in terms of microbicidal or tumoricidal mechanisms remains to be determined.     

Enhancement of lipopolysaccharide-stimulated cyclooxygenase-2 mRNA expression and prostaglandin E2 production in gingival fibroblasts from individuals with Down syndrome

It is well known that Down syndrome (DS) is a premature ageing syndrome. Periodontal disease in individuals with DS develops rapidly and extensively in a relatively younger age bracket compared with that in healthy controls. The mechanisms involved in the periodontal inflammatory processes in DS patients are not fully understood. In the present study, the non-inflamed gingival fibroblasts isolated from seven patients with DS (DGF) and seven healthy controls (NDGF) were stimulated with lipopolysaccharide (LPS) derived from Actinobacillus actinomycetemcomitans (A. a.). We measured the level of prostaglandin E2 (PGE2) production by DGF and NDGF by radioimmunoassay, and also measured the mRNA expression of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) by using the real-time PCR method. We found the higher levels of LPS-stimulated COX-2 mRNA expression and PGE2 production in DGF when compared with those in NDGF. This study may indicate that overexpression of LPS-stimulated COX-2 induced a greater ability of DGF to produce PGE2, and that these phenomena may be responsible for the severer periodontal disease in DS patients.     

Frozen-section services by telepathology: Experience of 100 cases in the San-in District, Japan

The early experience is reported here of the use of Intra-operative frozen-section service by telepathology using the Integrated Service Digital Network (ISDN), a commercially available system that is being connected between the Department of Pathology of Tottori University and Matsue City Hospital, a distance of 30 km. The transfer rate is currently 64kbit/s. The frozen-section service was conducted for a total of 117 tissue specimens (organs) from 100 patients between August 1993 and May 1995. The average time taken for examination of each specimen of frozen section was 13min, ranging between 2 and 42min. The average number of transmitted Images was 6.2. Six cases necessitated more than 11 transmitted Images to make a diagnosis, while 13 cases could be diagnosed from two images only. Correct and permissible diagnoses were obtained in 109 (93.2%) out of 117 specimens when comparing the telepathology diagnosis with that of direct microscopy. Improper or misdiag-nosis was made for eight cases (specimens), which were misinterpreted as papillary carcinoma in Basedow's disease, adenoma and hyperplasia in two pheochromocytomas, solid-tubular carcinoma in phyilodes tumor, mastopathy in invasive carcinoma, metastatic carcinoma in astrocytoma, follicular lymphoma in reactive hyperplasia, and lymphadenitis in follicular lymphoma. in retrospect, diagnosis of these cases should have been deferred. From the results, it was concluded that the Intraoperatlve frozen-section service by telepathology may be a worthwhile substitute for hospitals with limited accessibility to local pathology service, in spite of pitfalls in some cases. Well prepared, high-quality frozen sections, sufficient verbal communication with surgeons, and a rather conservative attitude on the part of a well-trained pathologist seem to be the essential Ingredients for reaching an accurate decision when using telepathology.