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Thrombin, a multifunctional protein, has been found to be involved in cellular mitogenesis, tumor growth, and metastasis, in addition to its well known effects on the initiation of platelet aggregation and secretion and the conversion of fibrinogen to fibrin to form blood clots. These properties of thrombin rely on its action as a serine protease, which cleaves the N-terminal region of a 7-transmembrane G protein receptor (protease-activated receptor, PAR-1), thus exposing a tethered end hexapeptide sequence capable of activating its receptor. Little is known about its effect on genes that regulate the cell cycle. This study was undertaken to investigate the possible mechanisms by which thrombin regulates tumor cell growth in several tumor cell lines: human CHRF megakaryocyte, DU145 prostate, MDAMB231 and MCF7 breast, U3A fibrosarcoma, and 2 murine fibroblast cell lines, MEFp53(-/-) and CD STAT(-/-). We have found that thrombin under the conditions of culture employed inhibits cell growth by both up-regulation of p21(waf/cip1) and induction of caspases via its PAR-1 receptor. The increased expression of p21(waf/cip1) by thrombin was p53 independent, STAT1 dependent, and protein synthesis independent. This was associated with tyrosine phosphorylation of JAK2 and STAT1, and nuclear translocation of STAT1. Induction of apoptosis is also PAR-1-specific, STAT1-dependent, and associated with up-regulation of caspases 1, 2, and 3. Our study establishes, for the first time, a link between PAR-1 receptor activation with the STAT signal pathway, which leads to cell cycle control and apoptosis. This observation broadens our understanding of the mechanism of PAR-1 activation and its effect on cell growth, and could possibly lead to therapeutic approaches for the treatment of cancer.

The urofacial (Ochoa) syndrome (UFS) is a rare autosomal recessive disorder characterized by abnormal facial expression and urinary abnormalities. Previously, we mapped the gene to a genomic interval of approximately 1 cM on chromosome region 10q23-24, using families from Columbia. Here we demonstrate genetic homogeneity of the syndrome through homozygosity mapping in American patients with Irish heritage. We established a physical map and identified novel polymorphic markers in the UFS critical region. Haplotype analysis using the new markers mapped the UFS gene within one YAC clone of 1,410 kb. We also determined the precise location of the gene encoding for glutamate oxaloacetate transaminase (GOT1) within the new UFS critical region and determined its genomic structure. However, mutation analysis excluded GOT1 as a candidate for the UFS gene.

Thrombin-treated tumor cells induce a metastatic phenotype in experimental pulmonary murine metastasis. Thrombin binds to a unique protease-activated receptor (PAR-1) that requires N-terminal proteolytic cleavage for activation by its tethered end. A 14-mer thrombin receptor activation peptide (TRAP) of the tethered end induces the same cellular changes as thrombin. Four murine tumor cells (Lewis lung, CT26 colon CA, B16F10 melanoma, and CCL163 fibroblasts) contain PAR-1, as detected by reverse transcriptase-polymerase chain reaction (RT-PCR). B16F10 cells did not contain the two other thrombin receptors, PAR-3 and glycoprotein Ib. TRAP-treated B16F10 tumor cells enhance pulmonary metastasis 41- to 48-fold (n = 17). Thrombin-treated B16F10 cells transfected with full-length murine PAR-1 sense cDNA (S6, S7, S14, and S22) enhanced their adhesion to fibronectin 1.5- to 2.4-fold (n = 5, P.04), whereas thrombin-treated wild-type cells do not. S6 (adhesion index, 1.5-fold) and S14 (index, 2.4-fold) when examined by RT-PCR and Northern analysis showed minimal expression of PAR-1 for S6 over wild-type and considerable expression for S14. Immunohistochemistry showed greater expression of PAR-1 for S14 compared with wild-type or empty-plasmid transfected cells. In vivo experiments with the thrombin-treated S14 transfectant showed a fivefold to sixfold increase in metastases compared with empty-plasmid transfected thrombin-treated naive cells or S6 cells (n = 20, P =.0001 to .02). Antisense had no effect on thrombin-stimulated tumor mass. Thus, PAR-1 ligation and expression enhances and regulates tumor metastasis.

A human herpesvirus-8 (HHV-8) enzyme-linked immunosorbent assay (ELISA) with a whole virus lysate as antigen was developed and used to measure the seroprevalence rate and levels of IgG antibodies to HHV-8 in sera/plasma of various patient groups and blood donors. The virus antigen was prepared from the KS-1 cell line, which produces lytic virus, and therefore contains a broad array of viral proteins. Seroprevalence studies using this ELISA showed the following: 10 of 91 blood donors (11%) had an average HHV-8 antibody titer of 118; 67 of 72 (93%) classic Kaposi's sarcoma (KS) patients were positive with an average titer of 14,111; and 57 of 62 (92%) KS/human immunodeficiency virus (HIV) patients were positive with an average titer of 4,000. A study on a very limited number of serial serum samples from patients before and after diagnosis with KS showed highly elevated antibody titers to HHV-8 virus after KS lesions developed. Preliminary data show that 50% of the sera from HIV-1(+) homosexual patients contain IgG antibodies to HHV-8 suggesting that this population is at high risk for developing KS. Antibody results correlated well with the confirmatory immunofluorescent assays (IFA) using KS-1 cells as the substrate. This HHV-8 IgG antibody detection ELISA is sensitive and specific and does not cross-react with Epstein-Barr virus (EBV) or other human herpesviruses. The results of this HHV-8 antibody survey suggest that this rapid ELISA assay can be used to screen large numbers of sera to find those at risk for developing KS.

The DNA sequences of a novel human gamma-herpesvirus type 8 (HHV-8) have recently been detected in Kaposi's sarcoma (KS) lesions obtained from different populations in whom this neoplasm occurs, suggesting that this virus may be implicated in the etiology and/or pathogenesis of KS. To study the distribution and possible means of transmission of the putative viral agent, specimens of KS skin lesions, matched uninvolved skin, peripheral blood mononuclear cells (PB-MCs), and semen were collected from 12 HIV-positive homosexual men with acquired immune deficiency syndrome (AIDS)-related KS (AIDS-KS) and 2 human immunodeficiency virus (HIV)-negative homosexual men with KS. HHV-8 virus DNA was detected by polymerase chain reaction (PCR) studies in all 14 of these KS specimens and in 6 of 14 biopsies of normal-appearing skin distant from any KS lesions including 1 uninvolved skin specimen from an HIV-negative homosexual male with KS. In addition, 3 of 12 PBMC samples and 3 of 12 semen samples from the AIDS-KS patients were positive for HHV-8. The DNA sequences of HHV-8 were not detected in the matched semen and PBMC specimens obtained from 2 HIV-negative homosexual men with KS, 4 HIV-positive homosexual patients without KS, 2 HIV-seronegative healthy homosexual men, 5 HIV-positive heterosexual male intravenous drug users, or 5 healthy HIV-negative heterosexual donors. Using PCR in situ, positive signals for HHV-8 were demonstrated in the B lymphocyte subsets of PBMCs and/or in spermatozoa and mononuclear cells in the semen from some of the PCR-positive specimens from the AIDS-KS patients examined. These data show that HHV-8 is present in and could possibly be transmitted via semen and/or blood from some homosexual men with AIDS-KS.

We have undertaken a large-scale study of various tissues from normal controls and patients with Kaposi's sarcoma (KS) or other malignancies, both with and without human immunodeficiency virus infection, to determine the prevalence of human herpesvirus 8 (HHV-8) DNA. A total of 566 specimens were analyzed by PCR for the presence of HHV-8 DNA. Of the samples tested, 251 were obtained from patients with KS and 315 were obtained from patients without KS. HHV-8 DNA was detected in 103 (41%) of the 251 samples from patients with KS. In particular, 92% of KS tumor specimens were positive. None of the tissues from patients without KS showed evidence of HHV-8 DNA. Sequencing and phylogenetic analyses indicate a high degree of conservation (97.5 to 100%) among the HHV-8 strains tested.

Thirty one biopsy samples of fresh Kaposi's sarcoma (KS) skin lesions were examined for alteration of the K-ras oncogene by differential PCR, SSCP and nucleic acid sequencing. Three KS specimens showed amplification of K-ras and 7 were found to have various mutations at exon 1 of the K-ras gene. Eight of the 10 K-ras gene alterations detected were found in more advanced nodular stage KS lesions, while only 2 were observed in the plaque stage KS tumors. Activation of the K-ras oncogene may play a role in the malignant progression of this unusual neoplasm.

Autoimmune mechanisms may be involved in the pathogenesis of insulin dependent diabetes mellitus (IDDM). Multiple autoantibodies have been detected in patients with IDDM. Islet cell antibodies (ICA), complent-fixing islet cell antibodies (CF-ICA) and antibodies to an islet cell protein 64000 M(r) (64K antibodies) have been regarded as immunological markers in IDDM. ICA detection with immunohistochemistry requires fresh normal human pancreas (blood group O) which provides an antigen for measuring ICA in serum samples. In the present study ICA detection was first carried out by using avidin-biotin-peroxidase complex technique (ABC) method and paraffin sections of human pancreas (blood group O), Serum samples were obtained from 17 patients with IDDM, 20 with NIDDM and 20 without diabetes mellitus. In patients with IDDM, ICA were detected in 9 of the 17 (52.94%) while none of the patients with NIDDM and without diabetes mellitus were ICA positive. In comparison with other methods, the present one is more reliable, sensitive, specific and simple. Therefore, it may be widely used for ICA detection in clinical practice.

Fibroblast growth factors (FGF), such as basic FGF, have been implicated in the development of Kaposi sarcoma (KS) in vitro. The expression of several genes of the FGF family and their receptors in KS tumor lesions and KS-derived cells were evaluated.Cultures of KS-derived cells were established. The expression of FGF family members and their receptors in these cells and in fresh biopsies from KS tumors was evaluated by reverse transcription polymerase chain reaction (RTPCR). The RTPCR products were confirmed by nucleotide sequencing.The expression of basic FGF and FGF receptor-1 (flg) was detected in all the samples tested. Acidic FGF (aFGF) and FGF-5 were detected in two of six and four of six KS tumor specimens, respectively, whereas both of these growth factors were expressed in all of the cell cultures, including six KS-derived cell cultures and human endothelial cells and smooth muscle cells. FGF-6 was expressed in two of six KS tumor specimens, but was not expressed in any of the cultured KS cells. Like flg, bek was expressed in all tissue samples and KS-derived cell cultures except in one KS specimen obtained from the patient's tongue showing expression of a high level of FGF-6.These results suggest that the expression of FGF in KS tumors with the coexpression of FGF receptors may provide a basis for autocrine and paracrine mechanisms contributing to the development of KS.

Growth factors, such as bFGF, have been shown to exert autocrine and paracrine effects on the growth of Kaposi's Sarcoma (KS)-derived cells, which suggested that the inhibitors of angiogenesis may be promising for KS treatment. However, KS lesions have been found to continue to enlarge after patients had been treated with FGF binding antagonists such as suramin. We investigated the effect of protamine and suramine on the growth of KS derived cells in vitro. Although both of these agents which are FGF binding antagonists were found to inhibit the incorporation of 3H thymidine in KS-derived cultured cells, increased expression of bFGF, FGF5 and the FGF receptor was observed after the KS cells were exposed to these substances. These results might explain the clinical observation that FGF binding antagonists such as suramin caused an apparent stimulation of KS tumor growth when administered systemically to patients with AIDS-related KS.

CD57, a glycoprotein, is a specific antigen presented on the surface of human natural killer cells and killer cells. The monoclonal antibody to CD57 has been available. In this study, the avidin-biotin-peroxidase complex (ABC) technique visualized with glucose oxidase-DAB-nickle (GDN) was used to detect the expression of CD57 in endocrine cells of human pancreas. The results showed that the CD57 immunoreactive (CD57-IR) cells were scattered throughout human pancreas. Most of positive cells were localized in the pancreatic islets. Grouped or single cells occurred intercalated between acinar cells, as well as in the epithelium of pancreatic ducts. The anti-CD57-monoclonal antibody stained the myelin sheaths of nerve fibers in all tissues investigated. Alternative sections immunostained with antibodies against bioactive peptides proved the coexistence of CD57 and Glu, SS, HPP or PS in endocrine cells. The significance of the expression of CD57 immunoreactivity in endocrine cells of human pancreas was discussed.

Full-valve annuloplasty of superficial femoral venous valves was performed successfully in 139 extremities affected with primary deep venous incompetence of the lower limb. Chronic ulcerations in 43 affected extremities healed after operation. Postoperative venography showed that the recovery rate of valvular function was 97%, and no serious complication was noted. Follow-up for a mean of 3.5 years showed no varicosed vein and no recurrence of ulceration. The principles of operation, the optimum site, number and degree of annuloplasty, indications and results of the operation were discussed.

It has been reported that the prevalence of cataract is much higher in plateau than that in plain regions. But the data are incomplete and can not be compared because of different examination methods and different diagnostic criteria. A small group of ophthalmologists was sent to Ganzi, Sichuan Province at the altitude of 3.393 m, and to Qinpu in the suburb of Shanghai which is at the sea level. In Ganzi 3,905 (91.97%) people including 822 Hans and 3,083 Zang and in Qinpu 3,101 (93.20%) were studied. Age-related cataract was diagnosed when there are cortical or nuclear, or mixed opacities; and aphakia due to previous extraction of age-related cataract. Direct method was used to standardize the data. The prevalence of age-related cataract in Ganzi is about 82% higher than that in Qinpu in all age groups; from age 16-60, the prevalence of age-related cataract in Ganzi is about 180% (in Hans) and 160% (in Zangs) higher than that in Qinpu. There is no significant difference in the prevalence of age-related cataract between the ethnic groups in Ganzi. In Ganzi, the sunshine hours per year are 1.28 times that in Qinpu, and the global radiation in Ganzi is 1.47 times that in Qinpu. The higher prevalence of cataract in Ganzi than in Qinpu may be due to the greater exposure to the sunshine on the Tibetan plateau. The differences in prevalence between males and females and between indoor and outdoor work are discussed.

Sister chromatid exchange (SCE) and chromosome aberration have been used to examine the peripheral lymphocytes of 20 epileptic children on 6-52 months monotherapy with valproic acid (VPA), and two matched control groups were set for comparison. The frequency of SCE in the VPA treated group is significantly higher than two control groups (P less than 0.01). It seems that the increase in SCE frequency is not related to epilepsy. SCE was also examined in 10 epileptic children before and after intake of only sodium valroat for 6-7 months, there was statistically significant change of SCE following VPA. 9 normal children's lymphocytes were exposed in vitro to sodium valproate (5-20 micrograms/ml), all showed a significant increase in SCE. These results suggested that VPA and its derivatives could damage the DNA of child users; however slightly higher rates of chromosome aberration were not significantly different from two control groups.

The valuable contributions of Maragoudakis and coworkers on thrombin and angiogenesis have always been recognized by our group and frequently quoted in our publications. Their work was quoted three times in the present paper [1], three times in our paper on thrombin and the induction of vascular endothelial growth factor (VEGF) [2] and three times in our paper on the induction of angiopoietin-2 [3]. Maragoudakis and coworkers object to our statement quoting their work in which we stated that an attempt to measure thrombin-induced angiogenesis was made by Maragoudakis et al . We believe that this was a fair statement. In their most recently reported publication on this subject, their measurement was indirect, employing C-proline incorporation into collagen in the presence of hydrocortisone (to inhibit inflammation) to measure neoangiogenesis [4]. They found a 34% increase in collagen synthesis using 1 U mL thrombin and interpreted this to represent an increase in neoangiogenesis. Our paper describes a direct observation on newly formed blood vessels on the CAM by microscopic examination following the addition of 0.1 U mL thrombin resulting in a 2–3-fold increase in new blood vessel development. We further showed that VEGF and angiopoietin-2 were upregulated in the CAM during this stimulation—and inhibited by soluble angiogenesis receptors Tie2-Fc and KDR-Fc as well as pertussis toxin (G coupled receptor) and inhibitors of protein kinase C, MAPkinase and PI3kinase. We believe that this is a more rigorous demonstration of the induction of neoangiogenesis by thrombin on a physiological membrane.

To clarify the rule of monoamine in cerebral ischemia, 3-methoxy-4-hydroxyphenylglycol (MHPG), 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA) in cerebrospinal fluid (CSF) of 33 patients and 18 controls were measured with high performance liquid chromatography. Results showed all the three metabolites increased in patients with severe ischemia but only MHPG and 5-HIAA increased significantly. MHPG changed more quickly and regularly in cerebral ischemia than the other two. A positive correlation between any couple of the metabolites was found in both the controls and patients in the first week after stroke, but it was disturbed at the end of the second week. Computer-assisted multivariate analysis indicated that 5-HIAA and MHPG are more closely related to the state of illness in the acute stage while HVA the least. Possible explanations for the changes of CSF levels of amine metabolites were discussed.