GHOST: global hepatitis outbreak and surveillance technology.

BACKGROUND: Hepatitis C is a major public health problem in the United States and worldwide. Outbreaks of hepatitis C virus (HCV) infections associated with unsafe injection practices, drug diversion, and other exposures to blood are difficult to detect and investigate. Effective HCV outbreak investigation requires comprehensive surveillance and robust case investigation. We previously developed and validated a methodology for the rapid and cost-effective identification of HCV transmission clusters. Global Hepatitis Outbreak and Surveillance Technology (GHOST) is a cloud-based system enabling users, regardless of computational expertise, to analyze and visualize transmission clusters in an independent, accurate and reproducible way. RESULTS: We present and explore performance of several GHOST implemented algorithms using next-generation sequencing data experimentally obtained from hypervariable region 1 of genetically related and unrelated HCV strains. GHOST processes data from an entire MiSeq run in approximately 3 h. A panel of seven specimens was used for preparation of six repeats of MiSeq libraries. Testing sequence data from these libraries by GHOST showed a consistent transmission linkage detection, testifying to high reproducibility of the system. Lack of linkage among genetically unrelated HCV strains and constant detection of genetic linkage between HCV strains from known transmission pairs and from follow-up specimens at different levels of MiSeq-read sampling indicate high specificity and sensitivity of GHOST in accurate detection of HCV transmission. CONCLUSIONS: GHOST enables automatic extraction of timely and relevant public health information suitable for guiding effective intervention measures. It is designed as a virtual diagnostic system intended for use in molecular surveillance and outbreak investigations rather than in research. The system produces accurate and reproducible information on HCV transmission clusters for all users, irrespective of their level of bioinformatics expertise. Improvement in molecular detection capacity will contribute to increasing the rate of transmission detection, thus providing opportunity for rapid, accurate and effective response to outbreaks of hepatitis C. Although GHOST was originally developed for hepatitis C surveillance, its modular structure is readily applicable to other infectious diseases. Worldwide availability of GHOST for the detection of HCV transmissions will foster deeper involvement of public health researchers and practitioners in hepatitis C outbreak investigation.

Increased Mitochondrial Genetic Diversity in Persons Infected With Hepatitis C Virus.

BACKGROUND & AIMS: The host genetic environment contributes significantly to the outcomes of hepatitis C virus (HCV) infection and therapy response, but little is known about any effects of HCV infection on the host beyond any changes related to adaptive immune responses. HCV persistence is associated strongly with mitochondrial dysfunction, with liver mitochondrial DNA (mtDNA) genetic diversity linked to disease progression. METHODS: We evaluated the genetic diversity of 2 mtDNA genomic regions (hypervariable segments 1 and 2) obtained from sera of 116 persons using next-generation sequencing. RESULTS: Results were as follows: (1) the average diversity among cases with seronegative acute HCV infection was 4.2 times higher than among uninfected controls; (2) the diversity level among cases with chronic HCV infection was 96.1 times higher than among uninfected controls; and (3) the diversity was 23.1 times higher among chronic than acute cases. In 2 patients who were followed up during combined interferon and ribavirin therapy, mtDNA nucleotide diversity decreased dramatically after the completion of therapy in both patients: by 100% in patient A after 54 days and by 70.51% in patient B after 76 days. CONCLUSIONS: HCV infection strongly affects mtDNA genetic diversity. A rapid decrease in mtDNA genetic diversity observed after therapy-induced HCV clearance suggests that the effect is reversible, emphasizing dynamic genetic relationships between HCV and mitochondria. The level of mtDNA nucleotide diversity can be used to discriminate recent from past infections, which should facilitate the detection of recent transmission events and thus help identify modes of transmission.

Hepatitis C virus antigenic convergence.

Vaccine development against hepatitis C virus (HCV) is hindered by poor understanding of factors defining cross-immunoreactivity among heterogeneous epitopes. Using synthetic peptides and mouse immunization as a model, we conducted a quantitative analysis of cross-immunoreactivity among variants of the HCV hypervariable region 1 (HVR1). Analysis of 26,883 immunological reactions among pairs of peptides showed that the distribution of cross-immunoreactivity among HVR1 variants was skewed, with antibodies against a few variants reacting with all tested peptides. The HVR1 cross-immunoreactivity was accurately modeled based on amino acid sequence alone. The tested peptides were mapped in the HVR1 sequence space, which was visualized as a network of 11,319 sequences. The HVR1 variants with a greater network centrality showed a broader cross-immunoreactivity. The entire sequence space is explored by each HCV genotype and subtype. These findings indicate that HVR1 antigenic diversity is extensively convergent and effectively limited, suggesting significant implications for vaccine development.

Sex-determining region Y box 4 is a transforming oncogene in human prostate cancer cells.

Prostate cancer is the most commonly diagnosed noncutaneous neoplasm and second most common cause of cancer-related mortality in western men. To investigate the mechanisms of prostate cancer development and progression, we did expression profiling of human prostate cancer and benign tissues. We show that the SOX4 is overexpressed in prostate tumor samples compared with benign tissues by microarray analysis, real-time PCR, and immunohistochemistry. We also show that SOX4 expression is highly correlated with Gleason score at the mRNA and protein level using tissue microarrays. Genes affected by SOX4 expression were also identified, including BCL10, CSF1, and NcoA4/ARA70. TLE-1 and BBC3/PUMA were identified as direct targets of SOX4. Silencing of SOX4 by small interfering RNA transfection induced apoptosis of prostate cancer cells, suggesting that SOX4 could be a therapeutic target for prostate cancer. Stable transfection of SOX4 into nontransformed prostate cells enabled colony formation in soft agar, suggesting that, in the proper cellular context, SOX4 can be a transforming oncogene.

RU486 inhibits expression of lysophosphatidic acid induced glycodelin.

OBJECTIVE: This study was undertaken to provide evidence for the mode of action of RU486 on glycodelin produced in K562 cells. To show that histiocytes may be a source of glycodelin in leiomyoma. STUDY DESIGN: With the use of K562, a leukemia cell line, the effect of lysophosphatidic acid (LPA), RU486, antioxidants, and ZK112,993 on glycodelin protein and gene expression was studied. Immunocytochemistry for glycodelin and HAM-56 (macrophage) was performed on leiomyoma and myometrium. RESULTS: Incubation of K562 cells with LPA, progesterone, ZK112,933 and RU486 significantly induced the expression of glycodelin protein and messenger RNA. The addition of RU486 to LPA activated cells markedly reduced expression of glycodelin. Addition of ZK112,993, an antiprogestin without antioxidant properties, to LPA activated cells did not reduce glycodelin. Histiocytes in leiomyoma and myometrium co-localize with glycodelin. CONCLUSION: RU486, partly acting as an antioxidant, markedly reduces LPA stimulated glycodelin production. Histiocytes in leiomyoma and myometrium immunostain for glycodelin and suggests a source for glycodelin in leiomyoma.

Loss of HOXC6 expression induces apoptosis in prostate cancer cells.

We have performed whole genome expression profiling of 28 patient prostate tumor samples and 12 normal prostate samples and identified 55 upregulated and 60 downregulated genes significantly changed in prostate tumor samples compared to normal prostate tissues. Among the members of the upregulated gene set was the developmental transcription factor Homeobox C6 (HOXC6). Silencing of HOXC6 expression using small-interfering RNA (siRNA) resulted in decreased proliferation rates for both androgen-dependent LnCaP cells and the LnCaP-derived androgen-independent C4-2 cell line. Flow cytometry and immunoblotting for the caspase-cleaved form of poly-ADP ribose polymerase (PARP) determined that the decrease in cell numbers was due to increased apoptosis. To validate the specificity of the siRNA-induced apoptosis, LnCaP cells were cotransfected with siRNA specific to the HOXC6 3'UTR and a mammalian expression vector containing the HOXC6 open reading frame, but lacking the 3'UTR. Overexpression of HOXC6 rescued the LnCaP cells from HOXC6 siRNA-induced apoptosis, and increased growth of control GFP siRNA-transfected cells. Expression profiling of HOXC6 siRNA transfections and HOXC6 overexpression identified neutral endopeptidase (NEP) and insulin-like growth factor binding protein-3 (IGFBP-3) as potential proapoptotic repression targets of HOXC6. Our data suggest that HOXC6 may be a novel potential therapeutic target for prostate cancer.

Signaling and transcriptional changes critical for transformation of human cells by simian virus 40 small tumor antigen or protein phosphatase 2A B56gamma knockdown.

One set of genes sufficient for transformation of primary human cells uses the combination of Ha-Ras-V12, the telomerase catalytic subunit hTERT, SV40 large tumor antigen (LT), and SV40 small tumor antigen (ST). Whereas SV40 LT inactivates the retinoblastoma protein and p53, the contribution of ST is poorly understood. The essential helper function of ST requires a functional interaction with protein phosphatase 2A (PP2A). Here we have identified changes in gene expression induced by ST and show that ST mediates these changes through both PP2A-dependent and PP2A-independent mechanisms. Knockdown of PP2A B56gamma subunit can substitute for ST expression to fully transform cells expressing LT, hTERT, and Ras-V12. We also identify those genes affected similarly in two cell lines that have been fully transformed from a common parental line by two alternative mechanisms, namely ST expression or PP2A B56gamma subunit knockdown. ST altered expression of genes involved in proliferation, apoptosis, integrin signaling, development, immune responses, and transcriptional regulation. ST reduced surface expression of MHC class I molecules, consistent with a need for SV40 to evade immune detection. ST expression enabled cell cycle progression in reduced serum and src phosphorylation in anchorage-independent media, whereas B56gamma knockdown required normal serum levels for these phenotypes. Inhibitors of integrin and src signaling prevented anchorage-independent growth of transformed cells, suggesting that integrin and src activation are key ST-mediated events in transformation. Our data support a model in which ST promotes survival through constitutive integrin signaling, src phosphorylation, and nuclear factor kappaB activation, while inhibiting cell-cell adhesion pathways.

Enhanced protein profiling arrays with ELISA-based amplification for high-throughput molecular changes of tumor patients' plasma.

PURPOSE: The purpose of this study is to develop a high-throughput approach to detect protein expression from hundreds and thousands of samples and to apply this technology to profile circulating angiogenic factor protein levels in patients with gynecological tumors. EXPERIMENTAL DESIGN: Analytes containing a mixture of protein are immobilized onto antibody-coated surface of support in array format. The presence of protein in analytes is detected with biotin-labeled antibody coupled with an enhanced chemiluminescence or fluorescence detection system. The exact amount of protein can be quantitatively measured. The expression levels of five angiogenic factors (angiogenin, interleukin 8, vascular endothelial growth factor, platelet-derived growth factor, and epidermal growth factor) from 157 samples were quantitatively measured using this novel protein array technology and were statistically analyzed. The expression patterns of angiogenic factors were analyzed using two-way hierarchical cluster analysis approach. RESULTS: A novel protein array technology, which can simultaneously and quantitatively measure few protein levels from hundreds and thousands of samples was developed. Only minute amounts of sample are required for the assay. This approach also features high sensitivity and specificity. Using this novel protein array approach, we analyzed the plasma expression levels of five angiogenic factors in 137 patients diagnosed with a tumor and 20 controls. Statistical analysis reveals different expression levels of angiogenic factors between patients and controls. Cluster analysis suggests a possible classification of normal subjects from patients. CONCLUSIONS: Enhanced protein profiling arrays provide a high-throughput and sensitive system to detect one or few protein from hundreds and thousands of samples. Such an approach should have broad application in biomedical discovery.

Oxidants and antioxidants affect the expression of glycodelin.

Glycodelin is a glycoprotein that has immunosuppressive activity. We have shown that K562 cells, hematopoitic progenitor cells, are capable of synthesizing glycodelin peptide (Gp) and, perhaps, contribute to Gp in tissues. In addition, several reproductive and nonreproductive tissues themselves are capable of synthesis of glycodelin. In this study, we report that lipid peroxides induce the synthesis of Gp. Antioxidants vitamin E and pyrrolidine dithiocarbamate (PDTC) and antioxidizing enzymes catalase and superoxide dismutase (SOD) effectively blocked phorbol myristate acetate- (PMA-) and lyso phosphatidic acid- (LPA-) induced synthesis of Gp. Dioctanoin (a mimic of diacylglycerol) activated Gp synthesis, and an inhibitor of protein kinase C (PKC) downregulated the response. Based on these observations, we postulate that oxidants by way of PKC might potentiate the angiogenic process.

Induction of monocyte chemotactic protein-1 in peritoneal mesothelial and endometrial cells by oxidized low-density lipoprotein and peritoneal fluid from women with endometriosis.

OBJECTIVE: To elucidate the effect of oxidized low-density lipoprotein (LDL) and peritoneal fluid of women with endometriosis on monocyte chemotactic protein-1 (MCP-1) production by peritoneal mesothelial cells and endometrial cells. DESIGN: In vitro study. SETTING: University medical center. PATIENT(S): Five women undergoing surgery for pelvic pain, infertility, or endometriosis; five women without endometriosis who were undergoing tubal ligation were the controls. INTERVENTION(S): Mesothelial cells and endometrial cells in culture were treated with oxidized LDL and peritoneal fluid from control and endometriosis patients, then MCP-1 levels were measured. MAIN OUTCOME MEASURE(S): ELISA was used to measure MCP-1 in the culture supernatants exposed to oxidized LDL and peritoneal fluid from control and endometriosis patients. Cellular MCP-1 messenger RNA expression was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) assay. RESULT(S): Treatment with oxidized LDL caused an increase in accumulation of immunoreactive MCP-1 in the medium of cultured mesothelial and endometrial cells (primary endometrial stromal cells and endometrial cell line EM42). The mesothelial cells secreted more MCP-1 than did endometrial cells under the culture condition. The EM42 cells cultured in the presence of peritoneal fluid from endometriosis patients secreted more MCP-1 than those cultured with peritoneal fluid from normal women. However, no differences were found in MCP-1 levels in the supernatant of endometrial stromal cells cultured with peritoneal fluid. CONCLUSION(S): This is the first report of MCP-1 expression in mesothelial cells induced by oxidized LDL, and provides direct evidence of inflammatory action of peritoneal fluid of women with endometriosis.

Glycodelin levels in uterine flushings and in plasma of patients with leiomyomas and polyps: implications for implantation.

BACKGROUND: Glycodelin, a glycoprotein, is present in both blood plasma and uterine flushings. It has been implicated in the process of implantation and angiogenesis. During the secretory phase, progesterone secretion is related to glycodelin production. METHODS AND RESULTS: We obtained uterine flushings, prospectively, from 47 infertile patients during the proliferative phase. Patients were recruited from our university practice. Transvaginal ultrasound and sonohysterography permitted the stratification of patients into control, leiomyoma or polyp groups. Total plasma and uterine flushing glycodelin was measured with enzyme-linked immunosorbent assay. Blood was also analysed for progesterone. Uterine flushing glycodelin levels were significantly increased in patients with polyps when compared with controls. An increase in uterine flushing glycodelin levels was noted in patients with leiomyomas compared with controls, though not statistically significant. Plasma glycodelin levels were significantly increased in patients with leiomyomas and polyps when separately compared with controls. There was a significant relationship between plasma glycodelin production and progesterone levels in patients with polyps. CONCLUSIONS: Leiomyomas and polyps are growing tumours and thus produce significant plasma glycodelin levels. Uterine glycodelin flushings are elevated in patients with both polyps and leiomyomas. Elevated glycodelin levels in the follicular and peri-ovulatory period may impair fertilization and implantation.

Dietary oxidized fatty acids may enhance intestinal apolipoprotein A-I production.

Apolipoprotein (apo)A-I, the major protein component of HDL, is synthesized principally in the small intestine and liver. Recently we observed an increase in plasma apoA-I level in humans who were on an oxidized fat diet. To test whether oxidized fatty acids could affect apoA-I synthesis, we incubated day 4 (undifferentiated) and day 14 (differentiated) Caco-2 cells with varying concentrations of oxidized linoleic acid (ox-linoleic acid) (5, 10, and 25 microM) and unoxidized linoleic acid for 24 h. Ox-linoleic acid caused a dose-dependent increase in the levels of apoA-I protein in both differentiated and undifferentiated Caco-2 cells as assessed by ELISA and Western blot analysis. Whereas apoB production was not increased by ox-linoleic acid in both day 4 and day 14 Caco-2 cells. The mRNA expression for apoA-I paralleled the protein expression when measured by RT-PCR. We also found that both day 4 and day 14 Caco-2 cells did express peroxisomal proliferator-activated receptor-gamma (PPAR-gamma). mRNA and PPAR-gamma ligand could increase apoA-I secretion in these cells. Therefore we propose that the mechanism for the induction of apoA-I might include PPAR-gamma for which oxidized fatty acid is a ligand.

Lysophosphatidic acid induces glycodelin gene expression in cancer cells.

Glycodelin is a glycoprotein that has been suggested to be important in normal pregnancy and in malignancy. The regulation of its synthesis has not been studied. In this study, we report the induction of glycodelin gene expression by lysophosphatidic acid (LPA). We studied the effect of LPA (5, 10 and 25 microM) on glycodelin production in breast (MDA-MB-231), cervical (Hela), endometrial (RL-95), ovarian cancer (OVCAR-3) and erythroleukemia (K562) cells. There was a dose-dependent (5-25 microM) induction of glycodelin gene and protein expression in these cell types. LPA is a mimic of phorbol myristate acetate (PMA) action and is found to be elevated in high concentrations in the serum of cancer subjects. As glycodelin is an angiogenic protein with a potential immunosuppressive role, control of LPA synthesis might offer a potential target for intervention.