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Research on processing procedures conducted to extract enteric viruses from oysters for PCR detection is presented. The method included improves the gastroenteritis outbreak response.

Genotyping of F(super +) RNA coliphages by nucleic acid hybridization with nonradioactive oliginucleotide probes is an effective and more economical method to group them. Genotyping of F(super +) RNA coliphages is practically viable for field samples. Serotyping of F(super +) RNA coliphages is necessary to distinguish between human fecal contamination and that of animal to determine the risk of infection from coliphage from different sources. Results obtained from genotyping compare very well with those from serotyping.

Dysregulation of γ-synuclein (SNCG) has been reported in many cancers; however, its role in cancer development is still controversial. Here, we examined the potential involvement of DNA methylation in regulating SNCG and its role in oral squamous cell carcinoma (OSCC). We used 8 OSCC cell lines to investigate SNCG methylation and expression. SNCG methylation was examination by methylation-specific polymerase chain reaction and bisulfate sequencing. Cells showing a high degree of SNCG methylation were treated with 5-aza (methylation inhibitor), and changes in their methylation and expression profiles were analyzed. Functional effects of SNCG in OSCC were examined by its overexpression and knockdown. Additionally, methylation and expression of SNCG in OSCC tissues were investigated and correlated with clinicopathologic features. All OSCC cells showed detectable SNCG expression at the mRNA and protein levels. Methylation-specific polymerase chain reaction and bisulfate sequencing revealed high SNCG expression in SCC25 cells with the unmethylated allele, and their 15 CpG islands were unmethylated. The methylated allele was detected only in OEC-M1 cells exhibiting low SNCG expression, and their CpG islands were partially methylated. 5-aza treatment in OEC-M1 cells attenuated methylation and restored SNCG expression. SNCG overexpression increased colony forming, migration, and invasion abilities in OEC-M1 cells. Silencing SNCG in SCC25 cells suppressed these behaviors. All 25 tumor-adjacent normal tissues were negative for SNCG immunostaining. SNCG upregulation was frequently observed in dysplastic and OSCC tissues. Positive SNCG expression was found in 45% (37 of 82) OSCC tissues. Positive SNCG expression in OSCC significantly correlated with cancer staging and lymph node metastasis. However, SNCG methylation did not correlate with its expression and clinicopathologic variables in OSCC tissues. DNA methylation may participate in regulating SNCG expression in some OSCC cells. SNCG upregulation could be involved in OSCC progression. [ABSTRACT FROM AUTHOR]

Aberrant Wnt signaling pathway is a common feature of tumors and also plays important roles in tumor progression and metastasis of many cancer types. Various lines of evidence suggest that genetic defects affect Wnt pathway components, as well as epigenetic mechanisms that modulate the suppressors of Wnt pathway in oral squamous cell carcinoma. Recently, the newly discovered microRNAs are important molecular regulators in gene expression through transcription and translation repression. They play fundamental roles in a wide spectrum of biological functions, including cancer. In this review, we aim to accumulate recent research findings on the roles of Wnt/β-catenin signaling and discuss how microRNAs affect Wnt/β-catenin signaling in oral squamous cell carcinoma tumorigenesis. Apparently, investigations into the role of microRNAs with regard to the Wnt pathway in oral squamous cell carcinoma may help in the development of better strategies for tumor treatment. [ABSTRACT FROM AUTHOR]

Recovery efficiencies of enteric bacteriophages (F+ RNA coliphages, somatic coliphages, and Salmonella phages) as alternative fecal indicators were determined from ground beef and chicken breast meat using amino acid eluants (glycine and threonine) and a complex eluant (3% beef extract). Levels of F+ RNA coliphages (MS2, GA, Qbeta, FI, and SP), the somatic coliphage phiX174, and three environmental isolates of Salmonella phages (isolated from raw sewage) were assayed using three respective hosts: Escherichia coli Famp, E. coli C, and Salmonella Typhimurium. When 8% polyethylene glycol and 0.1 M NaCl were used to precipitate bacteriophages eluted with five different eluants, the highest recoveries of the three phage groups were with 0.5 M threonine and 0.25 M glycine-threonine. The average recoveries of F+ RNA coliphages, somatic coliphages, and the Salmonella phages from ground beef and chicken meat were 100, 69, and 65%, respectively, with threonine (0.5 M, pH 9.0) as the eluate. Of eight market food samples tested, F+ RNA coliphages were detected in five (63%) and somatic coliphages were detected in seven (88%). The overall detection sensitivity of the method was 3 PFU/100 g of ground beef or chicken meat. Levels of bacteriophages and bacterial indicators on chicken carcass surfaces were determined at identified critical control points at a poultry plant. Through the processing steps of evisceration, washing, and chilling, the levels of F+ RNA coliphages and fecal coliforms were reduced by 1.6 and 1.9 log10 PFU or CFU/100 g, respectively. F+ RNA coliphages and perhaps other enteric bacteriophages may be effective candidate indicators for monitoring the microbiological quality of meat, poultry, and perhaps other foods during processing. The bacteriophage concentration method developed provides a simple, rapid, and practical tool for the evaluation of fecal contamination levels in ground beef and processed chicken meat.

Abnormalities of the tumor-suppressor p53 gene have been discovered in human hepatocellular carcinoma (HCC). It is unclear, however, whether HCC related to chronic viral hepatitis is associated with p53 gene alterations. In this study, we have examined p53 abnormalities in HCC associated with hepatitis C and B virus (HCV and HBV) infections. Tissues from 18 HCC patients from several hospitals throughout the United States were collected (9 were HCV-infected, 5 were HBV-infected, 1 was HCV/HBV-infected, and 3 were non-virus-associated). Immunostaining with monoclonal pAb 1801 revealed expression of p53 protein in tumor-cell nuclei in one HCV-associated HCC, and in no case of HBV-associated HCC, while the nuclei of adjacent hepatocytes were negative. Using Hae III-digestion of chromosomal DNA, mutations at codon 249 were not found in any of 18 HCC tissues studied. Direct sequencing demonstrated a mutated codon 244 and a wild-type codon 249 in the conserved regions (exon 5-8) of p53 gene from the tumor tissue with nuclear p53 expression. By reverse-transcription-polymerase chain reaction (RT-PCR), the expression of p53 mRNA was demonstrated in tumor cells from 10 out of 16 HCC tissues. In conclusion, the specific mutation at codon 249 with G to T transversion was not observed in the HCCs associated with HCV or HBV infections. In HBV or non-virus-associated HCCs studied, no other p53 gene abnormalities were found. A point mutation at codon 244 with G to A transition of p53 gene was detected in only one of 10 HCV-associated HCCs, which suggests that p53 mutations may not play a significant role in HCV- or HBV-associated hepatocarcinogenesis.

Background: Successful treatment of chronic hepatitis C with interferon alfa is frequently followed by relapse. Because loss of hepatitis C viral RNA (HCV-RNA) in serum is not predictive of sustained response, the loss of HCV-RNA in liver as a predictor of sustained response was investigated., Methods: Twenty-one patients with chronic hepatitis C treated with recombinant interferon alpha had HCV-RNA sequences determined in frozen liver tissue before and after treatment and in serum at the end of treatment. Reverse double polymerase chain reaction was used to detect sequences to the 5' nontranslated region of the HCV genome using double nested primers., Results: HCV-RNA disappeared in the liver in 10 of 11 (91%) complete responders whereas it remained detectable in the liver or serum of 7 of 8 (87%) nonresponders. Five complete responders relapsed biochemically during 6 month's follow-up; 4 of these had no detectable HCV-RNA in liver at end of treatment., Conclusions: Disappearance of HCV-RNA in liver correlates with initial clinical outcome, but as previously reported with serum HCV-RNA, this loss does not necessarily allow prediction of a sustained response.

Recurrent or intercurrent hepatitis C represents significant problems in patients with liver transplants and must be differentiated from hepatic allograft rejection and other conditions affecting allografts. Often, the currently available anti-hepatitis C virus (HCV) tests are not helpful in the differential diagnosis, because preexisting anti-HCV may persist after transplantation or its development may be delayed. We determined the presence of HCV RNA by the reverse double polymerase chain reaction in biopsy specimens of liver allografts from nine patients with acute or chronic hepatitis of uncertain origin and from three patients with cellular allograft rejection. The NS3 region sequences of HCV were detected in seven of nine liver allograft biopsy specimens 6 weeks to 20 months after transplantation. Hepatitis C virus RNA was not detected in two patients. One of these patients was anti-HCV positive, showing mild acute hepatitis 5 weeks after transplantation. Anti-HCV was present in three patients with detectable HCV RNA in the liver but was absent from four other patients with HCV RNA. These findings suggest that HCV is a major cause of acute and chronic hepatitis following liver transplantation, that detection of HCV RNA by polymerase chain reaction in the liver biopsy specimen represents a reliable method for the diagnosis of hepatitis C in liver allografts, and that in some patients HCV may be acquired during transplantation while in others it may represent a recurrent infection.

Several serologic studies suggest that infection by hepatitis C virus (HCV) may be associated with the development of hepatocellular carcinoma (HCC). Therefore, we examined tumor tissue and/or the surrounding liver of 20 patients for viral sequences by the polymerase chain reaction (PCR). In 12 cases, liver and tumor tissues were separable for extraction. RNA was extracted from frozen tissues and used as a template for reverse transcription followed by double PCR with nested primers for the 5'-untranslated (NT) and nonstructural NS3 regions of HCV. In addition, the tissue extracts were tested by single PCR for X gene and S gene sequences of hepatitis B virus (HBV). NT region sequences of HCV were detected in the available tumor tissue of all anti-HCV-positive patients except for one. Negative (replicative) strands of HCV RNA were found in the same tissues as positive (genomic) strands at almost the same relative amounts, suggesting replication of HCV in the tumor tissue rather than contamination by HCV-positive blood. HBV X and S sequences were demonstrated in two tumors, but were absent from three tumors that were surrounded by liver tissues with HBV X sequences. One patient had nucleic acids of both viruses in tumor tissue. These observations suggest that in addition to HBV, HCV may play a role in the development of hepatocellular carcinoma.

Currently, the most reliable method for the diagnosis of hepatitis C virus (HCV) infection is the detection of viral sequences by the reverse transcription double polymerase chain reaction (RT/PCR) in serum or liver samples. We demonstrate here that noncoding region sequences (NT) of HCV were amplifiable by RT/PCR in guanidinium extracts of formalin-fixed (for 6 to 48 h), paraffin-embedded liver sections of patients with chronic hepatitis C. In contrast, core and nonstructural region sequences of HCV were not detectable in fixed tissues by PCR amplification. Boiling of routinely processed tissue sections in water containing Chelex-100, a method for extraction of amplifiable hepatitis B virus DNA, was not successful. The amount of nucleic acid extracts from fixed liver sections needed for amplification of NT region sequences was over 1000 times larger than that of extracts from frozen tissue. This method will be useful for diagnostic and investigative studies of HCV infection.

Although sensitive assays for serum antibodies to hepatitis C virus (HCV/anti-HCV) have been developed recently, the relation of anti-HCV to HCV infection of the liver has not been clarified. Therefore, we determined the presence of HCV RNA by the reverse transcription-polymerase chain reaction (PCR) in liver biopsy specimens of 21 patients with chronic liver disease and 5 control patients. RNA was extracted from frozen liver tissues by the guanidinium method, HCV cDNA was synthesized by reverse transcription, and core region and NS3 region sequences were amplified by PCR. The sensitivity and specificity of the reaction was significantly enhanced by double PCR with nested primers followed by Southern blotting with an HCV specific oligomer probe. NS3 region sequences were detected in the liver specimens of 12 out of 15 anti-HCV positive patients. Core region sequences were detected in 9 patients, all of whom were also positive for NS3 region sequences. HCV sequences were not detected in 11 anti-HCV negative patients. In all cases, the integrity of the extracted RNA was demonstrated by successful amplification of albumin mRNA as internal control. Our findings demonstrate the feasibility of the reverse transcription-double PCR method followed by Southern blotting for the detection of HCV sequences in liver tissues. In this system, the detection rate of NS3 region sequences is higher than that of core region sequences. There is a statistically significant correlation between high titer anti-HCV antibodies in serum and NS3 region sequences in liver tissue. However, not all anti-HCV positive patients had HCV positive hepatitis. The reverse transcription-polymerase chain reaction for HCV sequences on liver tissue extracts may reveal valuable information on the diagnosis of HCV infection and the pathogenesis of chronic hepatitis C.

The polymerase chain reaction (PCR) is an extremely sensitive technique that has been used for detection of DNA sequences in formalin-fixed, paraffin-embedded tissues. In order to verify that hepatitis B virus (HBV) DNA sequences are adequately preserved in routinely processed liver tissues, we performed PCR with five different primer pairs for HBV sequences on DNA extracted by two different methods from paraffin and frozen liver sections. The amount of PCR products obtained with DNA templates extracted by the proteinase K-SDS method from frozen sections was significantly larger than that from paraffin sections. However, boiling of deparaffinized sections in water containing Chelex-100 resulted in ample amounts of PCR products irrespective of the primers used. On Southern blots, the location of the bands of amplified DNA obtained by the different methods was consistent with the predicted size, suggesting that the viral sequences had not been altered by processing. Although freezing of fresh tissue yields quantitatively more HBV DNA, formalin fixation qualitatively preserves the viral DNA sequences adequately for detection by PCR. Therefore, formalin-fixed, paraffin-embedded tissues may be used for the detection of viral DNA sequences by PCR. Application of the described procedure to routinely processed tissues significantly broadens the applicability of this powerful diagnostic and investigative method.

Bacteriophage of Lactococcus lactis ssp. lactis and ssp. cremoris, isolated in the United States and Argentina, were compared with respect to host range, adsorption, latent period, burst size and immunological cross-reactivity. Only 1 out of 13 U.S. culture isolates was sensitive to Argentinian phage. Argentinian L. lactis ssp. lactis C2 mutants were resistant to 13 U.S. phage isolates (4 prolate and 9 isometric). While Argentinian phage Stl-3 multiplied on U.S. culture isolate 59-1, low adsorption (38%) and insignificant burst size and latent period data were evident. Antisera prepared against U.S. phage D59-1 (prolate) and F4-1 (isometric) neutralized the lytic activities of all Argentinian prolate phage although the F4-1 antiserum was less effective. The data suggest homology especially between U.S. phage D59-1 and the Argentinian phage.