CAT25 defines microsatellite instability in colorectal cancer by high-resolution melting PCR.

BACKGROUND: CAT25 (T25mononucleotide repeat of the Caspase 2 gene), is a promising DNA marker for detecting microsatellite instability (MSI) in colorectal cancer. CAT25 has the potential to be incorporated into the Bethesda panel, a commonly used panel of DNA microsatellites, or replace it in its entirety. We aimed to develop and validate a high-resolution melting-PCR (HRM-PCR) method for CAT25 instability detection in clinical samples. METHODS: The instability of CAT25, BAT25 (a poly(A) tract occurring in c-kit) and BAT26 (a poly(A) tract localized in hMSH2) microsatellites were assessed in DNA from tumour and peripheral blood obtained from 110 patients with colorectal cancer using HRM-PCR and capillary electrophoresis. Immunohistochemistry (IHC) staining for MSH2, MSH6, MLH1, and PMS2 enzymes was performed on tumours with jigj MSI. Allelic size variation of CAT25 was analysed on peripheral blood DNA from 208 healthy volunteers. RESULTS: The HRM-PCR for CAT25 was validated in clinical samples. CAT25 showed a tight range of 64-66 base pairs. Of 110 tumours, 11 had High MSI, later confirmed by IHC. CAT25 defines MSI alone as well as when used together with BAT25 and BAT26. CAT25 results provided 100% predictive values and p < 0.0001 to classify a tumour as having high MSI. CONCLUSIONS: We developed and validated a new HRM-PCR assay to detect CAT25 instability. Our findings showed a limited allelic size variation of CAT25 and highlighted to CAT25 as a promising marker for MSI analysis.

Differentiation of distinct long-lived memory CD4 T cells in intestinal tissues after oral Listeria monocytogenes infection.

Mucosal antigen-specific CD4 T-cell responses to intestinal pathogens remain incompletely understood. Here we examined the CD4 T-cell response after oral infection with an internalin A 'murinized' Listeria monocytogenes (Lm). Oral Lm infection induced a robust endogenous listeriolysin O (LLO)-specific CD4 T-cell response with distinct phenotypic and functional characteristics in the intestine. Circulating LLO-specific CD4 T cells transiently expressed the 'gut-homing' integrin α4ß7 and accumulated in the intestinal lamina propria and epithelium where they were maintained independent of interleukin (IL)-15. The majority of intestinal LLO-specific CD4 T cells were CD27- Ly6C- and CD69+ CD103- while the lymphoid LLO-specific CD4 T cells were heterogeneous based on CD27 and Ly6C expression and predominately CD69-. LLO-specific effector CD4 T cells transitioned into a long-lived memory population that phenotypically resembled their parent effectors and displayed hallmarks of residency. In addition, intestinal effector and memory CD4 T cells showed a predominant polyfunctional Th1 profile producing IFNγ, TNFα, and IL-2 at high levels with minimal but detectable levels of IL-17A. Depletion of CD4 T cells in immunized mice led to elevated bacterial burden after challenge infection highlighting a critical role for memory CD4 T cells in controlling intestinal intracellular pathogens.

Use of Trichomonas vaginalis clinical isolates to evaluate correlation of gene expression and metronidazole resistance.

We investigated whether variations in gene expression of enzymes associated with anaerobic resistance of laboratory-derived strains of Trichomonas vaginalis could be detected in a group of 28 clinical isolates with variations in metronidazole sensitivity. We compared isolates by real-time PCR because this method allows for highly sensitive quantification of mRNA and for evaluation of several genes simultaneously. We found that PFOR gene A mRNA levels were highly correlated with PFOR gene B levels, as well as the D subunit of malic enzyme and ferrodoxin. Ferrodoxin mRNA expression was also significantly correlated with that of malic enzyme and hydrogenase. However, when we evaluated relationships between these enzymes and resistance to metronidazole, we found no significant correlations between aerobic or anaerobic in vitro sensitivity to drug and mRNA levels of any of the enzymes tested. Similarly, using a Student's t-test, no significant differences in enzyme mRNA levels were observed between isolates separated by metronidazole resistance or susceptibility. The lack of correlation between gene expression and resistance or susceptibility could be the result of differences in expression at the protein level or because other biochemical pathways or genes are involved in the resistance observed in clinical settings.

Effects of overexpression of HBP1 upon growth and differentiation of leukemic myeloid cells.

HMG-box containing protein 1 (HBP1) is a member of the high mobility group (HMG) of chromosomal proteins. Since HBP1 exhibits tumor-suppressor activity in nonmyeloid tissues, we examined the effects of ectopic overexpression of HBP1 upon the growth and differentiation of myeloid cells. We prepared transient and stable transfectants of the myeloblast cell line K562, which overexpress HBP1 mRNA and protein. HBP1 transfectants displayed slower growth in cell culture and reduced colony formation in soft agar, retardation of S-phase progression, reduced expression of cyclin D1 and D3 mRNAs and increased expression of p21 mRNA. HBP1 transfectants also underwent increased apoptosis, as demonstrated by morphology and binding of Annexin V. Fas ligand mRNA levels were increased in HBP1 transfectants, suggesting involvement of the Fas/Fas ligand pathway. HBP1 overexpression enhanced differentiation of K562 cells towards erythroid and megakaryocyte lineages, as evidenced by increased hemoglobin and CD41a expression. Overexpression of HBP1 modulated mRNA levels for myeloid-specific transcription factors C/EBPalpha, c-Myb, c-Myc, and JunB, as well as lineage-specific transcription factors PU.1, GATA-1, and RUNX1. These findings suggest that in myeloid cells HBP1 may serve as a tumor suppressor and a general differentiation inducer and may synergize with chemical differentiating agents to enhance lineage-specific differentiation.

Seroprevalence of human herpesvirus 6 in Andino Puneños (Argentina).

We carried out a seroepidemiological survey to define the prevalence of human herpesvirus 6 (HHV6) infection in an aboriginal population (Andino Puneños) from a remote region in north-west Argentina. Antibodies against HHV6 (total IgG and the 4 subclasses of IgG) were studied in 84 serum samples (collected in 1995 and stored at -70 degrees C), using core blood mononuclear cells infected with HHV6 in an immunofluorescence assay. Of the 84 samples, 70 (83%; 95% confidence interval, 75-91%) exhibited IgG antibodies against HHV6. No significant differences in the frequency of humoral immunity were found among the 4 age-groups studied (mean ages 13, 31, 47 and 70 years) namely, 75%, 89.7%, 79.2% and 100%, respectively. HHV6-specific IgG1 was found in all the positive serum samples tested but none of them contained specific IgG2, IgG3 and IgG4. These results confirmed a high rate of infection with HHV6 within this aboriginal group in Argentina and an IgG1 anti-HHV6 activity compatible with a maintenance of immunity.