The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) causes resistance to nitrosoureas in various human cancers. In this study, we analyzed the correlation between canine lymphomas and MGMT in vitro. Two of five canine lymphoma cell lines required higher concentrations of lomustine to inhibit cell growth by 50%, but their sensitivity to the drug increased when they were cultured with an MGMT inhibitor. Fluorometric oligonucleotide assay and real-time polymerase chain reaction of these cell lines revealed MGMT activity and high MGMT mRNA expression, respectively. We analyzed the methylation status of the CpG islands of the canine MGMT gene by the bisulfite-sequencing method. Unlike human cells, the canine lymphoma cell lines did not show significant correlation between methylation status and MGMT suppression levels. Our results suggest that in canine lymphoma MGMT activity may influence sensitivity to nitrosoureas; thus, inhibition of MGMT activity would benefit nitrosourea-resistant patients. Additional studies are necessary to elucidate the mechanism of regulation of MGMT expression.

OBJECTIVE To evaluate the effects of dietary nonphytate phosphorus (NPP) content on ileal lymphocyte subpopulations and cytokine expression in the cecal tonsils and spleen of hens that were or were not inoculated with Salmonella Typhimurium. ANIMALS 64 Salmonella-free hens. PROCEDURES Hens were fed a diet with 0.22% (control; n = 32) or 0.42% (high-P; 32) NPP for 6 weeks and then orally inoculated with S Typhimurium (5 × 107 CFUs) or PBSS. Tissues were obtained from 8 S Typhimurium–inoculated and 8 PBSS-inoculated hens from each group at 2 and 7 days postinoculation (DPI). Percentages of ileal CD4+ and CD8+ lymphocytes were determined by flow cytometry. Cytokine mRNA expression was determined by quantitative real-time PCR assays. RESULTS For S Typhimurium–inoculated hens, plasma parathyroid hormone concentration was significantly increased and 1,25-dihydroxyvitamin D3 concentration was decreased in hens fed the high-P diet, compared with values in hens fed the control diet. Salmonella Typhimurium inoculation caused an increase in the percentage of ileal CD8+ lymphocytes and the expression of interleukin (IL)-1β, IL-6, IL-8, interferon-γ, IL-12, and IL-18 in the cecal tonsils and spleen and a decrease in the expression of IL-4 and IL-10 in the cecal tonsils. Hens fed the high-P diet had significantly increased splenic expression of interferon-γ at 2 DPI and IL-1β, IL-6, IL-12, and IL-18 at 7 DPI, compared with hens fed the control diet. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested there was a T-helper 1 cytokine reaction in the cecal tonsils and spleen of S Typhimurium–inoculated hens, and dietary NPP content altered calcium regulation hormone concentrations and affected splenic cytokine expression.

Brucella melitensis is the Brucella species most frequently associated with brucellosis in humans. It is also the causative agent of the disease in goats and other ruminants. Although significant aspects of the pathogenesis of infection by this intracellular pathogen have been clarified, several events during invasion of host cells remain to be elucidated. In this study, infections of human macrophages from the THP-1 monocyte cell line were conducted with B. melitensis Bm133 wild-type strain and a strain of Salmonella serovar Enteritidis as a control. A multiplicity of infection of 100 was used in trials focused on defining the relative expression of syntaxin 4 (STX4), a soluble N-ethylmaleimide-sensitive factor attachment protein receptor, in the early events of phagocytosis (at 15, 30, 45, and 60 min). Immunoblot assays were also done to visualize expression of the protein in cells infected with either bacterial strain. The expression of STX4 was not significantly different in cells infected with B. melitensis strain Bm133 compared to that observed in cells infected with S. Enteritidis. When the expression of STX4 mRNA was inhibited with short or small interfering, or silencing, RNA in the THP-1 cells, the survival of B. melitensis was significantly reduced at time 0, when gentamicin treatment of cultures was begun (after 1 h of phagocytosis), and also at 2 h and 12 h after infection.