Lack of crucial role of mast cells in pathogenesis of experimental colitis in mice

Mast cell alterations have been implicated in the pathogenesis of chronic ulcerative colitis (UC). We studied the effect of mast cell deficiency on the severity of inflammation in a murine model of colitis. Colitis was induced in mice using dextran sodium sulfate (DSS). Mast-cell-deficient mice (WBB6F₁/J-W/W^v;N=17) and normal littermates (WBB6F₁/J-+/+;N=17) were administered DSS 4% w/v for seven days, then water alone for one week, followed by 5% DSS for six days. Animals were sacrificed at the end of the protocol. Segments of proximal, mid-, and distal colon of each animal were processed for histopathological examination. Mortality and morbidity (diarrhea and weight loss) for each group were assessed. There was no significant difference between the two groups in either their clinical parameters (mortality and morbidity) or the severity of colitis as graded histopathologically. Our findings suggest that mast cells are not crucial for the development of DSS-induced colitis.     

The -159C/T polymorphism in the promoter region of the CD14 gene is associated with advanced liver disease and higher serum levels of acute-phase proteins in heavy drinkers

BACKGROUND: Innate inflammatory responses to endotoxin (lipopolysaccharide) contribute to the development of alcoholic liver disease (ALD). A single-nucleotide polymorphism (-159C/T) in the promoter region of the gene coding for CD14 (a lipopolysaccharide receptor) could be associated with the development of ALD. We sought too investigate the relationship between the CD14/-159C/T polymorphism and advanced ALD and acute-phase protein levels in heavy drinkers. METHODS: A total of 138 heavy drinkers consecutively admitted to an Internal Medicine department were genotyped for the CD14/-159C/T polymorphism. Serum samples were analyzed for lipopolysaccharide-binding protein (LBP), soluble CD14 (sCD14), C-reactive protein (CRP), and immunoglobulin (Ig) A, IgG, and IgM. Patients with ascites or liver encephalopathy (n = 35) were classified as having advanced ALD. RESULTS: After adjusting for potential confounding variables, the CD14/-159TT genotype was positively associated with advanced ALD (odds ratio, 2.99; 95% confidence interval, 1.09-8.24, p = 0.03) and serum LBP (p = 0.01) and sCD14 (p = 0.04) levels. The CD14/-159C/T polymorphism was not associated with serum levels of CRP, IgA, IgG, or IgM. CONCLUSIONS: Our results support the notion that CD14/-159TT homozygous heavy drinkers have higher levels of the LPS-binding acute-phase proteins (LBP and sCD14) than do carriers of the CD14/-159C allele. Also, the CD14/-159TT genotype may be a risk factor for advanced ALD.     

Serum TNF-alpha levels in relation to alcohol consumption and common TNF gene polymorphisms

Tumor necrosis factor-alpha (TNF-alpha) mediates alcohol-induced organ dysfunction, including alcoholic hepatitis. Variations in the TNF-alpha gene may underlie the individual predisposition to alcoholic liver disease. Measurement of serum TNF-alpha levels has become a routine in clinical practice. The study was aimed at investigating the level of serum TNF-alpha levels in adults and analyzing its relationship with different levels of alcohol consumption, as well as the potential interaction between alcohol consumption and common TNF-alpha gene polymorphisms in relation to TNF-alpha levels and liver disease. Serum TNF-alpha was measured in a random sample of 459 individuals from a general adult population and in 137 hospital-admitted alcoholics. Three common TNF-alpha gene polymorphisms (-238G>A, -308G>A, and -857C>T) were investigated in 419 of these individuals. In the general adult population, the TNF-alpha levels were similar in alcohol abstainers and alcohol drinkers. Alcoholics admitted to the hospital showed the highest TNF-alpha levels, which were correlated with liver dysfunction. We found no evidence of an interaction between alcohol consumption and TNF-alpha gene polymorphisms in relation to TNF-alpha levels. Carriers of the TNF -238A allele tended to have a higher prevalence of advanced liver disease than -238G homozygotes, confirming previous reports. In conclusion, light-to-moderate drinking had no significant effect on the levels of serum TNF-alpha levels. Serum TNF-alpha levels are elevated in alcoholics independently of common TNF gene polymorphisms.     

Reliability of a dietary questionnaire on food habits,eating behaviour and nutritional knowledge of adolescents

OBJECTIVE: To develop a dietary questionnaire on food habits, eating behaviour and nutrition knowledge of adolescents and to examine its reliability. DESIGN: A cross-sectional baseline survey. The questionnaire was self-administered to study participants twice with 7 days between each administration. SETTING: A school community in Pavia, Italy. SUBJECTS: A group of students (n=72, aged 14-17 y, both sexes) studying in a secondary school in the second year of the course were invited to compile a dietary questionnaire during school time. Informed written consent was obtained from each subject and their parents. Subjects were initially recruited for a nutrition intervention; recruitment was opportunistic and school based. STATISTICAL ANALYSES: Reliability was assessed using the Cronbach's alpha and the Pearson correlation coefficients. RESULTS: Cronbach's alpha ranges from a minimum of 0.55 to a maximum of 0.75, indicating that only two sections have a poor internal consistency. The Pearson correlation coefficients range from a minimum of 0.78 to a maximum of 0.88, indicating a very good temporal stability of the questionnaire. All the Pearson correlation coefficients are statistically significant with P<0.01. CONCLUSIONS: The present questionnaire has the potential to measure the effects of nutrition interventions on adolescents because of its stability in making comparisons over time. The instruments is low in cost and easy to administer and analyse; moreover, it could be modified appropriately to fit the needs of other populations as well.     

A simple and robust quantitative PCR assay to determine CYP21A2 gene dose in the diagnosis of 21-hydroxylase deficiency

BACKGROUND: Correct diagnosis of 21-hydroxylase deficiency (21OHD) requires the identification of CYP21A2 gene deletions and CYP21A1P/CYP21A2 chimeric genes, which are disease-causing alleles, and gene duplications, which can lead to false-positive 21OHD allele results. Because lack of suitable CYP21A2 dosage assessment methods hampers correct 21OHD diagnosis, we developed a new assay based on the relative quantification of the CYP21A2 gene using the DSP gene as a reference. METHODS: The assay to determine CYP21A2 copy number is based on real-time PCR. The method also detects the presence of the CYP21A1P/CYP21A2 chimeric gene. We used a duplex PCR to coamplify the DSP gene, included as an internal control, along with CYP21A2. The difference in threshold cycles between CYP21A2 and DSP genes (DeltaCt) was used to assess CYP21A2 copy number. RESULTS: The DeltaCt values obtained from 24 samples used to set up the method clearly differentiated 3 nonoverlapping intervals, which corresponded to the number of CYP21A2 copies: -1.35 to -0.25 defined 2 gene copies, +0.20 to +2.00 defined 1 copy, and -2.50 to -1.50 defined 3 copies. With these intervals we were able to assess the gene copy number in 24 additional samples. CONCLUSIONS: This new method for gene copy assessment detects homozygous and heterozygous CYP21A2 gene deletions, CYP21A1P/CYP21A2 chimeric genes, and gene duplications. Moreover, the method is robust, fast, and easy to use in a molecular diagnosis laboratory. This method together with CYP21A2 gene sequencing can provide a definitive system for the detection of almost all, common as well as rare, 21OHD alleles.     

Effect of treatment, during primary infection, on establishment and clearance of cellular reservoirs of HIV-1

Patients in whom virologic suppression is achieved with highly active antiretroviral therapy (HAART) retain long-lived cellular reservoirs of human immunodeficiency virus type 1 (HIV-1); this retention is an obstacle to sustained control of infection. To assess the impact that initiating treatment during primary HIV-1 infection has on this cell population, we analyzed the decay kinetics of HIV-1 DNA and of infectivity associated with cells activated ex vivo in 27 patients who initiated therapy before or <6 months after seroconversion and in whom viremia was suppressed to <50 copies/mL. The clearance rates of cellular reservoirs could not be distinguished by these techniques (median half-life, 20 weeks) during the first year of HAART. The clearance of HIV-1 DNA slowed significantly during the subsequent 3 years of treatment (median half-life, 70 weeks), consistent with heterogeneous cellular reservoirs being present. Total cell-associated infectivity (CAI) after 1 year of treatment was undetectable (<0.07 infectious units/million cells [IUPM]) in most patients initiating treatment during primary infection either before (9/9) or <6 months after (6/8) seroconversion. In contrast, all 17 control patients who initiated HAART during chronic infection retained detectable CAI after 3-6 years of treatment (median reservoir size, 1.1 IUPM; P<.0005). These results suggest that treatment <6 months after seroconversion may facilitate long-term control of cellular reservoirs that maintain HIV-1 infection during treatment.