Abstract: Cytogenetic analysis still remains the gold standard method for prenatal diagnosis of trisomy 21 (Down syndrome, DS). Nevertheless, the conventional cytogenetic analysis needs live cultured cells and is too time-consuming for clinical application. In contrast, molecular methods such as FISH, QF-PCR, MLPA and quantitative Real-time PCR are rapid assays with results available in 24h. In the present study, we have successfully used a novel MGB TaqMan probe-based real time PCR assay for rapid diagnosis of trisomy 21 status in Down syndrome samples. We have also compared the results of this molecular method with corresponding results obtained by the cytogenetic analysis. Blood samples obtained from DS patients (n=25) and normal controls (n=20) were tested by quantitative Real-time PCR in parallel to standard G-banding analysis. Genomic DNA was extracted from peripheral blood lymphocytes. A high precision TaqMan probe quantitative Real-time PCR assay was developed to determine the gene dosage of DSCAM (target gene on 21q22.2) relative to PMP22 (reference gene on 17p11.2). The DSCAM/PMP22 ratio was calculated according to the formula; ratio=2 -ΔΔCT. The quantitative Real-time PCR was able to distinguish between trisomy 21 samples and normal controls with the gene ratios of 1.49±0.13 and 1.03±0.04 respectively (p value
Abstract: In a 10-week (May – August, 2008) Phase I trial, 840, 1+ rainbow trout, Oncorhynchus mykiss, received a commercial oral immunomodulator, Fin Immune™, at four different dosages (0, 10, 20 and 30 mg g-1) to evaluate immune response and growth. The overall objective of was to determine an optimal dosage of this product for rainbow trout that provides enhanced immunity with maximal growth and health. Biweekly blood samples were taken from 10 randomly selected fish in each tank (30 samples per treatment) to evaluate the duration of enhanced immunity conferred by Fin-Immune™. The immunological assessment included serum white blood cell (lymphocyte, neutrophil) densities and blood hematocrit (packed cell volume %). Of these three variables, only lymphocyte density increased significantly among trout fed Fin- Immune™ at 20 and 30 mg g-1 which peaked at week 6. At week 7, all trout were switched to regular feed (lacking Fin-Immune™) and by week 10, lymphocyte levels decreased among all levels but were still greater than at week 0. There was growth impairment at the highest dose of Fin-Immune™ tested (30 mg g-1) which can be associated with a physiological compensatory mechanism due to a dose-specific threshold level. Thus, our main objective of this Phase I study was achieved, the 20 mg g-1 dose of Fin-Immune™ should be the most efficacious (of those we tested) to use for a Phase II disease challenge trial.
Abstract: Oxidative stress is considered to be the cause for onset
and the progression of type 2 diabetes mellitus (T2DM) and
complications including neuropathy. It is a deleterious process that
can be an important mediator of damage to cell structures: protein,
lipids and DNA. Data suggest that in patients with diabetes and
diabetic neuropathy DNA repair is impaired, which prevents effective
removal of lesions. Objective: The aim of our study was to evaluate
the association of the hOGG1 (326 Ser/Cys) and XRCC1 (194
Arg/Trp, 399 Arg/Gln) gene polymorphisms whose protein is
involved in the BER pathway with DNA repair efficiency in patients
with diabetes type 2 and diabetic neuropathy compared to the healthy
subjects. Genotypes were determined by PCR-RFLP analysis in 385
subjects, including 117 with type 2 diabetes, 56 with diabetic
neuropathy and 212 with normal glucose metabolism. The
polymorphisms studied include codon 326 of hOGG1 and 194, 399
of XRCC1 in the base excision repair (BER) genes. Comet assay was
carried out using peripheral blood lymphocytes from the patients and
controls. This test enabled the evaluation of DNA damage in cells
exposed to hydrogen peroxide alone and in the combination with the
endonuclease III (Nth). The results of the analysis of polymorphism
were statistically examination by calculating the odds ratio (OR) and
their 95% confidence intervals (95% CI) using the ¤ç2-tests. Our data
indicate that patients with diabetes mellitus type 2 (including those
with neuropathy) had higher frequencies of the XRCC1 399Arg/Gln
polymorphism in homozygote (GG) (OR: 1.85 [95% CI: 1.07-3.22],
P=0.3) and also increased frequency of 399Gln (G) allele (OR: 1.38
[95% CI: 1.03-1.83], P=0.3). No relation to other polymorphisms
with increased risk of diabetes or diabetic neuropathy. In T2DM
patients complicated by neuropathy, there was less efficient repair of
oxidative DNA damage induced by hydrogen peroxide in both the
presence and absence of the Nth enzyme. The results of our study
suggest that the XRCC1 399 Arg/Gln polymorphism is a significant
risk factor of T2DM in Polish population. Obtained data suggest a
decreased efficiency of DNA repair in cells from patients with
diabetes and neuropathy may be associated with oxidative stress.
Additionally, patients with neuropathy are characterized by even
greater sensitivity to oxidative damage than patients with diabetes,
which suggests participation of free radicals in the pathogenesis of
neuropathy.
Abstract: The aim of this work was to study the in vitro effects
of δ-lactam 1 and its 4-chlorophenyl derivative 2, on the proliferative
responses of human lymphocytes and Th1 and Th2 cytokine
secretion. The possible protective role of vitamin E on intracellular
stress oxidative induced by these compounds was also investigated.
Peripheral blood lymphocytes were isolated using differential
centrifugation on a density gradient of Histopaque. They were
cultured with mitogen concanavalin A, vitamin E (10 μM) and with
different concentrations of the compounds 1 and 2 (0.1 to 10 μM).
Proliferation (MTT assay), IL-2, INFγ and IL-4 (Elisa kits),
intracellular superoxide anion were determined. 1 and 2 were
immunostimulant and increased cytokine secretion with a shift away
from Th1 response to Th2. These properties were however
accompanied by an increase in intracellular oxidative stress. The
presence of vitamin E exhibited protective effects by reducing δ-
lactam-induced superoxide anion generation in lymphocytes.