Establishment of a sequence-based typing system for BoLA-DQA1 exon 2

In cattle, bovine leukocyte antigens (BoLAs) have been extensively used as markers for bovine diseases and immunological traits. Here, we developed a rapid, high-resolution sequence-based typing (SBT) system for BoLA-DQA1. We amplified 355 bp of BoLA-DQA1 by fully nested polymerase chain reaction (PCR) using the newly constructed primers and then performed direct sequencing of each product. Using this method, we investigated the locus in 51 animals whose BoLA haplotypes had been characterized at the Fifth International BoLA Workshop. We identified 15 distinct DQA1 alleles, and there is no conflict between the typing result of PCR-SBT and restriction fragment length polymorphism analysis. Together with the previously developed method for typing BoLA-DRB3, the PCR-SBT for BoLA-DQA1 clearly provides a useful tool for detailed class IIa haplotype analysis.     

Vaccination of mice with MUC1 cDNA suppresses the development of lung metastases

C57BL/6 mice were immunized intradermally with various doses of purified pCEP4 plasmid DNA containing full-length MUC1 cDNA (22 tandem repeats). Mice immunized with MUC1 DNA three times at weekly intervals had serum antibodies to a synthetic peptide corresponding to the tandem repeats of MUC1. The antibody titer correlated with the plasmid DNA dose. After the third immunization mice were injected intravenously with 5×10⁵ B16-F10 melanoma cells that had been stably transfected with MUC1 cDNA (F10-MUC1-C8 clone cells). The number of lung metastatic nodules three weeks after inoculation of F10-MUC1-C8 cells was significantly lower in mice immunized with MUC1 plasmid DNA than in mice immunized with the vector DNA alone. Thus, the suppression of lung metastasis was antigen-specific. In vivo depletion of lymphocyte subpopulations by specific antibodies revealed that natural killer cells are the major effector cells responsible for the suppression of lung metastasis. CD4⁺ cells and CD8⁺ cells apparently played some roles too. This revised version was published online in July 2006 with corrections to the Cover Date.     

Metallo-beta-lactamase VIM-2 in clinical isolates of Pseudomonas aeruginosa from Portugal

Resistance to carbapenems is emerging, and it is a great problem to therapeutics. Three isolates of Pseudomonas aeruginosa from a Portuguese hospital identified in urine and sputum, in 1995, presented a high-level resistance to imipenem (> 32 mg/L). Afterward, one isolate of P. aeruginosa recovered from urine of an ambulatory patient in 1998 showed high resistance to imipenem and meropenem. The resistance to carbapenems in these strains was associated with the production of a class B beta-lactamase, as was demonstrated by imipenem hydrolysis and inhibition by EDTA. Using primers described for bla(IMP) and bla(VIM), the amplification of the latter was observed in all isolates and a VIM-2 metallo-enzyme was identified. The pulsed-field gel electrophoresis (PFGE) patterns of these isolates were indistinguishable, suggesting dissemination to the community of this VIM-2 producer.     

Prevalence of genetic markers for thrombophilia in recurrent pregnancy loss

BACKGROUND: The genetic predispositions to venous thrombosis such as factor V Leiden (FVL) mutation (Arg 506 Gln), prothrombin (FII) gene mutation (G20210A), and mutation of the methylenetetrahydrofolate reductase (MTHFR) gene (C677T) have been reported to be associated with recurrent pregnancy loss. This paper examines the prevalence of markers for genetic thrombophilias in women with recurrent miscarriage. METHODS: The prevalence of FVL, FII G20210A and MTHFR C677T was compared in 108 women with three or more pregnancy losses either exclusively in the first trimester, or mixed first and second trimester losses, with the prevalence found in 82 fertile parous control women without miscarriages. Markers for the thrombophilias were assessed by PCR analysis. RESULTS: Twenty-three of the 108 patients (21.3%), had thrombophilia markers, which was similar to the proportion of patients in the control group (20.7%) with these markers. The prevalences of FVL and FII G20210A were lower in the study group than in the control group (3.7 versus 6.1% for FVL and 4.6 versus 6.1% for FII respectively); however, the difference was not statistically significant. In contrast, the prevalence of MTHFR C677T was higher in the study group than the control population (13 versus 8.5% respectively), but this difference was not statistically significant. There was no statistically significant prevalence of any particular thrombophilia in patients with previous first and second trimester pregnancy losses compared with patients with first trimester losses alone. CONCLUSION: Thrombophilia was not found to be associated with recurrent pregnancy loss.     

Bronchiolar expression of aquaporin-3 (AQP3) in rat lung and its dynamics in pulmonary oedema

Aquaporins (AQPs) are water channel proteins that permit osmotically driven water movement. To determine their dynamics in pulmonary oedema, we examined the expression of mRNA and protein for AQP1, AQP3, AQP4, and AQP5 in the lungs of normal and thiourea-treated rats. In the thiourea group, lung water content increased significantly (vs. controls) with the peak at around 4 h. Semi-quantitative RT-PCR showed that AQP3 mRNA in the thiourea group rose significantly, peaking at around 4–8 h. The expression of AQP1, AQP4, AQP5, ENaC and CFTR mRNA each decreased significantly some time after the peak in lung water content. Immunoblot analysis showed that glycosylated AQP3 protein was increased 4–10 h after treatment. Expression of the other AQP proteins was not significantly altered, except for that of AQP4. Immunohistochemical examination revealed that AQP1 was expressed in endothelia, AQP3 in the basal cells of the large airways and in cuboidal cells in the bronchioles, AQP4 in the basolateral membrane of airway cells and AQP5 in type-I pneumocytes. Our results suggest that AQP3 is expressed not only in large airways, but also in bronchioles, and is related to water movement in pulmonary oedema.     

Linkage disequilibrium in young genetically isolated Dutch population

The design and feasibility of genetic studies of complex diseases are critically dependent on the extent and distribution of linkage disequilibrium (LD) across the genome and between different populations. We have examined genomewide and region-specific LD in a young genetically isolated population identified in the Netherlands by genotyping approximately 800 Short Tandem Repeat markers distributed genomewide across 58 individuals. Several regions were analyzed further using a denser marker map. The permutation-corrected measure of LD was used for analysis. A significant (P<0.0004) relation between LD and genetic distance on a genomewide scale was found. Distance explained 4% of the total LD variation. For fine-mapping data, distance accounted for a larger proportion of LD variation (up to 39%). A notable similarity in the genomewide distribution of LD was revealed between this population and other young genetically isolated populations from Micronesia and Costa Rica. Our study population and experiment was simulated in silico to confirm our knowledge of the history of the population. High agreement was observed between results of analysis of simulated and empirical data. We conclude that our population shows a high level of LD similar to that demonstrated previously in other young genetic isolates. In Europe, there may be a large number of young genetically isolated populations that are similar in history to ours. In these populations, a similar degree of LD is expected and thus they may be effectively used for linkage or LD mapping.     

Administration of yoghurt or lactobacillus casei to malnourished mice: Comparative effect on lymphoid cells and mucosal reconditioning of the intestine

The comparative effect of the administration of viable Lactobacillus casei and yoghurt on mucosal immunity, body weight and the recovery of intestinal microvilli was studied in malnourished mice. L. casei and yoghurt induced an increase in the number of cells producing IgA and IgM, yoghurt being more effective than L. casei. In both treatments, the number of CD4⁺ or CD8⁺ T‐lymphocytes did not reach the levels found in well‐nourished mice. However, yoghurt administration induced a slight increase in the number of CD8⁺cells and a significant increase in CD4⁺ helper T‐cells, compared with the malnourished control. Yoghurt was more effective than L. casei in improving the condition of intestinal microvilli and in the stimulation of mucus production. Yoghurt administration also induced an increase in body weight and avoided bacterial translocation of the normal intestinal microflora.     

Regulation of enhanced cerebrovascular expression of proinflammatory mediators in experimental subarachnoid hemorrhage via the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathway

Background

Subarachnoid hemorrhage (SAH) is associated with high morbidity and mortality. It is suggested that the associated inflammation is mediated through activation of the mitogen-activated protein kinase (MAPK) pathway which plays a crucial role in the pathogenesis of delayed cerebral ischemia after SAH. The aim of this study was first to investigate the timecourse of altered expression of proinflammatory cytokines and matrix metalloproteinase in the cerebral arteries walls following SAH. Secondly, we investigated whether administration of a specific mitogen-activated protein kinase kinase (MEK)1/2 inhibitor, U0126, given at 6 h after SAH prevents activation of the MEK/extracellular signal-regulated kinase 1/2 pathway and the upregulation of cerebrovascular inflammatory mediators and improves neurological function.

Methods

SAH was induced in rats by injection of 250 μl of autologous blood into basal cisterns. U0126 was given intracisternally using two treatment regimens: (A) treatments at 6, 12, 24 and 36 h after SAH and experiments terminated at 48 h after SAH, or (B) treatments at 6, 12, and 24 h after SAH and terminated at 72 h after SAH. Cerebral arteries were harvested and interleukin (IL)-6, IL-1β, tumor necrosis factor α (TNF)α, matrix metalloproteinase (MMP)-9 and phosphorylated ERK1/2 (pERK1/2) levels investigated by immunohistochemistry. Early activation of pERK1/2 was measured by western blot. Functional neurological outcome after SAH was also analyzed.

Results

Expression levels of IL-1β, IL-6, MMP-9 and pERK1/2 proteins were elevated over time with an early increase at around 6 h and a late peak at 48 to 72 h post-SAH in cerebral arteries. Enhanced expression of TNFα in cerebral arteries started at 24 h and increased until 96 h. In addition, SAH induced sensorimotor and spontaneous behavior deficits in the animals. Treatment with U0126 starting at 6 h after SAH prevented activation of MEK-ERK1/2 signaling. Further, U0126 significantly decreased the upregulation of inflammation proteins at 48 and 72 h following SAH and improved neurological function. We found no differences between treatment regimens A and B.

Conclusions

These results show that SAH induces early activation of the MEK-ERK1/2 pathway in cerebral artery walls, which is associated with upregulation of proinflammatory cytokines and MMP-9. Inhibition of the MEK-ERK1/2 pathway by U0126 starting at 6 h post-SAH prevented upregulation of cytokines and MMP-9 in cerebral vessels, and improved neurological outcome.

     

Molecular epidemiology of betanodaviruses isolated from sea bass and sea bream cultured along the Tunisian coasts

Viral nervous necrosis (VNN) is a serious viral disease affecting farmed sea bass (Dicentrarchus labrax). Only scarce molecular data are available on the disease-causing betanodavirus populations in Tunisia. Therefore, we carried out the first molecular survey of betanodaviruses in farmed sea bass and sea bream (Sparus aurata) along the Tunisian coasts. Among 81 samples from five farms, 20 tested positive with RT-PCR, not only in clinical cases but also in asymptomatic fish before and during outbreaks. Positive fish were found in all farms, except in one farm investigated in the south of Tunisia. Sequencing the fragments of both genomic components (RNA1 and RNA2) in 16 isolates revealed that the Tunisian viruses were related to the red-spotted grouper nervous necrosis virus (RGNNV) genotype. Furthermore, the newly sequenced isolates were generally highly related to one another suggesting a recent common ancestor. They also showed high identities with other isolates obtained from wild fishes in the Mediterranean, but were slightly more divergent from strains recently obtained from farmed fishes in the Mediterranean. The poor genetic diversity of the viral population along the Tunisian coasts is striking. One hypothesis is that it is the result of the maintenance of a homogenous genetic pool among infected wild fish, groupers for instance and subsequent dissemination to farmed fish over the seasons.