cDNA probes for the diagnosis of bovine torovirus (Breda virus) infection.

A genomic cDNA library of RNA from Breda virus (BRV), a bovine torovirus, was prepared. The nucleotide sequence of the 3' end of the genome was found to be highly conserved (93% identical) between BRV and Berne virus, the torovirus prototype. Cross-hybridization experiments were performed to select Berne virus cDNA clones for use as probes in a dot hybridization assay; the objective was to detect heterologous torovirus RNA in fecal material. A rapid RNA extraction method was employed to make the test applicable for routine diagnosis. Samples from calves after experimental and natural infection with BRV were assayed to establish the sensitivity and specificity of the test and to compare the test with the enzyme-linked immunosorbent assay (ELISA) for antigen detection. For this purpose, 53 samples from seven infected calves were tested with both methods. In the ELISA, BRV was detected in six fecal samples from three inoculated calves. By use of the hybridization test, 16 samples from seven calves reacted positively. With one exception, only postinoculation samples were found positive in hybridization. No signal was seen in feces from uninoculated calves or from calves infected with rotavirus or coronavirus.     

Risk groups for clinical complications of norovirus infections: an outbreak investigation

Norovirus infections have been described as self-limiting diseases of short duration. An investigation of a norovirus outbreak in a university hospital provided evidence for severe clinical features in patients with several underlying diseases. Clinical outcomes of norovirus infection were defined. Risk-factor analysis targeting underlying diseases and medication was performed using multivariate analyses. In five outbreak wards, 84 patients and 60 nurses were infected (an overall attack rate of 32% in patients, and 76% in nurses). The causative agent was the new variant Grimsby virus. Severe clinical features, including acute renal failure, arrhythmia and signs of acute graft organ rejection in renal transplant patients, were observed in seven (8.3%) patients. In multivariate analyses, cardiovascular disease (OR 17.1, 95% CI 2.17-403) and renal transplant (OR 13.0, 95% CI 1.63-281) were risk-factors for a potassium decrease of >20%. Age >65 years (OR 11.6, 95% CI 1.89-224) was a risk-factor for diarrhoea lasting >2 days. Immunosuppression (OR 5.7, 95% CI 1.78-20.1) was a risk-factor for a creatinine increase of >10%. Norovirus infections in patients with underlying conditions such as cardiovascular disease, renal transplant and immunosuppressive therapy may lead to severe consequences typified by decreased potassium levels, increased levels of C-reactive protein and creatine phosphokinase. In the elderly, norovirus infection may lead to an increased duration of diarrhoea. Therefore patients at risk should be hospitalised early and monitored frequently. Strict preventional measures should be implemented as early as possible to minimise the risk of nosocomial outbreaks.     

The decrease in nonsplenic interleukin-6 (IL-6) production after splenectomy indicates the existence of a positive feedback loop of IL-6 production during endotoxemia in dogs.

The spleen is involved in endotoxin-induced interleukin-6 (IL-6) production. To quantitate the relative contribution of the spleen to endotoxin-induced IL-6 production, we studied the effect of endotoxin (1.0 microg/kg of body weight) in control dogs (n = 7) and splenectomized dogs (n = 7). Blood for analysis of tumor necrosis factor (TNF) and IL-6 was sampled from the femoral artery and the portal, hepatic, and splenic (only in controls) veins. Arterial plasma endotoxin and cortisol levels were also measured. Whole-body IL-6 production was calculated by a deconvolution technique. Splenic IL-6 production in control dogs was measured from splenic blood flow and arteriovenous concentration differences. Endotoxin levels were higher in splenectomized dogs (P < 0.05) because of a decreased distribution volume (P < 0.05) and decreased clearance of endotoxin (P < 0.05). Endotoxin-induced plasma IL-6 levels were decreased by approximately 75% in splenectomized dogs (P < 0.01), and whole-body IL-6 production rates were severalfold lower (median of 8.7 mg/4 h and range of 3.9 to 11.4 mg/4 h versus a median of 32.3 mg/4 h and a range of 22.7 to 70.2 mg/4 h) (P < 0.05). However, in control dogs splenic IL-6 production (0.6 +/- 0.2 mg/4 h) was only approximately 2% of whole-body IL-6 production. Plasma TNF levels increased in both groups (P < 0.01) but were not different between the groups. Plasma cortisol levels were slightly higher in splenectomized dogs than in control dogs (P < 0.05). In conclusion, splenectomy decreases the distribution volume and clearance rate of endotoxin. Splenectomy results in decreased endotoxin-induced IL-6 production, which is caused not by the absence of splenic IL-6 production, but by a decrease in nonsplenic IL-6 production. Therefore, the spleen is an important mediator in the complete activation of nonsplenic IL-6 production by endotoxin.     

Rapid detection of Candida albicans in clinical blood samples by using a TaqMan-based PCR assay

We describe a rapid and reproducible PCR assay for quantitation of the Candida albicans ribosomal DNA (rDNA) in clinical blood samples based on the TaqMan principle (Applied Biosystems), in which a signal is generated by cleavage of a template-specific probe during amplification. We used two fluorogenic probes based on universal, fungus-specific primers, one for the detection of C. albicans species DNA and one for the detection of all Candida genus DNA. C. albicans blastoconidia mixed with whole blood in a titration experiment yielded a linear PCR signal over a range of 3 orders of magnitude. The TaqMan-based PCR assay for C. albicans exhibited a low limit of detection (5 CFU/ml of blood) and an excellent reproducibility (96 to 99%). While the C. albicans species-specific probe had 100% specificity for C. albicans, all Candida genus-specific probes cross-reacted with other organisms likely to coinfect patients with C. albicans infections. On the basis of these data, we determined the C. albicans loads with a species-specific probe from 122 blood samples from 61 hematology or oncology patients with clinically proven or suspected systemic Candida infections. Eleven positive samples exhibited a wide range of C. albicans loads, extending from 5 to 100,475 CFU/ml of blood. The sensitivity and specificity of the present assay were 100 and 97%, respectively, compared with the results of blood culture. These data indicate that the TaqMan-based PCR assay for quantitation of C. albicans with a species-specific probe provides an attractive alternative for the identification and quantitation of C. albicans rDNA in pure cultures and blood samples.     

Detection of serum antibodies to bovine norovirus in veterinarians and the general population in the Netherlands

The close genetic relationship of human and animal strains of norovirus has raised the possibility of transmission of noroviruses from animals to humans and may explain the emergence of certain norovirus strains. To assess if exposure to bovine noroviruses (NoV) might result in infection in humans, an enzyme immunoassay (EIA) was designed and validated in order to detect antibodies against bovine norovirus. This and two other EIAs were used to test sera from 210 veterinarians and 630 matched population controls for IgG and IgA antibodies to recombinant capsid protein of bovine NoV (rBoV), Norwalk virus (rNV), and Lordsdale virus (rLDV). Of 840 participants, IgG reactivity to rBoV was found in 185 (22%), to rNV in 638 (76%) and to rLDV in 760 (90%). IgG reactivity to rBoV was more common in veterinarians (58/210: 28%) than in controls (127/630: 20% [P = 0.03]). IgA reactivity to rBoV was similar in both veterinarians and controls. Cross-reactivity of IgA and IgG antibodies to rBoV and rNV was seen, but 26% of all specimens positive rBoV antibodies showed high IgG reactivity to rBoV but low reactivity to rNV, suggesting a specific response to bovine antigen. No evidence of overall cross-reactivity of antibodies to rBoV and rLDV was seen. Among veterinarians, youth spent on farm (Odds Ratio [OR] = 1.8) and membership of the bovine practitioners' society (OR = 2.7) were significantly associated with IgG seroreactivity to rBoV. These data indicate that bovine strains of NoV may infect humans though less frequently than human strains.     

Minor histocompatibility antigen DDX3Y induces HLA-DQ5-restricted T cell responses with limited TCR-Vbeta usage both in vivo and in vitro.

In vitro stimulation of human female T cells with male HLA-identical dendritic cells resulted in the generation of HLA-DQB1*0501/0502-restricted minor histocompatibility H-Y antigen-specific CD4(+) T cell clones. Two clones generated from different HLA-identical pairs were analyzed. Use of HLA-DQ5-expressing female Epstein-Barr virus transformed B lymphoblastoid cell lines transfected with various H-Y genes and loaded with overlapping peptides demonstrated that both T cell clones are specific for a peptide encoded by DDX3Y. Previously, an HLA-DQ5-restricted T cell clone specific for the same peptide was isolated from a patient with graft-versus-host disease. Thus, we compared the T cell receptor (TCR) rearrangements of the 2 in vitro generated T cell clones and the ex vivo isolated T cell clone. All 3 clones shared the same TCRBV5-4* gene segment and 2 of 3 clones also used similar TCR-Valpha segments. Our results suggest that T cells recognizing the HLA-DQ5/DDX3Y T cell epitope might be characterized by a relatively limited TCR-beta repertoire. The differences in the junctional TCR-beta region had no effect on the antigen specificity, but altered the capacity of the TCR to distinguish the HLA-DQ5/DDX3Y complex from its allelic counterpart. The results also demonstrate that in vitro stimulation of T cells with allogeneic HLA-identical dendritic cells may facilitate the characterization of in vivo, potentially relevant HLA class II-restricted minor H epitopes.     

Comparison of gastric, duodenal and jejunal contributions to the inhibition of food intake in the rat

Rats equipped with tubes leading to their stomach, duodenum or jejunum were infused with a liquid diet for 9 hr (4 ml/hr) and were allowed to eat during the last 8 hr of infusion. All rats ate significantly less on diet infusion days than on saline or no infusion days. A second study showed that a taste aversion could not be conditioned to flavored water associated with diet infusion. Apparently, intrajejunal injection of nutrients produces satiety and not discomfort. Infusion of the diet for 5 consecutive days into the stomach, duodenum or jejunum consistently and significantly lowered food intake by reducing meal size, not meal frequency. Results suggest that the small intestine below the infusion site contributes to normal satiety.     

Adding salmeterol to an inhaled corticosteroid reduces allergen-induced serum IL-5 and peripheral blood eosinophils

BACKGROUND: Adding a long-acting beta(2)-agonist to inhaled corticosteroids results in better symptomatic asthma control than increasing the dose of inhaled corticosteroids. OBJECTIVE: Investigating whether adding the long-acting beta(2)-agonist salmeterol to the inhaled corticosteroid fluticasone propionate has an effect on allergen-induced allergic inflammation in asthma. METHODS: Bronchial allergen challenges were performed in 26 patients with allergic asthma, pretreating them with a single dose of either fluticasone/salmeterol (100/50 microg) or fluticasone alone (100 microg), in a double-blind, randomized, cross-over design. Sputum and serum markers of bronchial inflammation were measured after allergen challenge, as well as lung function parameters. Primary outcomes were sputum eosinophil numbers and eosinophil cationic protein. RESULTS: Asthmatic responses after allergen challenge were significantly reduced after pretreatment with fluticasone/salmeterol relative to fluticasone alone. Sputum inflammatory markers after allergen challenge were not significantly affected by fluticasone/salmeterol pretreatment. By contrast, serum IL-5 was significantly reduced (geometric mean serum IL-5 [SEM]: 0.5 [0.3] vs 1.1 [0.3] pg/mL 1 hour and 0.6 [0.3] vs 1.1 [0.3] pg/mL 6 hours after challenge with fluticasone/salmeterol vs fluticasone alone pretreatment, respectively; P values < .05). Also, peripheral blood eosinophils were significantly reduced (geometric mean number x 10(6)/L [SEM]: 172 [0.1] vs 237 [0.1] at 6 hours and 271 [0.1] vs 351 [0.1] at 24 hours with fluticasone/salmeterol vs fluticasone alone pretreatment, respectively; P < .05). CONCLUSION: Adding salmeterol to fluticasone reduces allergen-induced serum IL-5 and peripheral blood eosinophils. This phenomenon may contribute to the improved clinical outcomes that result from adding a long-acting beta(2)-agonist to inhaled corticosteroids.