Molecular characterization and determination of the coding capacity of the genome of equine herpesvirus type 2 between the genome coordinates 0.235 and 0.258 (theEcoRI DNA fragment N; 4.2 kbp)

The complete DNA nucleotide sequence of theEcoRI DNA fragment N (0.235 to 0.258 viral map units) of equine herpes virus type 2 (EHV-2) strain T400/3 was determined. This DNA fragment comprises 4237 bp with a base composition of 55.23% G+C and 44.77% A+T. Nineteen open reading frames (ORFs) of 50-287 amino acid (aa) residues were detected. ORF number 10 is located between the nucleotide position 2220 and 2756 coding for a protein of 179 amino acid residues. This protein shows significant homology to the cytokine synthesis inhibitory factor (CSIF; interleukin 10) of human (76.4%) and mouse (68.5%), and to the Epstein-Barr virus (EBV) protein BCRF1 (70.6%). The existence of an interleukin 10 (IL-10) analogous gene within the genome of the EHV-2 was confirmed by screening the genome of nine EHV-2 strains using specific oligonucleotide primers corresponding to the 5 and 3 region of this particular gene by polymerase chain reaction. In all experiments an 870 bp DNA product was amplified. The specifity of the amplified DNA fragments obtained from individual EHV-2 strains was confirmed by DNA-DNA hybridization experiments. The DNA sequence analysis of the amplified DNA products of the EHV-2 strain LK was carried out. This analysis revealed the identity of the corresponding IL-10 gene (540 bp) of this strain to the IL-10 gene of EHV-2 strain T400/3. The presented data indicate that the EHV-2 genome harbors a viral interleukin 10-like gene. This is further evidence that the IL-10 gene can be present in the genomes of members of the Herpesviridae family.     

Correction of EOG Artifacts in Event-Related Potentials of the EEG: Aspects of Reliability and Validity

Correction of EOG artifacts using a regression approach is evaluated in terms of reliability and validity. Transmission rates are estimated for eight EEG channels in 67 subjects. The trimmed group means of these rates are shown to provide reliable measures. Eye artifact correction based on these group means is superior to the conventional rejection in terms of reducing correlation between EOG and EEG.     

Quantitation of cytomegalovirus (hCMV) DNA and β-actin DNA by duplex real-time fluorescence PCR in solid organ (liver) transplant recipients

Even now rare human cytomegalovirus (hCMV) reactivation is still a life-threatening complication after solid organ transplantation. Although PCR techniques are regarded as the most sensitive detection methods for hCMV, their accuracy and reproducibility are limited. This is a major disadvantage with quantitative PCR assays, which are thought to provide valuable information about hCMV latency or active viral replication in transplant patients. To enhance the diagnostic safety of quantitative hCMV PCR, we developed a duplex real-time fluorescence PCR that is capable of quantifying hCMV DNA and beta-actin DNA as internal control simultaneously within one reaction. By the use of 6-carboxyfluorescein and hexa-chloro-6-carboxyfluorescein as reporter fluorophores and 4-(4'-dimethylamino-phenylazo) benzoic acid as dark quencher dye, hCMV DNA and beta-actin DNA could be quantified in parallel in a wide linear range from 10(1) to 10(7) copies, each. To test the clinical applicability of this approach, we investigated hCMV DNA kinetics in peripheral leukocytes of three hCMV antigen-positive and four antigen-negative patients after liver transplantation, as assessed by intracellular hCMV pp65 alkaline phosphate-anti-alkaline phosphate (APAAP) complex. While all APAAP-negative individuals remained PCR negative, kinetics of HCMV DNA in leukocyte DNA samples of APAAP-positive patients correlated closely with hCMV antigen tests. Here, comparison of separate and simultaneous target quantitation revealed identical results. It is of interest that, while single hCMV antigen positivity is commonly not regarded as a reliable parameter of viral reactivation, in our study a viral load greater than 10(4) copies/2x10(5) beta-actin DNA copies clearly indicated a subsequent increase in APAAP-positive leukocytes. We conclude that with the presented method the reliability of hCMV quantitation via real-time PCR can be substantially increased and may be used to monitor hCMV kinetics in vivo.     

The effect of anti-IgE treatment on in vitro leukotriene release in children with seasonal allergic rhinitis

BACKGROUND: Binding of allergens with IgE to the IgE receptors on mast cells and basophils results in the release of inflammatory mediators as sulfidoleukotrienes (SLTs), triggering allergic cascades that result in allergic symptoms, such as asthma and rhinitis. OBJECTIVE: We sought to investigate whether anti-IgE (Oma-lizumab), a humanized monoclonal anti-IgE antibody, in addition to specific immunotherapy (SIT) affects the leukotriene pathway. METHODS: Ninety-two children (age range, 6-17 years) with sensitization to birch and grass pollens and with seasonal allergic rhinitis were included in a phase III, placebo- controlled, multicenter clinical study. All subjects were randomized to one of 4 treatment groups. Two groups subcutaneously received birch SIT and 2 groups received grass SIT for at least 14 weeks before the start of the birch pollen season. After 12 weeks of SIT titration, placebo or anti-IgE was added for 24 weeks. The primary clinical efficacy variable was symptom load (ie, the sum of daily symptom severity score and rescue medication score during pollen season). Blood samples taken at baseline and at the end of study treatment after the grass pollen season were used for separation of leukocytes in this substudy. After in vitro stimulation of the blood cells with grass and birch pollen allergens, SLT release (LTC4, LTD4, and LTE4) was quantified by using the ELISA technique. RESULTS: Before the study treatment, SLT release to birch and grass pollen exposure did not differ significantly among the 4 groups. Under treatment with anti-IgE + SIT-grass (n = 23), a lower symptom load occurred during the pollen season compared to placebo + SIT-grass (n = 24, P =.012). The same applied to both groups receiving birch SIT (n = 23 and n = 22, respectively; P =.03). At the end of treatment, the combination of anti-IgE plus grass SIT, as well as anti-IgE plus birch SIT, resulted in significantly lower SLT release after stimulation with the corresponding allergen (416 ng/L [5th-95th percentile, 1-1168] and 207 ng/L [1-860 ng/L], respectively) compared with placebo plus SIT (2490 ng/L [384-6587 ng/L], P =.001; 2489 ng/L [1-5670 ng/L], P =.001). In addition, treatment with anti-IgE was also followed by significantly lower SLT releases to the allergens unrelated to SIT (grass SIT: 300 ng/L [1-2432 ng/L] in response to birch allergen; birch SIT: 1478 ng/L [1-4593 ng/L] in response to grass pollen) in comparison with placebo (grass SIT: 1850 ng/L [1-5499 ng/L], P =.001; birch SIT: 2792 ng/L [154-5839 ng/L], P =.04]. CONCLUSION: Anti-IgE therapy reduces leukotriene release of peripheral leukocytes stimulated with allergen in children with allergic rhinitis undergoing allergen immunotherapy independent of the type of SIT allergen used.     

Observation of a nematic phase displayed by a polysiloxane with trinitrofluorenones as side groups

The synthesis and the results of the structural study of two copolysiloxanes with laterally fixed trinitrofluorenone (TNF) units is reported. The two copolysiloxanes having 2,4 ( 1a ) and 5,3 ( 1b ) dimethylsiloxane comonomer units per TNF side group differ significantly in their phase behaviour as evident from optical microscopy, differential scanning calorimetry and X-ray scattering: 1b shows a nematic mesophase whereas 1a is an amorphous material. The different phase behaviour is discussed in terms of microphase separation between the siloxane backbone and TNF side groups.     

Alcohol use, urinary cortisol, and heart rate variability in apparently healthy men: Evidence for impaired inhibitory control of the HPA axis in heavy drinkers.

Alcoholism and heavy drinking are associated with a number of physiological, behavioral, affective, and cognitive problems. One such problem involves dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis, with alcoholics showing higher basal cortisol levels and reduced inhibitory feedback control. In addition, alcohol consumption is associated with decreased heart rate variability (HRV). In the present study we examined the relationships among alcohol consumption, cortisol excretion, and HRV in 542 apparently healthy men. Men in the top tertile of self-reported alcohol consumption had higher cortisol levels and lower HRV compared to men in the lower two tertiles of alcohol consumption. In addition, the inverse relationship between cortisol and HRV was greatly attenuated in the heavy drinking group even after accounting for a number of potential confounding factors. These results support prior research on the HPA axis dysregulation in alcoholics and suggest impaired inhibitory control of the HPA axis in heavy drinkers. The findings are consistent with the neurovisceral integration model, which links central and peripheral processes, and may provide a comprehensive framework for the future investigation of the complex mix of physiological, behavioral, affective, and cognitive factors which comprise the heavy drinking phenotype.     

Inhibition of the development of immediate hypersensitivity by staphylococcal enterotoxin B

We investigated the ability of staphylococcal enterotoxin B (SEB) to modify the immediate hypersensitivity response induced in BALB/c mice following sensitization to ovalbumin (OVA), a response mediated by OVA-reactive Vβ8 T cells. Mice were sensitized by skin painting with OVA every second day over a period of 2 weeks. SEB, a potent activator of Vβ8⁺ T cells, was administered at the same site where OVA was applied (skin of the lower abdomen) following two different protocols. In protocol (A) SEB was injected intradermally 1 day before painting with OVA and on day 7; in protocol B, SEB was injected each time OVA was applied to the skin (eight times). SEB (but not SEA) altered the development of immediate hypersensitivity to OVA, as demonstrated by the reduction in allergen-specific IgE, decreased OVA-specific immediate skin test responsiveness, and prevented the development of increased airways responsiveness after bronchial challenge with OVA. Injections of SEB did not alter the proliferative responses of local draining lymph node cells or spleen mononuclear cells to OVA, indicating that administration of SEB did not inhibit the sensitization to OVA, but shifted the immune response away from an immediate type response (IgE/IgG₁) to IgG_2a, IgG_2b and IgG₃. Although both protocols of SEB treatment did not lead to a major deletion of the Vβ8 T cell population, they did reduce the proliferative response of Vβ8⁺ T cells to OVA. These data indicate that the bacterial toxin SEB is capable of modifying the immediate hypersensitivity response induced by OVA by altering the functional capacity of antigen-reactive Vβ8 T cells.