A comprehensive survey for polymorphisms in the bovine IFN-gamma gene reveals a highly polymorphic intronic DNA sequence allowing improved genotyping of Bovinae.

This study aimed to identify interferon-gamma (IFN-gamma) gene variants in cattle for diagnostic purposes. Therefore, the entire bovine IFN-gamma gene (BoIFNG) and 2605 bp of its promoter DNA were sequenced. The BoIFNG DNA sequence conforms to the published part of Bo-IFN-gamma cDNA. Primer extension experiments show the presence of a 5' extension of exon 1 by 42 nucleotides (nt). One SINE element (Bov-A2) is located in the 5'-region, and two SINE elements (Bov-tA, Bov-B) are contained in the 3'-region of BoIFNG. The variants were detected by comparative sequence analysis of PCR amplicons from different bovine species. Four polymorphic mononucleotide repeats are situated in the promoter and in intron 1. Four distinct series of single nucleotide polymorphisms (SNP) were found in functionally important regions of BoIFNG. The region between the two intron 1 microsatellites contains the highest density of SNPs in Bos taurus breeds. One G-T transversion in the coding region of exon 1 causes a Gly(14) to Val(14) exchange in the BoIFNG signal peptide of different bovine species. A G-A transition in exon 2 encodes a Ser(19) to Asn(19) change in the mature protein of the Tibetan yak. Genotyping of randomly sampled Holstein Friesian cows at selected SNPs and of both intron 1 microsatellites revealed two dominant BoIFNG microhaplotypes. The detected SNPs improve the recently reported genotyping system of cattle.     

Molecular cloning of a vaccine antigen against infection with the larval stage of Echinococcus multilocularis

Alveolar and cystic hydatidosis are caused by infection with the larval stages of Echinococcus multilocularis and Echinococcus granulosus, respectively. A host-protective antigen has been identified in E. granulosus. Here we identify the presence of a closely related protein in E. multilocularis, characterize and express a cDNA encoding the antigen (designated EM95), determine the structure of the em95 gene, and demonstrate that the EM95 recombinant protein can be used to induce significant levels of protection against challenge infection with E. multilocularis eggs in mice.     

Influence of selected wound dressings on PMN elastase in chronic wound fluid and their antioxidative potential in vitro

Exudates from non-healing wounds contain elevated levels of proteolytic enzymes, like elastase from polymorphonuclear granulocytes (PMN elastase), reactive oxygen species (ROS) and reactive nitrogen species (RNS). The overproduction of proteolytic enzymes leads to reduced concentrations of growth factors and proteinase inhibitors, resulting in an imbalance between degradation and remodelling processes. Thus, the reduction of protein-degrading enzymes and scavenging of ROS and RNS seem to be suitable ways to support the healing process of chronic stagnating wounds. The aim of this study was to test selected wound dressings from different biomaterials (collagen, oxidized regenerated cellulose (ORC) and ORC/collagen mixture), regarding their antioxidative potential in vitro and their influence on the concentration and activity of PMN elastase in chronic wound fluid. Antioxidant capacity of the investigated wound dressing was determined by a pholasin-based chemiluminescent assay. PMN elastase concentration was determined by means of ELISA. Enzyme activities could be measured by a fluorescence assay. As the presented data demonstrates, all tested materials showed antioxidant capacity. In addition, the investigated materials were able to reduce the concentration and activity of PMN elastase. Beside other aspects, such as biocompatibility, biodegradability, fluid absorption and clinical effects (e.g. angiogenesis and microcirculation), the understanding of these properties may help to support the further refinement of wound dressings for improved wound healing.     

Diagnostic work-up of rectal bleeding in general practice

BACKGROUND: GPs have many patients with gastrointestinal discomfort. Among bowel-related complaints, the sign of rectal bleeding is of particular importance in patients aged 50 years and above, as it can be an early sign for serious bowel diseases such as colon carcinoma. Despite many guidelines offered to GPs for screening and early detection of colorectal carcinomas, there is very little information about the actual diagnostic approach to the sign of rectal bleeding. AIM: The aim of the study was to collect data concerning treatment strategies used by GPs who treat patients presenting with rectal bleeding. DESIGN OF STUDY: Prospective data collection. SETTING: General practices in Germany. METHOD: Over the course of a year, GPs recorded their treatment strategies in patients presenting with rectal bleeding and associated symptoms. Using a digital practice patient file, physicians participating in the study were able to continuously transmit data electronically to the researchers of the study about diagnostics, referrals, hospital admissions, and final diagnoses. RESULTS: During the course of 1 year, 94 participating physicians collected data on 1584 patients. Information about treating rectal bleeding was recorded for 422 patients; 60% of the patients were referred to specialists in internal medicine or gastroenterologists for further diagnostics. A colonoscopy was the most frequently performed diagnostic procedure (46.2%). Twenty-two per cent (n = 93) of the patients--54 of them aged 50 years and above--were exclusively treated by their GP without conducting a colonoscopy or cooperating with specialists. For these patients, GPs diagnosed less severe diseases like haemorrhoids or other proctologic diseases. CONCLUSION: By using a study that allows GPs to transmit electronically their findings and data, it is possible to draw a picture of treatment strategies of GPs in patients presenting with rectal bleeding. The high percentage of patients who received medical treatment in consultation with specialists underscores the significance of the sign of rectal bleeding in general practice. The need for further diagnostic measures in patients who have been treated exclusively by GPs has to be discussed.     

Determination of penetration profiles of topically applied substances by means of tape stripping and optical spectroscopy: UV filter substance in sunscreens

Penetration profiles of topically applied drugs and cosmetic products provide important information on their efficacy. The application of tape stripping in combination with UV/VIS spectroscopy is checked to determine the local position of topically applied substances inside the stratum corneum, the penetration profile. The amount of corneocytes removed with each tape strip is quantified via the particle-dependent absorption, the pseudoabsorption, in the visible spectral range. The concentration of a typical UV filter substance, 4-methylbenzylidene camphor, is determined by optical spectroscopy using the tape strips removed originally. In this case, a time-dependent increase in the absorbance must be taken into account. Laser scanning microscopic investigations confirm that the nonhomogeneous distribution of the filter substance, on the strips, can explain this spectroscopic behavior. When reaching a homogeneous distribution, the UV spectroscopic signal reflects the correct concentration. These spectroscopic values are compared with high performance liquid chromatography (HPLC) data. The values obtained with both methods for the concentrations of 4-methylbenzylidene camphor are in good agreement. The data obtained are used to illustrate the determination of a penetration profile of a UV filter substance. The results demonstrate that the described protocol is well suited to characterize, in a simple manner, topically applied substances that have a characteristic UV/VIS absorption band.     

Initial characterization of Chlamydophila (Chlamydia) pneumoniae cultured from the late-onset Alzheimer brain

Previous studies from this laboratory provided evidence that the intracellular bacterial pathogen Chlamydophila (Chlamydia) pneumoniae is present in the late-onset Alzheimer's disease (AD) brain. Here we report culture of the organism from two AD brain samples, each of which originated from a different geographic region of North America. Culturable organisms were detectable after one and two passages in HEp-2 cells for the two samples. Both isolates, designated Tor-1 and Phi-1, were demonstrated to be authentic C. pneumoniae using PCR assays targeting the C. pneumoniae-specific genes Cpn0695, Cpn1046, and tyrP. Assessment of inclusion morphology and quantitation of infectious yields in epithelial (HEp-2), astrocytic (U-87 MG), and microglial (CHME-5) cell lines demonstrated an active, rather than a persistent, growth phenotype for both isolates in all host cell types. Sequencing of the omp1 gene from each isolate, and directly from DNA prepared from several additional AD brain tissue samples PCR-positive for C. pneumoniae, revealed genetically diverse chlamydial populations. Both brain isolates carry several copies of the tyrP gene, a triple copy in Tor-1, and predominantly a triple copy in Phi-1 with a minor population component having a double copy. This observation indicated that the brain isolates are more closely related to respiratory than to vascular/atheroma strains of C. pneumoniae.     

PCR detection of human papillomavirus of the mucosa: comparison between MY09/11 and GP5+/6+ primer sets

BACKGROUND AND OBJECTIVES: Human papillomaviruses (HPV) are the causal agent for the development of carcinomas in the cervix uteri and further pathological changes of the skin including mucosa, particularly warts, condylomas and dysplasias. Therefore, we investigated the efficacy of different consensus primers pairs for HPV detection by PCR using brushed samples from the oral cavity in comparison with samples from the cervix uteri. STUDY DESIGN: In the present study, we used two well-established sets of PCR primers in different combinations for the detection of HPV DNA in 106 non-invasive brush biopsy samples of the oral mucosa and 56 samples from the cervix uteri. Direct sequencing of PCR products in all cases determined HPV genotype and specificity. RESULTS: Overall, HPV was detected in 69 of 106 oral mucosa samples. HPV specific amplicons were obtained in 35.8% (N = 38) when using GP5+/6+ primers. The positivity rate was increased to 65.1% in a GP5+/6+ auto-nested PCR approach. In contrast, MY9/11 PCR and nested PCR with MY9/11 outer followed by GP5+/6+ inner primers yielded 2.2% and 16.1%, respectively. In gynaecological samples, PCR results were similar independent of the primer combination used. Thus, DNA quality and DNA content could be additional factors influencing the rate of positivity. CONCLUSION: For oral mucosa samples, auto-nested GP5+/6+ PCR is in our hands the most suitable approach for epidemiological studies because of its high sensitivity, high reliability and reproducibility as well as its relatively simple laboratory procedure.     

Mapping translocation breakpoints by next-generation sequencing

Balanced chromosome rearrangements (BCRs) can cause genetic diseases by disrupting or inactivating specific genes, and the characterization of breakpoints in disease-associated BCRs has been instrumental in the molecular elucidation of a wide variety of genetic disorders. However, mapping chromosome breakpoints using traditional methods, such as in situ hybridization with fluorescent dye-labeled bacterial artificial chromosome clones (BAC-FISH), is rather laborious and time-consuming. In addition, the resolution of BAC-FISH is often insufficient to unequivocally identify the disrupted gene. To overcome these limitations, we have performed shotgun sequencing of flow-sorted derivative chromosomes using "next-generation" (Illumina/Solexa) multiplex sequencing-by-synthesis technology. As shown here for three different disease-associated BCRs, the coverage attained by this platform is sufficient to bridge the breakpoints by PCR amplification, and this procedure allows the determination of their exact nucleotide positions within a few weeks. Its implementation will greatly facilitate large-scale breakpoint mapping and gene finding in patients with disease-associated balanced translocations.     

Bordetella Pertussis Toxin does not induce the release of pro-inflammatory cytokines in human whole blood

Pertussis Toxin (PTx) is one of the most important virulence factors of Bordetella pertussis, the cause of whooping cough. Therefore, the inactivated toxin is an obligatory constituent of acellular pertussis vaccines. It is described in the literature that both native PTx and recombinant Pertussis Toxin (PTg) activate human monocytes whereas others report an inhibition of mammalian monocytes during pertussis infection. B. pertussis, as a Gram-negative bacterium, harbours naturally lipopolysaccharide (LPS, also known as endotoxin), one of the strongest stimulators of monocytes. The latter is triggered via the interaction of endotoxin with inter alia the surface receptor CD14. Consequently, it is necessary to consider a potential contamination of Pertussis Toxin preparations with LPS. First, we determined the LPS content in different preparations of PTx and PTg. All preparations examined were contaminated with LPS; therefore, possible PTx- and PTg-driven monocyte activation independently of LPS was investigated. To meet these aims, we examined monocyte response to PTx and PTg while blocking the LPS receptor CD14 with a specific monoclonal antibody (anti-CD14 mAb). In addition, all toxin preparations examined underwent an LPS depletion. Our results show that it is contaminating LPS, not Pertussis Toxin, which activates human monocytes. Blocking the CD14 receptor prevents Pertussis Toxin-mediated induction of pro-inflammatory cytokines in human monocytes. The depletion of LPS from Pertussis Toxin leads to the same effect. Additionally, the PTx toxicity after LPS depletion procedure was confirmed by animal tests. In contrast, the original Pertussis Toxin preparations not treated as mentioned above generate strong monocyte activation. The results in this publication allow the conclusion that purified Pertussis Toxin preparations do not induce the release of pro-inflammatory cytokines in human whole blood.     

Ahnak1 abnormally localizes in muscular dystrophies and contributes to muscle vesicle release

Ahnak1 is a giant, ubiquitously expressed, plasma membrane support protein whose function in skeletal muscle is largely unknown. Therefore, we investigated whether ahnak would be influenced by alterations of the sarcolemma exemplified by dysferlin mutations known to render the sarcolemma vulnerable or by mutations in calpain3, a protease known to cleave ahnak. Human muscle biopsy specimens obtained from patients with limb girdle muscular dystrophy (LGMD) caused by mutations in dysferlin (LGMD2B) and calpain3 (LGMD2A) were investigated for ahnak expression and localization. We found that ahnak1 has lost its sarcolemmal localization in LGMD2B but not in LGMD2A. Instead ahnak1 appeared in muscle connective tissue surrounding the extracellular site of the muscle fiber in both muscular dystrophies. The entire giant ahnak1 molecule was present outside the muscle fiber and did only partially colocalize with CD45-positive immune cell infiltration and the extracelluar matrix proteins fibronectin and collagenVI. Further, vesicles shedded in response to Ca(2+) by primary human myotubes were purified and their protein content was analysed. Ahnak1 was prominently present in these vesicles. Electron microscopy revealed a homogenous population of vesicles with a diameter of about 150?nm. This is the first study demonstrating vesicle release from human myotubes that may be one mechanism underlying abnormally localized ahnak1. Taken together, our results define ahnak1 in muscle connective tissue as a novel feature of two genetically distinct muscular dystrophies that might contribute to disease pathology.