Retinoids and spermatogenesis: lessons from mutant mice lacking the plasma retinol binding protein.

Using Rbp4-null mice as models, we have established for the first time the kinetics of the spermatogenetic alterations during vitamin A deficiency (VAD). Our data demonstrate that the VAD-induced testicular degeneration arises through the normal maturation of germ cells in a context of spermatogonia differentiation arrest. They indicate that retinoic acid (RA) appears dispensable for the transition of premeiotic to meiotic spermatocytes, meiosis, and spermiogenesis. They confirm that RA plays critical roles in controlling spermatogonia differentiation, spermatid adhesion to Sertoli cells, and spermiation, and suggest that the VAD-induced arrest of spermatogonia differentiation results from simultaneous blocks in RA-dependent events mediated by RA receptor gamma (RARgamma) in spermatogonia and by RARalpha in Sertoli cells. They also provide evidence that expression of major RA-metabolizing enzymes is increased in mouse Sertoli cells upon VAD and that vitamin A-deficient A spermatogonia differ from their RA-sufficient counterparts by the expression of the Stra8 gene.     

Clone-based systematic haplotyping (CSH): a procedure for physical haplotyping of whole genomes

We present a novel methodology to determine the phase of single-nucleotide polymorphisms (SNPs) on a chromosome, which we term clone-based systematic haplotyping (CSH). The CSH procedure is based on separating the allelic chromosomes of a diploid genome by fosmid/cosmid cloning, and subsequent SNP typing of 96 clone pools, each representing approximately 10% of the genome. The pools are screened by PCR for the sequence of interest, followed by SNP typing on the PCR products using the GOOD assay. We demonstrate that by CSH, the haplotype of SNPs separated by more than 50 kilobases can definitely be assigned. We propose this method as being suitable for constructing maps of ancestral haplotypes, analysis of complex diseases, and for diagnosis of rare defects in which the molecular haplotype is crucial. In addition, by amplifying the initial DNA by many orders of magnitude, the original DNA resource is effectively immortalized, enabling the haplotyping of hundreds of thousands of SNPs per individual.     

Direct detection and differentiation of Legionella spp. and Legionella pneumophila in clinical specimens by dual-color real-time PCR and melting curve analysis

A dual-color LightCycler PCR assay targeting the 16S rDNA gene of Legionella spp. was established. By using two pairs of hybridization probes, Legionella spp. and Legionella pneumophila could be detected and differentiated simultaneously. With 26 culture-positive and 42 culture-negative respiratory specimens from patients with atypical pneumonia, 100% sensitivity and specificity was observed for L. pneumophila.     

Changes in blood lactate and respiratory gas exchange measures in sports with discontinuous load profiles

This study compares two different sport events (orienteering = OTC; tennis = TEC) with discontinuous load profiles and different activity/recovery patterns by means of blood lactate (LA), heart rate (HR), and respiratory gas exchange measures (RGME) determined via a portable respiratory system. During the TEC, 20 tennis-ranked male subjects [age: 26.0 (3.7) years; height: 181.0 (5.7) cm; weight: 73.2 (6.8) kg; maximal oxygen consumption (V˙O₂max): 57.3 (5.1) ml·kg⁻¹·min⁻¹] played ten matches of 50 min. During the OTC, 11 male members of the Austrian National Team [age: 23.5 (3.9) years; height: 183.6 (6.8) cm; weight: 72.4 (3.9) kg; V˙O₂max: 67.9 (3.8) ml·kg⁻¹·min⁻¹] performed a simulated OTC (six sections; average length: 10.090 m). In both studies data from the maximal treadmill tests (TT) were used as reference values for the comparison of energy expenditure of OTC and TEC. During TEC, the average V˙O₂ was considerably lower [29.1 (5.6) ml^·kg^−1·min⁻¹] or 51.1 (10.9)% of VO₂max and 64.8.0 (13.3)% of V˙O₂ determined at the individual anaerobic threshold (IAT) on the TT. The short high-intensity periods (activity/recovery = 1/6) did not result in higher LA levels [average LA of games: 2.07 (0.9) mmol·l⁻¹]. The highest average V˙O₂ value for a whole game was 47.8 ml·kg^−1·min⁻¹ and may provide a reference for energy demands required to sustain high-intensity periods of tennis predominately via aerobic mechanism of energy delivery. During OTC, we found an average V˙O₂ of 56.4 (4.5) ml·kg⁻¹·min⁻¹ or 83.0 (3.8)% of V˙O₂max and 94.6 (5.2)% of V˙O₂ at IAT. In contrast to TEC, LA were relatively high [5.16 (1.5) mmol·l⁻¹) although the average V˙O₂ was significantly lower than V˙O₂ at IAT. Our data suggest that portable RGEM provides valuable information concerning the energy expenditure in sports that cannot be interpreted from LA or HR measures alone. Portable RGEM systems provide valuable assessment of under- or over-estimation of requirements of sports and assist in the optimization and interpretation of training in athletes. Electronic Publication     

T-cell-mediated autoimmunity: novel techniques to characterize autoreactive T-cell receptors

Histological samples of autopsy or biopsy tissue provide the best available evidence that autoreactive T cells are involved in the immunopathogenesis of many autoimmune diseases. However, morphology alone does not provide information on the antigen-specific T-cell receptor (TCR) of these cells, let alone on their antigen specificity. In this review article we discuss a number of emerging possibilities for identifying TCR sequences directly from biopsy tissue. We also review the methods for expressing presumably autoreactive TCR molecules and speculate on how the expressed TCR might be used to identify target antigens. Such information should eventually provide new insights into disease pathogenesis which lead to better therapies.     

Ferret odor as a processive stress model in rats: neurochemical, behavioral, and endocrine evidence

Predator odors have been shown to elicit stress responses in rats. The present studies assessed the use of domestic ferret odor as a processive stress model. Plasma corticosterone and adrenocorticotropin hormone levels were higher after 30 min of exposure to ferret odor (fur/skin) but not control odors, ferret feces, urine, or anal gland secretions. Behavioral differences were also found between ferret and the control odors as tested in a defensive withdrawal paradigm. In addition, c-fos messenger RNA expression in several brain areas previously associated with processive stress was significantly higher in ferret odor-exposed rat brains than in control odor-exposed brains. These results suggest that ferret odor produces a reliable unconditioned stress response and may be useful as a processive stress model.     

Impact of M49, Mrp, Enn, and C5a Peptidase Proteins on Colonization of the Mouse Oral Mucosa by Streptococcus pyogenes

Resistance to phagocytosis is a hallmark of virulent Streptococcus pyogenes (group A streptococcus). Surface-bound C5a peptidase reduces recruitment of phagocytes to the site of infection, and hyaluronic acid capsules and/or the M protein limit the uptake of streptococci. In this study the relative impact of M and M-like proteins and the C5a peptidase on the virulence of a serotype M49 strain was assessed. The capacities of isogenic strains with an insertion mutation in emm49; with a deletion mutation in scpA49 (C5a peptidase gene); and with a deletion that removes all three M-like genes, mrp49, emm49, and enn49, to colonize mice and resist phagocytosis were compared. Experiments confirmed results obtained in an earlier study, which showed that the M49 protein was not required for in vitro resistance to phagocytosis, and also showed that the M protein was not required for colonization of mice. Failure to produce all three M-like proteins, M49, Mrp, and Enn49, significantly reduced the ability of these streptococci to resist phagocytosis in vitro but did not significantly alter the persistence of streptococci on the oral mucosa. In vitro experiments indicate that M⁺ streptococci are phagocytized by polymorphonuclear leukocytes that have been activated with phorbol-12-myristate 13-acetate or recombinant human C5a. This observation may explain the finding that expression of M49 protein is not essential for short-term colonization of the mouse oral mucosa.     

Randomized Controlled Clinical Trial of Blood Glucose Awareness Training (BGAT III) in Switzerland and Germany

Although both diabetes and the efficacy of medical management are international issues, psycho-educational interventions might be culturally bound. Blood Glucose Awareness Training (BGAT) is a psycho-educational program for patients with type 1 diabetes mellitus. It is focused on improving recognition and management of extreme blood glucose levels, and is the best documented American psycho-educational program for this purpose. A randomized controlled clinical trial of BGAT's long-term benefits in a non-American setting has been lacking. One hundred and eleven adults with type 1 diabetes mellitus from Switzerland and Germany participated. After a 6 months baseline assessment, subjects were randomly assigned to receive either 2 months of BGAT (n = 56) or a physician-guided self-help control intervention (n = 55). BGAT improved recognition of low (p = 0.008), high (p = .03), and overall blood glucose (p = 0.001), and reduced frequency of severe hypoglycemia (p = 0.04), without compromising metabolic control. BGAT reduced both the external locus of control (p < 0.02) and fear of hypoglycemia (p < 0.02). BGAT was efficacious in reducing adverse clinical events and achieving clinically desirable goals in a European, as well as American setting.     

Longitudinally orientated smooth muscle cells in rabbit arteries

Intima formation in vessels, spontaneous or experimentally induced, is generally characterized by the presence of longitudinally orientated smooth muscle cells (LSMC). During an experiment of neo-intima induction in carotid arteries in rabbits, by application of a nonconstrictive silastic cuff, a study was performed to investigate the presence of LSMC in the systemic and pulmonary circulations, in both elastic and muscular arteries. Three patterns could be distinguished: intimai cushions in muscular arteries, single or small groups of LSMC in the intima in elastic and larger muscular arteries, and intra-medially located layers or columns of LSMC in the aorta, the pulmonary artery, at the bifurcation of the aorta and around orifices of branches. In order to understand this peculiar orientation a biomechanical approach was used: this showed that near the lumen the circumferential stress is 4.5 times higher than the longitudinal. Because the cell surface of the smooth muscle cells exposed to this stress per unit vessel length is much less in the longitudinal than in the circular direction we conclude that the LSMC align in the direction which allows them to cope most effectively with the mechanical stresses.     

Selective targeting of antibody-conjugated nanoparticles to leukemic cells and primary T-lymphocytes

In the present study, surface-modified nanoparticles based on biodegradable material were used for antibody coupling in order to get a selective drug carrier systems. Gelatin nanoparticles were prepared by a desolvation process. Sulfhydryl groups were introduced which enabled the linkage of NeutrAvidin (NAv). Antibodies specific for the CD3 antigen on lymphocytic cells were conjugated to the nanoparticles surface. The binding of biotinylated anti-CD3 antibody was achieved by NAv-biotin-complex formation. Cellular binding and uptake were determined by flow cytometry and confocal laser scanning microscopy (CLSM). Cell-type-specific targeting of anti-CD3-conjugated nanoparticles into CD3-positive human T-cell leukemia cells and primary T-lymphocytes could be shown. Celluar uptake and effective internalization of antibody-conjugated nanoparticles into CD3 expressing cells were demonstrated. Uptake rates of about 84% into T-cell leukemia cells were observed. To confirm selectivity of T-cell targeting, competition experiments were carried out adding excessive free anti-CD3 prior to nanoparticle incubation leading to significantly reduced cellular uptake of antibody-conjugated nanoparticles. Further analysis on the mechanism of uptake confirmed a receptor-mediated endocytotic process. Protein-based nanoparticles conjugated with an antibody against a specific cellular antigen hold promise as selective drug delivery systems for specific cell types.