Identification of MHC class II restricted T-cell-mediated reactivity against MHC class I binding Mycobacterium tuberculosis peptides

Major histocompatibility complex (MHC) class I restricted cytotoxic T lymphocytes (CTL) are known to play an important role in the control of Mycobacterium tuberculosis infection so identification of CTL epitopes from M. tuberculosis is of importance for the development of effective peptide-based vaccines. In the present work, bioinformatics technology was employed to predict binding motifs of 9mer peptides derived from M. tuberculosis for the 12 HLA-I supertypes. Subsequently, the predicted peptides were synthesized and assayed for binding to HLA-I molecules in a biochemically based system. The antigenicity of a total of 157 peptides with measured affinity for HLA-I molecules of K(D) ≤ 500 nM were evaluated using peripheral blood T cells from strongly purified protein derivative reactive healthy donors. Of the 157 peptides, eight peptides (5%) were found to induce T-cell responses. As judged from blocking with HLA class I and II subtype antibodies in the ELISPOT assay culture, none of the eight antigenic peptides induced HLA class I restricted CD8(+) T-cell responses. Instead all responses were blocked by pan-HLA class II and anti-HLA-DR antibodies. In addition, CD4(+) T-cell depletion before the 10 days of expansion, resulted in total loss of reactivity in the ELISPOT culture for most peptide specificities. FACS analyses with intracellular interferon-γ staining of T cells expanded in the presence of M. tuberculosis peptides confirmed that the responsive cells were indeed CD4(+). In conclusion, T-cell immunity against HLA-I binding 9mer M. tuberculosis-derived peptides might in many cases turn out to be mediated by CD4(+) T cells and restricted by HLA-II molecules. The use of 9mer peptides recognized by both CD8(+) and CD4(+) T cells might be of importance for the development of future M. tuberculosis peptide-based vaccines.     

Structural basis for the constitutive activity and immunomodulatory properties of the Epstein-Barr virus-encoded G protein-coupled receptor BILF1

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Numerical Simulation and Experimental Validation of Blood Flow in Arteries with Structured-Tree Outflow Conditions

Blood flow in the large systemic arteries is modeled using one-dimensional equations derived from the axisymmetric Navier–Stokes equations for flow in compliant and tapering vessels. The arterial tree is truncated after the first few generations of large arteries with the remaining small arteries and arterioles providing outflow boundary conditions for the large arteries. By modeling the small arteries and arterioles as a structured tree, a semi-analytical approach based on a linearized version of the governing equations can be used to derive an expression for the root impedance of the structured tree in the frequency domain. In the time domain, this provides the proper outflow boundary condition. The structured tree is a binary asymmetric tree in which the radii of the daughter vessels are scaled linearly with the radius of the parent vessel. Blood flow and pressure in the large vessels are computed as functions of time and axial distance within each of the arteries. Comparison between the simulations and magnetic resonance measurements in the ascending aorta and nine peripheral locations in one individual shows excellent agreement between the two. © 2000 Biomedical Engineering Society. PAC00: 8719Uv     

A Common Variant of Staphylococcal Cassette Chromosome mec Type IVa in Isolates from Copenhagen,Denmark, Is Not Detected by the BD GeneOhm Methicillin-Resistant Staphylococcus aureus Assay

Rapid tests for detection of methicillin-resistant Staphylococcus aureus (MRSA) carriage are important to limit the transmission of MRSA in the health care setting. We evaluated the performance of the BD GeneOhm MRSA real-time PCR assay using a diverse collection of MRSA isolates, mainly from Copenhagen, Denmark, but also including international isolates, e.g., USA100-1100. Pure cultures of 349 MRSA isolates representing variants of staphylococcal cassette chromosome mec (SCCmec) types I to V and 103 different staphylococcal protein A (spa) types were tested. In addition, 53 methicillin-susceptible Staphylococcus aureus isolates were included as negative controls. Forty-four MRSA isolates were undetectable; of these, 95% harbored SCCmec type IVa, and these included the most-common clone in Copenhagen, spa t024-sequence type 8-IVa. The false-negative MRSA isolates were tested with new primers (analyte-specific reagent [ASR] BD GeneOhm MRSA assay) supplied by Becton Dickinson (BD). The ASR BD GeneOhm MRSA assay detected 42 of the 44 isolates that were false negative in the BD GeneOhm MRSA assay. Combining the BD GeneOhm MRSA assay with the ASR BD GeneOhm MRSA assay greatly improved the results, with only two MRSA isolates being false negative. The BD GeneOhm MRSA assay alone is not adequate for MRSA detection in Copenhagen, Denmark, as more than one-third of our MRSA isolates would not be detected. We recommend that the BD GeneOhm MRSA assay be evaluated against the local MRSA diversity before being established as a standard assay, and due to the constant evolution of SCCmec cassettes, a continuous global surveillance is advisable in order to update the assay as necessary.Methicillin-resistant Staphylococcus aureus (MRSA) is a common nosocomial pathogen in countries all over the world. In recent years, community-associated MRSA (CA-MRSA) has become increasingly prevalent and has shown potential to cause health care-associated bloodstream infections (8, 26). Screening and isolation of MRSA-positive patients is essential to control the transmission of MRSA in hospitals (16, 24). However, conventional detection of MRSA by culture takes at least 48 h before a preliminary result is available, and as patients in many countries are only isolated when they are recognized as MRSA positive, the risk of having already transmitted MRSA is high. The real-time PCR BD GeneOhm MRSA assay (Becton Dickinson [BD] Diagnostics GeneOhm; San Diego, CA), formerly called IDI-MRSA, is one of a number of commercial kits for rapid MRSA detection directly from nasal swabs (7) and is based on primers developed by Huletsky et al. (18). The forward primers bind to the J3 region of the staphylococcal cassette chromosome mec (SCCmec), and the reverse primer binds in the orfX region that is specific for Staphylococcus aureus. At least seven SCCmec types are known (types I to VII) (3), and several subtypes, especially of type IV, have been described (21, 27).The BD GeneOhm MRSA assay has been tested in a number of studies (4, 5, 10, 11, 13-15, 22, 23, 25, 29-31). Most studies screened hospitalized patients, but only two studies described the SCCmec types of their MRSA isolates (15, 25). Therefore, it is possible that only a few predominant hospital clones with the same SCCmec types were tested. In Denmark, different CA-MRSA clones dominate and MRSA isolates mainly harbor SCCmec types IV (85%) and V (6%) (2). In-house testing with the Huletsky primers (18) revealed that they did not amplify a PCR fragment from our most-common MRSA clone, spa t024-sequence type 8 (ST8)-IVa. Based on this finding and with the knowledge of the high number of type IV subtypes known, we were interested in finding out whether the BD GeneOhm MRSA assay could detect MRSA isolates from a collection that included mainly CA-MRSA strains. We tested 349 MRSA isolates representing variants of SCCmec types I to V. Furthermore, we chose MRSA isolates of different staphylococcal protein A (spa) types to have a broad range of genetic backgrounds, testing the hypothesis that the same SCCmec type might have minor differences in different MRSA lineages and that these differences could be in the primer regions of the assay.     

No cytotoxicity or genotoxicity of graphene and graphene oxide in murine lung epithelial FE1 cells in vitro

Graphene and graphene oxide receive much attention these years, because they add attractive properties to a wide range of applications and products. Several studies have shown toxicological effects of other carbon‐based nanomaterials such as carbon black nanoparticles and carbon nanotubes in vitro and in vivo. Here, we report in‐depth physicochemical characterization of three commercial graphene materials, one graphene oxide (GO) and two reduced graphene oxides (rGO) and assess cytotoxicity and genotoxicity in the murine lung epithelial cell line FE1. The studied GO and rGO mainly consisted of 2–3 graphene layers with lateral sizes of 1–2 µm. GO had almost equimolar content of C, O, and H while the two rGO materials had lower contents of oxygen with C/O and C/H ratios of 8 and 12.8, respectively. All materials had low levels of endotoxin and low levels of inorganic impurities, which were mainly sulphur, manganese, and silicon. GO generated more ROS than the two rGO materials, but none of the graphene materials influenced cytotoxicity in terms of cell viability and cell proliferation after 24 hr. Furthermore, no genotoxicity was observed using the alkaline comet assay following 3 or 24 hr of exposure. We demonstrate that chemically pure, few‐layered GO and rGO with comparable lateral size (> 1 µm) do not induce significant cytotoxicity or genotoxicity in FE1 cells at relatively high doses (5–200 µg/ml). Environ. Mol. Mutagen. 57:469–482, 2016. © 2016 The Authors. Environmental and Molecular Mutagenesis Published by Wiley Periodicals, Inc.     

Androgens and estrogens in relation to hot flushes during the menopausal transition.

In this paper, the association of hormones to vasomotor complaints during the menopausal transition is discussed. Fifty-seven regularly menstruating women without history of hormone replacement therapy (HRT) were selected for a longitudinal, prospective study around the menopausal transition. The mean age at the start of the study was 51.3 (+/-2.0) years. At intervals of 12 months all women went through a semi-structured interview and filled in questionnaires. Venous blood samples were collected every 12-month for analyses of estradiol (E2), testosterone, androstendione, dehydroepiandrosterone-sulphate (DHEA-S), follicle stimulating hormone (FSH), thyrotropin (TSH), and luteinizing hormone (LH). Vasomotor complaints were tested using questions about hot flushes and bouts of sweating in terms of occurrence, frequency and degree of distress. Forty-six percent of the subjects reported hot flushes and bouts of sweating before menopause, increasing to 67% during the first year after menopause and 49% in the second year postmenopause. Low levels of estradiol and high levels of FSH were associated with vasomotor complaints before menopause. During menopause high levels of TSH were related to vasomotor complaints. The first year after menopause, women, who at this point achieved hot flushes, were characterised by high levels of E2, but declining and low levels of FSH, but increasing. Postmenopausal, high levels of testosterone and DHEA-S seemed to protect against vasomotor symptoms. Our most important finding was, that among women who achieved hot flushes at the first assessment postmenopause, the high androgen levels was a significant predictor of recovery from hot flushes at the last assessment, 1 year later.     

Evaluation of PCR-based preimplantation genetic diagnosis applied to monogenic diseases: a collaborative ESHRE PGD consortium study

Preimplantation genetic diagnosis (PGD) for monogenic disorders currently involves polymerase chain reaction (PCR)-based methods, which must be robust, sensitive and highly accurate, precluding misdiagnosis. Twelve adverse misdiagnoses reported to the ESHRE PGD-Consortium are likely an underestimate. This retrospective study, involving six PGD centres, assessed the validity of PCR-based PGD through reanalysis of untransferred embryos from monogenic-PGD cycles. Data were collected on the genotype concordance at PGD and follow-up from 940 untransferred embryos, including details on the parameters of PGD cycles: category of monogenic disease, embryo morphology, embryo biopsy and genotype assay strategy. To determine the validity of PCR-based PGD, the sensitivity (Se), specificity (Sp) and diagnostic accuracy were calculated. Stratified analyses were also conducted to assess the influence of the parameters above on the validity of PCR-based PGD. The analysis of overall data showed that 93.7% of embryos had been correctly classified at the time of PGD, with Se of 99.2% and Sp of 80.9%. The stratified analyses found that diagnostic accuracy is statistically significantly higher when PGD is performed on two cells versus one cell (P=0.001). Se was significantly higher when multiplex protocols versus singleplex protocols were applied (P=0.005), as well as for PGD applied on cells from good compared with poor morphology embryos (P=0.032). Morphology, however, did not affect diagnostic accuracy. Multiplex PCR-based methods on one cell, are as robust as those on two cells regarding false negative rate, which is the most important criteria for clinical PGD applications. Overall, this study demonstrates the validity, robustness and high diagnostic value of PCR-based PGD.     

Further immunohistochemical characterization of <Emphasis Type="Italic">BRD1</Emphasis> a new susceptibility gene for schizophrenia and bipolar affective disorder

We have recently shown that the gene BRD1 is associated with schizophrenia and bipolar affective disorder and that the BRD1 protein (BRD1) which is expressed in neurons may occur in a short and a long variant. The aim of the study was to generate polyclonal antibodies against new BRD1 epitopes enabling discrimination between the long and short BRD1 variants, and elucidate the BRD1 distribution in several human tissues, including the CNS. Polyclonal rabbit antibodies were raised against three different BRD1 epitopes. One (67) was specific for the long BRD1 variant, whereas the two others (63/64 and 65/66) like the original monoclonal mouse antibody (K22) were predicted to stain both variants. Immunohistochemical staining procedures were subsequently performed on paraffin-embedded human cerebral cortex and microarray slides containing 30 different human tissues. Western blotting confirmed the predicted specificity of the developed antibodies. K22, 63/64 and 65/66 displayed a similar neuronal staining pattern characterized by a distinct but weak nuclear staining, while the surrounding cytoplasm and proximal dendrites were more intensely stained. Interestingly, staining with 67 generated in contrast primarily an intense nuclear staining. The new antibodies resulted, furthermore, in a prominent neuroglial reaction characterized by staining of cell bodies, nuclei and glial processes. The tissue microarray analysis revealed that BRD1 was widely distributed in human tissues. The particular expression profile, e.g., the degree of nuclear and/or cytoplasmatic staining, seemed, however, to be highly tissue dependent. These results suggest a general role of BRD1 in the cell and stress that the two BRD1 variants may play different roles in the etiology of psychiatric disease.     

Histological evidence of testicular dysgenesis in contralateral biopsies from 218 patients with testicular germ cell cancer

This study was prompted by a hypothesis that testicular germ cell cancer may be aetiologically linked to other male reproductive abnormalities as a part of the so-called 'testicular dysgenesis syndrome' (TDS). To corroborate the hypothesis of a common association of germ cell cancer with testicular dysgenesis, microscopic dysgenetic features were quantified in contralateral testicular biopsies in patients with a testicular germ cell tumour. Two hundred and eighty consecutive contralateral testicular biopsies from Danish patients with testicular cancer diagnosed in 1998-2001 were evaluated retrospectively. Two hundred and eighteen specimens were subsequently included in this study, after 63 patients who did not meet inclusion criteria had to be excluded. The presence of carcinoma in situ (which is believed to originate from transformed gonocytes) was detected in 8.7% of biopsies. The incidence of other dysgenetic features was immature tubules with undifferentiated Sertoli cells, 4.6%; microcalcifications (microliths), 6.0%; and the presence of a Sertoli-cell-only pattern in at least a few tubules, 13.8%. The cumulative incidence of one or more signs of testicular dysgenesis was 25.2%. In a few patients, areas with immature and morphologically distorted tubules were also noted. Spermatogenesis was qualitatively normal in 51.4%, whereas 11.5% had very poor or absent spermatogenesis. It is concluded that microscopic testicular dysgenesis is a frequent feature in contralateral biopsies from patients presenting with testicular germ cell neoplasms of the adolescent and young type. The findings therefore support the hypothesis that this cancer is part of a testicular dysgenesis syndrome. The presence of contralateral carcinoma in situ was higher in the present study than previously reported.     

Unusual Distribution of Burkholderia cepacia Complex Species in Danish Cystic Fibrosis Clinics May Stem from Restricted Transmission between Patients

Forty-four of 48 Burkholderia cepacia complex strains cultured from Danish cystic fibrosis patients were Burkholderia multivorans, a distribution of species that has not been reported before. Although cases of cross infections were demonstrated, no major epidemic clone was found. The species distribution may represent the sporadic acquisition of bacteria from the environment.Burkholderia cepacia and related bacteria have emerged as significant pathogens in cystic fibrosis (CF) patients due to the risk of cepacia syndrome (a fatal necrotizing pneumonia with bacteremia), the organism''s innate multiresistance to antibiotics, and the transmissibility of bacterial strains between patients by social contact (10, 15). The genus Burkholderia encompasses more than 50 validly published species that can be divided into four groups (21). Strains colonizing the respiratory tract of CF patients are predominantly members of the B. cepacia complex (BCC), with 17 formally named species (23). Chronic infections typically involve a single strain, although strain displacements have been demonstrated (24).Most or all species of the BCC can colonize the lower airways of CF patients, although some of them are infrequently demonstrated. Studies from North America, Europe, and Australasia have shown that Burkholderia cenocepacia is the dominant species being recovered from 46 to 90% of colonized patients (4, 6, 13, 17, 19, 20). Different situations have been described in Lisbon, where contamination of saline solutions used in inhalant therapy by CF patients has resulted in a predominance of B. cepacia (5), and in France, where a small excess of B. multivorans (52%) over B. cenocepacia (45%) has been reported (3).Danish CF patients are treated in two centers, and respiratory cultures are routinely performed at the monthly visit to the outpatient clinic. Four hundred thirty-one patients were alive by 1 January 2007, and 24 (5.6%) were chronically infected with BCC species. A chronic infection was defined as the isolation of BCC bacteria from more than half of sputum cultures for more than 6 months (modified “Leeds criteria” for chronic Pseudomonas aeruginosa infection [14]), and/or the development of ≥2 precipitins measured by crossed immunoelectrophoresis (18). A total of 52 Danish CF patients are known to have been intermittently (n = 11) or chronically (n = 41) infected with BCC bacteria (Fig. ​(Fig.1).1). Intermittently colonized patients may be underrepresented in data from before the routine use of colistin-containing selective agar plates (9), and some of the recent BCC acquisitions may be reclassified as chronic infections with time. In retrospect, the first Danish patient was chronically infected with BCC in the late 1970s (18), but few cases were identified until 1990. The increased rate of BCC colonization after 1990 may be secondary to the widespread use of inhaled colistin for P. aeruginosa infection, which was introduced in the 1980s (11). Since 1993, the rate has stabilized at around three new cases per year (43 BCC acquisitions during 14 years) (Fig. ​(Fig.1).1). In the same time period, 174 Danish patients have been diagnosed with CF (on average, 12.4 ± 4.4 [mean ± standard deviation] new patients per year; range, 6 to 22).Open in a separate windowFIG. 1.Cumulative numbers of Danish CF patients experiencing a first-time isolation of BCC bacteria, separated by status (open bars, chronic infections; gray bars, intermittent colonizations).BCC isolates from 9 intermittently colonized and 39 chronically infected patients were available for characterization. One isolate per patient, cultured between 1994 and 2006, was included in the study. Allocation to species within the BCC was performed by partial atpD and recA sequencing (1); occasional isolates with no PCR product from either amplification were subjected to partial sequencing of fur (16). Two independent sequence-based identifications were thus obtained for all BCC isolates. Only three species were identified in Danish patients, and B. multivorans accounted for more than 90% of the isolates (Table ​(Table1).1). Pulsed-field gel electrophoresis (PFGE) genotypes were assessed after digestion with Xba and SpeI and interpreted as described previously (22). Thirty-eight BCC genotypes were disclosed by both enzymes, and five of the genotypes were identified in more than one patient (two to four patients). Some of these small clusters were epidemiologically related and probably reflect cases of cross infections. Two pairs of siblings each carried the same strain, and one additional patient harbored the same genotype as the two siblings treated in that CF center. Between 1994 and 2003, chronic infections with BCC of a single genotype were established in 4 patients treated in one center. A fourth cluster was composed of patients treated at both of the two Danish CF centers; a possible epidemiological relationship between these three patients was unknown. No patient-to-patient transmission could have occurred in the fifth cluster, where the same genotype was intermittently detected in two patients in 1994 and 1999, respectively, and established a chronic infection in a third patient in 2005. All BCC genotypes identified in more than one patient were B. multivorans.

TABLE 1.

Specific identification of 48 BCC strains isolated from Danish CF patients