High risk HPV load estimated by Hybrid Capture II correlates with HPV16 load measured by real-time PCR in cervical smears of HPV16-infected women.

BACKGROUND: High risk human papillomavirus (HR-HPV) load determined by quantitative methods has already been considered as highly predictive of future development of high grade cervical lesions. Some studies also demonstrated that Hybrid Capture II (HCII) results can be considered as a reflection of HPV DNA load, while others did not. HCI assay, well suited for routine HR-HPV screening, is not especially dedicated for quantitative use. However, we have recently shown that women with high viral loads assessed by HCII were at increased risk of cervical precancer. OBJECTIVES: The aim of the study was to determine if the values given by the HCII assay can be considered as quantitative. STUDY DESIGN: We used a real-time PCR allowing precise quantification of both HPV16 genome and albumin gene to normalize the measuring HPV16 load in cervical cells and to compare the data with those obtained by HCIIin a series of 40 HR-HPV positive samples. RESULTS: Reproducibility of the HPV16 real-time PCR, assessed from nine independent experiments of serial dilutions of SiHa cell DNA, was reflected in coefficients of variation for standard curves of crossing point (Cp) values below 5%. The HPV16 loads with a broad individual variability were significantly related to the cumulative load estimated by HCII and did not depend on the cellularity of samples. CONCLUSIONS: We assume that the HCII values can be used as a quantitative measure of HR-HPV DNA, so long as cervical specimens are collected using standardized protocols.     

Bacterial and epidemiologic study of the resistance to oxyiminocephalosporins in Escherichia coli in a French hospital

Objective: To understand the mechanisms and epidemiology of resistance to oxyiminocephalosporins in Escherichia coli over a 2-year period in a French hospital.
Methods: Forty-four strains, resistant or intermediately resistant to one of the oxyiminocephalosporins or aztreonam, were collected from 35 patients. MIC determinations were carried out for the 44 isolates using a panel of β-lactam antibiotics, and characterization of the β-lactamases they produced by isoelectric focusing and catalytic activity measurement. Extended-spectrum β-lactamase production was studied by use of the double disk diffusion test. Conjugation experiments were used to search for plasmidic cephalosporinase. An epidemiologic study was then performed, by use of molecular typing of the strains with an ERIC-PCR method and a case-control analysis.
Results: Less than 1% of all the E. coli isolates at our hospital showed decreased susceptibility to oxyiminocephalosporins. Only three of the 44 isolates showed synergy between clavulanate and a third-generation cephalosporin and produced an extended-spectrum β-lactamase. For the other strains, a β-lactamase with a highly basic isoelectric point was detected. Spectrophotometric measures confirmed that most of these isolates were AmpC hyper-producers. No plasmidic cephalosporinase could be detected by conjugation experiments. Molecular typing showed all isolates to be different, except for two strains isolated in two patients of the same hospital unit, and for the repeated isolates of some patients. When 20 case patients were compared to 40 randomly selected control patients, prior receipt of an antimicrobial and more specifically of a β-lactam agent was significantly associated with case patients.
Conclusions: Although it appears to be very rare, the resistance to broad-spectrum cephalosporins needs our attention, because of the high frequency of E. coli infections and β-lactam use in their treatment.     

Novel autoantibody markers for early and seronegative rheumatoid arthritis

Approximately one-third of rheumatoid arthritis (RA) patients are seronegative for the 2 serological RA markers, rheumatoid factor (RF) and antibodies against cyclic citrullinated peptides (ACCP). Moreover, the sensitivities of both markers are lower in the diagnostically important early disease phase. The aim of this study was to identify additional autoantibody markers for early RA and for RF-negative, ACCP-negative (seronegative) RA.We screened an RA synovium cDNA phage display library with autoantibodies in plasma from 10 early (symptoms of maximum 1 year) and 10 seronegative (RF-negative, ACCP-negative) RA patients with validation in 72 additional RA patients and 121 controls (38 healthy controls, 43 patients with other inflammatory rheumatic diseases, 20 osteoarthritis patients and 20 subjects with mechanical joint complaints). Fourteen novel autoantibodies were identified that showed a 54% sensitivity and 90% specificity for RA. For 11 of these autoantibodies, an exclusive presence was demonstrated in RA patients (100% specificity, 37% sensitivity) as compared to controls. All early RA patients were positive for at least one of the identified autoantibodies and antibody-positivity was associated with a shorter disease duration (P = 0.0087). 52% of RA patients who initially tested negative for RF and ACCP, tested positive for at least one of the 14 novel autoantibodies, resulting in a 19% increase in sensitivity compared to current serological testing. Moreover, 5 identified autoantibodies were detected more frequently in seronegative RA patients, indicating that these autoantibodies constitute novel candidate markers for this RA subtype. We demonstrated that the targets of 3 of these 5 autoantibodies had an increased expression in RA synovial tissue compared to control synovial tissue, pointing towards a biological rationale for these auto antibody targets in RA.In conclusion, we identified novel candidate autoantibody markers for RA that can be detected in early and seronegative RA patients indicating the potential added value for RA diagnostics.     

Age-associated cerebral atrophy in mouse lemur primates

We assessed the regional brain atrophy in mouse lemur primates from 4.7 T T2-weighted magnetic resonance images. Thirty animals aged from 1.9 to 11.3 years were imaged. Sixty-one percent of the 23 animals older than 3 years involved in the study displayed an atrophy process. Cross-sectional analysis suggests that the atrophy follows a gradual pathway, starting in the frontal region then involving the temporal and/or the parietal part of the brain and finally the occipital region. Histological evaluation of five animals selected according to various stages of atrophy suggested that extracellular amyloid deposits and tau pathology cannot explain by themselves this atrophy and that intracellular amyloid deposition is more closely linked to this pathology. This study suggests that most of the age-related atrophy occurring in mouse lemurs is caused by one clinical, evolving, pathological process. The ability to follow this pathology non-invasively by MRI will allow to further characterize it and evaluate its relationship with neuropathological lesions that are involved in human diseases such as Alzheimer.     

Detection of 95 novel mutations in coagulation factor VIII gene F8 responsible for hemophilia A: results from a single institution

Hemophilia A (HA) is an X‐linked hereditary bleeding disorder defined by a qualitative and/or quantitative factor VIII (FVIII) deficiency. The molecular diagnosis of HA is challenging because of the high number of different causative mutations that are distributed throughout the large F8 gene. The putative role of the novel mutations, especially missense mutations, may be difficult to interpret as causing HA. We identified 95 novel mutations out of 180 different mutations responsible for HA in 515 patients from 406 unrelated families followed up at a single hemophilia treatment center of the Bicêtre university hospital (Assistance Publique‐Hôpitaux de Paris [AP‐HP], Le Kremlin‐Bicêtre). These 95 novel mutations comprised 55 missense mutations, 12 nonsense mutations, 11 splice site mutations, and 17 small insertions/deletions. We therefore developed a mutation analysis based on a body of proof that combines the familial segregation of the mutation, the resulting biological and clinical HA phenotype, and the molecular consequences of the amino acid (AA) substitution. For the latter, we studied the putative biochemical modifications: its conservation status with cross‐species FVIII and homologous proteins, its putative location in known FVIII functional regions, and its spatial position in the available FVIII 3D structures. The usefulness of such a strategy in interpreting the causality of novel F8 mutations is emphasized. Hum Mutat 27(7), 676–685, 2006. © 2006 Wiley‐Liss, Inc.     

Oppositional COMT Val158Met effects on resting state functional connectivity in adolescents and adults

Prefrontal dopamine levels are relatively increased in adolescence compared to adulthood. Genetic variation of COMT (COMT Val158Met) results in lower enzymatic activity and higher dopamine availability in Met carriers. Given the dramatic changes of synaptic dopamine during adolescence, it has been suggested that effects of COMT Val158Met genotypes might have oppositional effects in adolescents and adults. The present study aims to identify such oppositional COMT Val158Met effects in adolescents and adults in prefrontal brain networks at rest. Resting state functional connectivity data were collected from cross-sectional and multicenter study sites involving 106 healthy young adults (mean age 24 ± 2.6 years), gender matched to 106 randomly chosen 14-year-olds. We selected the anterior medial prefrontal cortex (amPFC) as seed due to its important role as nexus of the executive control and default mode network. We observed a significant age-dependent reversal of COMT Val158Met effects on resting state functional connectivity between amPFC and ventrolateral as well as dorsolateral prefrontal cortex, and parahippocampal gyrus. Val homozygous adults exhibited increased and adolescents decreased connectivity compared to Met homozygotes for all reported regions. Network analyses underscored the importance of the parahippocampal gyrus as mediator of observed effects. Results of this study demonstrate that adolescent and adult resting state networks are dose-dependently and diametrically affected by COMT genotypes following a hypothetical model of dopamine function that follows an inverted U-shaped curve. This study might provide cues for the understanding of disease onset or dopaminergic treatment mechanisms in major neuropsychiatric disorders such as schizophrenia and attention deficit hyperactivity disorder.     

Value of Microimmunofluorescence for Diagnosis and Follow-up of Bartonella Endocarditis

Bartonella endocarditis is a disease of emerging importance that causes serious complications and high rates of mortality. Due to the fastidious nature of Bartonella species and their high degrees of antibiotic susceptibility, cultures of clinical samples most often remain sterile and valvular biopsy specimens, the best specimens for PCR amplification, are seldom available. Therefore, serology appears to be the easiest diagnostic tool. In order to determine the best cutoff value for serology and its predictive values for the detection of Bartonella endocarditis, we studied 48 patients with culture- and/or PCR-confirmed Bartonella endocarditis. We also applied these serological criteria to 156 patients with blood culture-negative endocarditis. Furthermore, we compared the kinetics of the antibody responses to Bartonella spp. in order to estimate the value of serology for prediction of the occurrence of relapses. A titer of ≥1:800 for immunoglobulin G antibodies to either Bartonella henselae or B. quintana has a positive predictive value of 0.810 for the detection of chronic Bartonella infections in the general population and a value of 0.955 for the detection of Bartonella infections among patients with endocarditis. When this cutoff was applied to 156 patients with blood culture-negative endocarditis, we were able to diagnose Bartonella infections in an additional 45 patients with definite endocarditis for whom a positive Bartonella serology was the only evidence of infection. On follow-up, the kinetics of the decrease in antibody titers were significantly delayed in two patients with relapses. In conclusion, we recommend the determination of antibodies to both B. quintana and B. henselae and the use of a cutoff value of 1:800 for the diagnosis of Bartonella endocarditis. We propose that this criterion, which may also help with the detection of late relapses, be included as a major criterion in the Duke criteria for the diagnosis of infective endocarditis.     

The sortase SrtA of Listeria monocytogenes is involved in processing of internalin and in virulence

Listeria monocytogenes is an intracellular gram-positive human pathogen that invades eucaryotic cells. Among the surface-exposed proteins playing a role in this invasive process, internalin belongs to the family of LPXTG proteins, which are known to be covalently linked to the bacterial cell wall in gram-positive bacteria. Recently, it has been shown in Staphylococcus aureus that the covalent anchoring of protein A, a typical LPXTG protein, is due to a cysteine protease, named sortase, required for bacterial virulence. Here, we identified in silico from the genome of L. monocytogenes a gene, designated srtA, encoding a sortase homologue. The role of this previously unknown sortase was studied by constructing a sortase knockout mutant. Internalin was used as a reporter protein to study the effects of the srtA mutation on cell wall anchoring of this LPXTG protein in L. monocytogenes. We show that the srtA mutant (i) is affected in the display of internalin at the bacterial surface, (ii) is significantly less invasive in vitro, and (iii) is attenuated in its virulence in the mouse. These results demonstrate that srtA of L. monocytogenes acts as a sortase and plays a role in the pathogenicity.     

Molecular aspects of human FcgammaR interactions with IgG: functional and therapeutic consequences

The binding of IgG antibodies to receptors for the Fc region of IgG (FcgammaR) is a critical step for the initiation and/or the control of effector immune functions once immune complexes have been formed. Site-directed and random mutagenesis as well as domain-swapping, NMR and X-ray cristallography have made it possible to get detailed insights in the molecular mechanisms that govern IgG/FcgammaR interactions and to define some of the structural determinants that impact IgG binding to the various FcgammaR. It has demonstrated the role of particular stretches and individual residues located in the lower hinge region of the CH2 domain and in the CH2 and CH3 domains of the Fc region. The importance of the sugar components linked to asparagine 297 in the binding properties of IgG1, the human IgG isotype the most widely used in antibody-based therapies, has been also highlighted. These data have led to the engineering of a new generation of monoclonal antibodies for therapeutic use with optimized effector functions.     

A CFSE based assay for measuring CD4+CD25+ regulatory T cell mediated suppression of auto-antigen specific and polyclonal T cell responses

CD4(+)CD25(+) regulatory T cells (Tregs) are considered to play a key role as suppressors of immune mediated reactions. The analysis of Treg function in patients with autoimmune, allergic or oncogenic diseases has emerged over the past years. In the present study we describe a CFSE based protocol to measure Treg mediated suppression of CD4(+) T cells. Measuring Treg suppressive capacity towards proliferation of anti-CD3 Ab stimulated CD4(+)CD25(-) T cells in coculture experiments by means of a CFSE based and a classical [(3)H]thymidine incorporation assay gave similar results, provided that CD4(+)CD25(+) T cells were anergic. However, when CD4(+)CD25(+) T cells proliferated upon mitogenic stimulation, data obtained by the CFSE assay allowed the detection of a significant Treg suppression whereas this was clearly underestimated using the [(3)H]thymidine assay. In addition, an indirect CFSE based method was developed to analyze antigen specific responses of total CD4(+) T cells and Treg depleted CD4(+) T cells (i.e. CD4(+)CD25(-) T cells). Our results indicate that, in healthy individuals, CD4(+) T cell responses against the multiple sclerosis (MS) auto-antigens, myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG), were increased in Treg depleted CD4(+) T cells as compared to total CD4(+) T cells. Our initial data suggest that Tregs in MS patients show an impaired suppression of myelin reactive T cells when compared to healthy controls. Moreover, this experimental setup permits the measurement of cytokine production of the antigen proliferated CFSE(low) T cells by additional flow cytometric analyses. In conclusion, the described CFSE based Treg suppression assay is a valuable tool to study suppressor T cells in (auto)immune disorders.