Novel paradigm enables accurate monthly gestational screening to prevent congenital toxoplasmosis and more.

Background: Congenital toxoplasmosis is a treatable, preventable disease, but untreated causes death, prematurity, loss of sight, cognition and motor function, and substantial costs worldwide. Methods/Findings: In our ongoing USA feasibility/efficacy clinical trial, data collated with other ongoing and earlier published results proved high performance of an Immunochromatographic-test(ICT) that enables accurate, rapid diagnosis/treatment, establishing new paradigms for care. Overall results from patient blood and/or serum samples tested with ICT compared with gold-standard-predicate-test results found ICT performance for 4606 sera/1876 blood, 99.3%/97.5% sensitive and 98.9%/99.7% specific. However, in the clinical trial the FDA-cleared-predicate test initially caused practical, costly problems due to false-positive-IgM results. For 58 persons, 3/43 seronegative and 2/15 chronically infected persons had false positive IgM predicate tests. This caused substantial anxiety, concerns, and required costly, delayed confirmation in reference centers. Absence of false positive ICT results contributes to solutions: Lyon and Paris France and USA Reference laboratories frequently receive sera with erroneously positive local laboratory IgM results impeding patient care. Therefore, thirty-two such sera referred to Lyon's Reference laboratory were ICT-tested. We collated these with other earlier/ongoing results: 132 of 137 USA or French persons had false positive local laboratory IgM results identified correctly as negative by ICT. Five false positive ICT results in Tunisia and Marseille, France, emphasize need to confirm positive ICT results with Sabin-Feldman-Dye-test or western blot. Separate studies demonstrated high performance in detecting acute infections, meeting FDA, CLIA, WHO ASSURED, CEMark criteria and patient and physician satisfaction with monthly-gestational-ICT-screening. Conclusions/Significance: This novel paradigm using ICT identifies likely false positives or raises suspicion that a result is truly positive, rapidly needing prompt follow up and treatment. Thus, ICT enables well-accepted gestational screening programs that facilitate rapid treatment saving lives, sight, cognition and motor function. This reduces anxiety, delays, work, and cost at point-of-care and clinical laboratories. Author's Summary: Toxoplasmosis is a major health burden for developed and developing countries, causing damage to eyes and brain, loss of life and substantial societal costs. Prompt diagnosis in gestational screening programs enables treatment, thereby relieving suffering, and leading to > 14-fold cost savings for care. Herein, we demonstrate that using an ICT that meets WHO ASSURED-criteria identifying persons with/without antibody to Toxoplasma gondii in sera and whole blood with high sensitivity and specificity, is feasible to use in USA clinical practice. We find this new approach can help to obviate the problem of detection of false positive anti- T.gondii IgM results for those without IgG antibodies to T.gondii when this occurs in present, standard of care, predicate USA FDA cleared available assays. Thus, this accurate test facilitates gestational screening programs and a global initiative to diagnose and thereby prevent and treat T.gondii infection. This minimizes likelihood of false positives (IgG and/or IgM) while maintaining maximum sensitivity. When isolated IgM antibodies are detected, it is necessary to confirm and when indicated continue follow up testing in ∼2 weeks to establish seroconversion. Presence of a positive ICT makes it likely that IgM is truly positive and a negative ICT makes it likely that IgM will be a false positive without infection. These results create a new, enthusiastically-accepted, precise paradigm for rapid diagnosis and validation of results with a second-line test. This helps eliminate alarm and anxiety about false-positive results, while expediting needed treatment for true positive results and providing back up distinguishing false positive tests.

The role of musical training in emergent and event-based timing.

INTRODUCTION: Musical performance is thought to rely predominantly on event-based timing involving a clock-like neural process and an explicit internal representation of the time interval. Some aspects of musical performance may rely on emergent timing, which is established through the optimization of movement kinematics, and can be maintained without reference to any explicit representation of the time interval. We predicted that musical training would have its largest effect on event-based timing, supporting the dissociability of these timing processes and the dominance of event-based timing in musical performance. MATERIALS AND METHODS: We compared 22 musicians and 17 non-musicians on the prototypical event-based timing task of finger tapping and on the typically emergently timed task of circle drawing. For each task, participants first responded in synchrony with a metronome (Paced) and then responded at the same rate without the metronome (Unpaced). RESULTS: Analyses of the Unpaced phase revealed that non-musicians were more variable in their inter-response intervals for finger tapping compared to circle drawing. Musicians did not differ between the two tasks. Between groups, non-musicians were more variable than musicians for tapping but not for drawing. We were able to show that the differences were due to less timer variability in musicians on the tapping task. Correlational analyses of movement jerk and inter-response interval variability revealed a negative association for tapping and a positive association for drawing in non-musicians only. DISCUSSION: These results suggest that musical training affects temporal variability in tapping but not drawing. Additionally, musicians and non-musicians may be employing different movement strategies to maintain accurate timing in the two tasks. These findings add to our understanding of how musical training affects timing and support the dissociability of event-based and emergent timing modes.

Health insurance as a requirement to undergo cardiac transplantation: a national survey of transplant program practices.

BACKGROUND: Recent limitations in Medicaid coverage of transplantation in Arizona jeopardized transplantation of potential recipients in that state and called attention to financial barriers inherent in the present organ allocation system. Policies of cardiac transplant centers regarding insurance requirements for transplantation have not been previously assessed. We sought to determine the policies of adult cardiac transplant programs nationwide regarding insurance requirements for evaluation and listing for cardiac transplantation. METHODS: From December 15, 2008 to November 16, 2010, all active adult cardiac transplant programs in the United States were surveyed regarding insurance requirements for evaluation and listing for cardiac transplantation. RESULTS: Surveys were completed by 62 of 101 programs, accounting for 71% of adult cardiac transplants in 2007. Only 2% of recipients were uninsured. Insurance was required by 48% of programs to evaluate and 84% to list for transplantation. For uninsured patients, 81% of programs required a large amount of money upfront (median, $200,000; interquartile range, $10,000-$300,000) to list for transplantation and often (84%) educated patients about fundraising to acquire these resources. CONCLUSIONS: Adult cardiac transplant programs generally require candidates to have adequate health insurance to undergo transplantation. Uninsured patients typically need a significant amount of money upfront to be listed for transplantation and often are advised to fundraise to gather such resources.

Purification and identification of bovine cheese whey fatty acids exhibiting in vitro antifungal activity.

Milk lipids contain several bioactive factors exhibiting antimicrobial activity against bacteria, viruses, and fungi. In the present study, we demonstrate that free fatty acids (FFA) derived from the saponification of bovine whey cream lipids are active in vitro at inhibiting the germination of Candida albicans, a morphological transition associated with pathogenicity. This activity was found to be significantly increased when bovine FFA were enriched in non-straight-chain FFA. At low cell density, this non-straight-chain FFA-enriched fraction was also found to inhibit in a dose-dependant manner the growth of both developmental forms of C. albicans as well as the growth of Aspergillus fumigatus. Using an assay-guided fractionation, the main components responsible for these activities were isolated. On the basis of mass spectroscopic and gas chromatographic analysis, antifungal compounds were identified as capric acid (C10:0), lauroleic acid (C12:1), 11-methyldodecanoic acid (iso-C13:0), myristoleic acid (C14:1n-5), and gamma-linolenic acid (C18:3n-6). The most potent compound was gamma-linolenic acid, with minimal inhibitory concentration values of 5.4 mg/L for C. albicans and 1.3 mg/L for A. fumigatus, in standardized conditions. The results of this study indicate that bovine whey contains bioactive fatty acids exhibiting antifungal activity in vitro against 2 important human fungal pathogens.

Molecular analysis of Desulfitobacterium frappieri pcp-1 involved in reductive dehalogenation of pentachlorophenol.

Desulfitobacterium are Gram positive, spore-forming, strictly anaerobic bacteria, that belong to the Firmicutes, Clostridia, Clostridiales, and Peptococcaceae. Most known members of the genus Desulfitobacterium have the ability to dechlorinate several halogenated compounds by a mechanism of reductive dehalogenation and use them as electron acceptors to generate energy (halorespiration). Desulfitobacteria are therefore perfect candidates to be used in bioremediation treatments of environment polluted with halogenated compounds. Understanding the physiology and the molecular mechanisms of these bacteria will help to develop better bioremediation systems. This report summarizes works that have been done in our laboratories with D. frappieri PCP-1 on reductive dehalogenases, genes encoding these dehalogenases and their expression, and the development of lab-scale PCP-degrading reactors using this bacterium.

Class II transactivator-induced expression of HLA-DO(beta) in HeLa cells.

HLA-DO is an intracellular nonclassical MHC class II molecule expressed in the endocytic pathway of B lymphocytes. It shapes the repertoire of peptides bound to classical class II molecules such as HLA-DR by regulating the activity of HLA-DM. Using a peptide corresponding to the cytoplasmic tail of HLA-DO(beta), we have developed a mouse monoclonal antibody, HKC5. Immunofluorescence microscopy revealed that HKC5 recognizes HLA-DO molecules present in the endoplasmic reticulum as well as those in vesicular compartments of the endocytic pathway. In addition, the antibody detects the isolated beta chain on Western blots. Using mutants of the DO(beta) cytoplasmic tail fused to a reporter molecule and expressed in epithelial cells, we showed by flow cytometry that the antibody epitope includes one or both of the leucine residues forming the lysosomal sorting signal. Finally, we have used HKC5 to evaluate the presence of the HLA-DO(beta) chain in HeLa cells expressing the class II transactivator protein CIITA. Our flow cytometry and confocal microscopy analyses showed a marked expression of DO(beta) suggesting that HLA-DO could accumulate under the influence of CIITA in non-B cells.

Validity and reliability of simple nutrition screening tools adapted to the elderly population in healthcare facilities.

This study was conducted to assess the validity and the reliability of simple tools to screen the protein-energy malnutrition (PEM) risk among the elderly population in healthcare facilities. An initial screening tool, made up of nine PEM risk factors, was previously developed to be validated. This tool was quite complex and showed low validity results. A stepwise regression analysis determined significant risk factors (P < or = 0.05) among those included in the initial tool. These were the foundation to develop two simplified screening tools. One included Body Mass Index (BMI) and % weight loss over time. The second included BMI and albumin. Both tools classified subjects in low or high PEM risk levels. In the present study, the simple tools were assessed in a sample of 142 elderly subjects divided into two categories: acute care elderly (ACE, n=72) and long-term care elderly (LTCE, n=70). The simple tools were administered by a dietetic technician and a nurse with the purpose of assessing inter-rater and test-retest reliabilities. The criterion validity of the simple tools were assessed in comparison to in-depth nutritional assessments carried out by a dietitian. The validity results were ranked between 60.5% and 91.7%. The reliability scores showed levels of agreement of 70.8% to 93.1% and kappa coefficients ranking between 0.59(+/-0.07) and 0.79(+/-0.05). Simple tools are now available for efficiently screening the PEM risk among the elderly population on a healthcare facility-wide basis.

Quantitative relationship between MHC class II-superantigen complexes and the balance of T cell activation versus death.

The binding of bacterial superantigens (SAgs) is profoundly affected by the nature of the MHC class II-associated antigenic peptide. It was proposed that this limitation in the density of SAgs displayed at the surface of APCs is important for efficient TCR serial triggering as well as for preventing apoptosis of the responding T lymphocytes. Here, we have addressed quantitatively the size of this SAg-receptive pool of HLA-DR molecules that are available to bind and present staphylococcal enterotoxin A (SEA) at the surface of B lymphocytes. Our binding curves, depletion experiments, and quantitative immunoprecipitations show that about half the HLA-DR class II molecules on B cells are refractory to SEA binding. Yet, as compared with typical nominal Ags, an unusually high amount of class II-SAg complexes can be presented to T cells. This characteristic appears to be necessary for SAg-induced T cell apoptosis. When <0.3% of the total cell surface MHC class II molecules are occupied by SEA, T cells undergo a normal sequence of early activation events. However, presentation of a ligand density beyond this threshold results in T cell activation that is readily aborted by apoptosis but only after a few cell divisions. Thus, we confirm the existence of MHC class II subsets that are structurally unable to present SEA and provide a quantitative framework to account for the ability of bacterial SAgs to induce peripheral activation vs tolerance in the host.

Functional characterization of a lysosomal sorting motif in the cytoplasmic tail of HLA-DObeta.

HLA-DO is an intracellular non-classical class II major histocompatibility complex molecule expressed in the endocytic pathway of B lymphocytes, which regulates the loading of antigenic peptides onto classical class II molecules such as HLA-DR. The activity of HLA-DO is mediated through its interaction with the peptide editor HLA-DM. Here, our results demonstrate that although HLA-DO is absolutely dependent on its association with DM to egress the endoplasmic reticulum, the cytoplasmic portion of its beta chain encodes a functional lysosomal sorting signal. By confocal microscopy and flow cytometry analysis, we show that reporter transmembrane molecules fused to the cytoplasmic tail of HLA-DObeta accumulated in Lamp-1(+) vesicles of transfected HeLa cells. Mutagenesis of a leucine-leucine motif abrogated lysosomal accumulation and resulted in cell surface redistribution of reporter molecules. Finally, we show that mutation of the di-leucine sequence in DObeta did not alter its lysosomal sorting when associated with DM molecules. Taken together, these results demonstrate that lysosomal expression of the DO-DM complex is mediated primarily by the tyrosine-based motif of HLA-DM and suggest that the DObeta-encoded motif is involved in the fine-tuning of the intracellular sorting.

Alternative proteolytic processing of mouse mammary tumor virus superantigens.

Mouse mammary tumor viruses express a superantigen essential for their life cycle. It has been proposed that viral superantigens (vSags) require processing by prohormone convertases (PCs) for activity. We now observe, using a panel of mutant forms of potential PC cleavage sites and in vitro cleavage assays, that only the CS1 (position 68 to 71) and CS2 (position 169 to 172) sites are utilized by furin and PC5. Other members of the convertase family that are expressed in lymphocytes are not endowed with this activity. Furthermore, mutant forms of two different viral superantigens, vSag7 and vSag9, which completely abrogated in vitro processing by convertases, were efficient in functional presentation to responsive T-cell hybridomas. This effect was observed in both endogenous presentation and paracrine transfer of the vSag. Processing by convertases thus appears not to be essential for vSag function. Finally, we have identified the purified endosomal protease cathepsin L as another protease that is able to cleave convertase mutant vSag in vitro, yielding fragments similar to those detected in vivo, thus suggesting that proteases other than convertases are involved in the activation of vSags.

Understanding the mechanism of action of bacterial superantigens from a decade of research.

In the face of the unique diversity and plasticity of the immune system pathogenic organisms have developed multiple mechanisms in adaptation to their hosts, including the expression of a particular class of molecules called superantigens. Bacterial superantigens are the most potent stimulators of T cells. The functional consequences of the expression of superantigens by bacteria can be extended not only to T lymphocytes, but also to B lymphocytes and to cells of the myeloid compartment, including antigen-presenting cells and phagocytes. The biological effects of bacterial superantigens as well as their molecular aspects have now been studied for a decade. Although there is still a long way to go to clearly understand the role these molecules play in the establishment of disease, recently acquired knowledge of their biochemistry now offers unique experimental opportunities in defining the molecular rules of T-cell activation. Here, we present some of the most recent functional and molecular aspects of the interaction of bacterial superantigens with MHC class II molecules and the T-cell receptor.

Cellular distribution of a mixed MHC class II heterodimer between DRalpha and a chimeric DObeta chain.

Human MHC class II antigens include HLA-DR, -DQ, and -DP molecules that present antigens to CD4+ T cells, as well as the non-classical molecules HLA-DM and -DO. HLA-DM promotes peptide binding to class II molecules in endocytic compartments and HLA-DO, which is physically associated with HLA-DM in B lymphocytes, regulates HLA-DM function. Antibodies specific for the DObeta chain were obtained by immunization of mice with a heterodimer consisting of a chimeric DObeta chain (DR/DObeta), containing 18 N-terminal residues of DRbeta, paired with the DRalpha chain and isolated from transfected murine fibroblasts. The specificity of this serum for the DObeta chain and the lysosomal expression of the HLA-DO protein was confirmed using mutant human B cell lines lacking DR or DO molecules. The lysosomal localization of HLA-DO in human B cells contrasts with the cell surface expression of the mixed pair in transfected murine fibroblasts and raises questions concerning the role of the putative targeting motifs in HLA-DO. Transfection of the chimeric DR/DObeta chain along with DRalpha into human epithelial HeLa cells resulted in high levels of expression of the mixed isotypic pair at the surface of transfectants as well as in lysosomes. The same pattern was observed in HeLa cells transfected with the DObeta chimera and a DRa chain lacking the cytoplasmic tail. Taken together, these results suggest that functional sorting motifs exist in the DObeta chain but that the tight compartmentalization of HLA-DO observed inside B lymphocytes is controlled by the HLA-DOalpha chain and HLA-DM.

Conserved structural features between HLA-DO beta and -DR beta.

HLA-DO is a non-classical MHC class II molecule presumed to play a specialized role in the antigen processing pathway. We have modeled the HLA-DO beta-chain and found its overall structure compatible with the one of DR beta. Functional studies further highlighted the similarity between these beta-chains of the class II family of proteins. Indeed, a mixed heterodimer composed of the DR alpha and a chimeric DO beta-chains presented bacterial superantigens to T cells and was shown to interact with CD4. The implications of such structural conservation for the in vivo functions of HLA-DO are discussed.

Selective binding of bacterial toxins to major histocompatibility complex class II-expressing cells is controlled by invariant chain and HLA-DM.

Bacterial superantigens (SAgs) bind to major histocompatibility complex (MHC) class II molecules and activate T cells in a Vbeta-restricted fashion. We recently identified subsets of HLA-DR1 molecules that show selectivity for SAgs. Here, we extend these observations by showing that different cell lineages demonstrate distinct SAg-binding specificities although they all express HLA-DR1. Indeed, B cells bind staphylococcal enterotoxin A (SEA) and toxic shock syndrome toxin 1 (TSST-1) with high affinity while staphylococcal enterotoxin B (SEB) binding is barely detectable. In contrast, DR1-transfected HeLa cells show efficient binding of SEB, but not of SEA or TSST-1. We investigated the class II maturation events required for efficient interaction with SAgs and found that the ability of cells to bind and present the toxins can be drastically modulated by coexpression of the class II-associated invariant chain (Ii) and HLA-DM. SEA binding to DR1 molecules required coexpression of Ii, whereas TSST-1 binding was selectively enhanced by DM. Binding of SEB was affected by cell type-specific factors other than Ii or DM. The selectivity of SAgs for different MHC class II populations was minimally affected by HLA-DR intrinsic polymorphism and could not be explained by binding to alternative sites on DR molecules. Our results indicate that SAgs are sensitive to structural heterogeneity in class II molecules, which is consequent to the differential regulation of expression of antigen processing cofactors. Therefore, we speculate that Staphylococcus aureus have retained the ability to express numerous SAgs in adaptation to the micro-heterogeneity displayed by MHC class II molecules and that this may relate to their ability to infect different tissues.

Breast cancer survival: a phenomenological inquiry.

This inquiry describes the experience of 45 female breast cancer survivors using Schutz's interpretation of phenomenology as the theoretical framework. The research design is a multicase, comparative situational analysis. Breast cancer survival is explored from the points of view of all study participants. A dialectic is formed that juxtaposes etic and emic views of survival to enhance understanding of the meaning of breast cancer survival. Hermeneutic analysis yielded commonalities in meanings, situations, and life experiences. Analysis was further divided into thematic analysis, depiction of exemplars, and paradigm cases to provide clarity and vividness to the multifaceted phenomenon of breast cancer survival.

MHC class II-dependent peptide antigen versus superantigen presentation to T cells.

T lymphocytes expressing the CD4 coreceptor can be activated by two classes of major histocompatibility complex (MHC) class II-bound ligands. The elaboration of a conventional T-cell mediated immune response involves recognition of an antigenic peptide bound to the MHC class II molecules by a T-cell receptor (TCR) specific to that particular antigen. Conversely, superantigens (SAgs) also bind to MHC class II molecules and activate T cells, leading to a completely different functional outcome; indeed, SAg-responsive T cells die through apoptosis following stimulation. Superantigens are proteins that are secreted by various bacteria. They interact with the TCR using molecular determinants that are distinct from the residues involved in the recognition of nominal antigenic peptides. Despite the similarities between the recognition of the two classes of ligands by the TCR, considerable structural difference is observed. Here, we discuss the current knowledge on the presentation of SAgs to T cells and compare the different aspects of the SAg response with the recognition of antigenic peptide/MHC complexes.

Molecular characterization and role in T cell activation of staphylococcal enterotoxin A binding to the HLA-DR alpha-chain.

Superantigens bind to MHC class II-positive cells and stimulate T lymphocytes expressing specific V beta regions of the TCR. Two distinct regions of staphylococcal enterotoxin A superantigen (SEA) have been shown to affect the binding to MHC class II molecules. Results presented here demonstrate for the first time that the SEA-DR interaction can be affected by mutations on the class II alpha-chain. Furthermore, we have precisely mapped the interaction of the SEA N-terminal domain with the alpha1 domain of HLA-DR. Scatchard analysis using DAP cells transfected with mutant class II molecules showed a role for residue DR alpha K39 in the binding of SEA. Also, complementation experiments using mutant SEA molecules revealed an interaction between SEA residue F47 and position alphaQ18 on an outer loop of HLA-DR. These interactions between SEAF47 and the DR alpha-chain are critical, as they allow the recognition by an otherwise nonreactive V beta1+ T cell hybridoma and induction of tyrosine phosphorylation through the TCR.

Paracrine transfer of mouse mammary tumor virus superantigen.

Transfer of vSAG7, the endogenous superantigen encoded in the Mtv7 locus, from MHC class II to MHC class II+ cells has been suggested to occur both in vivo and in vitro. This transfer usually leads to the activation and deletion of T cells expressing responsive V beta s. However, there is no direct molecular evidence for such a transfer. We have developed an in vitro system which confirms this property of vSAGs. vSAG7 was transfected into a class II murine fibroblastic line. Coculture of these cells with class II+ cells and murine T cell hybridomas expressing the specific V beta s led to high levels of IL-2 production which was specifically inhibited by vSAG7- and MHC class II-specific mAbs. Moreover, injection of vSAG7+ class II+ cells in mice led to expansion of V beta 6+ CD4+ cells. We show that this transfer activity is paracrine but does not require cell-to-cell contact. Indeed, vSAG7 was transferred across semi-permeable membranes. Transfer can occur both from class II+ and class II+ cells, indicating that MHC class II does not sequester vSAG7. Finally, competition experiments using bacterial toxins with well defined binding sites showed that the transferred vSAG7 fragment binds to the alpha 1 domain of HLA-DR.

V alpha domain modulates the multiple topologies of mouse T cell receptor V beta20/staphylococcal enterotoxins A and E complexes.

The superantigens staphylococcal enterotoxin A and E (SEA and SEE) both contact major histocompatibility complex (MHC) class II molecules on two sites located on the alpha and beta chains. We have investigated the role of the T cell receptor (TCR) alpha chain in the modulation of the various topologies of TCR/SEA (or SEE)/class II complexes. For this purpose, we have used three mouse V beta20 T cell lines expressing different V alpha domains and two T cell hybridomas expressing mouse V beta1 or V beta11 segments. The response of these T cells to SEA and SEE was studied in the context of presentation by wild-type human MHC class II molecules; or by mutants on MHC, in each of the two superantigen binding sites (position alpha39K and beta81H) to which the superantigens can still bind but with an altered conformation. Although V beta20 T cell lines are efficiently stimulated using SEA and SEE presented by wild-type HLA-DR1 molecules, our results show that the nature of the TCR V alpha domain can affect differently the recognition of the toxins bound to mutant class II molecules. This suggests that various functional topologies exist for both SEA and SEE/class II complexes and that the T cell response to each of these complexes can be modulated by the V alpha domain of the TCR. Interestingly, the recognition of SEA and SEE is achieved in different fashions by a given V beta20 T cell line.