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.

T cell receptor-major histocompatibility complex class II interaction is required for the T cell response to bacterial superantigens.

Bacterial and retroviral superantigens (SAGs) stimulate a high proportion of T cells expressing specific variable regions of the T cell receptor (TCR) beta chain. Although most alleles and isotypes bind SAGs, polymorphisms of major histocompatibility complex (MHC) class II molecules affect their presentation to T cells. This observation has raised the possibility that a TCR-MHC class II interaction can occur during this recognition process. To address the importance of such interactions during SAG presentation, we have used a panel of murine T cell hybridomas that respond to the bacterial SAG Staphylococcal enterotoxin B (SEB) and to the retroviral SAG Mtv-7 when presented by antigen-presenting cells (APCs) expressing HLA-DR1. Amino acid substitutions of the putative TCR contact residues 59, 64, 66, 77, and 81 on the DR1 beta chain showed that these amino acids are critical for recognition of the SAG SEB by T cells. TCR-MHC class II interactions are thus required for T cell recognition of SAG. Moreover, Mtv-7 SAG recognition by the same T cell hybridomas was not affected by these mutations, suggesting that the topology of the TCR-MHC class II-SAG trimolecular complex could be different from one TCR to another and from one SAG to another.

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.

Binding sites for bacterial and endogenous retroviral superantigens can be dissociated on major histocompatibility complex class II molecules.

Bacterial and retroviral superantigens (SAGs) interact with major histocompatibility complex (MHC) class II molecules and stimulate T cells upon binding to the V beta portion of the T cell receptor. Whereas both types of molecules exert similar effects on T cells, they have very different primary structures. Amino acids critical for the binding of bacterial toxins to class II molecules have been identified but little is known of the molecular interactions between class II and retroviral SAGs. To determine whether both types of superantigens interact with the same regions of MHC class II molecules, we have generated mutant HLA-DR molecules which have lost the capacity to bind three bacterial toxins (Staphylococcus aureus enterotoxin A [SEA], S. aureus enterotoxin B [SEB], and toxic shock syndrome toxin 1 [TSST-1]). Cells expressing these mutated class II molecules efficiently presented two retroviral SAGs (Mtv-9 and Mtv-7) to T cells while they were unable to present the bacterial SAGs. These results demonstrate that the binding sites for both types of SAGs can be dissociated.

Cross-linking of major histocompatibility complex class II molecules by staphylococcal enterotoxin A superantigen is a requirement for inflammatory cytokine gene expression.

Staphylococcal enterotoxin A (SEA) has two distinct binding sites for major histocompatibility complex (MHC) class II molecules. The aspartic acid located at position 227 (D227) in the COOH terminus of SEA is one of the three residues involved in its interaction with the DR beta chain, whereas the phenylalanine 47 (F47) of the NH2 terminus is critical for its binding to the DR alpha chain. Upon interaction with MHC class II molecules, SEA triggers several cellular events leading to cytokine gene expression. In the present study, we have demonstrated that, contrary to wild-type SEA, stimulation of the THP1 monocytic cell line with SEA mutated at position 47 (SEAF47A) or at position 227 (SEAD227A) failed to induce interleukin 1 beta and tumor necrosis factor-alpha messenger RNA expression. Pretreatment of the cells with a 10-fold excess of either SEAF47A or SEAD227A prevented the increase in cytokine messenger RNA induced by wild-type SEA. However, cross-linking of SEAF47A or SEAD227A bound to MHC class II molecules with F(ab')2 anti-SEA mAb leads to cytokine gene expression, whereas cross-linking with F(ab) fragments had no effect. Taken together, these results indicate that cross-linking of two MHC class II molecules by one single SEA molecule is a requirement for cytokine gene expression.

HLA-DR polymorphism affects the interaction with CD4.

Major histocompatibility complex (MHC) class II molecules are highly polymorphic and bind peptides for presentation to CD4+ T cells. Functional and adhesion assays have shown that CD4 interacts with MHC class II molecules, leading to enhanced responses of CD4+ T cells after the activation of the CD4-associated tyrosine kinase p56lck. We have addressed the possible contribution of allelic polymorphism in the interaction between CD4 and MHC class II molecules. Using mouse DAP-3-transfected cells expressing different isotypes and allelic forms of the HLA-DR molecule, we have shown in a functional assay that a hierarchy exists in the ability of class II molecules to interact with CD4. Also, the study of DR4 subtypes minimized the potential contribution of polymorphic residues of the peptide-binding groove in the interaction with CD4. Chimeras between the DR4 or DR1 molecules, which interact efficiently with CD4, and DRw53, which interacts poorly, allowed the mapping of polymorphic residues between positions beta 180 and 189 that can exert a dramatic influence on the interaction with CD4.

Subsets of HLA-DR1 molecules defined by SEB and TSST-1 binding.

Superantigens bind to major histocompatibility complex class II molecules on antigen-presenting cells and stimulate T cells. Staphylococcus aureus enterotoxin B (SEB) and toxic shock syndrome toxin-1 (TSST-1) bind to the same region of human lymphocyte antigen (HLA)-DR1 but do not compete with each other, which indicates that they bind to different subsets of DR1 molecules. Here, a mutation in the peptide-binding groove disrupted the SEB and TSST-1 binding sites, which suggests that peptides can influence the interaction with bacterial toxins. In support of this, the expression of the DR1 molecule in various cell types differentially affected the binding of these toxins.

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.

Expression in Escherichia coli of the 36 kDa domain of poly(ADP-ribose) polymerase and investigation of its DNA binding properties.

We have expressed in Escherichia coli the 36 kDa domain of the human poly(ADP-ribose) polymerase. This polypeptide comprises the C-terminal part of the DNA binding domain, as well as the automodification region of the enzyme, but lacks the zinc-finger motifs of the N-terminal region and the C-terminal catalytic domain. By probing the crude E. coli protein extracts with radioactive DNA probes (South-Western blots), we have shown that the 36 kDa domain binds a DNA probe of 222 bp but does not bind a shorter probe of 66 bp. This interaction is stronger when the polypeptide is fused to the 55 kDa catalytic domain of the enzyme.

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.

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.

Selection of mouse cells with amplified metallothionein genes retaining their glucocorticoid inducibility.

Two new mouse cell mutants, resistant to either 80 or 100 mM CdCl2, were isolated to study the regulation of transcription by the glucocorticoid hormones. Their metallothionein mt-1% and mt-2+ genes were amplified coordinately to a maximum of 30 copies per cell. By Southern blot analysis, no gross rearrangement was detectable near the mt+ loci. Contrary to other mutants previously isolated, the metallothionein-specific mRNAs of these mutants are inducible by dexamethasone.

Structural analysis of the putative regulatory region of the rat gene encoding poly(ADP-ribose) polymerase.

A lambda EMBL3 clone containing the first three exons along with part of the 4th exon of the rat poly(ADP-ribose) polymerase gene was isolated from a genomic DNA library. This clone also contains 6.6 kbp of upstream sequences. Nucleotide sequence analysis of the proximal 5' 670 nucleotides flanking the major RNA start site of the rat gene does not reveal significant global homology with the same region of the human gene, but a series of short sequences are identical. Among these sequences are found two putative Sp1 binding sites along with a decanucleotide sequence responsible for the attachment of the transcription factor AP-2.

Expression in E. coli of the catalytic domain of rat poly(ADP-ribose)polymerase.

A 2 kilobase pair cDNA coding for the entire C-terminal catalytic domain of rat poly(ADP-ribose)polymerase has been expressed in E. coli. The overproduced 55 kDa polypeptide is active in synthesizing poly(ADP-ribose) and the 4 kDa N-terminal region of this domain is recognized by the monoclonal antibody C I,2 directed against the calf enzyme. Also, the minor alpha-chymotrypsin cleavage site found in the human catalytic domain is not present in the rat enzyme as revealed by the absence of the 40 kDa specific degradation product in the E. coli cells expressing the rat domain. The expression of this partial rat cDNA should thus permit the rapid purification and subsequent crystallization of the catalytic domain of the enzyme.

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.

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.

Evolution of a nursing center.

The collaborative spirit that characterized the project period resulted in continuation of the nursing center after the expiration of the contract. The town purchased the modular unit and provides ongoing maintenance, housekeeping services, and utilities. A secretary is funded through a program providing employment for older adults. The University provides the salary for one full-time certified NP to manage the center, and students and faculty together provide care and continue the services of the center. An advisory committee composed of representatives from the town, senior center, and university oversees the ongoing activities of the center. Although the center has evolved from a small room to a fully equipped modular unit, the purpose of providing high-quality nursing care through education, practice, and research remains the same.

Ocular timolol levels after drug withdrawal: an experimental model.

Carbon 14-labelled timolol maleate was instilled into both eyes of 12 pigmented rabbits daily for 42 days. Drug levels in the aqueous humour and ocular tissues were measured up to 42 days after drug withdrawal. The results indicate that timolol concentrates mainly in melanotic tissues, with slow release. Even 42 days after withdrawal the drug was still present in pigmented ocular tissues. Timolol was detected in the aqueous up to 5 days after withdrawal. These findings explain the long-term depressant effect of topically administered timolol on aqueous production. We conclude that lower or less frequent doses of timolol should be considered in patients with glaucoma.

Moving toward a nursing model in advanced practice.

The nurse practitioner focus has been evolving from a medical to a nursing orientation since the inception of the role in 1965. The purpose of this study was to examine relationships between role attitudes and values, confidence about practice knowledge and skills, and orientation to a medical or nursing model to guide practice. A national random sample of 482 nurse practitioners completed the Attitudes and Values Scale, the Confidence in Skills Scale, and a demographic survey. Findings indicate that nurse practitioners are very confident about their practice skills and knowledge and have a very strong nursing orientation. There is a direct positive correlation between level of confidence and degree of nursing orientation. Nurses in the sample also rated themselves as more confident about hands-on skills than indirect role components such as utilization of research, change theory, and evaluation of practice outcomes. The discussion interweaves this study's findings with role theory and offers a comparison and contrast to the existing body of knowledge.

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.