The two membrane proximal domains of CD4 interact with the T cell receptor.

During T cell activation, CD4 is intimately involved in colocalizing the T cell receptor (TCR) with its specific peptide ligand bound to class II molecules of the major histocompatibility complex (MHC). Previously, the COOH-terminal residues, Trp62/63, which flank the immunodominant epitope of hen egg lysozyme (HEL 52-61), were shown to have a profound effect on TCR recognition. CD4 maintains the fidelity of this interaction when short peptides are used. To determine which portion of CD4 was responsible for this effect, a series of CD4 mutants were made and transfected into CD4 loss variants of two HEL 52-61-specific T cell hybridomas. Surprisingly, some CD4 mutants that failed to interact with MHC class II molecules (D2 domain mutant) or with p56kk (cytoplasmic-tailless mutant) restored responsiveness. Nevertheless, a significant reduction in association between cytoplasmic-tailless CD4 and the TCR, as determined by fluorescence resonance energy transfer, was observed. Thus, neither colocalization of CD4 and the TCR nor signal transduction via CD4 was solely responsible for the functional restoration of these T cell hybridomas by wild-type CD4. However, substitution of the two membrane proximal domains of murine CD4 (D3 and D4) with domains from human CD4 or intercellular adhesion molecule 1 not only abrogated its ability to restore function, but also substantially reduced its ability to associate with the TCR. Furthermore, the mouse/human CD4 chimera had a potent dominant negative effect on T cell function in the presence of equimolar concentrations of wild-type CD4. These data suggest that the D3/D4 domains of CD4 may interact directly or indirectly with the TCR-CD3 complex and influence the signal transduction processes. Given the striking structural differences between CD4 and CD8 in this region, these data define a novel and unique function for CD4.

Simultaneous quantitation of 15 cytokines using a multiplexed flow cytometric assay.

Several methods have been developed to quantify cytokines and chemokines in biological fluids and tissue culture samples, including bioassays, enzyme-linked immunosorbent assay (ELISA), intracellular staining, ribonuclease protection assay (RPA) and polymerase chain reaction (PCR). However, each of these techniques possesses one or more significant limitations. Here, we describe a new multiplexed assay, using the FlowMetrix system, that can quantify multiple cytokines simultaneously in a small sample volume. This assay was found to be more accurate, sensitive and reproducible than the conventional microtitre ELISA procedure. Furthermore, the time and cost involved are comparable to, or less than, the ELISA. A key feature of the FlowMetrix assay is its ability to multiplex: here, we show that this assay can accurately quantitate 15 cytokines in a 100 microl sample volume while the same analysis by ELISA requires 1.5 ml (100 microl for each cytokine assay). By using this Flow Metrix assay, we could demonstrate that only T helper 1 (T(H)1)-deviated cells produce detectable levels of interleukin (IL)-2, while only T(H)2-deviated cells produce significant amounts of IL-4. Six other cytokines were produced by both T cell subsets, with the T(H)1 population producing more IL-3, granulocyte-monocyte colony stimulating factor (GM-CSF) and interferon (IFN)-gamma, and the T(H)2 population producing more IL-5, IL-10, and IL-13. Seven other cytokines were not produced in detectable amounts. This assay should prove to be a powerful tool in the quantitation of cytokines, or any other soluble product for which antibody pairs are available. It will also provide a more complete picture of the plethora of cytokines secreted during an immune response.

Population-based time preferences for future health outcomes.

CONTEXT: Time preference (how preference for an outcome changes depending on when the outcome occurs) affects clinical decisions, but little is known about determinants of time preferences in clinical settings. OBJECTIVES: To determine whether information about mean population time preferences for specific health states can be easily assessed, whether mean time preferences are constant across different diseases, and whether under certain circumstances substantial fractions of the patient population make choices that are consistent with a negative time preference. DESIGN: Self-administered survey. SETTING: Family physician waiting rooms in four states. PATIENTS: A convenience sample of 169 adults. INTERVENTION: Subjects were presented five clinical vignettes. For each vignette the subject chose between interventions maximizing a present and a future health outcome. The options for individual vignettes varied among the patients so that a distribution of responses was obtained across the population of patients. MAIN OUTCOME MEASURE: Logistic regression was used to estimate the mean preference for each vignette, which was translated into an implicit discount rate for this group of patients. RESULTS: There were marked differences in time preferences for future health outcomes based on the five vignettes, ranging from a negative to a high positive (116%) discount rate. CONCLUSIONS: The study provides empirical evidence that time preferences for future health outcomes may vary substantially among disease conditions. This is likely because the vignettes evoked different rationales for time preferences. Time preference is a critical element in patient decision making and cost-effectiveness research, and more work is necessary to improve our understanding of patient preferences for future health outcomes.

Psychometric validation of symptom severity measures in irritable bowel syndrome with constipation.

BACKGROUND: Historically, measures of symptom severity of irritable bowel syndrome with constipation (IBS-C) in clinical trials have not met the evidence requirements described in the FDA guidance on patient-reported outcomes (PROs), which describes the evidentiary requirements and review criteria for patient-reported outcome measures intended to support product approval or labelling claims. AIM: Data from two phase 3 trials (N = 1608) of linaclotide for the treatment of IBS-C were analysed to evaluate the psychometric properties of patient-reported outcome measures assessing changes in the severity of abdominal and bowel symptoms. METHODS: A set of patient-reported outcome assessments addressing abdominal and bowel symptoms, the IBS-C Symptom Severity Measures, were administered daily using interactive voice response system technology. Intraclass correlation coefficients (ICCs), Pearson correlations, factor analyses, F-tests and effect sizes were computed to evaluate the reliability, construct validity, discriminating ability and responsiveness of the IBS-C Symptom Severity Measures in a clinical trial context. RESULTS: The IBS-C Symptom Severity Measures showed highly satisfactory test-retest reliability (ICCs ranging from 0.79 to 0.95) and construct validity. Factor analyses indicated one factor for abdominal symptoms and another for bowel symptoms. Known-groups F-tests comparing subgroups based on various responder definitions were statistically significant and in the expected direction, substantiating the discriminating ability of the IBS-C Symptom Severity Measures. Responsiveness statistics (ranging from 0.6 to 2.1) demonstrated these measures are also capable of detecting change. CONCLUSIONS: The psychometric analysis results strongly support the reliability, construct validity, discriminating ability and responsiveness of the IBS-C Symptom Severity Measures and substantiate the conclusion of linaclotide treatment benefit.

An evaluation of the FDA responder endpoint for IBS-C clinical trials: analysis of data from linaclotide Phase 3 clinical trials.

BACKGROUND: Our objective was to evaluate the performance of the Food and Drug Administration (FDA) Responder Endpoint for clinical trials in IBS-C, using data from two large Phase 3 clinical trials of linaclotide. The FDA interim endpoint requires that, for 50% of trial weeks, patients report ≥30% decrease in Abdominal Pain at its worst and (in the same week) an increase in Complete Spontaneous Bowel Movements (CSBMs) of ≥1 from baseline. METHODS: Anchor-based methodology was used to estimate thresholds of clinically meaningful change using symptom-specific patient rating of change questions (PRCQs) and symptom severity questions. The diagnostic accuracy of the FDA Responder Endpoint was assessed using sensitivity/specificity-based methods. KEY RESULTS: Using anchor-based methods, the estimates of the clinically meaningful improvement thresholds for Abdominal Pain ranged from 25.9% to 32.4% and thresholds for increase in weekly CSBM rate ranged from 1.4 to 1.6 CSBMs per week. Compared with the symptom-specific PRCQs for patient rating of relief, the FDA Responder Endpoint has a sensitivity of 60.7%, a specificity of 93.5%, and an accuracy of 82.0%. Changing the number of weeks required to be a responder or the percentage improvement in the Abdominal Pain criteria did not result in notable improvement in the accuracy of the FDA Responder Endpoint. CONCLUSIONS & INFERENCES: The FDA Responder Endpoint for IBS-C clinical trials represents clinically meaningful improvements in IBS-C symptoms for patients with excellent specificity and reasonable sensitivity.

Baseline risk and preference for reductions in risk-to-life.

The typical model of individual attitudes toward risk-to-life suggests that an individual's willingness to pay for a reduction in mortality risk increases with the baseline risk. The higher-baseline hypothesis has been the subject of several empirical tests but results have so far been mixed. Using survey evidence, we present a situation in which subjects do prefer to reduce risks for which the baseline is higher. This finding is robust to several alternative explanations. Survey responses reflect subjects' concerns about government effectiveness in risk reduction, environmental effects associated with the various hazards, and other idiosyncratic elements of the risks; however, these concerns appear to occur in addition to, not in lieu of, the preference to reduce higher risks.

Influence of Gm allotype on the IgG subclass response to streptococcal M protein and outer membrane proteins of Moraxella catarrhalis.

The IgG antibody response to streptococcal M protein is distributed between the IgG1 and IgG3 subclasses, however individual sera vary with respect to the relative amounts of these two subclasses. The basis of this variation was investigated. Sera were also analysed for IgG subclass antibodies to the outer membrane proteins (OMP) of Moraxella catarrhalis, as these have also been reported to have a major IgG3 component. The mean percentage of IgG3 was higher in the antibody response to OMP and there was less variability between sera for this antigen than was seen for M protein. Non-specific binding of IgG3 in ELISA, which has been reported for some bacterial proteins (including M protein of some serotypes) was excluded as an explanation for the apparent IgG3 bias of these antibodies. The relative amount of IgG3 antibody to the two antigens showed a positive correlation, suggesting that some individuals tended to make a greater IgG3 response to unrelated antigens. Serial bleeds from two individuals maintained a relatively constant subclass profile over several months, suggesting that time since infection did not play a major role in determining the proportion of IgG1 and IgG3. Gm allotypes for the sera were determined, and found to correlate with both total serum IgG3 concentrations and with IgG subclass composition of specific antibodies. Mean serum IgG3 concentrations were highest in sera typed as Gm(fb/fb) homozygous and lowest in sera typed as Gm(ag/ag) homozygous. Similarly, in the M protein-specific antibodies, the mean percentage of IgG3 was much lower in the Gm(ag/ag) sera than in the Gm(fb/fb) homozygous sera. Sera which typed as Gm(fb/ag) heterozygous were not significantly different from the Gm(fb/fb) homozygous sera for either total serum IgG3 or for M protein-specific IgG3. Moreover, both Gm(fb/fb) homozygous and Gm(fb/ag) heterozygous sera included samples in which IgG1 was the predominant antibody subclass and the percentage of IgG3 was very low. In contrast to the M protein-specific antibodies, for the OMP-specific antibodies there was no correlation between Gm phenotype and the proportion of IgG3. The data suggest that Gm allotype may influence the IgG subclass composition of antibody responses to bacterial surface protein, but that other factors are also likely to be involved.

HEL-Flu: an influenza virus containing the hen egg lysozyme epitope recognized by CD4+ T cells from mice transgenic for an alphabeta TCR.

Reverse genetics was used to modify the influenza virus genome by inserting the p46-63 sequence of hen egg lysozyme (HEL) into the neuraminidase stalk of the virus. The resulting virus, HEL-Flu, contained the epitopes recognized by CD4+ T cells from 3A9-TCR transgenic mice (C3HTg). Here, we show that HEL-Flu was infectious in the respiratory tract of both C3H and C3HTg mice, the latter animals showing an early, transient morbidity. Splenic dendritic cells and certain cloned populations of splenic macrophages and brain microglia constitutively presented infectious and inactivated HEL-Flu to the T cells in an Ag-specific and MHC class II-restricted manner. These results demonstrate the utility of HEL-Flu in assessing the APC activity for naive T cells; they also extend the previous studies showing that discrete populations of macrophages and microglia constitutively process and present Ag to naive T cells.

T cell receptor recognition of MHC class II-bound peptide flanking residues enhances immunogenicity and results in altered TCR V region usage.

Naturally processed MHC class II-bound peptides possess ragged NH2 and COOH termini. It is not known whether these peptide flanking residues (PFRs), which lie outside the MHC anchor residues, are recognized by the TCR or influence immunogenicity. Here we analyzed T cell responses to the COOH-terminal PFR of the H-2A(k) immunodominant epitope of hen egg lysozyme (HEL) 52-61. Surprisingly, the majority of T cells were completely dependent on, and specific for, the COOH-terminal PFR of the immunogen. In addition, there were striking correlations between TCR V beta usage and PFR dependence. We hypothesize that the V alpha CDR1 region recognizes NH2-terminal PFRs, while the V beta CDR1 region recognizes COOH-terminal PFRs. Last, peptides containing PFRs were considerably more immunogenic and mediated a greater recall response to the HEL protein. These results demonstrate that PFRs, which are a unique characteristic of peptides bound to MHC class II molecules, can have a profound effect on TCR recognition and T cell function. These data may have important implications for peptide-based immunotherapy and vaccine development.

Immunoregulation of Th cells by naturally processed peptide antagonists.

Th cells recognize protein Ags as short peptides bound to MHC class II molecules. Altered peptide ligands can antagonize (inhibit) T cell responses to stimulatory peptides. Peptides generated by APC may contain peptide flanking residues (PFR), which lie outside the minimal binding epitope and can be recognized by the TCR. Our data show that PFR-dependent T cells were found to be potently antagonized by peptides that lack PFR and responded poorly to native protein or the immunogenic epitope delivered by a recombinant influenza virus. These data provide the first evidence that Ag processing generates both stimulatory and antagonist peptides from a single immunogenic epitope, an observation that may have important implications for T cell immunoregulation and autoimmunity.

Requirement for Stat4 in interleukin-12-mediated responses of natural killer and T cells.

Signal transducers and activators of transcription (STATs) are activated by tyrosine phosphorylation in response to cytokines and mediate many of their functional responses. Stat4 was initially cloned as a result of its homology with Stat1 (refs 4, 5) and is widely expressed, although it is only tyrosine-phosphorylated after stimulation of T cells with interleukin (IL)-12 (refs 6,7). IL-12 is required for the T-cell-independent induction of the cytokine interferon (IFN)-gamma, a key step in the initial suppression of bacterial and parasitic infections. IL-12 is also important for the development of a Th1 response, which is critical for effective host defence against intracellular pathogens. To determine the function of Stat4 and its role in IL-12 signalling, we have produced mice that lack Stat4 by gene targeting. The mice were viable and fertile, with no detectable defects in haematopoiesis. However, all IL-12 functions tested were disrupted, including the induction of IFN-gamma, mitogenesis, enhancement of natural killer cytolytic function and Th1 differentiation.

Lack of IL-4-induced Th2 response and IgE class switching in mice with disrupted Stat6 gene.

Signal transducers and activators of transcription (Stats) are activated by tyrosine phosphorylation in response to cytokines, and are thought to mediate many of their functional responses. Stat6 is activated in response to interleukin (IL)-4 and may contribute to various functions including mitogenesis, T-helper cell differentiation and immunoglobulin isotype switching. To evaluate the role of Stat6, we generated Stat6-null mice (Stat6 -/-) by gene disruption in embryonic stem cells. The mice were viable, indicating the lack of a non-redundant function in normal development. Although naive lymphoid cell development was normal, Stat6 -/- mice were deficient in IL-4-mediated functions including Th2 helper T-cell differentiation, expression of cell surface markers, and immunoglobulin class switching to IgE. In contrast, IL-4-mediated proliferation was only partly affected.