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Meiotic drivers are selfish alleles that subvert gametogenesis to increase their transmission into progeny. Drivers impose a fitness cost, putting pressure on the genome to evolve suppressors. Here we investigate the wtf gene family from Schizosaccharomyces pombe, previously shown to contain meiotic drivers in wild isolates. We discovered that wtf13 found in lab stocks is a meiotic driver. wtf13 kills spores that do not inherit it by generating both a diffusible poison and a spore-specific antidote. Additionally, we demonstrate that wtf13 is suppressed by another wtf gene, wtf18-2, that arose spontaneously in the lab and makes only an antidote. Wtf18-2 does not act indiscriminately to prevent spore destruction. Instead, it rescues only the spores that inherit wtf18-2. In this way, wtf18-2 selfishly gains a transmission advantage of its own while dampening the drive of wtf13. This establishes a novel paradigm for meiotic drive suppressors and provides insight into the mechanisms and evolution of drive systems. [ABSTRACT FROM AUTHOR]

Influenza virus immune human T-lymphocyte clones maintained in continuous culture in TCGF were analysed for helper activity and interleukin-2 (IL-2) production. The clones that functioned as helper cells in the production of specific antibody failed to release detectable amounts of IL-2. Conversely, the T cells that produced IL-2 were unable to provide either specific or non-specific helper function. These findings indicated the IL-2 is not an essential component for helper activity. However, phenotypic analysis revealed that both the functional subsets of T-cell clones expressed the helper phenotype in that they were T4+, T3+ and T11+ Nevertheless analysis with other antibodies revealed differences in that the IL-2 releasing clone showed greater staining with the anti-T-cell subset antibodies 9.3 and Leu 8, confirming that there is phenotype as well as functional heterogeneity within the helper inducer T-cell population. [ABSTRACT FROM AUTHOR]

A T-cell line (H3) was established by culturing human peripheral blood mononuclear cells with influenza virus A/X31 and maintained in long term culture with Interleukin-2 (TCGF). Supernatants were prepared by culturing these cells overnight in the absence of Interleukin-2 but with A/X31 and irradiated autologous E rosette negative cells as a source of antigen presenting cells, and harvesting by centrifugation. The supernatants were shown to replace T cells in helping E- (B) cells to produce antibody specific to A/X31 which was measured by enzyme immunoassay (EIA). Although maximal help was obtained with autologous or semi allogeneic B cells (in the latter case bearing HLA-DR 3 loci) there was still significant antibody production with allogeneic combinations. The supernatants were subsequently fractionated into specific and non-specific helper activities by gel filtration, giving an approximate tool. wt of 50–70,000 and 10–30,000 for each respectively. The specific HF was shown to be genetically restricted in its action upon B cells and also to generate antibody to A/X31 only. The lower molecular weight material acted on any responding B cell regardless of H LA-DR type and produced antibody non-specifically in culture with E- cells even in the absence of antigen. The apparent lack of restriction was therefore due to the masking effect of non-specific and non-restricted HF(s) on the genetically restricted specific HF produced by this line. [ABSTRACT FROM AUTHOR]

Helper factors specifically stimulating cooperative antibody responses by normal mouse spleen cells to a dinitrophenylated protein antigen from Streptococcus mutans (DNP-SA) were produced in vitro from monkey peripheral blood leucocytes and mouse spleen cells. The factors were partially characterized by gel filtration on Sephadex G-75, isoelectric focusing, treatment with heat and degradative enzymes and binding to specific immunoadsorbents. Gel filtration of both the monkey and mouse factors showed coelution with human serum albumin, suggesting a molecular weight of approximately 70,000. The isoelectric points fell within the range of 4·9-5·2 for monkey and 6·4-6·7 for the mouse helper factors. The glycoprotein nature of both factors was suggested by their lability to heat and sensitivity to pronase and neuraminidase. The factors carried a small fragment of the stimulating antigen and showed specific binding to SA but not to keyhole limpet haemocyanin (KLH). Monkey factor bound to rabbit antisera directed against the Fc portion of monkey IgM, but not to the IgG or IgA isotypes. The mouse factor contained determinants coded for by the I-Ak but not I-Jk subregion of the MHC. Both factors were absorbed by an antisenim to helper factor raised in rabbits against a KLH-specific mouse helper factor as immunogen. A corresponding antisenim to suppressor factor failed to adsorb either factor. This emphasizes the specific identities of helper and suppressor factors and suggests an evolutionary relationship between those derived from monkey and mouse leucocytes. [ABSTRACT FROM AUTHOR]

Non-human primates have been used to study immune function to a much lesser extent than readily available strains of inbred rodents. Nevertheless, in situations where it might be desirable, but impossible, to study human immune responses in vivo, lower primates could provide an acceptable alternative. In order to extend the knowledge of T- and B-lymphocyte function in lower primates, the common marmoset Callithrix jaccus was used as an experimental model. The functional similarities between this species and humans at the level of T-B co-operation in the antibody response were examined, and xenoreactive T-lymphocyte clones were obtained from marmoset spleen cells using Epstein—Barr virus (EBV)-transformed human B cells as stimulators. These clones could act as helper cells when co-cultured with human B lymphocytes, inducing the secretion of both IgM and IgG. Lymphokine production by mitogen-stimulated marmoset T-cell clones was also examined. Interleukins (IL) 2 and 4 activities were detected in clone supernatants using bioassays and interferon-gamma (IFN-γ) was detected using a solid-phase ELISA system. However, SDS—PAGE analysis of biosynthetically labelled marmoset and human T-cell clone supernatant proteins revealed major differences between the soluble T-cell products of the two species. The proliferative responses of marmoset T and B cells to recombinant human IL-2 and IL-4 were also examined. Stimulation of [3H]thymidine uptake was detected in both T cell- and anti-IgM-stimulated B-cell cultures with both of the lymphokines. These results suggests that the key components of the antibody response are functionally conserved between lower primates and man and that the common marmoset may be useful as an in vivo model of immune function, particularly with regard to the role of interleukins such as IL-2 and IL-4. [ABSTRACT FROM AUTHOR]

Peripheral blood mononuclear cells (PBMC) from a patient suffering from the hyper IgE syndrome were used to generate phytohaemagglutinin (PHA)-expanded T-cell clones (all CD4+, CD8-, CD23-). A selection of the clones was tested for their ability to help IgE secretion by culturing with normal B cells in the presence of solid-phase antibody to CD3. Supernatants were harvested on Day 7 and assayed by ELISA for IgE, IgG and IgM. Lymphokine secretion by the clones was assessed by culturing clones for 24 hr with solid-phase antibody to CD3 followed by assay of the supernatants for IL-2, IL-4 and interferon-gamma (IFN-γ) production. In addition, clones were analysed by flow cytometry for CDw29 and CD45R expression. Initial experiments with seven clones indicated that those clones that could help IgE secretion also stimulated optimal IgG and IgM responses. All clones appeared to secrete IL-2, IL-4 and IFN-γ, although the mounts of each varied. These results confirm recent findings that human T-cell clones do not fall into Tinf (Th1) and Th (Th2) type subsets as described in the muse. There was no clear correlation between the lymphokines secreted by the clones and their capacity to help IgE production. However, the helper function of the clones for all isotypes, including IgE, appeared to be related to the level of expression of the surface antigen CDw29. [ABSTRACT FROM AUTHOR]

An allergen-dependent in vitro model of immunoglobulin E (IgE) synthesis by human B cells is reported. Using this model, it is demonstrated that polyclonal T cells and CD4+ Dermatophagoides spp. (house dust mite)-specific 1-cell clones derived from an atopic, house dust mite (HDM)-allergic individual are able to support IgE synthesis by autologous B cells. The helper activity was interleukin- 4 (IL-4) dependent as only cloned T cells expressing detectable mRNA for IL-'I were able to induce IgE synthesis without the addition of exogenous IL-4. Peripheral and cloned I cells reactive with HDM could also be identified from a non-atopic individual but neither population was able to support IgE production even in the presence of exogenous IL-4. [ABSTRACT FROM AUTHOR]

The complex pleiotropic effects of the T-cell derived lymphokine interleukin-4 (IL-4) are becoming increasingly well documented; however, functional studies have been hampered by the lack of reagents directed against the receptor for this factor. In this report, we present data which suggest that the monoclonal antibody MR6 binds to the human interleukin-4 receptor (IL-4R). Addition of MR6 to cultures of T cells proliferating in response to IL-4 inhibited this response in a dose-dependent fashion, giving total inhibition at 10 μg/ml. Similarly, the IL-4-dependent production of specific antigen-induced IgE by B-cell populations was completely abrogated by MR6. Flow cytometric studies of the modulation of cell surface molecules after T-cell activation suggest that expression of the molecule detected by MR6 (p145-MR6) correlates inversely with that of the interleukin-2 receptor (IL-2R). These data, together with the previously determined molecular weight and tissue distribution of this molecule, strongly indicate that MR6 binds to the human IL-4R.. [ABSTRACT FROM AUTHOR]

Cloned human T lymphocytes and a mitogenic monoclonal antibody (UCHT1) that binds to the T3 antigen complex were used to study the role of inositol lipid hydrolysis in T-cell activation. Binding of the T3 molecular complex with anti-T3 antibody induced the generation of inositol trisphosphate after a lag of 1 min. While this is commensurate with the rise in cytosolic Ca2+ in these cells, examination of the inositol lipid revealed that phosphorylation of phosphatidylinositol 4-phosphate to phosphatidylinositol 4,5-bisphosphate occurred before the generation of inositol trisphosphate. Thus, activation of an inositol lipid kinase appears to be one of the primary signals in cellular activation in human T lymphocytes. [ABSTRACT FROM AUTHOR]

The development of Functional Genomics technologies has opened new avenues to investigate the complexity of the immune system. Microarray technology has been particularly successful because of its relatively low cost and high genome coverage. Consequently to our ability to monitor the expression of a significant proportion of an organism genome, our understanding of the molecular dynamics behind cell differentiation and cell response has greatly improved. Molecular signatures associated to immune cells have provided important tools to investigate the molecular basis of diseases and have been often associated to diagnostic and prognostic markers. The availability of such large collection of data has stimulated the application of complex machine learning techniques in the attempt to link molecular signatures and cell physiology. Here we review the most recent developments in the analysis of molecular signatures in the immune system.

In most protocols aimed at testing balance abilities, patients are generally required to close their eyes in order to gain insight about proprioceptive cues and the way the central nervous system (CNS) uses this information. However, one should not exclude possible interaction with the physiological mechanisms involved in eyelid closure, thus leading to a biased neurological evaluation. To assess this possible involvement, 15 healthy adults were required to keep their eyes open in the dark (YOn), to close normally (YF) and forcibly their eyelids (YFF), respectively in random order. The analysis was focused on elementary motions computed from the complex center of pressure (CP) trajectories, i.e. the horizontal motions of the center of gravity (CG(h)) and the difference between the CP and the vertical projection of the center of gravity (CP-CG(v)). The former is recognized as the main variable in postural control whilst several interesting features can be extracted from the latter: their link with the horizontal accelerations communicated to the CG, the level of muscular activity and the implied ankle stiffness expressed by their frequential distribution. The results indicate that the amplitudes of the CP-CG(v) spectra are statistically reduced in YOn when compared to the YF condition, especially in the antero-posterior direction. On the other hand, no shift in the frequential bandwidth was observed on these spectra, signifying a constancy in ankle stiffness over all conditions. Interestingly, the CNS does not really seem to gain from these reduced horizontal accelerations at the CG level since the CG(h) amplitudes are only slightly reduced. However, it is important to emphasize that the CP trajectories alone are not able to demonstrate any statistical trend. It can thus be hypothesized that, despite the fact that visual information is still unavailable, the proprioceptive cues nonetheless continue to play a minor role in the detection-correction process aimed at controlling body sway. Past studies on the physiology of eyes blinking have suggested the probability that the cerebellar cortex or brain stem structures (such as the reticular formation) are involved in these commands. Because of its dual facilitating-inhibiting function, the latter is indeed a fair candidate for modulating the descending command operating through the postural muscles. These data are of interest for the practician in order to assess with precision the role played by proprioceptive and visual cues in possible balance disfunctioning.

The recent publications in Nature and Science by the Human Genome Consortium and Celera Genomics, respectively, while being landmark achievements in themselves, have also given pause for thought. A definitive catalogue of human genes is still not available but the broad picture of how humans compare with lower organisms at the genomic level is becoming clearer. The full impact of these findings on the practice of medicine is hard to predict, but research being conducted now, in the early years of the 21st century, will form the basis of future advances in the diagnosis and treatment of disease. Exactly what this will entail is the subject of intense debate, but there are some common starting points that were discussed at this meeting in Munich. The main theme to emerge was the need to move beyond the human genome sequence towards an understanding of proteins and their interactions in complex biological pathways, thereby increasing opportunities for drug discovery through the identification of new targets. The majority of the talks were therefore devoted to the description of technological advances in the analysis of gene and protein expression (and interaction) and in the use of various methods of gene deletion in order to validate individual proteins as drug targets. Perhaps it will still be a few years before it will be possible to report on the application of genomic analyses to routine medical practice at the first point of care for patients but when that happens, the research efforts described here will have been worthwhile.

Synovial fluid proteins from microliter volumes of synovial fluid were resolved by two-dimensional polyacrylamide gel electrophoresis and detected by silver staining to investigate the feasibility of using two-dimensional (2D) electrophoresis in the clinical research setting and provide global disease information of disease progression. Several hundred proteins could be resolved as spots, many of which displayed the characteristic pattern of plasma-derived glycoproteins. The lowest level of detection was approximately 0.2 ng from a total of 50 microg of protein loaded. Most of the proteins could be identified on the basis of pI and molecular weight when compared with plasma protein maps on the World Wide Web. Unknown proteins were characterized by mass spectrometry of tryptic digests and by comparison with peptide databases. Synovial fluids from patients with rheumatoid arthritis were analyzed using this technique. Each subject received a fixed dose of antibody to CD4 as part of a phase II clinical trial to determine the efficacy of this immunosuppressive treatment in modifying disease activity. Synovial fluid was removed at day 0, followed by administration of antibody. Subsequent removal of synovial fluid and additional administration of antibody were carried out at different times thereafter. Changes in levels of acute-phase proteins were quantified by densitometry of silver-stained 2D polyacrylamide gels. Other parameters of disease progression such as serum C-reactive protein and physician's global assessment of clinical condition were used for comparison. In this way, changes in acute-phase proteins towards normal levels, as measured by 2D polyacrylamide gel electrophoresis, could be correlated with clinical improvement and conventional clinical chemistry measurements. Thus, the system can be used for quantitative analysis of protein expression in sites of autoimmune disease activity such as the synovial fluid of rheumatoid arthritis patients.

Many drug targets are components of complex signalling pathways, and in order to understand the true biological consequences of modulating these targets it is necessary to understand the biology of the system in great detail. Genomics research can contribute some of the tools to achieve this, for example through the use of cDNA microarrays. Since activation of signalling pathways leads to mRNA expression, microarray technology can be used to provide a detailed quantitative assessment of the consequences of this activation, often providing a completely new biological perspective on well established cellular systems. This review will discuss some of the results obtained using mRNA profiling of yeast and mammalian cells to analyse signalling pathways of relevance to inflammation and cancer, and will point towards the future applications of this exciting approach to drug target evaluation.

Subtractive hybridization of cDNAs generated from synovial RNA which had been isolated from patients with rheumatoid arthritis (RA) or normal controls was used in conjunction with high-density array hybridization to identify genes of immunological interest. The method was designed to detect gene expression in small needle biopsy specimens by means of a prior amplification of nanogram amounts of total RNA to full-length cDNA using PCR. The latter was cut with Rsa I, ligated with adapters, hybridized with unmodified driver cDNA, and subjected to suppression subtraction PCR. Differentially expressed products were cloned into E. coli and picked into 384 well plates. Inserts were obtained by PCR across the multiple cloning site, and the products arrayed at high density on nylon filters. The subtracted cDNAs were also labelled by random priming for use as probes for library screening. The libraries chosen were the subtracted one described above and a set of 45,000 ESTs from the I.M. A.G.E consortium. Clones showing positive hybridization were identified by sequence analysis and homology searching. The results showed that the subtracted hybridization approach could identify many gene fragments expressed at different levels, the most abundant being immunoglobulins and HLA-DR. The expression profile was characteristic of macrophage, B cell and plasma cell infiltration with evidence of interferon induction. In addition, a significant number of sequences without matches in the nucleotide databases were obtained, this demonstrates the utility of the method in finding novel gene fragments for further characterisation as potential members of the immune system. Although RA was studied here, the technology is applicable to any disease process even in cases where amounts of tissue may be limited.

Interleukin-5 (IL-5) plays a crucial role in the proliferation, differentiation and activation of eosinophils. The IL-5 receptor is composed of an IL-5-specific alpha subunit, which is expressed by eosinophils and basophils, and a beta c-subunit shared with the receptors for IL-3 and GM-CSF. We identified an AP-1 element which is important for IL-5R alpha promoter activity in eosinophilic HL60 cells. The AP-1 site and the previously identified EOS1 site cooperate, since single mutation of either of the sites decreased promoter activity. We show that the AP-1 site of the IL-5R alpha promoter binds multiple proteins, including cJun, CREB, and CREM.

Interleukin-5 is a T cell-derived cytokine with actions restricted to the eosinophil/basophil lineage and a subset of murine B cells. High affinity receptors have been identified and shown to comprise an IL-5-specific alpha chain (IL-5R alpha) in association with a beta chain which is shared with the receptors for IL-3 and GM-CSF. Nothing is currently known of the factors which regulate the transcription and subsequent expression of the IL-5 receptor alpha chain; this study was undertaken, therefore, in order to identify agents which modulate IL-5R alpha mRNA levels, with the goal of understanding the regulation of this gene in vivo. The human IL-5-dependent erythroleukemia TF-1 was used as a source of mRNA which was analysed by northern blotting using a cDNA probe for IL-5R alpha. A range of cytokines and pharmacological agents were used in 20 hour cultures of TF-1 followed by northern analysis. Of these, only TGF-beta 1 and PMA showed any effect, which was a selective downregulation, although the PMA displayed some cytotoxicity over the long culture period. The remainder (interleukins 1 to 11, G-CSF, GM-CSF, LIF, SCF, erythropoetin, IFN-gamma, RANTES, MIP-1 alpha, FGF, EGF, PDGF, dexamethasone, forskolin, retinoic acid and cyclosporin A) failed to alter expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Intravenous injection of goat antibodies to mouse IgD (GAMD) into BALB/c mice has been shown to induce vigorous T-cell dependent immunoglobulin responses, particularly of the IgG1 and IgE isotypes. We have confirmed these findings and show that IgA responses are also triggered in this model. Since the study of IgE regulation in allergic individuals is concerned with secondary and subsequent T- and B-cell responses, we boosted GAMD-primed mice with goat antibodies to IgE or IgA in an attempt to specifically retrigger IgE- and IgA-bearing memory B cells. However, we found that secondary IgG1, IgE and IgA production could be elicited equally well by either antibody preparation or by normal goat IgG (GIg). As with the primary response, GIg primed and boosted mice produced very low or undetectable IgG1, IgE and IgA responses. These data suggest that GAMD is very efficient at priming T cells specific for GIg epitopes and that once primed they can be readily re-triggered by GIg. Spleen cells taken 7 days after boosting GAMD-primed mice were found to spontaneously produce much higher levels of interleukin-6 (IL-6) in culture than cells from unboosted or GIg primed and boosted mice. In contrast to primary responses, where IgE levels return to background (less than 40 ng/ml) very quickly, circulating IgE levels in boosted mice initially declined before reaching a plateau level (approximately 1 microgram/ml) which was maintained for at least 148 days. IgG1 and IgA levels continued to fall over this same time period. Mice which had been primed (but not boosted) 10 months earlier were all found to have detectable IgE in their blood, despite the fact that following priming IgE becomes undetectable within 2-3 weeks. Since only a part of the IgE response was directed towards the antigen (GIg), these observations suggest the possibility that B cells initially primed to make IgE can be non-specifically retriggered in vivo.

Non-human primates have been used to study immune function to a much lesser extent than readily available strains of inbred rodents. Nevertheless, in situations where it might be desirable, but impossible, to study human immune responses in vivo, lower primates could provide an acceptable alternative. In order to extent the knowledge of T- and B-lymphocyte function in lower primates, the common marmoset Callithrix jaccus was used as an experimental model. The functional similarities between this species and humans at the level of T-B co-operation in the antibody response were examined, and xenoreactive T-lymphocyte clones were obtained from marmoset spleen cells using Epstein-Barr virus (EBV)-transformed human B cells as stimulators. These clones could act as helper cells when co-cultured with human B lymphocytes, inducing the secretion of both IgM and IgG. Lymphokine production by mitogen-stimulated marmoset T-cell clones was also examined. Interleukins (IL) 2 and 4 activities were detected in clone supernatants using bioassays and interferon-gamma (IFN-gamma) was detected using a solid-phase ELISA system. However, SDS-PAGE analysis of biosynthetically labelled marmoset and human T-cell clone supernatant proteins revealed major differences between the soluble T-cell products of the two species. The proliferative responses of marmoset T and B cells to recombinant human IL-2 and IL-4 were also examined. Stimulation of [3H]thymidine uptake was detected in both T cell- and anti-IgM-stimulated B-cell cultures with both of the lymphokines. These results suggests that the key components of the antibody response are functionally conserved between lower primates and man and that the common marmoset may be useful as an in vivo model of immune function, particularly with regard to the role of interleukins such as IL-2 and IL-4.