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Signal transducer and activator of transcription (Stat)6 is a transcription factor important for the development of Th2 cells and regulation of gene expression by IL-4 and IL-13. It is known that Stat6 is rapidly activated in response to IL-4; however, the fate of activated Stat6 is less clear. We examined the fate of activated Stat6 and found that during continuous exposure to IL-4, Stat6 activity was sustained for 72 h and that the maintenance of a constant level of activated Stat6 did not require new protein synthesis. In contrast, when cells were pulsed with IL-4 and then incubated in the absence of IL-4, the half-life of Stat6 phosphorylation and DNA binding activity was less than 1 h. Stat6 did not accumulate in the nucleus, and protein degradation did not play a major role in the disappearance of activated Stat6. Inhibition of kinase activity by staurosporine or the JAK inhibitor, AG490, revealed that maintenance of Stat6 activation in the continuous presence of IL-4 required ongoing phosphorylation of latent cytoplasmic Stat6 molecules. Cells treated with an inhibitor of nuclear export, leptomycin B, were unable to maintain Stat6 activation. Thus, the maintenance of Stat6 activation requires a constant cycle of activation, deactivation, nuclear export, and reactivation.

Asthma is a complex polygenic disease. Many studies have implicated the importance of IL-4Rα in the development of allergic inflammation and its gene has been implicated in the genetics of asthma and atopy. In this study, we examined the functional consequences of two of the human IL-4Rα allelic variants that have been found to associate with asthma and atopy. We examined the effects of each variant alone and in combination on IL-4-dependent gene induction. We found that neither the Q576R nor the I75V variants affected IL-4-dependent CD23 expression. However, the combination of V75R576 resulted in expression of an IL-4Rα with enhanced sensitivity to IL-4. We next examined the genetics of five of the known IL-4Rα allelic variants in asthmatic and nonatopic populations. Strikingly, the association of V75/R576 with atopic asthma was greater than either allele alone and the association of R576 with atopic asthma was dependent on the coexistence of V75. A haplotype analysis revealed a single IL-4Rα haplotype that was associated with allergic asthma, VACRS, further confirming the importance of the V75 and R576 combination in the genetics of asthma. This is the first report demonstrating that a functional alteration in IL-4Rα requires the coexistence of two naturally occurring single nucleotide polymorphisms (snps) in combination; neither snp alone is sufficient. These data illustrate the importance of studying snps in combination, because the functional significance of a given snp may only be evident in a specific setting of additional snps in the same or different genes.

Stroke can result in Hemiparesis, often resulting in the disuse of impaired limbs and the increased use of the unimpaired limbs. Disuse leads to muscle atrophy, contracture and stiffening of the joints, which impedes daily living and quality of life. Physical therapy can maintain or increase motor function following stroke, but the impaired limb may still have less motor function than the unimpaired limb, leading to further disuse. Thus negating the gains accomplished by therapy. The development of devices that assist motor function of the impaired limbs during daily activities would promote use and help maintain any progress from physical therapy. Automated motion assistance can overcome spasticity by allowing a user to perform a specific task multiple times, thus reducing the stiffness developed in the fingers. The purpose of this study was to develop prototype modules of a glove-like system for hand grasping assistance. The full system could be used during therapy sessions or worn to assist with activities of daily living. The force sensors positioned in the glove on the front and back of each fingertip would be used to determine whether an attempt is being made to grasp or to let go of an object. The system controls motors that wind or unwind cables that run along the forearm and connect independently to the glove for each finger. The modules were designed and assembled for testing. A simulation model was built for analysis. The early results appear promising, but further testing will be required to make developments toward the full system to assist with hand motor function.

Asthma and other atopic disorders affect a large percentage of the population. While many factors contribute to the phenotype of asthma, there is a strong genetic predisposition. IL-4 is a central mediator of allergic inflammation. Along with IL-13, it is the major cytokine responsible for the induction of IgE synthesis. Furthermore, IL-4 acts on Th0 cells and promotes their differentiation into Th2 cells resulting in the production of more IL-4 and IL-13, thereby propagating the allergic cascade. Both IL-4 and IL-13 utilize IL-4Ralpha as a component of their cognate receptor complexes. Eight polymorphisms of the IL-4Ralpha gene resulting in amino acid changes in the coding sequence have been described, and several have been associated with asthma. The central objective of this study was to elucidate the role of the Ser786Pro polymorphism in asthma and its impact on IL-4R function. One-hundred ninety-six individuals with asthma and 53 controls were genotyped for Pro786. Pro786 occurred infrequently in the general population with an allele frequency of 1.8% and, thus, is unlikely to play a major role in atopy or asthma. The Pro786 allele frequency was 1.5% in the asthma group and 2.8% in the control group. The asthma group was subdivided into allergic and nonallergic asthma, and the Pro786 allele frequencies were 1.7 and 1.0%, respectively. The data suggested linkage disequilibrium between Ser786Pro and the Gln576Arg allele, which is associated with atopy. In order to study the impact of the polymorphism on receptor signaling function, we transfected a mouse B lymphoma cell line with the wild-type and Pro786 variants of human IL-4Ralpha. The Ser786Pro polymorphism in isolation did not affect IL-4R function.

Sulfhydryl-2 domain-containing tyrosine phosphatase-1 (SHP-1) has an important role in the negative regulation of many receptors including the interleukin (IL)-4 receptor. Motheaten mice (me/me) have a homozygous mutation in SHP-1 and do not possess functional SHP-1. Pre-B-cell lines derived from me/me mice have been reported to display prolonged IL-4-dependent activation of signal transducer and activator of transcription-6 (Stat6). We evaluated IL-4-dependent Stat6 activation and Fcɛ receptor 1 (FcɛRI) modulation in bone marrow-derived mast cells (BMMCs) from me/me and wild-type mice. IL-4 down-regulated FcɛRI expression in wild-type BMMCs but had no effect on FcɛRI expression in me/me BMMCs. Furthermore, me/me mast cells did not exhibit enhanced or prolonged IL-4-induced Stat6 activation compared with wild-type cells, indicating that mast cells possess alternative tyrosine phosphatases that are responsible for down-regulating Stat6 or can substitute for SHP-1. Thus, SHP-1 is not a negative regulator of IL-4 signaling in BMMCs. These results demonstrate the complexity and cellular specificity of these signaling pathways and indicate a previously unrecognized role for SHP-1 in murine mast cells.

IL-13 is a Th2-derived pleiotropic cytokine that recently was shown to be a key mediator of allergic asthma. IL-13 mediates its effects via a complex receptor system, which includes the IL-4R α-chain, IL-4Rα, and at least two other cell surface proteins, IL-13Rα1 and IL-13Rα2, which specifically bind IL-13. IL-13 has been reported to have very limited effects on mouse B cells. It was unclear whether this was due to a lack of receptor expression, a disproportionate relative expression of the receptor components, or an additional subunit requirement in B cells. To determine the requirements for IL-13 signaling in murine B cells, we examined IL-13-dependent Stat6 activation and CD23 induction in the murine B cell line, A201.1. A201.1 cells responded to murine IL-4 via the type I IL-4R, but were unresponsive to IL-13, and did not express IL-13 receptor. B220+ splenocytes also failed to signal in response to IL-13 and did not express IL-13 receptor. We transfected A201.1 cells with human IL-4Rα, IL-13Rα1, or both. Transfectants expressing either human IL-4Rα or human IL-13Rα1 alone were unable to respond or signal to IL-13. Thus, human IL-13Rα1 could not combine with the endogenous murine IL-4Rα to generate a functional IL-13R. However, cells transfected with both human IL-4Rα and IL-13Rα1 responded to IL-13. Thus, the relative lack of IL-13 responsiveness in murine B cells is due to a lack of receptor expression. Furthermore, the heterodimeric interaction between IL-4Rα and IL-13Rα1 is species specific.

The major surface glycoprotein (MSG) of Pneumocystis carinii f. sp. carinii consists of a heterogeneous family of proteins that are encoded by approximately 100 unique genes. A genomic expression library was screened with a panel of MSG-specific monoclonal antibodies (MAbs) to identify conserved and rare epitopes. All of the antibodies reacted with epitopes that are encoded within the 5′ end of MSG. The results from the expression screening identified antibodies that recognize highly conserved, moderately conserved, and rare epitopes. Four MAbs (MAbs RA-F1, RA-E7, RA-G10, and RB-E3) reacted with a maltose binding protein–MSG-B fusion protein ( MBP MSG-B 41–1065 ) by immunoblotting and enzyme-linked immunosorbent assay. Three of the MAbs (MAbs RA-F1, RA-G10, and RA-E7) reacted with the same continuous epitope that was localized to amino acids 278 to 290 of MSG-B. Comparison of the sequence of the RA-F1-, RA-G10-, and RA-E7-reactive epitope to the deduced amino acid sequences of multiple MSGs demonstrated that it is highly conserved. The reactivity of RB-E3 with MSG-B was shown to be dependent on amino acids 184 to 192, which may comprise a portion of a discontinuous epitope.

Pneumocystis carinii is a common cause of life-threatening pneumonia in immunodeficient patients. Pulmonary surfactant protein A (SP-A), an alveolar glycoprotein containing collagen-like and carbohydrate recognition domains (CRD), binds P. carinii and enhances adherence to alveolar macrophages. In this study, we examined the structural basis of the interaction between SP-A and the major surface glycoprotein of P. carinii (MSG). Rat SP-A bound to purified rat P. carinii-derived MSG in a saturable and calcium-dependent manner, which was partially reversible by coincubation with excess monosaccharides, or pretreatment of MSG with N-glycanase. Mutant recombinant SP-As with neutral amino acid substitutions for the predicted calcium- and carbohydrate-coordinating residues of the CRD were synthesized in insect cells using baculovirus vectors and tested for binding to MSG. Substitutions of negatively charged (Glu195, Glu202, and Asp215) and polar residues (Asn214) of the CRD with alanine but not substitution of the Arg197 with glycine reduced the binding of SP-A to mannose-Sepharose beads and to MSG. Deletion of the N-linked oligosaccharides from SP-A by mutagenesis of the consensus sequences for glycosylation had no effect on binding. We conclude that the CRD mediates the binding of SP-A to oligosaccharides attached to MSG.

Pneumocystis carinii are coated by abundant heterogenous major surface glycoproteins (MSGs), which facilitate interaction with the host. We have produced MSG-specific T-cell clones from the spleens of P. carinii-exposed Lewis rats and analyzed five for antigen specificity to native MSG and a recombinant form of MSG, cell surface markers, and cytokine profiles. All five of the clones were CD4+. All of the clones proliferated specifically to both the native MSG and the recombinant MSG only in the presence of antigen presenting cells demonstrating that the response is antigen/driven rather than mitogen/driven. All five of the clones secreted IL-2 and IFN-gamma, although in differing amounts, implicating a Th1 response. Only one of the clones produced any detectable IL-4. This is the first report of T cell clones responsive to a specific antigen of P. carinii, MSG. We conclude that the T cell clones will be helpful in mapping protective epitopes present in MSG and in functional studies of MSG.

Signal transducer and activator of transcription (Stat) 6 is vital to interleukin (IL)-4 and IL-13 responses and the generation of Th2 immunity. We investigated the cellular location of phosphorylated Stat6 and Stat6 DNA binding activity in A201.1 murine B cells and primary splenocytes. Phosphorylated Stat6 was present in cytoplasmic and nuclear extracts from IL-4-treated cells. Confocal microscopy confirmed the presence of phosphorylated Stat6 in the cytoplasm of IL-4-treated cells. In contrast, Stat6 DNA binding activity was present in nuclear extracts, but not in cytoplasmic extracts. Thus, cytoplasmic extracts from IL-4-stimulated cells were devoid of Stat6 DNA binding activity despite the presence of phosphorylated Stat6. Addition of cytoplasmic extracts to nuclear extracts did not inhibit Stat6 DNA binding present in the nuclear extracts. Detergent treatment restored Stat6 DNA binding activity in cytoplasmic extracts of IL-4-stimulated cells. Thus, DNA binding activity of cytoplasmic phosphorylated Stat6 is masked by a factor dissociable by detergent treatment.

Signal transducer and activator of transcription (Stat)6 is a transcription factor important for the development of Th2 cells and regulation of gene expression by IL-4 and IL-13. It is known that Stat6 is rapidly activated in response to IL-4; however, the fate of activated Stat6 is less clear. We examined the fate of activated Stat6 and found that during continuous exposure to IL-4, Stat6 activity was sustained for 72 h and that the maintenance of a constant level of activated Stat6 did not require new protein synthesis. In contrast, when cells were pulsed with IL-4 and then incubated in the absence of IL-4, the half-life of Stat6 phosphorylation and DNA binding activity was less than 1 h. Stat6 did not accumulate in the nucleus, and protein degradation did not play a major role in the disappearance of activated Stat6. Inhibition of kinase activity by staurosporine or the JAK inhibitor, AG490, revealed that maintenance of Stat6 activation in the continuous presence of IL-4 required ongoing phosphorylation of latent cytoplasmic Stat6 molecules. Cells treated with an inhibitor of nuclear export, leptomycin B, were unable to maintain Stat6 activation. Thus, the maintenance of Stat6 activation requires a constant cycle of activation, deactivation, nuclear export, and reactivation.

Pneumocystis carinii is a major opportunistic pathogen and a leading cause of morbidity in patients with AIDS. CD4+ cells have been shown to be important in host defenses against P. carinii, but the antigen(s) involved with this response have not been identified. We undertook the present study to determine whether the major surface glycoprotein (MSG) of P. carinii contains epitopes that can elicit a protective cellular immune response. Spleen cells and purified CD4+ cells isolated from Lewis rats, pulsed 1-4 d with MSG, and injected into corticosteroid-treated Lewis rats with pneumocystosis resulted in significant reduction in the P. carinii burden, as judged by organism quantitation and lung histology. The protective response demonstrated by the donor cells was dependent on previous exposure to P. carinii, cell concentration, and time of incubation with MSG. In addition, reconstitution with MSG-specific CD4+ cells resulted in an early hyperinflammatory response within the lungs of these animals with a high percentage of mortality. Thus, in this model, MSG can elicit an immune response mediated by CD4+ cells, which has a harmful as well as helpful effect on the host, and these responses occur despite the presence of corticosteroids.

Naturally derived T-cell responses by rats to a 120-kDa major surface glycoprotein (MSG) of rat-derived Pneumocystis carinii were analyzed in vitro. Specific cytokines elicited by the T-cell response to the MSG were also identified. MSG was purified from rat-derived P. carinii by three different techniques: lectin affinity chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by electroelution, and size-exclusion high-performance liquid chromatography. The cell-mediated immunity of spleen cells isolated from Lewis rats with and without natural exposure to P. carinii to the purified MSG was studied. Exposure to P. carinii was monitored by the presence or absence of serum antibodies to P. carinii antigens by Western blotting (immunoblotting). A T-cell proliferative response to the MSG was identified only with spleen cells isolated from rats exposed to P. carinii and peaked at 4 days. Flow cytometric analysis revealed that the percentage of CD4 cells was significantly increased during the proliferative response to MSG. MSG also elicited secretion of tumor necrosis factor alpha, interleukin-1, and interleukin-2, with peak activity of these cytokines occurring after 12, 24, and 48 h, respectively, of culture. These findings suggest that MSG is important in host T-cell recognition of and immune response to P. carinii by recruitment of inflammatory cells and cytokine production.