The mythic function of narcissism.

The paper makes some critical comments on the use Freud makes of myth and examines some of the inconsistencies and contradictions in his conceptualization of narcissism. Using some of the ideas of Bachelard and Hillman on the role of the imaginary, the authors theorize a function of myth that is independent of and not subordinated to the reality function. They suggest that narcissism must be seen not only in terms of individual history but that it has a mythic function; narcissism facilitates the creation of a relationship between the ego and the Self through the mediation of the imaginal world and through the prospective value of images.

Signaling lymphocytic activation molecule expression and regulation in human intracellular infection correlate with Th1 cytokine patterns.

Induction of Th1 cytokines, those associated with cell-mediated immunity, is critical for host defense against infection by intracellular pathogens, including mycobacteria. Signaling lymphocytic activation molecule (SLAM, CD150) is a transmembrane protein expressed on lymphocytes that promotes T cell proliferation and IFN-gamma production. The expression and role of SLAM in human infectious disease were investigated using leprosy as a model. We found that SLAM mRNA and protein were more strongly expressed in skin lesions of tuberculoid patients, those with measurable CMI to the pathogen, Mycobacterium leprae, compared with lepromatous patients, who have weak CMI against M. leprae. Peripheral blood T cells from tuberculoid patients showed a striking increase in the level of SLAM expression after stimulation with M. leprae, whereas the expression of SLAM on T cells from lepromatous patients show little change by M. leprae stimulation. Engagement of SLAM by an agonistic mAb up-regulated IFN-gamma production from tuberculoid patients and slightly increased the levels of IFN-gamma in lepromatous patients. In addition, IFN-gamma augmented SLAM expression on M. leprae-stimulated peripheral blood T cells from leprosy patients. Signaling through SLAM after IFN-gamma treatment of Ag-stimulated cells enhanced IFN-gamma production in lepromatous patients to the levels of tuberculoid patients. Our data suggest that the local release of IFN-gamma by M. leprae-activated T cells in tuberculoid leprosy lesions leads to up-regulation of SLAM expression. Ligation of SLAM augments IFN-gamma production in the local microenvironment, creating a positive feedback loop. Failure of T cells from lepromatous leprosy patients to produce IFN-gamma in response to M. leprae contributes to reduced expression of SLAM. Therefore, the activation of SLAM may promote the cell-mediated immune response to intracellular bacterial pathogens.

Functional cloning of Src-like adapter protein-2 (SLAP-2), a novel inhibitor of antigen receptor signaling.

In an effort to identify novel therapeutic targets for autoimmunity and transplant rejection, we developed and performed a large-scale retroviral-based functional screen to select for proteins that inhibit antigen receptor-mediated activation of lymphocytes. In addition to known regulators of antigen receptor signaling, we identified a novel adaptor protein, SLAP-2 which shares 36% sequence similarity with the known Src-like adaptor protein, SLAP. Similar to SLAP, SLAP-2 is predominantly expressed in hematopoietic cells. Overexpression of SLAP-2 in B and T cell lines specifically impaired antigen receptor-mediated signaling events, including CD69 surface marker upregulation, nuclear factor of activated T cells (NFAT) promoter activation and calcium influx. Signaling induced by phorbol myristate acetate (PMA) and ionomycin was not significantly reduced, suggesting SLAP-2 functions proximally in the antigen receptor signaling cascade. The SLAP-2 protein contains an NH2-terminal myristoylation consensus sequence and SH3 and SH2 Src homology domains, but lacks a tyrosine kinase domain. In antigen receptor-stimulated cells, SLAP-2 associated with several tyrosine phosphorylated proteins, including the ubiquitin ligase Cbl. Deletion of the COOH terminus of SLAP-2 blocked function and abrogated its association with Cbl. Mutation of the putative myristoylation site of SLAP-2 compromised its inhibitory activity and impaired its localization to the membrane compartment. Our identification of the negative regulator SLAP-2 demonstrates that a retroviral-based screening strategy may be an efficient way to identify and characterize the function of key components of many signal transduction systems.

Signaling lymphocytic activation molecule (SLAM) regulates T cellular cytotoxicity.

Signaling lymphocytic activation molecule (SLAM) is a CD2-related surface receptor expressed by activated T cells and B cells. SLAM is a self ligand and enhances T cellular proliferation and IFN-gamma production. A defective SLAM associated protein (SAP) causes X-linked lymphoproliferative syndrome (XLP), a frequently lethal mononucleosis based on the inability to control EBV. We report that SLAM augments TCR-mediated cytotoxicity. In normal CD4(+) and CD8(+) T cells, SLAM enhanced TCR-mediated cytotoxicity. In CD4(+) and CD8(+) Herpesvirus saimiri (H.saimiri) infected T cells, SLAM engagement alone triggered cytotoxicity. Using H.saimiri-transformed T cells as a model system we found that SLAM-engagement promotes the release of lytic granules and a CD95-independent killing that requires extracellular Ca(2+), cytoskeletal rearrangements, and signaling mediated by mitogen-activated protein kinase kinases MEK1/2. SLAM-enhanced cytotoxicity implies an immunoregulatory function by facilitating the elimination of APC and a role in overcoming infections with pathogens requiring a cytotoxic immune response.

Transmembrane-sequence-dependent overexpression and secretion of glycoproteins in Saccharomyces cerevisiae.

Protein expression using the secretory pathway in Saccharomyces cerevisiae can lead to high amounts of overexpressed and secreted proteins in culture supernatants in a short period of time. These post-translational modified expression products can be purified up to >90% in a single step. The overexpression and secretion of the transmembrane glycoprotein signaling lymphocytic activation molecule (SLAM) was studied. SLAM belongs to the immunoglobulin superfamily and its engagement results in T-cell expansion and INF-gamma production. The molecule is composed of an extracellular, a single-span transmembrane and a cytoplasmatic domain. The extracellular part may be relevant for stimulation studies in vitro since SLAM is a high-affinity self-ligand. Therefore several fragments of this region have been expressed as Flag-fusions in S. cerevisiae: a full-length fragment containing the transmembrane region and the autologous signal sequence, another without the transmembrane region, and two fragments without the autologous signal sequence with and without the transmembrane region. By molecular cloning, the different deletion mutants of the cDNA encoding the full-length construct have been inserted in a yeast episomal plasmid. Upstream of the cDNA, the alpha-leader sequence of a yeast mating pheromone has been cloned to direct the fusion proteins into the secretory protein maturation pathway. All four fragments were expressed but yield, location, and maturation were highly influenced by the transmembrane domain and the autologous signal sequence. Only the fragment without autologous signal sequence and transmembrane domain could be efficiently secreted. High-mannose glycosylation was analyzed by lectin mapping and digestion with specific glycosidases. After enzyme treatment, a single band product with the theoretical size could be detected and identified as SLAM by a specific monoclonal antibody. The fusion protein concentration in the supernatant was 30 microg/ml. The affinity-purified and deglycosylated protein is a tool for further biochemical and biophysical characterization of SLAM.

Molecular and functional characterization of mouse signaling lymphocytic activation molecule (SLAM): differential expression and responsiveness in Th1 and Th2 cells.

Optimal T cell activation and expansion require engagement of the TCR plus costimulatory signals delivered through accessory molecules. SLAM (signaling lymphocytic activation molecule), a 70-kDa costimulatory molecule belonging to the Ig superfamily, was defined as a human cell surface molecule that mediated CD28-independent proliferation of human T cells and IFN-gamma production by human Th1 and Th2 clones. In this study, we describe the cloning of mouse SLAM and the production of mAb against it which reveal its expression on primary mouse T and B cells. Mouse SLAM is expressed on highly polarized Th1 and Th2 populations, and is maintained on Th1, but not on Th2 clones. Anti-mouse SLAM mAb augmented IFN-gamma production by Th1 cells and Th1 clones stimulated through the TCR, but did not induce IFN-gamma production by Th2 cells, nor their production of IL-4 or their proliferation. Mouse SLAM is a 75-kDa glycoprotein that upon tyrosine phosphorylation associates with the src homology 2-domain-containing protein tyrosine phosphatase SHP-2, but not SHP-1. Mouse SLAM also associates with the recently described human SLAM-associated protein. These studies may provide new insights into the regulation of Th1 responses.

Altered signaling lymphocytic activation molecule (SLAM) expression in HIV infection and redirection of HIV-specific responses via SLAM triggering.

Signaling lymphocytic activation molecule (SLAM) is a transmembrane lymphocytic receptor which gets rapidly upregulated following cell activation. SLAM engagement augments T cell expansion and interferon-gamma (IFN-gamma) production independently of CD28. SLAM signaling is regulated by the SLAM-associated protein. We evaluated the expression and function of SLAM on CD4(+) and CD8(+) lymphocytes in HIV-infected individuals with either recently acquired infection (Group A) or asymptomatic HIV infection (Group B) and in healthy controls (HC). Soluble antigen (HIV env peptides and tetanus toxoid)- and mitogen-stimulated proliferation and IFN-gamma and IL-10 production upon SLAM costimulation were also measured. Results showed that: (1) SLAM-expressing CD4(+) and CD8(+) lymphocytes diminish in group A patients compared to both group B patients and HC; (2) SLAM expression on CD4(+) lymphocytes is preferentially associated with the lack of CD7 on cell surface (CD4(+)CD7(-) produce IL-10 but not IFN-gamma); (3) SLAM engagement increases HIV env peptide-stimulated, but neither tetanus toxoid- nor PHA-stimulated proliferation of peripheral blood mononuclear cells (PBMC) in patients but not in HC; and (4) SLAM engagement augments IFN-gamma and reduces IL-10 production by env peptide-stimulated PBMC of HIV-infected individuals. These results demonstrate that early HIV infection results in an altered SLAM expression which correlates with a time-limited impairment of cell-mediated immunity. Furthermore, they show that triggering via SLAM potentiates HIV-specific proliferative responses with simultaneous downregulation of IL-10 and redirection of the response to TH0/TH1.

Receptors and cytokines involved in allergic TH2 cell responses.

Cytokines produced by allergen-specific TH2 cells play a central role in the induction and maintenance of allergic responses. Therefore antagonizing TH2 differentiation and TH2 effector functions will provide an effective way to intervene in allergic diseases. This article discusses that antagonizing the effects of IL-4 and IL-13 by IL-4Ralpha antagonists inhibits human TH2 development and IgE synthesis. In addition, it is shown that the activation of allergen-specific TH2 cells with an agonistic anti-CDw150 mAb redirects the cytokine production profile of these TH2 cells to a TH0 phenotype.

IFN-gamma is only partially restored by co-stimulation with IL-12, IL-2, IL-15, IL-18 or engagement of CD28.

BACKGROUND: Although it is well established that T cells derived from patients with atopic diseases produce low levels of interferon-gamma (IFN-gamma), the mechanisms responsible for this phenomenon are poorly understood. OBJECTIVES: To elucidate whether IFN-gamma production may be restored by co-stimulatory molecules known to increase IFN-gamma production in vitro. Further, to investigate whether deficient IFN-gamma production is associated with disease activity. METHODS: Purified peripheral T cells obtained from patients with severe atopic dermatitis (AD), individuals with a history but no symptoms of AD and healthy control subjects were activated with anti-CD3 MoAbs in the presence or absence of anti-CD28 MoAbs, interleukin (IL-) 12, IL-2, IL-15 or IL-18. IFN-gamma production was determined at the single cell level by flow cytometry, as well as by ELISA. RESULTS: Activated T cells from patients with severe AD produced less IFN-gamma than T cells from healthy control individuals. IL-12 or engagement of CD28 enhanced IFN-gamma production in both healthy and atopic T cells. However, absolute values of IFN-gamma were still different. IL-2, IL-15 and IL-18 did not restore IFN-gamma production. T cells from individuals with a history of AD produced more IFN-gamma than those from subjects with severe AD, but less than T cells from healthy individuals. Atopic T cells expressed regular levels of CD3, CD28 and Stat4, the main signal transducer and activator of transcription for IL-12. IL-4, IL-10 and TGF-beta production by T cells were not different between healthy and atopic individuals. CONCLUSION: IFN-gamma deficiency in atopic T cells is not due to a lack of responsiveness to CD28, IL-12, IL-2, IL-15 or IL-18. T cell-derived cytokines able to antagonize IFN-gamma do not contribute to decreased IFN-gamma production. The extent of IFN-gamma deficiency seems to be dependent on disease activity.

Expression of soluble human signaling lymphocytic activation molecule in vivo.

BACKGROUND: Signaling lymphocytic activation molecule (SLAM) is a novel glycoprotein expressed on activated T and B cells. Ligation of cell surface SLAM, either by anti-SLAM mAbs or the recombinant soluble form of SLAM (sSLAM), enhanced the proliferation of T and B cells in vitro. In addition, the engagement of SLAM on T cells preferentially induced IFN-gamma production even by allergen-specific TH2 clones. OBJECTIVE: In this study we investigated the expression of sSLAM in vivo in healthy individuals and in disease conditions that are associated with increased TH1 - or TH2 -cell responses. METHODS: The expression of mRNA encoding sSLAM in peripheral blood and synovial fluid (SF) lymphocytes was studied by using reverse transcriptase-PCR, and the presence of sSLAM protein in serum and SF samples was investigated by using a specific ELISA. RESULTS: Lymphocytes from patients with rheumatoid arthritis (RA) and healthy individuals consistently expressed mRNA encoding sSLAM. In addition, sSLAM protein was present in 38% of serum and 54% of SF samples from patients with RA and in 47% of serum samples from healthy individuals. The levels of sSLAM in positive serum and SF samples from patients with RA and in positive serum samples from healthy individuals were not significantly different. In contrast, the levels of sSLAM were significantly lower in patients with reactive arthritis or in patients with elevated IgE levels than in patients with RA. Similarly, the frequency of positive SF samples was significantly lower in reactive arthritis (28%) than in RA (54%). CONCLUSION: These results indicate that sSLAM is present in serum and SF, further suggesting that sSLAM regulates T- and B-cell function in vivo. Moreover, these data suggest an association between low sSLAM production and the occurrence of TH2 responses in vivo.

Subconjunctival mitomycin C for the treatment of ocular cicatricial pemphigoid.

PURPOSE: The authors performed a prospective evaluation of the efficacy of treating ocular cicatricial pemphigoid (OCP) with subconjunctival mitomycin C. DESIGN: Unmasked, prospective, internally controlled case series. METHODS: Patients were eligible for treatment with subconjunctival mitomycin C under three criteria: (1) significant complications of systemic immunosuppressant therapy; (2) markedly asymmetric conjunctival disease; and (3) end-stage OCP. All patients received monocular subconjunctival injections of 0.25 ml of 0.2 mg/ml mitomycin C to both the superior and inferior bulbar conjunctivae in the eye with the more severe disease. RESULTS: Nine eyes of nine patients (mean age, 74 years) were treated with subconjunctival mitomycin C to the more-involved eye and were followed for a mean of 23.5 months (range, 12-40 months). Eight of nine patients showed quiescence of their OCP in the treated eye based on serial evaluation of conjunctival cicatrization and grading of conjunctival erythema. Five of the nine untreated eyes showed progression of the conjunctival disease. One patient required concomitant systemic immunosuppressive therapy after subconjunctival mitomycin C. Two patients underwent successful visual rehabilitative surgery in the mitomycin C-treated eye. CONCLUSION: The use of subconjunctival mitomycin C may be effective in preventing progression of conjunctival cicatrization and erythema in patients with OCP. No complications of mitomycin C treatment were noted. Long-term follow-up and further investigation into the efficacy of subconjunctival mitomycin C in the management of OCP is warranted.

Identification of functional human splenic memory B cells by expression of CD148 and CD27.

Memory B cells isolated from human tonsils are characterized by an activated cell surface phenotype, localization to mucosal epithelium, expression of somatically mutated immunoglobulin (Ig) variable (V) region genes, and a preferential differentiation into plasma cells in vitro. In spleens of both humans and rodents, a subset of memory B cells is believed to reside in the marginal zone of the white pulp. Similar to tonsil-derived memory B cells, splenic marginal zone B cells can be distinguished from naive follicular B cells by a distinct cell surface phenotype and by the presence of somatic mutations in their Ig V region genes. Although differences exist between human naive and memory B cells, no cell surface molecules have been identified that positively identify all memory B cells. In this study, we have examined the expression of the receptor-type protein tyrosine phosphatase CD148 on human B cells. CD148(+) B cells present in human spleen exhibited characteristics typical of memory B cells. These included an activated phenotype, localization to the marginal zone, the expression of somatically mutated Ig V region genes, and the preferential differentiation into plasma cells. In contrast, CD148(-) B cells appeared to be naive B cells due to localization to the mantle zone, the expression of surface antigens typical of unstimulated B cells, and the expression of unmutated Ig V region genes. Interestingly, CD148(+) B cells also coexpressed CD27, whereas CD148(-) B cells were CD27(-). These results identify CD148 and CD27 as markers which positively identify memory B cells present in human spleen. Thus, assessing expression of these molecules may be a convenient way to monitor the development of memory B cell responses in immunocompromised individuals or in vaccine trials.

CD148: a receptor-type protein tyrosine phosphatase involved in the regulation of human T cell activation.

Following ligation of the TCR and costimulatory molecules such as CD28, T cells proliferate and secrete cytokines. Several other cell surface molecules have been identified that are capable of augmenting activation mediated via the TCR. These include CD2, CD27, CD40 ligand, and signaling lymphocytic activation molecule. Here, we have characterized the expression and function of CD148, a recently identified receptor-type protein tyrosine phosphatase. CD148 is expressed at low levels on resting T cells, but is up-regulated following in vitro activation. Cross-linking CD148 with immobilized anti-CD148 mAb induced vigorous proliferation of anti-CD3 mAb-activated, highly purified peripheral blood T cells in an IL-2-dependent, cyclosporin A-sensitive manner. This effect was greatest after 8 days of in vitro culture, suggesting that this molecule is involved in the latter stages of a T cell response. CD148-induced proliferation was significantly greater for CD8+ T cells than for CD4+ T cells. Thus, CD148 is a receptor-type protein tyrosine phosphatase involved in the activation of T lymphocytes.

Negative regulation of human T cell activation by the receptor-type protein tyrosine phosphatase CD148.

T cell activation represents a balance between positive and negative signals delivered via distinct cell surface molecules. Many cytoplasmic protein tyrosine phosphatases are involved in regulating cellular responses by antagonizing the action of protein tyrosine kinases. CD148 is a receptor-type protein tyrosine phosphatase expressed by all human mononuclear cells. We have investigated the effect of CD148 on TCR-mediated activation of human T cells. Overexpression of wild-type, but not a phosphatase-deficient, CD148 in Jurkat T cells inhibited TCR-mediated activation, evidenced by reduced expression of the early activation Ag CD69, inhibition of tyrosine phosphorylation of many intracellular proteins including the critical protein tyrosine kinase ZAP-70, and impairment of mitogen-activated protein kinase activation. Taken together, these results suggest that CD148 is an important phosphatase involved in negatively regulating the proximal signaling events during activation of Ag-specific T cells.

The X-linked lymphoproliferative-disease gene product SAP regulates signals induced through the co-receptor SLAM.

In addition to triggering the activation of B- or T-cell antigen receptors, the binding of a ligand to its receptor at the cell surface can sometimes determine the physiological outcome of interactions between antigen-presenting cells, T and B lymphocytes. The protein SLAM (also known as CDw150), which is present on the surface of B and T cells, forms such a receptor-ligand pair as it is a self-ligand. We now show that a T-cell-specific, SLAM-associated protein (SAP), which contains an SH2 domain and a short tall, acts as an inhibitor by blocking recruitment of the SH2-domain-containing signal-transduction molecule SHP-2 to a docking site in the SLAM cytoplasmic region. The gene encoding SAP maps to the same area of the X chromosome as the locus for X-linked lymphoproliferative disease (XLP) and we found mutations in the SAP gene in three XLP patients. Absence of the inhibitor SAP in XLP patients affects T/B-cell interactions induced by SLAM, leading to an inability to control B-cell proliferation caused by Epstein-Barr virus infections.

Topical Cyclosporine A in the management of postkeratoplasty glaucoma and corticosteroid-induced ocular hypertension (CIOH) and the penetration of topical 0.5% cyclosporine A into the cornea and anterior chamber.

PURPOSE: To evaluate the effect on intraocular pressure (IOP) of substituting topical Cyclosporine A 0.5% for topical corticosteroids in patients with postkeratoplasty glaucoma and corticosteroid-induced ocular hypertension (CIOH). We also sought to determine the penetration of topical 0.5% Cyclosporine A into the cornea and anterior chamber. METHODS: Topical Cyclosporine A 0.5% was prospectively substituted for topical corticosteroids in 47 patients (52 eyes) with postkeratoplasty glaucoma and CIOH in order to eliminate the IOP-elevating effect of topical corticosteroids, while maintaining protection against allograft rejection. Ten patients received 0.5% topical Cyclosporine before keratoplasty. Their corneal tissue and aqueous samples were evaluated by high pressure liquid chromatography for Cyclosporine levels. RESULTS: Forty-eight of 52 eyes (92.3%) demonstrated a reduction of IOP at first followup (mean: -7.9 mmHg; range: -19 to +2). Mean followup was 10.3 months, ranging from 1 to 37 months. At last follow-up, mean IOP was -8.2 mm Hg. There were six allograft rejections, five of which were reversed with the reintroduction of topical corticosteroids. Graft clarity was maintained in 46 of 52 eyes (88%). The mean cornea Cyclosporine concentration was 3679 ng/gm (range: 1980 to 5520 ng/ gm) and aqueous humor mean concentration was 6.05 ng/mL (range: 0.4 to 15.5 ng/mL). CONCLUSIONS: Topical Cyclosporine A 0.5% may be substituted for topical corticosteroids to aid in the management of postkeratoplasty glaucoma and CIOH. However, the use of Cyclosporine in place of corticosteroids may be associated with an increased risk of immune rejections. The corneal penetration of topical Cyclosporine is excellent while the penetration into the anterior chamber is poor.

Cytokine production and surface marker expression in acute and stable multiple sclerosis: altered IL-12 production and augmented signaling lymphocytic activation molecule (SLAM)-expressing lymphocytes in acute multiple sclerosis.

Ag-stimulated IL-2 production and mitogen-stimulated type 1 and type 2 cytokine production by PBMC, as well as expression of Th1- and Th2-associated phenotypical markers, of B7-1, B7-2, and CD95 (Fas) on the surface of immune cells, and the serum concentration of soluble Apo-1/Fas were evaluated in multiple sclerosis (MS) patients with either acute (AMS) or stable (SMS) disease and in healthy controls (HC). Results showed that 1) Ag-stimulated IL-2 production is reduced in MS patients compared with that in HC; 2) mitogen-stimulated type 1 cytokine production is increased, and IL-10 production is reduced in MS patients compared with those in HC, and in AMS patients compared with those in SMS; 3) whereas production of the metabolically active p70 heterodimers is comparable in SMS, AMS, and HC, production of the p70 heterodimer and the p40 chains (total IL-12) is increased in SMS compared with that in AMS and HC; 4) CD4+, CD4+ SLAM+, and CD4+ CD7+ lymphocytes (preferentially type 1 cytokine-producing lymphocytes) are increased in MS compared with levels in HC; 5) B7-2- as well as Fas+-expressing monocytes are augmented in MS compared with those in HC, and serum soluble Apo-1/Fas is augmented in AMS compared with SMS and HC. These results confirm that a complex imbalance in both cytokine production and the Fas system is present in MS and indicate that different cytokine profiles may be observed in patients with acute or stable disease. The data also suggest that peculiar phenotypic populations are over-represented in MS patients, and for the first time show that SLAM expression is correlated with dysregulation of type 1 and type 2 cytokine production in human pathology.

Antagonistic peptides specifically inhibit proliferation, cytokine production, CD40L expression, and help for IgE synthesis by Der p 1-specific human T-cell clones.

BACKGROUND: Allergic disorders are characterized by IgE antibody responses to a multitude of allergens as a result of the ability of these antibodies to specifically bind to high-affinity IgE receptors on mast cells and basophils. This interaction results in receptor activation and release of soluble mediators such as histamine and leukotrienes, which cause allergic reactions in various target organs. Because the synthesis of IgE is tightly regulated by cytokines and CD40 ligand (L) interactions, CD4+ helper T cells are obvious targets, with the aim to modulate allergen-induced IgE responses. OBJECTIVES: Because of the central role of allergen-specific T-helper type 2 (TH2) cells in the pathway leading to IgE synthesis in vitro and in vivo, we have evaluated the possibility of inhibiting allergen-induced activation of these cells by using allergen-derived peptides that have been modified by single amino acid substitutions. METHODS: Three cloned human TH2-like CD4+ T-cell lines, specific for Der p 1, the major allergen in house dust, were used in this study. Upon activation with Der p 1 or specific Der p 1-derived wild-type peptides, these T-cell clones produce high levels of IL-4 and IL-5 and low levels of interferon-gamma and IL-2, respectively, and furthermore give help to B cells for the production of IgE in vitro. Modified synthetic peptides were generated by the introduction of single amino acid substitutions into two different T-cell activation-inducing epitopes on Der p 1. The effects of these modified peptides were studied in Der p 1-induced proliferation, cytokine production, and in vitro IgE production assays. RESULTS: Several substituted Der p 1-derived peptides failed to induce T-cell proliferation, in contrast to the native peptides. In addition, some of these peptides acted as antagonists by strongly inhibiting wild-type peptide-induced proliferation as well as the production of interferon-gamma, IL-2, IL-4, and IL-5, although the production of the latter two cytokines was less affected than that of interferon-gamma, even at a 100-fold molar excess of the antagonistic peptides. In addition, the presence of an excess of each of the antagonistic peptides during the activation of Der p I-specific T-cell clones prevented induction of CD40L expression, resulting in a failure of these cells to give help to B cells for the production of IgE in vitro, even in the presence of exogenous IL-4. CONCLUSIONS: Substitution of certain amino acid residues in immunogenic Der p 1-derived peptides results in the generation of peptides that fail to induce proliferation of Der p 1-specific T-cell clones. In addition, these modified peptides have strong antagonistic activities on Der p 1-induced proliferation, cytokine production, and CD40L expression by allergen-specific T-cell clones as well as on T cell-mediated IgE production by B cells. These findings suggest that modified peptides interfere with allergen-induced activation of T cells, including the production of cytokines and the expression of surface molecules important for successful T cell-B cell interactions, and may therefore have therapeutic potential by inhibiting the expansion and function of allergen-specific TH2 cells.

Gene transfer with IL-4 and IL-13 improves survival in lethal endotoxemia in the mouse and ameliorates peritoneal macrophages immune competence.

Systemic anti-cytokine therapies have been unsuccessful in preventing mortality from gram-negative bacteremia in humans partly because of the failure to neutralize pro-inflammatory cytokines at sites of exaggerated production. In an attempt to deliver anti-inflammatory cytokines to organs directly, gene transfer was employed. Thirty-six BALB/c mice were injected intraperitoneally with cationic liposomes containing plasmids encoding the human interleukin-4 (hIL-4) or IL-13 gene. Both, hIL-4 and hIL-13 mRNA were detected by reverse transcription-polymerase chain reaction analysis in the liver and the spleen of the animals. Fourty-eight hours after the in vivo gene transfer, these 36 mice and 18 mock-transfected mice, were challenged with a lethal dose of E. coli lipopolysaccharide with D-galactosamine (D-GalN). Gene transfer with hIL-4 reduced the serum tumor necrosis factor (TNF)-alpha production in response to endotoxin/D-GalN by 80% from 113.1 pg/ml in mock-transfected animals to 22.2 pg/ml (p < 0.05); human IL-13 gene transfer reduced serum TNF-alpha levels by 90% (113.1 pg/ml to 11.6 pg/ml; p < 0.05). Survival was improved from 20% to over 83% in both treatment groups (p < 0.001). Our data demonstrate a potent in vivo anti-inflammatory action of both IL-4 and IL-13. In addition, the immune functions of peritoneal macrophages are significantly ameliorated in both treatment groups, with IL-13 demonstrating better macrophage immune modulation than IL-4 (p < 0.05).