Blocking antibodies induced by allergen-specific immunotherapy ameliorate allergic airway disease in a human/mouse chimeric model.

BACKGROUND: Allergen-specific immunotherapy (AIT) induces specific blocking antibodies (Ab), which are claimed to prevent IgE-mediated reactions to allergens. Additionally, AIT modulates cellular responses to allergens, for example, by desensitizing effector cells, inducing regulatory T and B lymphocytes and immune deviation. It is still enigmatic which of these mechanisms mediate(s) clinical tolerance. We sought to address the role of AIT-induced blocking Ab separately from cellular responses in a chimeric human/mouse model of respiratory allergy. METHODS: Nonobese diabetic severe combined immunodeficient γc-/- (NSG) mice received intraperitoneally allergen-reactive PBMC from birch pollen-allergic patients together with birch pollen extract and human IL-4. Engraftment was assessed by flow cytometry. Airway hyperresponsiveness (AHR) and bronchial inflammation were analyzed after intranasal challenges with allergen or PBS. Sera collected from patients before and during AIT with birch pollen were added to the allergen prior to intranasal challenge. The IgE-blocking activity of post-AIT sera was assessed in vitro. RESULTS: Human cells were detected in cell suspensions of murine lungs and spleens indicating successful humanization. Humanized mice displayed a more pronounced AHR and bronchial inflammation when challenged with allergen compared to negative controls. Post-AIT sera exerted IgE-blocking activity. In contrast to pre-AIT sera, the presence of heterologous and autologous post-AIT sera significantly reduced the allergic airway inflammation and matched their IgE-blocking activity determined in vitro. CONCLUSION: Our data demonstrate that post-AIT sera with IgE-blocking activity ameliorate allergic airway inflammation in a human/mouse chimeric model of respiratory allergy independently of AIT-induced cellular changes.

Characterization of the T-cell response to Dau c 1, the Bet v 1-homolog in carrot.

BACKGROUND: In contrast to other Bet v 1-related food allergens, the major carrot allergen, Dau c 1, has been suggested to induce food allergy independently from Bet v 1. As T cells are crucial in the sensitization process, we sought to characterize the T-cell response to Dau c 1 and its cross-reactivity with Bet v 1. METHODS: Dau c 1-specific T-cell lines (TCL) and clones (TCC) established from PBMC of birch pollen-allergic patients with carrot allergy were analyzed for reactivity to Bet v 1, epitope specificity, allergen-induced cytokine secretion, and expression of integrins α4ß7 and α4ß1, critical for gut and lung homing, respectively. mRNA expression of GATA3 and Tbet was analyzed in sorted CD3+ CD4+ CFSElow cells proliferating upon stimulation of PBMC with Dau c 1 or Bet v 1. Dau c 1 was incubated with endolysosomal proteases, and the resulting fragments were identified by mass spectrometry. RESULTS: Among 14 distinct T-cell-activating regions, Dau c 1139-153 was recognized by 55% of the patients. Only 6 of 15 (40%) Dau c 1-specific TCL and 9 of 21 (43%) TCC reacted with Bet v 1. Bet v 1-nonreactive TCC were mainly Th1-like and showed a higher expression of the integrin ß7 and a significantly lower expression of the integrin ß1 than Bet v 1-positive TCC. A Th1-like response was also detected in Dau c 1-reactive CD3+ CD4+ CFSElow cells. Full-length Dau c 1 was still detectable after 48 h of endolysosomal degradation. Proteolytic fragments of Dau c 1 matched its T-cell-activating regions. CONCLUSION: Dau c 1 displays several characteristics of sensitizing allergens, namely a major T-cell-activating region, low susceptibility to endolysosomal degradation, and induction of a Bet v 1-independent T-cell response. These cellular insights confirm that the major carrot allergen has a special status among Bet v 1-related food allergens.

Differential activation of dendritic cells by toll-like receptors causes diverse differentiation of naïve CD4+ T cells from allergic patients.

BACKGROUND: To avert the differentiation of allergen-specific Th2 cells in atopic individuals is a major goal in the prevention and therapy of IgE-mediated allergy. We aimed to compare different toll-like receptor (TLR) agonists regarding their effects on antigen-presenting cells and the differentiation of naïve T cells from allergic patients. METHODS: Monocytes and monocyte-derived dendritic cells (mdDC) from allergic patients were stimulated with Pam3CSK4 (TLR1/2 ligand), FSL-1 (TLR2/6 ligand), monophosphoryl lipid (MPL)-A, lipopolysaccharide (LPS, both TLR4 ligands), and flagellin (TLR5 ligand). Allergen uptake and upregulation of CD40, CD80, CD83, CD86, CD58, CCR7 and PD-L1 were analyzed by flow cytometry. Functional maturation of mdDC was tested in mixed leukocyte reactions, and the synthesis of proinflammatory cytokines, IL-10 and members of the IL-12 family was assessed. TLR-ligand-activated mdDC were used to stimulate naïve CD4(+) T cells, and cytokine responses were assessed in supernatants and intracellularly. RESULTS: All TLR ligands except flagellin enhanced allergen uptake. All TLR ligands induced functional maturation of mdDC with differential expression of surface molecules and cytokines and promoted the differentiation of IFN-γ-producing T cells. LPS-matured mdDC exclusively induced Th1-like responses, whereas mdDC stimulated with the other TLR ligands induced both Th1- and Th0-like cells. Pam3CSK4 and flagellin additionally induced Th2-like cells. Th1-like responses were associated with higher expression levels of co-stimulatory molecules, PD-L1, IL-6, TNF-α, and IL-12p70. None of the TLR-ligand-stimulated mdDC induced IL-10- or IL-17-producing T cells. CONCLUSION: Different TLR ligands differently influence T-cell responses due to varying activation of the three signals relevant for T-cell activation, that is, antigen presentation, co-stimulation and cytokine milieu.

A comprehensive and quantitative analysis of the major specificities in rabbit antithymocyte globulin preparations.

Antithymocyte globulin (ATG) preparations are used for treatment and prevention of graft rejection episodes, graft versus host disease and aplastic anemia. The immunomodulatory and immuosuppressive properties of ATGs are mediated by their interaction with a large variety of antigens expressed on immune and nonimmune cell populations. We have conducted a comprehensive analysis on antibody specificities contained in rabbit ATGs in clinical use, ATG-Fresenius (ATG-F) and Thymoglobulin (THG). We have used retroviral expression cloning to identify novel ATG antigens and demonstrate that together with ATG antigens described earlier, these molecules account for the majority of ATG antibodies directed to human cells. Moreover, we have employed cell lines engineered to express antigens at high levels to quantify the antibodies directed to each ATG antigen. We have used cell lines expressing the T cell receptor complex, CD2 and CD28 to remove antibodies to these antigens from ATG preparations and demonstrate that this treatment abrogated the ability of ATGs to induce activation and forkhead box P3 expression in T cells. Comprehensive information and differences on the antigens targeted by ATG-F and THG as well as novel approaches to assess their functional properties are the basis for a better understanding of their immunomodulatory capacities and might eventually translate into improved ATG-based regimen.

CMV late phase-induced mTOR activation is essential for efficient virus replication in polarized human macrophages.

Human cytomegalovirus (CMV) remains one of the most important pathogens following solid-organ transplantation. Mounting evidence indicates that mammalian target of rapamycin (mTOR) inhibitors may decrease the incidence of CMV infection in solid-organ recipients. Here we aimed at elucidating the molecular mechanisms of this effect by employing a human CMV (HCMV) infection model in human macrophages, since myeloid cells are the principal in vivo targets of HCMV. We demonstrate a highly divergent host cell permissiveness for HCMV with optimal infection susceptibility in M2 but not M1 polarized macrophages. Employing an ultrahigh purified HCMV stock we observed rapamycin-independent viral entry and induction of IFN-ß transcripts, but no proinflammatory cytokines or mitogen-activated protein kinases and mTOR activation early after infection. However, in the late infection phase, sustained mTOR activation was observed in HCMV-infected cells and was required for efficient viral protein synthesis including the viral late phase proteins pUL-44 and pp65. Accordingly, rapamycin strongly suppressed CMV replication 3 and 5 days postinfection in macrophages. In conclusion, these data indicate that mTOR is essential for virus replication during late phases of the viral cycle in myeloid cells and might explain the potent anti-CMV effects of mTOR inhibitors after organ transplantation.

Human blood basophils do not act as antigen-presenting cells for the major birch pollen allergen Bet v 1.

BACKGROUND: Several studies in mice have recently shown that basophils can act as antigen-presenting cells (APC) inducing Th2-mediated immune responses against parasites or protease allergens. The aim of this study was to investigate whether human basophils function as APC for the major birch pollen allergen Bet v 1. METHODS: Fluorescently labeled Bet v 1 was used to assess surface binding and internalization of allergen by basophils and different types of APC from birch pollen-allergic and nonallergic individuals. Sorted basophils were analyzed in terms of up-regulation of MHC class II and co-stimulatory molecules in the absence and presence of IL-3 and IFN-γ by flow cytometry. Expression of proteins crucial for antigen presentation, namely cathepsin S and invariant chain, was determined. Basophils were used as APC in co-culture experiments with Bet v 1-specific T-cell clones (TCCs). RESULTS: Basophils from birch pollen-allergic donors very efficiently bound Bet v 1 through IgE/FcεRI complexes on their surface. In contrast to professional APC, basophils did not internalize allergen and expressed marginal levels of cathepsin S and invariant chain. HLA-DP, HLA-DQ, CD80/CD86, and CD40 were absent from purified basophils even when stimulated with IL-3 plus IFN-γ. IL-3/IFN-γ marginally up-regulated HLA-DR. Bet v 1-pulsed basophils failed to induce proliferative and cytokine responses in Bet v 1-specific, HLA-DR-restricted TCCs. CONCLUSION: Human basophils neither internalize, process nor present Bet v 1. Because Bet v 1 is a highly relevant allergen, we conclude that basophils play no role as APC in IgE-mediated allergy in humans.

A hypoallergenic variant of the major birch pollen allergen shows distinct characteristics in antigen processing and T-cell activation.

BACKGROUND: BM4 is a novel genetically engineered variant of the major birch pollen allergen Bet v 1 that lacks the typical Bet v 1-like fold and displays negligible IgE-binding but strong T cell-activating capacity. The aim of this study was to elucidate possible differences between BM4 and Bet v 1 in internalization, antigen processing, and presentation. METHODS: Proliferative responses to BM4 and Bet v 1 of peripheral blood mononuclear cells and Bet v 1-specific T-cell clones were compared. Fluorescently labeled BM4 and Bet v 1 were used to study surface binding, endocytosis, and intracellular degradation by monocyte-derived DC (mdDC). Both proteins were digested by endolysosomal extracts of mdDC. BM4- and Bet v 1-pulsed mdDC were employed to assess the kinetics of activation of Bet v 1-specific T-cell clones and the polarization of naïve T cells. RESULTS: BM4 displayed a significantly stronger T cell-activating capacity than Bet v 1. Furthermore, BM4 showed increased surface binding and internalization as well as faster endolysosomal degradation compared with Bet v 1. BM4-pulsed mdDC induced enhanced proliferative responses at earlier time-points in Bet v 1-specific T-cell clones and promoted less IL-5 production in T cells than Bet v 1-pulsed mdDC. CONCLUSION: The loss of the Bet v 1-fold changes the protein's interaction with the human immune system at the level of antigen-presenting cells resulting in altered T-cell responses. By combining low IgE-binding with strong and modulating T cell-activating capacity, BM4 represents a highly interesting candidate for specific immunotherapy of birch pollen allergy.

Danon disease: case report and detection of new mutation.

Danon disease is an X-linked disorder resulting from mutations in the lysosome-associated membrane protein-2 (LAMP2) gene. We report a male patient with skeletal myopathy, mental retardation, and massive hypertrophic obstructive cardiomyopathy necessitating heart transplantation. Immunohistochemistry of skeletal muscle and leukocytes, western blot analysis of leukocytes and cardiac muscle, flow cytometry, and DNA sequencing were performed. Muscle biopsy revealed autophagic vacuolar myopathy and lack of immunohistochemically detectable LAMP-2. Diagnosis of Danon disease was confirmed by western blot analysis of myocardial tissue and peripheral blood sample of the patient showing deficiency of LAMP-2 in myocardium and leukocytes. Moreover, absence of LAMP-2 in lymphocytes, monocytes and granulocytes was shown by flow cytometric analysis. Genetic analysis of the LAMP2 gene revealed a novel 1-bp deletion at position 179 (c.179delC) at the 3' end of exon 2, resulting in a frameshift with a premature stop codon.

A microplate assay for the detection of oxidative products using 2',7'-dichlorofluorescin-diacetate.

A fluorometric microplate assay was established for the detection of respiratory burst activity in phagocytic cells by assessing oxidation of 2',7'-dichlorofluorescin-diacetate (DCFH-DA). This method is based on flow cytometric studies by Bass et al. (J. Immunol. 130 (1983) p. 1910) describing intracellular detection of DCFH oxidation due to the presence of hydrogen peroxides. In the present study we have adapted the assay for use in microtiter plates to determine the amount of extracellular reactive oxidative products. DCFH-DA, granulocytes and stimuli (phorbol myristate acetate, n-formyl-methionyl-leucylphenylalanine, concanavalin A) were added to microtiter plates and after incubation at 37 degrees C, the development of fluorescence intensity was read in a fluorescence concentration analyzer (FCA, Baxter). Calibration of fluorescence units recorded by the FCA was achieved by comparison with defined amounts of fluorescent DCF. The change in measured fluorescence was linear with cell density over the range of 2 x 10(5)-1 x 10(6) cells/well. Cumulative DCF generation in individual wells could be recorded non-destructively at frequent intervals for time course measurements. Results from FCA measurements correlated perfectly with the FACS analysis of the same samples (r = 0.99). In conclusion, this assay can be useful for screening monoclonal antibodies recognizing cell surface structures possibly involved in signal transduction as well as for testing phagocytes for their capacity to release reactive oxidative intermediates.

Evidence that sensitivity to cyclosporine is influenced by the HLA-DR phenotype of kidney graft recipients.

Clinical as well as experimental studies have shown a great interindividual variability in the immunosuppressive efficacy of CsA. Evaluating previous in vitro findings of a correlation between sensitivity of alloresponsiveness to CsA and the HLA-DR phenotype CsA levels were compared in kidney transplant recipients with and without rejections during the early posttransplant period and tested for a possible relationship to the HLA-DR phenotype of the recipient. In patients treated with CsA and prednisolone only, rejection frequency was significantly higher in HLA-DRw6 positive than in DRw6 negative graft recipients (77% vs. 53%, P = 0.045). In the DRw6 positive group incidence of rejection was independent of CsA blood levels, whereas in DRw6 negative patients frequency of rejection episodes decreased as a function of increasing CsA levels. Therefore the relative risk in developing graft rejection continuously increased in HLA-DRw6 positive patients. In HLA-DR2 positive graft recipients, however, a decrease in the relative risk could be observed with increasing CsA levels. Within patients with bioptically verified rejection episodes HLA-DR2 positive recipients had significantly lower CsA levels than DR2 negative patients (P = 0.01). In other HLA-DR phenotypes no association with CsA blood levels could be assessed. Also no statistically significant difference could be found in nonrejecting patients. These clinical findings demonstrate an association of sensitivity to immunosuppressive treatment and the HLA-DR phenotype of the graft recipient. Our results would indicate a very low CsA sensitivity of HLA-DRw6 positive graft recipients and thus might offer an explanation for previous findings about an increase in the incidence of rejection reported on those patients.

Influence of HLA phenotypes on the inhibition of in vitro alloreactivity by cyclosporine.

Interindividual variations in the immunosuppressive effect of Cyclosporine have been observed in clinical organ transplantation. Searching for an in vitro correlate we investigated a possible relation between inhibition of alloresponsiveness by CsA and the HLA phenotypes of the responder or stimulator in mixed lymphocyte reactions. Peripheral blood mononuclear cells from 28 healthy volunteers were used as responder or stimulator cells (gamma-irradiated) and the inhibitory effect of graded amounts of CsA was determined in 130 criss-cross combinations. Sensitivity of alloresponsiveness to the drug was expressed as the dose causing 50% inhibition (ED50) and was read from the inhibition curves generated after four-parameter logistic curve fitting. ED50 ranged from 0.35 ng/ml to 33.4 ng/ml and correlated only weakly with the magnitude of the response (r = 0.12). In MLC with HLA DR4-positive responder cells, ED50 was significantly lower (Pc = 0.0035, Kruskal Wallis) when compared with MLC with responder cells of other DR haplotypes. For HLA DR5-positive responder cells ED50 was significantly higher (Pc = 0.042) when compared with DR5-negative responder cells. No significant correlation between ED50 and any particular haplotype of the stimulator cells could be observed. Sensitivity to CSA did not differ in MLC with 1 or 2 mismatches in the HLA-DR locus. In summary, we found that sensitivity of in vitro alloreactivity was different for particular HLA DR phenotypes, which may have important implications for the immunosuppressive therapy of transplanted patients with cyclosporine.

Suppression of primary T-cell responses and induction of alloantigen-specific hyporesponsiveness in vitro by the Janus kinase inhibitor tyrphostin AG490.

BACKGROUND: Tyrphostin AG490 has recently been shown to block interleukin (IL)-2 receptor gamma-chain-associated Janus kinase 3. Here, we analyzed the effect of AG490 on T-cell alloresponses in vitro. METHODS: For the evaluation of T-cell activation, DNA synthesis, surface marker expression, cytokine secretion, intracellular calcium mobilization, early protein tyrosine phosphorylation, and apoptosis were measured. RESULTS: AG490 effectively inhibited T-cell proliferation in human mixed lymphocyte culture (MLC) even when added 4 days after culture initiation. Inhibition of IL-2-dependent proliferation in T-cell blasts and the incapability of IL-2 or IL-15 to restore proliferation in AG490-treated MLC suggests interference with cytokine receptor signaling. T-cell receptor-triggered early protein tyrosine phosphorylation, calcium mobilization, up-regulation of CD69, and initial CD25 expression were not affected. Interestingly, AG490 substantially inhibited production of IL-2 and interferon-gamma in T cells stimulated with alloantigen or via CD3 and CD28. In CD28-independent activation models (e.g., stimulation with phorbol myristate acetate plus ionomycin), however, cytokine secretion was not inhibited. Pretreatment of primary MLC with AG490 resulted in substantial down-regulation of secondary responses to cells from the original donor as opposed to third-party cells or phytohemagglutinin. Unresponsiveness was induced also in T cells stimulated with CD3 monoclonal antibody. Induction of apoptosis in polyclonally activated T cells and the incapability of IL-2 to reverse specific hyporesponsiveness, suggest programmed cell death as an important mechanism underlying antigen-specific down-regulation of alloresponses. CONCLUSIONS: We demonstrate that AG490 blocks different manifestations of T-cell activation. This and its ability to induce alloantigen-specific hyporesponsiveness point to a potential use for interfering with alloreactivities in vivo.

Role of humoral immune reactions as target for antirejection therapy in recipients of a spousal-donor kidney graft.

Excellent graft outcome has been reported for spousal-donor kidney transplantation. In husband-to-wife transplantation, however, a tendency toward inferior graft survival has been described for recipients who were previously pregnant. In our series of spousal-kidney transplantations (nine transplantations; three female recipients), actual graft survival is 100% (median observation time, 339 days). Five patients experienced early allograft rejection. In four transplant recipients, rejection was easily reversible by conventional antirejection therapy. In a multiparous recipient, however, mild interstitial allograft rejection associated with early graft dysfunction was resistant to anticellular treatment (antilymphocyte antibody, tacrolimus rescue therapy). The particular finding of polymorphonuclear neutrophils in peritubular capillaries and the finding of diffuse capillary deposits of the complement split product, C4d, in a posttransplantation biopsy specimen suggested a role of antibody-mediated graft injury. Retrospective flow cytometry cross-matching showed the presence of preformed immunoglobulin G (IgG) antibodies to HLA class I antigens that were not detectable by pretransplantation lymphocytotoxic cross-match testing or screening for panel reactive antibodies. After transplantation, however, complement-fixing antibodies, also presumably triggered by reexposure to spousal-donor HLA antigens, could be detected in the patient's serum. These findings suggested antibody-mediated allograft rejection and led to the initiation of immunoadsorption therapy (14 sessions) with staphylococcal protein A. Selective removal of recipient IgG resulted in complete reversal of graft dysfunction. Our findings suggest that in husband-to-wife transplantation, donor-specific antibodies, presumably triggered by previous pregnancies, might occasionally induce sustained allograft dysfunction. Thus, in this particular setting, a detailed immunologic and histopathologic work-up regarding antibody-mediated allograft dysfunction is warranted because immunoadsorption may be a highly effective treatment modality.

Mode of action of coumarin in immune cells.

In order to characterize further the mode of action of coumarin, binding studies were undertaken using human monocytes and radioactively labelled drug. Since coumarin is only a small compound and we wanted to exclude possible artefacts due to variations in size or conformation, the drug was produced by synthesis in the presence of radioactive 14C. Adding increasing amounts of a mixture of labelled and unlabelled drug to monocytes resulted in saturating conditions only at rather high concentrations. Performing Scatchard analysis demonstrated that binding sites for coumarin appeared to be present in relatively high numbers (7.5 x 10(8)/cell) but their affinity was rather low (K alpha approximately 2 x 10(2) M-1). Inhibition studies with 7-hydroxycoumarin revealed that an approximately four times higher molar concentration of the derivative was necessary to cause 50% displacement of coumarin from its binding site. These results indicate that binding of the drug to cells is characterized by high-capacity but low-affinity conditions. This would be compatible with the hypothesis that coumarin interacts with ubiquitous intracellular receptor proteins able to interact with aromatic hydrocarbons, which might form the basis for enzyme induction, and leads to the effects observed in vitro and in vivo.

Stable prodrugs of n-butyric acid: suppression of T cell alloresponses in vitro and prolongation of heart allograft survival in a fully allogeneic rat strain combination.

n-Butyric acid has previously been shown in vitro to suppress T cell alloresponses and beyond that to induce a state of alloantigen-specific hyporesponsiveness suggesting a potential relevance for suppressing alloresponses also in vivo. The clinical use of butyrate salt derivatives, however, is limited by an extremely short half-life due to rapid metabolism. This prompted us to investigate the effect of butyric acid derivatives with prolonged residence time in vivo on T cell alloresponses in vitro and further to explore the immunosuppressive capacity of esterified n-butyric acid in vivo. First, the effect of three butyric acid esters, i.e. glucose pentabutyrate, diacetone glucose butyrate and tributyrin on T cell proliferation in a human mixed lymphocyte culture (MLC) was evaluated. All three derivatives were found to inhibit T cell alloresponses in a concentration-dependent manner. Based on the ED50 values, glucose pentabutyrate was found to be most effective in inhibiting T cell alloreactivity in vitro (11 microM), followed by diacetone glucose butyrate (122 microM), tributyrin (146 microM) and sodium butyrate (539 microM). Because of its favourable in vitro properties, glucose pentabutyrate was chosen for in vivo experiments. To test the effect of this compound on allograft survival in vivo, in the second part of this study, heterotopic heart transplants were performed in a high responder fully allogeneic rat strain combination (Brown Norway to Lewis strain rats). We found that intraperitoneal (i.p.) injection of glucose pentabutyrate at 500 mg/kg/day (day 0 and daily up to 12 days posttransplant) induced a significant prolongation of allograft survival as compared to animals treated with vehicle (glycerol formal, i.p.) alone (14.1+/-6.3 versus 9.6+/-3.2 days, p = 0.036), whereby at lower dosage (100 mg/kg/day) no such effect was observed (10.2+/-2.1 days, p = 0.21). Our findings suggest that stable prodrugs of n-butyric acid might have potential clinical relevance for inhibiting alloresponses in vivo.

Complement-dependent acceleration of apoptosis in neutrophils by dialyzer membranes.

Recently, we have shown that cuprophan (CU) causes receptor modulation by a C5-dependent mechanism, which is activated by neutrophil-derived reactive oxygen intermediates. The objective of our study was to evaluate the contribution of dialyzer membranes to the induction of apoptosis in human neutrophils [polymorphonuclear cells (PMNs)]. PMNs harvested from healthy donors were incubated with hollow fibers from a biocompatible membrane polysulfone (PS) and a bioincompatible membrane CU, all in the presence of 25% human serum. After 4, 8, and 12 hours of incubation at 37 degrees C, apoptosis was quantitated by counting the numbers of cells showing features of apoptosis on cytospins by light microscopy and also by flow cytometry using propidium iodide nuclear staining. Compared with PMNs incubated with serum alone, cells cultured with fibers of PS demonstrated a higher percentage of apoptosis. Fibers from CU dialyzers led to a more pronounced induction of apoptosis in PMNs, which was significantly higher compared with PS. This effect was partly mediated by heat-sensitive serum products and depended on the presence of divalent cations. In contrast to the recently described C5-dependent pathway in PMN receptor modulation by CU, this effect seemed to depend on the presence of the complement factor C3. In conclusion, our results indicate that besides the well-known accelerated apoptosis of PMNs in uremia, both biocompatible and bioincompatible dialyzer material itself can accelerate apoptosis in human PMNs.

Demonstration of collagen type conversion in culture supernatants from human chondrocytes by an inhibition ELISA.

Culture supernatants from monolayer cultures of human chondrocytes were tested for the presence of collagen type I and type II during subpassaging using an inhibition Elisa. The sensitivity of the Elisa was 404 +/- 50 ng/ml (mean +/- SEM) for the determination of type I collagen and 112 +/- 16 ng/ml for type II collagen. Whereas using immunofluorescence techniques, type II collagen was observed in human chondrocytes cultured under monolayer conditions only up to the first subpassage (1), in culture supernatants from human chondrocytes type II collagen could be found up to the fourth subpassage. Type I collagen was detectable in supernatants from the beginning of primary cultures and was present up to the tenth subpassage in increasing concentrations. Variations in the amount of collagen present in the culture supernatants seemed in part to be due to different growth characteristics of the chondrocytes. Cell shape was not associated with the release of a particular collagen type. In summary, the collagen inhibition Elisa appears to be equivalent to biochemical methods with regard to sensitivity and specificity. Investigations on influencing the switch in collagen production in human chondrocytes may benefit from the use of both techniques described.

Rheumatoid factor isotypes and circulating immune complexes in rheumatoid arthritis.

Solid phase enzyme immunoassays were here used to quantify rheumatoid factors (RF) of the IgM, IgG and IgA classes and the immune complexes (IK) by their ability to bind to C1q or conglutinin in both the serum and synovial fluid of patients with rheumatoid arthritis (RA). Elevated serum levels of any RF isotype could be found in all patients with seropositive RA (IgM: 63%, IgG: 87%, IgA: 90%). Seronegative patients with RA presented to a significantly lesser extent with elevated levels of all the RF isotypes tested (IgM: 0%, IgG: 40%, IgA: 32%). Synovial fluid RF levels were significantly higher in SPRA patients than in SNRA patients with the exception of IgG-RF. All of the RF classes in both RA groups, however, were elevated when compared to RF in the synovial fluid of patients with osteoarthrosis. Both C1q binding and conglutinin binding immune complexes were significantly higher in the synovial fluid than in the serum of RA patients. The erythrocyte sedimentation rate and plasma iron levels were correlated with the levels of C1q binding immune complexes (IC) in the synovial fluid; total iron binding capacity showed an inverse relationship to synovial fluid IgG-RF levels. A radiographic index was also correlated with IgG-RF levels in the synovial fluid. Extraarticular manifestations were significantly more frequent in patients with elevated serum levels of IgM-RF or conglutinin binding IC. These findings indicate that IgG-RF in the synovial fluid and the formation of IC determined by their ability to bind C1q seem to be closely related to clinical features of local disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoimmunity and T-cell subpopulations in old age.

To investigate the interrelationship between T-cell-dependent immune functions and autoimmune phenomena in old age we determined T-cell subpopulations in 20 aged healthy individuals (80-96 years old) using monoclonal antibodies. These persons were also investigated as to humoral parameters such as antinuclear antibodies, rheumatoid factors (IgG-, IgA-, IgM-RF), antibodies to collagen types I-IV as well as autoantibodies to organ-specific antigens. In addition, immune complexes were determined. We found that aged individuals have an increased frequency of autoantibodies as compared to a young control population, each aged subject presenting with at least one autoantibody species. Immune complexes, however, were only rarely detected. Three individuals showed a slightly increased T-helper/T-suppressor cell ratio, four had a decreased ratio. An increased number of T-suppressor cells was significantly correlated with a lowered incidence of anticollagen antibodies. Other parameters tested by us: fibronectin, laminin, procollagen type III, C3 and C4 complement components, immunoglobulins and acid alpha 1-glycoprotein. Aged individuals have significantly higher serum levels of fibronectin, while laminin and procollagen concentrations are in the normal range. A large percentage of old individuals had increased serum levels of C3 and/or C4. The acute phase protein orosomucoid, however, was in the normal range.

[OKT 3 treatment of kidney transplant recipients]. / OKT 3-Therapie bei Nierentransplantatempfängern.

Anti-rejection therapy with the monoclonal antibody OKT 3 was effective in the treatment of acute rejection episodes in 66% (47/71) of OKT 3 treated kidney graft recipients. The success of OKT 3 therapy, however, appeared to be dependent on certain variables. Reversal of rejection differed significantly according to the indication, namely if patients were treated first line, as opposed to rescue therapy. Vascular components of rejection and/or severe interstitial rejection, as well as acute tubular necrosis were associated with a worse reversal rate. Non-responders to OKT 3 presented with significantly lower serum OKT 3 trough levels during the first three days of the treatment course. The presence of more than one of these risk factors was shown to decrease the reversal rate of rejection significantly. The variables appeared to exert their influence in an additive fashion. Administration of OKT 3 led to the induction of an acute phase reaction, which could be detected within one day after start of therapy. The incidence of both bacterial and viral infection was increased dramatically in OKT 3 treated patients as compared with graft recipients who were not treated with monoclonal or polyclonal antilymphocyte antibodies. The findings demonstrate that OKT 3 is of value in the treatment of acute rejection episodes but provide evidence of differential indications for OKT 3 therapy, which must be taken into account to optimize control of rejection and avoid unnecessary side effects where the drug is not indicated.