Occupational sensitization to ribosome-inactivating proteins in researchers.

BACKGROUND: Ribosome-inactivating proteins (RIPs) are expressed in many plants. Because of their anti-infectious and anti-proliferative effects, intensive research is going on for applying these toxins in therapy against viral infections or malignancies. Recently, we demonstrated that type I allergy against RIPs from elderberry can occur. OBJECTIVE: Stimulated by our study, a group of RIP researchers reported that some of the employees had suspected allergy to RIPs. METHODS AND RESULTS: We tested their sera in ELISA on natural RIPs. Specific IgE in four subjects were found against dianthin30, gelonin, momordin, PAP-S, saporin, ricin and volkensin. In contrast, asparin and lychnin did not show any IgE binding. When separating extracts of plants containing the toxins in SDS-PAGE, RIPs appeared to be the predominant constituents. Interestingly, among the other plant proteins, they were exclusively recognized by IgE in immunoblot. RIPs derived from close botanical families share high sequence homologies. Nevertheless, in IgE inhibition experiments with human sera, cross-reactivity between RIPs also derived from non-related plants could be demonstrated. CONCLUSION: We conclude that sensitization and IgE induction to RIPs may occur upon exposure. This has to be considered when applying them in therapy against malignancies or viral infections.

Heightened levels of circulating 20S proteasome in critically ill patients.

BACKGROUND: Recently, circulating proteasome core particles (20S proteasome) have been suggested as a marker of cell damage and immunological activity in autoimmune diseases. Aberrant leucocyte activation and increased lymphocyte apoptosis with consecutive T-cell unresponsiveness is deemed to play a pivotal role in the sepsis syndrome. Moreover sepsis-induced muscle proteolysis mainly reflects ubiqutin proteasome-dependent protein degradation. We therefore sought to investigate serum levels of 20S proteasome in critical ill patients. MATERIAL AND METHODS: Case-control-study at a university hospital intensive care unit; 15 patients recruited within 24-48 h of diagnosis of sepsis, 13 trauma patients recruited within 24 h of admission to the ICU, a control group of 15 patients who underwent abdominal surgery, and 15 healthy volunteers. ELISA was used to measure the concentration of 20S proteasome in the sera of the patients and controls. Data are given as mean +/- SEM. Mann-Whitney U-test was used to calculate significance and a P-value of 0.05 was considered to be statistically significant. RESULTS: Marked increase of 20S proteasome was detected in the sera of septic patients (33 551 +/- 10 034 ng mL-1) as well as in trauma patients (29 669 +/- 5750 ng mL-1). In contrast, significantly lower concentrations were found in the abdominal surgery group (4661 +/- 1767 ng mL-1) and in the healthy control population (2157 +/- 273 ng mL-1). CONCLUSION: Detection of 20S proteasome may represent a novel marker of immunological activity and muscle degradation in sepsis and trauma patients, and may be useful in monitoring the clinical effect of proteasome-inhibitors.

Alpha-Gal on bioprostheses: xenograft immune response in cardiac surgery.

BACKGROUND: The alpha-Gal (Galalpha1,3-Galbeta1-4GlcNAc-R) epitope is the major xenoantigen causing hyperacute rejection of pig organs transplanted into primates. Porcine bioprostheses are utilized in cardiac surgery. However, premature degeneration of bioprostheses has limited utilization in younger patients and the immune response remains elusive. We sought to investigate whether a specific alpha-Gal immune response may play a role in this clinical scenario. MATERIALS AND METHODS: We investigated the presence of alpha-Gal-epitope on native and fixed porcine valves by means of confocal laser scanning microscope (CLSM). ELISA was utilized to evidence whether implantation of bioprostheses elicits augmentation of pre-existing cytotoxic anti alpha-Gal IgM antibodies within 10 days of surgery. Patients who underwent coronary artery bypass grafting (CABG) or mechanical valve replacement served as controls (each group, n = 12). To corroborate the clinical relevance of the alpha-Gal immune response in vivo, we studied serum obtained before and after implantation of bioprostheses and its potency to lyse porcine alpha-Gal-bearing PK15 cells. RESULTS: We found the immunogenic alpha-Gal-epitope on fibrocytes interspersed in the connective tissue of porcine valves as determined by vimentin/IB4 lectin binding. Moreover, patients who were provided with a bioprostheses had developed a significant increase of naturally occurring cytotoxic IgM antibodies directed towards alpha-Gal after surgical intervention as compared with control patients (P < 0.0001, respectively). Sera obtained from the patients after the implantation of bioprostheses demonstrated an increased cytotoxicity against alpha-Gal-bearing PK-15 cells as compared with preoperative sera (P < 0.001). The specificity of the cytotoxic effects was proven as soluble Galalpha1-3Galbeta1-4GlcNAc markedly inhibited cell death of alpha-Gal-bearing PK15 cells (P < 0.001). CONCLUSION: Our data suggest that implantation of bioprostheses in cardiac surgery induces a xenograft-specific immune response. Procedures diminishing the presence of alpha-Gal on bioprostheses, such as utilization of genetically manipulated alpha-Gal-deficient xenograft or pretreatment with alpha-Galactosidase, might diminuate the immune response against bioprostheses and extend durability.

Interaction of human immunoglobulin G with CD16 on natural killer cells: ligand clearance, FcgammaRIIIA turnover and effects of metalloproteinases on FcgammaRIIIA-mediated binding, signal transduction and killing.

Human natural killer (NK) cells express low-affinity Fc immunoglobulin G (IgG) receptor (FcgammaRIIIA/CD16). The binding of monomeric IgG (mIgG) and F(ab')(2) fragments of 3G8 anti-CD16 monoclonal antibody (mAb) to FcgammaRIIIA was investigated by flow cytometry. Over 90% of NK cells bound endogenous IgG, and during incubation at 37 degrees C, the FcgammaRIIIA occupancy decreased slowly. Approximately 90% of NK cells bind mIgG or F(ab')(2) fragments of 3G8 anti-CD16 mAb. The calculated half-time (T(1/2)) of in vitro mIgG dissociation from FcgammaRIIIA was 130 min. By cross-linking the mIgG ligand with F(ab')(2) fragments of anti-human IgG antibody, the T(1/2) decreases to 85 min. In kinetics study, it has been shown that (125)I-mIgG bound to FcgammaRIIIA is slowly released in the culture supernatant, maybe eluted at acid pH, or partially internalized and degraded. The binding of IgG to FcgammaRIIIA was increased by 53.8% on cells cultured in the presence of RU36156, a matrix metalloproteinase (MMP) inhibitor. Furthermore, an increase in phosphorylation of Lyn tyrosine kinase, after cross-linking of mIgG-FcgammaRIIIA complex, was observed on NK cells treated with RU36156. When the FcgammaRIIIA was occupied by mIgG, the capacity of NK cells to kill K562 target cells was decreased by RU36156, because the MMP inhibitor protects CD16 from proteolysis. Our data demonstrate that binding of mIgG to human NK cells is followed by ligand dissociation and/or internalization, enzymatic degradation and exocytosis. The RU36156 MMP inhibitor protects FcgammaRIIIA from cleavage, augments NK-cell activation and may interfere in their killing capacity.

Allergen-loaded biodegradable poly(D,L-lactic-co-glycolic) acid nanoparticles down-regulate an ongoing Th2 response in the BALB/c mouse model.

BACKGROUND AND OBJECTIVE: Biocompatible and biodegradable microparticles have gained interest as antigen delivery systems during the recent years. We investigated whether biodegradable poly(d,l-lactic-co-glycolic) acid (PLGA) nanospheres could be used as allergen vehicles for few-shot therapy of type I allergy. METHODS: The major birch pollen allergen Bet v 1 was encapsulated in PLGA nanospheres (PLGA-Bet v 1). We examined the antigenicity and the immune response to PLGA-Bet v 1 in a BALB/c mouse model. RESULTS: The antigenicity of Bet v 1 was largely unaffected by PLGA entrapment. When BALB/c mice were immunized subcutaneously with PLGA-Bet v 1, they formed allergen-specific IgG antibodies, but did not develop hypersensitivity to Bet v 1, as shown by type I skin tests. To evaluate their therapeutic potential, PLGA-Bet v 1 with or without Al(OH)3 or non-entrapped Bet v 1 with Al(OH)3 were used for single-shot treatment of sensitized mice. Both groups treated with PLGA-Bet v 1 developed high levels of Bet v 1-specific IgG2a antibodies (P<0.01), whereas IgG1 levels decreased significantly (P<0.01). Moreover, T cells from mice treated with PLGA-Bet v 1 showed IFN-gamma and IL-10 production. The synthesis of these cytokines was enhanced in the groups where Al(OH)3 had been added to the vaccine formulation. CONCLUSION: Allergen-loaded PLGA nanoparticles modulate an ongoing Th2 response in the BALB/c mouse model, as demonstrated by down-regulation of IgG1 and production of IFN-gamma and IL-10. Our data strongly suggest that PLGA nanospheres can advantageously be used for formulations of allergen extracts or allergen derivatives for the few-shot treatment of type I allergy.

Increased serum concentrations of soluble CD95/Fas and caspase 1/ICE in patients with acute angina.

OBJECTIVES: To investigate the expression of death inducing receptors in the sera of patients with stable and unstable angina. DESIGN: 80 consecutive patients with stable (n = 40) or unstable (n = 40) angina pectoris were studied. Serum concentrations of soluble CD95 (sCD95), soluble CD95 ligand (sCD95L; CD178), tumour necrosis factor (TNF) alpha, soluble TNFalpha receptor type 1 (sTNFR1), and interleukin 1beta converting enzyme (ICE; caspase 1) were measured by enzyme linked immunosorbent assay (ELISA). RESULTS: Significant increases in the concentrations of sCD95 and ICE (p < 0.001 and p < 0.023, respectively) were found in the serum from patients with unstable angina relative to those with stable angina. There were no significant differences in the concentrations of sCD95L, TNF alpha, and sTNFR1 between the groups. CONCLUSIONS: These data provide the first evidence that sCD95 and ICE are important serological markers that may help to discriminate between stable and unstable angina. This observation may warrant further clinical study to elucidate the clinical impact of sCD95 and ICE in acute coronary syndromes.

Type I allergy to elderberry (Sambucus nigra) is elicited by a 33.2 kDa allergen with significant homology to ribosomal inactivating proteins.

BACKGROUND: Patients suffering from allergic rhinoconjunctivitis and dyspnoea during summer may exhibit these symptoms after contact with flowers or dietary products of the elderberry tree Sambucus nigra. OBJECTIVE: Patients with a history of summer hayfever were tested in a routine setting for sensitization to elderberry. Nine patients having allergic symptoms due to elderberry and specific sensitization were investigated in detail. We studied the responsible allergens in extracts from elderberry pollen, flowers and berries, and investigated cross-reactivity with allergens from birch, grass and mugwort. METHODS: Sera from patients were tested for IgE reactivity to elderberry proteins by one-dimensional (1D) and 2D electrophoresis/immunoblotting. Inhibition studies with defined allergens and elderberry-specific antibodies were used to evaluate cross-reactivity. The main elderberry allergen was purified by gel filtration and reversed-phase HPLC, and subjected to mass spectrometry. The in-gel-digested allergen was analysed by the MS/MS sequence analysis and peptide mapping. The N-terminal sequence of the predominant allergen was analysed. RESULTS: 0.6% of 3668 randomly tested patients showed positive skin prick test and/or RAST to elderberry. IgE in patients' sera detected a predominant allergen of 33.2 kDa in extracts from elderberry pollen, flowers and berries, with an isoelectric point at pH 7.0. Pre-incubation of sera with extracts from birch, mugwort or grass pollen rendered insignificant or no inhibition of IgE binding to blotted elderberry proteins. Specific mouse antisera reacted exclusively with proteins from elderberry. N-terminal sequence analysis, as well as MS/MS spectrometry of the purified elderberry allergen, indicated homology with ribosomal inactivating proteins (RIPs). CONCLUSION: We present evidence that the elderberry plant S. nigra harbours allergenic potency. Independent methodologies argue for a significant homology of the predominant 33.2 kDa elderberry allergen with homology to RIPs. We conclude that this protein is a candidate for a major elderberry allergen with designation Sam n 1.

Allergologic exploration of germins and germin-like proteins, a new class of plant allergens.

BACKGROUND: Germins and the related germin-like proteins (GLPs) are glycoproteins expressed in many plants in response to biotic and abiotic stress. To test the potential impact of germins and GLPs, recombinant germin from Triticum aestivum (tGermin) and GLPs from Arabidopsis thaliana (tGLP), both produced in transformed tobacco plants, were used. METHODS: Sera from 82 patients with type I allergy to birch, grass or mugwort pollen and/or wheat were tested in immunoblot for IgE binding to tGermin and tGLP, and the IgE reactivity after chemical and enzymatic deglycosylation was analysed. The biological activity of tGermin and tGLP was determined in a histamine release assay and in skin prick testing (SPT). RESULTS: In an immunoblotting assay, 24 out of 82 tested sera (29.26%) from allergic patients showed IgE-binding to tGermin, and 18 of these sera (21.95%) displayed also IgE-binding to tGLP. The deglycosylation experiments indicated that glycan moieties contribute significantly to the IgE-binding of tGermin and tGLP. Both tGermins and tGLP induced specifically histamine release in an in vitro assay as well as in SPT. CONCLUSION: Our in vitro and in vivo findings demonstrate that germin and GLPs are capable to bind IgE most likely via carbohydrate determinants, and represent allergenic molecules.

Activation-induced T cell death, and aberrant T cell activation via TNFR1 and CD95-CD95 ligand pathway in stable cardiac transplant recipients.

Specific blockade by antibodies (Abs) utilized in induction therapy may cause activation-induced cell death (AICD) in lymphocytes of transplant recipients, preactivated via CD95 and tumour necrosis factor-alpha receptor type 1 (TNFR1), and reduce allograft rejection frequency. Amongst 618 heart transplant (HTX) patients receiving antithymocytes globulin (ATG) therapy, 14 recipients with IVUS-verified freedom of transplant vasculopathy were studied. The control group contained 14 patients awaiting transplantation, classified by the New York Hearth Association heart failure as class IV. From 618 HTX patients 89% were free of rejection grade ISHLT > or =2-3 within 3-month post transplantation and 86% after one year. The death inducing receptors (DIR) such as CD95, CD95L and soluble TNFR1 were significantly increased in HTX recipients versus controls, as demonstrated by FACS, immunoblotting or ELISA (P < 0.001). The presence of increased DIR and in vivo apoptosis in HTX recipients, indicated by annexin-V binding, was further confirmed by the presence of high concentration of histones in the sera of patients. ATG, anti-IL-2R and OKT-3 Abs inhibited cell proliferation in a dose-dependent manner. The induction of apoptosis and/or necrosis was demonstrated in cells cultured with these Abs by annexin-V and 7-aminoactinomycin staining, respectively. Our findings demonstrate that T cells from HTX recipients express high level of CD95, CD95L and soluble TNFR1, and undergo apoptosis and AICD. These cells recognizing donor alloantigens may be selectively eliminated in vivo, and should be responsible for the observed immunological unresponsiveness, indicated by low rejection rates in our patient cohort treated by conventional triple therapy.

Death-inducing receptors and apoptotic changes in lymphocytes of patients with heart transplant vasculopathy.

The specific role of lymphocyte apoptosis and transplant-associated atherosclerosis is not well understood. The aim of our study was to investigate the impact of T cell apoptotic pathways in patients with heart transplant vasculopathy. Amongst 40 patients with cardiac heart failure class IV who have undergone heart transplantation, 20 recipients with transplant-associated coronary artery disease (TACAD) and 20 with non-TACAD were investigated one year postoperative. Expression of CD95 and CD45RO, and annexin V binding were measured by FACS. Soluble CD95, sCD95 ligand (sCD95L), tumour necrosis factor receptor type 1 (sTNFR1), and histones were measured in the sera by ELISA. The percentage of cells expressing CD3 and CD4 was significantly reduced in TACAD as well as in non-TACAD patients as compared with control volunteers. Interestingly, the proportion of CD19+ (B cells) and CD56+ (NK) cells was increased in TACAD groups (versus non-TACAD; P < 0.01, and P < 0.001, respectively). In contrast to sCD95, the expression of CD95 (APO-1/Fas) and CD45RO (memory T cells), and sCD95L were significantly increased in non-TACAD and TACAD patients. T cell activation via CD95 with consecutive apoptosis was increased in both groups. The concentration of sTNFR1, IL-10 and histones was significantly elevated in sera from TACAD than non-TACAD patients, and in both groups than in healthy controls. These observations indicate that the allograft may induce a pronounced susceptibility of CD4+ T cells to undergo apoptosis and antibody-driven activation-induced cell death. This data may suggest a paradox immune response similar to that seen in patients with autoimmune diseases.

Transitory immunologic response after implantation of the DeBakey VAD continuous-axial-flow pump.

BACKGROUND: The development of local and systemic infection is a significant risk factor associated with implantation of a ventricular assist device. The immunologic consequence of continuous-flow rotary blood pumps is not known. METHODS: Six male adult patients (mean age 47 plus minus 10.3) with end-stage left heart failure received a DeBakey VAD axial-flow pump for use as a bridge to transplantation. (Four patients underwent transplantation after a mean 115 plus minus 14 days; 2 patients are still waiting for the allograft.) RESULTS: We prospectively monitored T-cell populations and apoptosis-specific aberrant T-cell activation via CD95 triggering and annexin V binding to lymphocytes, identifying T cells undergoing early phases of apoptosis, within the first 10 weeks. Moreover, soluble death-inducing receptors soluble CD95 and soluble tumor necrosis factor-R1 were evaluated by enzyme-linked immunosorbent assay. CONCLUSION: Patients bridged to transplantation by a nonpulsatile ventricular assist device demonstrated an initial pronounced apoptosis-specific immune alteration by increased annexin V binding to CD3 T cells and death-inducing receptors soluble CD95/tumor necrosis factor-R1 (all P <.001). All parameters normalized after 7 weeks to baseline. No blood-borne sepsis was detected, as defined by blood culture, within the first 10 weeks of the cohort study. These results indicate a biphasic immunologic response in patients with end-stage heart failure treated with nonpulsatile ventricular assist devices.

Phage-displayed Bet mim 1, a mimotope of the major birch pollen allergen Bet v 1, induces B cell responses to the natural antigen using bystander T cell help.

BACKGROUND AND OBJECTIVE: In previous studies we have generated mimotopes of Bet v 1, the major birch pollen allergen, by biopannings of phage-display random peptide libraries. In the present study, we analysed the humoral and cellular immune response to Bet v 1-mimotopes. METHODS: The mimotope CFPYCYPSESA, designated Bet mim 1, was used for intraperitoneal immunizations of BALB/c mice in phage-displayed form. For examination of the humoral immune response, enzyme-linked immunosorbent assay (ELISA) experiments were applied. Stimulation capacities were investigated in cultured mouse splenocytes and in humoral Bet v 1-specific T cell clones. RESULTS: We demonstrated that the Bet mim 1-induced murine antibody response against Bet v 1 was predominated by the IgG1 isotype. In these mice only the phage-displayed mimotopes, but neither the allergen nor the synthetic Bet mim 1-mimotopes were able to stimulate proliferation of cultured splenocytes. Using Bet v 1-specific T cell clones of allergic patients, phage-displayed and synthetic mimotopes were unable to stimulate T cell proliferation. Moreover, tolerance induction to Bet v 1 in mice by intranasal administration of Bet mim 1-phages or Bet mim 1-peptide failed. CONCLUSION: Taking these results together, our data indicate that Bet mim 1 mimics a Bet v 1-epitope on the B cell but not on the T cell level. We suggest that the phage itself is responsible for the recruitment of T cells providing bystander help in the formation of a mimotope-specific humoral response.

Anti-Galectin-3 IgG autoantibodies in patients with Crohn's disease characterized by means of phage display peptide libraries.

Galectin-3, a member of beta-galactoside-binding lectins, is expressed and secreted by a variety of cell types including human intestinal epithelial cells. The presence of anti-galectin-3 antibody in the sera of patients was analyzed by immunoblotting using recombinant human galectin-3. A substantially higher percentage of sera from Crohn's disease patients contained anti-galectin-3 IgG autoantibodies than from patients with ulcerative colitis, primary biliary cirrhosis, or autoimmune hepatitis and of apparently healthy control volunteers. In Crohn's disease patients the titer of autoantibodies was high and interestingly correlated negatively with disease activity. To characterize and generate artificial epitopes (mimotopes), the anti-galectin-3 monoclonal antibodies A3A12 and B2C10 were used for biopannings of phage display nonapeptide libraries. These mimotopes interfered with the binding of autoantibodies to recombinant and native intestinal epithelial galectin-3. Our data may suggest that galectin-3 mimotopes could be used for the induction of IgG with desired specificity to regulate immune responses in Crohn's disease patients.

Monovalent fusion proteins of IgE mimotopes are safe for therapy of type I allergy.

By screening phage display random peptide libraries with purified immunoglobulin E (IgE) from birch pollen-allergic patients, we previously defined peptides mimicking natural IgE epitopes (mimotopes) of the major birch pollen allergen Bet v 1. The present study aimed to define a monovalent carrier for the IgE mimotopes to induce protective antibodies directed to the IgE epitopes, suitable for mimotope-specific therapy. We expressed the selected mimotopes as fusion proteins together with streptococcal albumin binding protein (ABP). The fusion proteins were recognized specifically by anti-Bet v 1 human IgE, which demonstrated that the mimotopes fused to ABP resemble the natural IgE epitope. Bet v 1-specific IgG was induced by immunization of BALB/c mice with fusion proteins. These IgG antibodies could inhibit IgE binding to Bet v 1. Skin testing of Bet v 1 allergic mice showed that the ABP mimotope constructs did not elicit type I skin reactions, although they possess IgE binding structures. Our data suggest that IgE mimotopes are safe for epitope-specific immunotherapy of sensitized individuals, when presented in a monovalent form. Therefore, ABP-fused mimotopes are promising candidates for a new type of immunotherapy based on the precise induction of blocking antibodies.

Impaired T cell proliferation, increased soluble death-inducing receptors and activation-induced T cell death in patients undergoing haemodialysis.

Haemodialysis is a widespread option for end-stage renal disease (ESRD). Long-term success of dialysis is, however, limited by a high rate of serious bacterial and viral infections. We compared T cell functions in ESRD patients undergoing haemodialysis (n = 20), or were not dialysed and received conventional medical treatment (n = 20). Healthy volunteers (n = 15) served as controls. The T cell phenotype was examined by immunofluorescence using fluorochrome-labelled monoclonal antibodies and FACS analysis. The concentration of soluble CD95/Fas and of tumour necrosis factor-alpha receptor type 1 (sTNFR1) in the sera was quantified by ELISA. Activation-induced programmed T cell death was triggered by anti-CD3/CD28 antibodies and measured by 7-AAD staining. All immunological tests were performed at least 1 month after dialysis initiation. T cell proliferation in response to phytohaemagglutinin or anti-CD3 monoclonal antibodies was moderately diminished in non-dialysed patients and markedly reduced in haemodialysis patients compared to healthy controls (P < 0.01 and P < 0.001, respectively). In a mixed lymphocyte culture the proliferative response of T cells from dialysed patients was significantly diminished (P < 0.001). T cells of both non-dialysed and dialysed patients have augmented CD95/Fas and CD45RO expression, increased sCD95/Fas and sTNFR1 release and spontaneously undergo apoptosis. Culture of T cells from haemodialysis patients with anti-CD3/CD28 antibodies increased the proportion of CD4(+) T cells committing activation-induced cell death by a mean 7.5-fold compared to T-helper cells from non-dialysed patients (P < 0.001). Renal failure and initiation of haemodialysis results in a reduced proliferative T cell response, an aberrant state of T cell activation and heightened susceptibility of CD4(+) T cells to activation-induced cell death.

A serine/alanine polymorphism in the nucleotide-binding fold-2 of the sulphonylurea receptor-1 (S1369A) is associated with enhanced glucose-induced insulin secretion during pregnancy.

The sulphonylurea receptor-1 (SUR-1) regulates glucose-induced insulin secretion by controlling K+-ATP channel activity of the pancreatic beta-cell membrane. In this study, we investigated the putative role of a T/G-polymorphism (exon 33, codon 1369; S1369A) in the adenosine diphosphate-sensing nucleotide-binding fold-2 (NBF-2) of the SUR-1 on glucose-induced insulin secretion during an oral glucose tolerance test in pregnant women (PW; n=182). Compared to PW with the T/T genotype, statistically significant elevated C-peptide concentrations were found 60 min after glucose intake in PW with the T/G and G/G genotype (T/T 9.0+/-0.4 ng/ml vs T/G 10.8+/-0.4 ng/ml or G/G 10.8+/-0.7 ng/ml, p=0.01). Furthermore, compared to PW with T/T genotype the deltaC-peptide (60/0 min) was significantly enhanced in PW with T/G or G/G genotype (T/T 6.7+/-0.3 vs T/G 8.9+/-0.4 or G/G 8.9+/-0.7, p=0.0009). A significant correlation of C-peptide concentrations with blood glucose (BG) 60 min after glucose intake was only found in PW with the T/T genotype (r=0.6, p<0.0004). Similarly, a significant correlation of insulin concentrations with BG 60 min after glucose intake was observed in PW with T/T genotype (r=0.5, p<0.0001) and T/G genotype (r=0.24, p<0.03) but not in PW with G/G genotype (r=0.01, p=0.9). From our data we conclude that in PW with the alanine substitution in the NBF-2 region, the insulin response of the pancreatic beta-cell after glucose intake is enhanced and does not correlate with actual BG levels.

Relationship between interleukin-6 plasma concentration in patients with sepsis, monocyte phenotype, monocyte phagocytic properties, and cytokine production.

Monocyte phenotype, their phagocytic capacity as well as the cytokine production from 10 patients with sepsis with low interleukin-6 (IL-6) serum concentrations (<1000 pg/mL) and 8 patients with sepsis with high IL-6 (> or = 1000 pg/mL) plasma concentrations were investigated within 24 hours of fulfilling the criteria for sepsis. Monocytes from patients with high IL-6 levels had higher levels of human leukocyte antigen (HLA)-DR, HLA-ABC, CD64, and CD71, and the production of tumor necrosis factor-alpha (TNF-alpha) and IL-8, as well as the capacity of monocytes to phagocytose, was significantly elevated. Of 8 patients with high levels of plasma IL-6, 4 patients died. In contrast, all 10 patients with low plasma IL-6 concentrations survived until day 28. Patients who died had constant high IL-6 concentrations during the first 3 days, whereas IL-6 levels in patients who survived decreased by 88%. Our data indicate that IL-6 levels are a better prognostic parameter in the early phase of sepsis than the monocyte HLA-DR expression.

Lipopolysaccharide causes atrophy of Peyer's patches and an increased expression of CD28 and B7 costimulatory ligands.

Intestinal mucosal dysfunction appears to contribute to infectious complications in critically ill patients. The current study was undertaken to investigate whether endotoxin affects lymphocyte subpopulations and the expression of costimulatory signals in Peyer's patches (PP). Female Balb/c mice were given an intraperitoneal injection of 25 microg LPS and sacrified 24 h or 72 h later to determine total cell yield, lymphocyte subpopulations (B-cells, total T-cells, CD4+- and CD8+-cells), the costimulatory molecules CD28, B7.1 (CD80) and B7.2 (CD86) and the percentage of apoptotic cells in PP and in the spleen as well as small intestinal IgA concentration. Lipopolysaccharide (LPS) challenge caused a significant decrease of total cell yield in PP at both time-points (-50+/-28% and -43+/-25%, respectively; P < 0.001). This decrease was significant for all measured lymphocyte subpopulations. In contrast, total cell yield was increased (P < 0.001) in the spleen 24 h (+52+/-13%) and 72 h (+130+/-22%) after LPS. The decrease of lymphocyte numbers in the PP was accompanied by an increased percentage of lymphocytes expressing costimulatory molecules. In this respect, an increased percentage of CD40+CD80+, CD40+CD86+, and of CD4+CD28+ could be demonstrated after LPS administration. In the spleen, the percentage of CD4+CD28+ was also elevated after LPS bolus, however, the percentage of CD40+CD80+ was reduced, and that of CD40+CD86+ was unaltered. The influence of LPS on apoptosis of lymphocytes was time-dependent. The percentage of apoptotic cells 24 h after LPS was increased in PP (P < 0.01), but was unchanged in the spleen. Seventy-two hours after LPS injection, the percentage of apoptotic cells returned to normal in PP. Luminal IgA levels remained unchanged after LPS challenge. In conclusion, our data show that LPS causes atrophy of PP which seems to be counterregulated by an enhanced expression of costimulatory molecules.