Simple determination of polysaccharide specific antibodies by means of chemically modified ELISA plates.

A new ELISA technique using Nunc CovaLink NH microtiter plates has been developed to measure anticapsular polysaccharide specific antibodies. Capsular polysaccharide (PS) of Haemophilus influenzae type b (PRP) and pneumococcal antigens types 3, 6, 8, 14, 19, 23 were immobilized on CovaLink NH. These are modified plates with secondary amino groups bound to their surface which, in the presence of a water-soluble carbodiimide as coupling reagent, facilitate the direct binding of polysaccharides. We compared the binding characteristics of PS antigens to CovaLink NH and a conventional polystyrene ELISA plate. Checkerboard titration of PS antigens between 0.04-30 micrograms/ml clearly demonstrated that with Covalink NH optimal binding of a pooled serum from immunized donors was achieved for all PS antigens tested at a concentration of 1 microgram/ml, while binding of PS to the conventional plate was rather poor even at concentrations of 30 micrograms/ml. The CVs for the ELISA ranged from 1.1 to 2.8% for intra-assay comparisons and from 3.6 to 7.3% for inter-assay comparisons. In addition, when PRP-IgG antibodies were determined with the CovaLink NH ELISA and compared with the Farr assay an acceptable correlation ( r = 0.89, p < 0.0001) was obtained. The technique described provides a simple and sensitive tool for evaluating specific immunity to PS antigens.

Differential immune response to influenza and pneumococcal vaccination in immunosuppressed patients after heart transplantation.

INTRODUCTION: Patients after solid organ transplantation are at an increased risk for microbial infections. Due to therapeutic immunosuppression, the response to active immunizations may be reduced. The serological efficacy of pneumococcal and influenza vaccination was studied in heart transplant recipients. PATIENTS AND METHODS: Sixteen patients over 1 year after heart transplantation and control patients were immunized with a 23-valent pneumococcal vaccine and a triple-split influenza vaccine. Pre- and postvaccinal antibody titers were serologically determined, including quantitation of specific antibodies against nine pneumococcal serotypes. RESULTS: Both vaccines were well tolerated without systemic reactions or infectious complications. Median postvaccinal pneumococcal antibody titers in the transplant patients were comparable to controls (5513 U/ml, range: 694-41007, vs. 5490 U/ml, range: 1088-38042; P=NS); vaccination was successful in 23/23 (100%) of controls and in 15/16 (94% plus 1 borderline positive case) of the transplant recipients. Specific antibody titers were similar for eight of nine serotypes; only the immune response against serotype 3 was reduced after transplantation. The efficacy of influenza vaccination was significantly impaired in transplant patients against all three virus strains (62% vs. 97%, P<0.01/50% vs. 94%, P<0.001/37% vs. 80%, P<0.01), but 9/16 (56%) of patients still showed a sufficient immune response to two out of three virus strains. No clinical or demographic predictors of successful vaccination could be established. CONCLUSIONS: Pneumococcal vaccination under cyclosporine-based immunosuppression after heart transplantation is safe and equally effective as in healthy controls. In contrast, the immune response to influenza vaccination is significantly reduced, although not completely abolished. This differential response might be accounted for by T cell-independent antibody production against polysaccharide antigens contained in the pneumococcal vaccine.

Studies of the ATP dependence of protein degradation in cells and cell extracts.

Experiments with metabolic inhibitors in vivo indicate that intracellular protein degradation requires the continuous production of ATP. We have established soluble cell-free preparations from rabbit reticulocytes, rat liver, and Escherichia coli that degrade abnormal protein in an ATP-dependent fashion. These enzymes appear to be responsible for the selective breakdown of abnormal protein that may result from mutations, biosynthetic errors or intracellular denaturation. Experiments with inhibitors indicate that this process and the degradation of many short-lived normal proteins does not occur in the lysosome. The cell-free extracts prepared from these crude extracts hydrolyse [14C] globin by a process stimulated 2--3-fold by ATP and to a lesser extent by GTP, CTP or UTP. These activities degrade globin to large peptides which are then cleaved by soluble peptidases. The ATP-stimulated protease that partially purified from rat liver cytoplasm is also stimulated by pyrophosphate. This protease has an apparent molecular weight of 480,000. In contrast, the E. coli enzyme has an apparent molecular weight of 115,000 and is completely dependent on ATP, after partial purification by ion exchange and gel chromatography. This enzyme can be distinguished from six other proteolytic enzymes from E. coli active at pH 7.8. E. coli contains, in addition, four proteases that are not stimulated by ATP and degrade globin to acid-soluble material. We have also demonstrated in E. coli and reticulocytes other proteases that appear specific for small protein substrates and may play a role in the later steps in protein breakdown. The ATP-stimulated endoproteases appear to catalyse the rate-limiting steps in intracellular protein breakdown. However, the actual role of ATP in the degradative process is not known.

Photoaffinity labeling the torpedo nicotinic acetylcholine receptor with [(3)H]tetracaine, a nondesensitizing noncompetitive antagonist.

Tetracaine (N,N-dimethylaminoethyl-4-butylaminobenzoate) and related N,N-dialkylaminoethyl substituted benzoic acid esters have been used to characterize the high-affinity binding site for aromatic amine noncompetitive antagonists in the Torpedo nicotinic acetylcholine receptor (nAChR). [(3)H]Tetracaine binds at equilibrium to a single site with a K(eq) value of 0.5 microM in the absence of agonist or presence of alpha-bungarotoxin and with a K(eq) value of 30 microM in the presence of agonist (i.e., for nAChR in the desensitized state). Preferential binding to nAChR in the absence of agonist is also seen for N,N-DEAE and N,N-diethylaminopropyl esters, both binding with 10-fold higher affinity in the absence of agonist than in the presence, and for the 4-ethoxybenzoic acid ester of N, N-diethylaminoethanol, but not for the 4-amino benzoate ester (procaine). Irradiation at 302 nm of nAChR-rich membranes equilibrated with [(3)H]tetracaine resulted in covalent incorporation with similar efficiency into nAChR alpha, beta, gamma, and delta subunits. The pharmacological specificity of nAChR subunit photolabeling as well as its dependence on [(3)H]tetracaine concentration establish that the observed photolabeling is at the high-affinity [(3)H]tetracaine-binding site. Within alpha subunit, >/=95% of specific photolabeling was contained within a 20-kilodalton proteolytic fragment beginning at Ser(173) that contains the M1 to M3 hydrophobic segments. With all four subunits contributing to [(3)H]tetracaine site, the site in the closed channel state of the nAChR is most likely within the central ion channel domain.

Immunogenicity and tolerance of a 7-valent pneumococcal conjugate vaccine in nonresponders to the 23-valent pneumococcal vaccine.

There is still a lack of effective vaccination strategies for patients with a deficient antibody response to bacterial polysaccharide antigens. In an open trial, we evaluated the immunogenicity and tolerance of a new 7-valent pneumococcal conjugate vaccine in 22 infection-prone nonresponders to pneumococcal polysaccharide vaccine and 21 controls. In the patient group, nonresponsiveness was confirmed by repeated vaccination with a 23-valent pneumococcal polysaccharide vaccine. The study protocol provided two doses of the pneumococcal conjugate vaccine, given 4 to 6 weeks apart, for both groups. The antibody response was determined before each vaccination and on follow-up by an enzyme-linked immunosorbent assay and compared to the response in a functional opsonophagocytosis assay. Patients showed a significantly lower postvaccination immune response for all serotypes than did controls. The postvaccination response was serotype dependent. A median titer of >1 microgram/ml in patients was recorded only for serotypes 4, 9V, 14, and 19F, which are known to be more immunogenic than serotypes 6B, 18C, and 23F. In the patient group, 70% responded to serotype 19F (Pnc 19F), 65% responded to Pnc 14 and 4, 60% responded to Pnc 9V, 55% responded to Pnc 18C, 50% responded to Pnc 23F, and 25% responded to Pnc 6B. In the control group >95% of individuals showed a titer of >1 microgram/ml to every serotype. The vaccine was tolerated well, and no major side effects have been reported. The new pneumococcal conjugate vaccine is clearly more immunogenic in previous nonresponders than is the 23-valent pneumococcal vaccine. Immunization with a pneumococcal conjugate vaccine should be considered as a strategy to protect high-risk patients.

Persistent hypogammaglobulinemia following mononucleosis in boys is highly suggestive of X-linked lymphoproliferative disease--report of three cases.

Hypogammaglobulinemia is a common symptom in different immunodeficiencies. It is, however, not usually associated with Epstein-Barr virus (EBV) infections. The X-linked lymphoproliferative disease (XLP) on the other hand shows immunological changes in response to the EBV. Here we report three previously healthy boys, all of which developed persistent hypogammaglobulinemia following severe acute infectious mononucleosis. All three patients revealed T-cell abnormalities including inverted CD4/CD8 and increased CD8(+) T-cell numbers. The number of IFN-gamma-producing T cells were markedly increased in the two patients studied so far. In addition, patient 2 showed mainly T cells, instead of B cells, to be infected with the EBV. Apart from an uncle of patient 3, who died of malignant lymphoma, family history was unremarkable in all cases. All three patients exhibited mutations in the SH2D1A gene, establishing the diagnosis of XLP. Protein expression was found on immunoblot analysis in one patient with a missense mutation. Development of persistent hypogammaglobulinemia after severe primary EBV infection seems to be a specific diagnostic sign for XLP even in males with unremarkable family history.

[Pneumococcal vaccination after heart and liver transplantation. Immune responses in immunosuppressed patients and in healthy controls]. / Pneumokokkenimpfung nach Herz- und Lebertransplantation. Immunantworten bei immunsupprimierten Patienten und gesunden Kontrollpersonen.

BASIC PROBLEM AND OBJECTIVE OF STUDY: Among other effects, therapeutic immunosuppression after organ transplantation impairs antibody formation. But because of the increased risk of infection, the efficacy of prophylactic immunization is of particular importance in patients after transplantation. For this reason the immunogenicity after immunization against pneumococcus was investigated in transplanted patients. PATIENTS AND METHODS: A 23-valent vaccine of capsular polysaccharides (Pneumovax 23) was administered to 31 patients 4-85 months after transplantation (16 hearts transplants, 15 liver transplants; age range 22-63 years). The same immunization was given to 23 healthy control subjects. The immune response was measured serologically in all groups. RESULTS: Immunization was well tolerated by all participants, and there were no infectious or systemic side effects. Mean postvaccinal global pneumococcus-specific antibody titres were comparable in the healthy and transplanted subjects (controls: 5490 U/ml, heart transplanted: 5513 U/ml, liver transplanted: 4148 U/ml; differences not significant). Analysis of individual titres for the nine most important serotypes showed comparable results for six serotypes, reduced titres being found for serotypes 3 and 8 after heart transplantation, and for serotypes 8 and 23 after liver transplantation. CONCLUSION: Safe antipneumococcal immunization is possible during therapeutic immunosuppression after heart or liver transplantation. The achieved immune response is comparable to that in healthy controls. The high efficacy of the pneumococcal vaccine, compared with other vaccines, in immunosuppressed patients may be due to T-cell dependent antibody production against polysaccharides.