Immunosurveillance of alglucerase enzyme therapy for Gaucher patients: induction of humoral tolerance in seroconverted patients after repeat administration.
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Alglucerase, a macrophage-targeted enzyme replacement therapy for Gaucher disease, has been successfully used for several years to improve clinical symptoms and reverse disease progression. As part of an immunosurveillance program, 1,122 Gaucher patients were monitored for antibody response to glucocerebrosidase, the active component of alglucerase. Seroconversion was detected in 142 patients (12.8%) by enzyme-linked immunosorbent assay (ELISA) and confirmed by radioimmunoprecipitation. The majority (75%) of the seroconverted population had no detectable levels of circulating inhibitory antibody as assessed by in vitro inhibition of enzymatic activity of the therapeutic molecule. Of the remaining patients with putative inhibitory antibodies, the majority had only low levels of serum inhibitory activity, which was transient. A very small number of patients were identified as developing true neutralizing antibodies, as defined by the development of antibodies that impacted clinical efficacy. Many of the patient antibody responses were also diminished with time. Eighty-two of the 142 seroconverted patients have stopped producing antibody to the molecule and appear tolerized. The mean time for humoral tolerization was 28 months from initiation of therapy. Of 64 seroconverted patients followed for at least 30 months of therapy, the tolerization rate was 93%. These results show that although 12.8% of the patients on therapy developed antibodies to the molecule, 90% of these patients became tolerized over time. (+info)
Autoreactive human T cell lines recognizing ribosomal protein L7.
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Sera of patients suffering from systemic lupus erythematosus (SLE) frequently contain oligoclonal IgG autoantibodies with high affinity for the ribosomal protein L7 (rpL7). The humoral autoimmune response to rpL7 apparently is driven by antigen and T cell dependent. In order to analyze the T cell response to rpL7 we cultured peripheral blood lymphocytes of healthy individuals and SLE patients in the presence of recombinant rpL7. After 10 days, the cytokine response to re-stimulation with rpL7 was examined using a spot-ELISA. Measuring IFN-gamma secretion, the T cells of two patients and four healthy donors showed a significant increase in the number of spots as compared to control cells. Secretion of IL-4 or IL-10 was not detected. From the antigen-stimulated primary cultures we established by limiting dilution cloning six rpL7-reactive, IFN-gamma-secreting T cell lines which show a CD3+CD4+CD8- phenotype. One line additionally was shown to be positive for HLA-DR and CD45R0, but negative for CD27 and CD31. The cell lines carry alphabeta TCR chains which differ from each other in sequence and specificity. rpL7 fragments rich in basic amino acids could be identified as epitopes recognized by the TCR of three cell lines. Recognition of rpL7 is HLA-DR6 restricted or respectively HLA-DP restricted in the two cell lines analyzed. (+info)
MHC class II gene associations with autoantibodies to U1A and SmD1 proteins.
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Autoantibodies against U small nuclear ribonucleoproteins (snRNP) are frequently present in the serum of patients with systemic rheumatic diseases, and have been reported to be associated with HLA-DR and -DQ genes. To better define the role of HLA genes in the production of such antibodies, we studied immunogenetic associations with autoantibodies reacting with U1 RNP, U1A and SmD1 proteins, and synthetic peptides containing immunodominant linear epitopes of these proteins. Only two out of the 15 overlapping peptides of U1A (i.e. peptides 35-58 and 257-282) and three of 11 peptides of SmD1 (i.e. peptides 1-20, 44-67 and 97-119) were significantly recognized by patients' sera selected on the basis of their antibody positivity with RNP in immunodiffusion. The distribution of DRB1, DQB1 and DPB1 alleles among the anti-RNP antibody-positive patients (n = 28) and healthy control subjects was similar. Antibodies against U1A (tested in Western immunoblotting with HeLa cell extracts) were positively associated to DRB1*06 allele; antibodies reacting with SmD1 peptide 44-67 were negatively associated to DRB1*02 and DQB1*0602 alleles. No association was found between DPB1 alleles and antibodies reacting with U1A and SmD1 antigens. This first study reporting an association between autoantibodies reacting with U1A and SmD1 proteins (and peptides of these proteins), and immunogenetic markers suggest that the production of antibody subsets directed against different components (or regions of these proteins) bound to the same snRNP particle is associated with distinct MHC class II alleles. (+info)
Type I IFN sets the stringency of B cell repertoire selection in the bone marrow.
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Locally produced type I interferon (IFN-I) enhances the sensitivity of bone marrow B cell to IgM receptor ligation. The establishment of B cell repertoires, on the other hand, seems to involve selective processes that are critically dependent on B cell receptor (BCR) ligation. In order to assess the importance of BCR triggering thresholds on the selection of polyclonal unmanipulated B cell populations, we compared VH gene expression and reactivity repertoires in various B cell compartments of wild-type and IFN-I receptor-deficient mice (IFN-I-R-/-). These analyses demonstrate that increased B cell sensitivity to BCR ligation mediated by IFN-I in the bone marrow (BM) has consequences on the stringency of B cell repertoire selection. Thus, the normal counter-selection of both VH7183 gene family expression and multireactivity was impaired among immature BM B cells from mutant mice. Furthermore, as a result of reduced efficiency of BCR ligation-dependent inhibition of terminal differentiation, IFN-I-R-/- animals produce, in BM and thymus, higher numbers of plasma cells secreting antibodies that are more multireactive than wild-type animals. Finally, mutant serum IgM natural antibodies display a more reactive repertoire than controls, a likely reflection of the BM resident plasma cell repertoire. The present observations demonstrate, therefore, that local modulation of BCR triggering thresholds leads to important modifications in the generation and/or selection of normal B cell populations. (+info)
Serum IGF-binding protein-6 and prostate specific antigen in breast cancer.
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OBJECTIVE: Recent studies have demonstrated the presence of the IGF-binding proteins (IGFBPs) and prostate specific antigen (PSA), an IGFBP protease. in human breast tissue. We sought to investigate the differences in serum IGFs, IGFBP-1, -3 and -6, and PSA between patients with surgically proven breast cancer and patients with benign breast disease. DESIGN AND METHODS: Concentrations of IGFs, IGFBP-1, -3 and -6, and PSA were determined in the sera from 57 patients with breast cancer (CA), and 46 women with benign breast disease (BBD) using immunoassays for IGFs and IGFBPs and an ultrasensitive ELISA for PSA. RESULTS: The mean (+/- S.E.M.) serum IGFBP-6 level in the CA group, 127 (16) ng/ml, was statistically significantly lower than in the BBD group, 157 (10) ng/ml (P = 0.016). Patients with CA had an elevated geometric mean serum PSA level of 0.018 (range: 0.0015-0.107) ng/ml, compared with 0.007 (range: 0.0015-0.019) ng/ml in women with BBD (P = 0.025). Mean serum IGFBP-1 concentrations were significantly lower in the CA group, 16 (2) ng/ml, versus 37 (4) ng/ml in the BBD group (P = 0.001). Mean serum IGFBP-3 concentrations were also lower in the CA group versus the BBD group, at 1981 (65) ng/ml, versus 2603 (140) ng/ml (P = 0.002) respectively. In the CA group, statistically significant correlations between PSA and IGFBP-6 (r = 0.413; P = 0.001), and between PSA and IGFBP-1 (r = -0.329; P = 0.021) were seen. Differences in IGF-I and -II between the two groups were not statistically significant. CONCLUSION: Lower serum concentrations of IGFBP-6, -3 and -1, but higher PSA concentrations were seen in the breast cancer group, and collectively these would suggest that there is an increase in bioavailable IGF-I in breast cancer. (+info)
Fatty acid binding protein in heart and skeletal muscles of the migratory barnacle goose throughout development.
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The long-distance migratory flights of birds are predominantly fueled by the oxidation of fatty acids, which are sourced primarily from extracellular adipose stores. These fatty acids have to be transported, via the circulatory system, to the mitochondria of the active muscles. An important facilitator of fatty acid transport within the cytoplasm of muscle cells is fatty acid binding protein (FABP), which serves as an intracellular carrier of long-chain fatty acids. In mammals, the muscular FABP content is related to the fatty acid oxidation capacity of the tissue. The aim of this study was to measure FABP in samples taken from the cardiac, pectoralis, and semimembranosus muscles of a long-distance avian migrant, the barnacle goose (Branta leucopsis), at various stages of development. Western blot analysis identified a single goose muscle protein of 15 kDa that was able to bind fatty acids and showed a 66% cross-reactivity with antibodies against human heart-type FABP. Captive goslings showed no significant changes in FABP content of either the heart (62.6 +/- 10.6 microgram/g wet wt) or the semimembranosus muscle (8.4 +/- 1.9 microgram/g wet wt) during development. However, in both peripheral and deep sites within the pectoralis muscle, FABP content of samples taken from captive goslings were approximately 10-fold higher throughout development and reached values of 30-40 microgram/g wet wt in fledging goslings at 7 wk of age. A further twofold higher value was seen in wild but not in captive goslings immediately before migration (12 wk of age). Similarly, FABP content was significantly higher in pectoralis samples taken from wild adults (94.3 +/- 3.6 microgram/g wet wt) compared with those from captive adults (60.5 +/- 3.6 micro/g wet wt). These results suggest that the experience of flight activity may be of critical importance in achieving maximal expression of FABP in the pectoralis muscles of postfledging and mature geese immediately before migration. (+info)
Qualitative and semiquantitative polymerase chain reaction testing for cytomegalovirus DNA in serum allows prediction of CMV related disease in liver transplant recipients.
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AIM: To identify cytomegalovirus (CMV) infection in liver transplant recipients by polymerase chain reaction (PCR) techniques and to separate the cases in which CMV related disease will occur, for whom treatment is indicated, from those in whom infection will remain innocuous. METHODS: The combination of qualitative and semiquantitative PCR of serum and urine was assessed to determine whether these assays can identify those at risk of CMV related disease and compared their performance with conventional approaches to diagnosis. RESULTS: Qualitative PCR of serum had superior specificity, sensitivity, and positive and negative predictive values compared with urine DEAFF (detection of early antigen fluorescent foci) and PCR of urine. All episodes of CMV related disease were associated with the presence of CMV DNA by PCR in serum or urine; CMV was detected before clinical onset in 70% and 60% of cases, respectively. The period over which CMV DNA could be detected was not correlated with CMV related disease. Both peak viral load and cumulative viral load estimated using a semiquantitative PCR method on serum samples positive by the qualitative method could be used to distinguish asymptomatic infection from CMV related disease with 100% specificity and sensitivity. In contrast semiquantitative PCR of urine was of little value. CONCLUSIONS: An approach based on PCR testing with a combination of qualitative and subsequently semiquantitative serum samples would improve the diagnosis of CMV infection and aid identification of those patients at risk of CMV related disease, allowing treatment to be targeted specifically. (+info)
Cigarette smoking decreases interleukin-8 secretion by human alveolar macrophages.
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Cigarette smoking can impair pulmonary immune function, and hence influences the development of lung diseases. Interleukin-8 (IL-8) is a proinflammatory peptide and a potent chemotactic factor for neutrophils, and is produced by both immune and non-immune cells including monocytes and alveolar macrophages (AM). We investigated the effect of cigarette smoking on the secretion of IL-8 by human AM. The IL-8 concentration in bronchoalveolar lavage fluid (BALF) was much higher in smokers than in non-smokers (18.4 +/- 3.9 vs 4.1 +/- 1.0 pg ml-1; P < 0.005). However, spontaneous IL-8 secretion by cultured AM was lower in smokers than in non-smokers (46.8 +/- 12.7 vs 124.1 +/- 24.0 ng ml-1; P < 0.01). When stimulated with lipopolysaccharide (LPS), AM from smokers secreted significantly less IL-8 than those from non-smokers at all tested concentrations of LPS. In contrast, the amount of IL-8 secreted by peripheral blood monocytes with or without LPS stimulation was comparable in smokers and non-smokers. These observations indicate that smoking decreases IL-8 secretion by AM, which may modify or decrease the inflammatory response in the lung. (+info)