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Sample records for human leucocyte antigen

  1. Neonatal alloimmune thrombocytopenia caused by human leucocyte antigen-B27 antibody.

    PubMed

    Thude, H; Schorner, U; Helfricht, C; Loth, M; Maak, B; Barz, D

    2006-04-01

    Neonatal alloimmune thrombocytopenia (NAIT) occurs when maternal alloantibodies to antigens presented on foetal platelets cause their immune destruction. Whether human leucocyte antigen (HLA) antibodies can cause NAIT is controversial. Here, a patient was described who suffered from a NAIT caused by an HLA-B27 antibody. Sera from the mother and the newborn were tested for human platelet antigen antibodies and HLA antibodies by monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay, solid phase-linked immunosorbent assay (ELISA), lymphocytotoxicity assay (LCT) and flow cytometric analysis. No antibodies against cluster designation (CD)109 and platelet glycoproteins of the father were found in patient's and mother's serum. However, HLA ELISA was used to identify HLA antibody in both sera. The antibody was specified as HLA-B27 antibody. Typing results showed that the father descended HLA-B27 antigen on patient and his brother. The mother was HLA-B27 negative. It is most conceivable that the previous pregnancy of the mother induced the production of anti-HLA-B27 antibody, which crossed the placenta and subsequently caused an NAIT in the case presented. PMID:16623921

  2. JC Polyomavirus Infection Is Strongly Controlled by Human Leucocyte Antigen Class II Variants

    PubMed Central

    Sundqvist, Emilie; Buck, Dorothea; Warnke, Clemens; Albrecht, Eva; Gieger, Christian; Khademi, Mohsen; Lima Bomfim, Izaura; Fogdell-Hahn, Anna; Link, Jenny; Alfredsson, Lars; Søndergaard, Helle Bach; Hillert, Jan; Oturai, Annette B.; Hemme, Bernhard

    2014-01-01

    JC polyomavirus (JCV) carriers with a compromised immune system, such as in HIV, or subjects on immune-modulating therapies, such as anti VLA-4 therapy may develop progressive multifocal leukoencephalopathy (PML) which is a lytic infection of oligodendrocytes in the brain. Serum antibodies to JCV mark infection occur only in 50–60% of infected individuals, and high JCV-antibody titers seem to increase the risk of developing PML. We here investigated the role of human leukocyte antigen (HLA), instrumental in immune defense in JCV antibody response. Anti-JCV antibody status, as a surrogate for JCV infection, were compared to HLA class I and II alleles in 1621 Scandinavian persons with MS and 1064 population-based Swedish controls and associations were replicated in 718 German persons with MS. HLA-alleles were determined by SNP imputation, sequence specific (SSP) kits and a reverse PCR sequence-specific oligonucleotide (PCR-SSO) method. An initial GWAS screen displayed a strong HLA class II region signal. The HLA-DRB1*15 haplotype was strongly negatively associated to JCV sero-status in Scandinavian MS cases (OR = 0.42, p = 7×10−15) and controls (OR = 0.53, p = 2×10−5). In contrast, the DQB1*06:03 haplotype was positively associated with JCV sero-status, in Scandinavian MS cases (OR = 1.63, p = 0.006), and controls (OR = 2.69, p = 1×10−5). The German dataset confirmed these findings (OR = 0.54, p = 1×10−4 and OR = 1.58, p = 0.03 respectively for these haplotypes). HLA class II restricted immune responses, and hence CD4+ T cell immunity is pivotal for JCV infection control. Alleles within the HLA-DR1*15 haplotype are associated with a protective effect on JCV infection. Alleles within the DQB1*06:03 haplotype show an opposite association. These associations between JC virus antibody response and human leucocyte antigens supports the notion that CD4+ T cells are crucial in the immune defence to JCV and lays

  3. JC polyomavirus infection is strongly controlled by human leucocyte antigen class II variants.

    PubMed

    Sundqvist, Emilie; Buck, Dorothea; Warnke, Clemens; Albrecht, Eva; Gieger, Christian; Khademi, Mohsen; Lima Bomfim, Izaura; Fogdell-Hahn, Anna; Link, Jenny; Alfredsson, Lars; Søndergaard, Helle Bach; Hillert, Jan; Oturai, Annette B; Hemmer, Bernhard; Hemme, Bernhard; Kockum, Ingrid; Olsson, Tomas

    2014-04-01

    JC polyomavirus (JCV) carriers with a compromised immune system, such as in HIV, or subjects on immune-modulating therapies, such as anti VLA-4 therapy may develop progressive multifocal leukoencephalopathy (PML) which is a lytic infection of oligodendrocytes in the brain. Serum antibodies to JCV mark infection occur only in 50-60% of infected individuals, and high JCV-antibody titers seem to increase the risk of developing PML. We here investigated the role of human leukocyte antigen (HLA), instrumental in immune defense in JCV antibody response. Anti-JCV antibody status, as a surrogate for JCV infection, were compared to HLA class I and II alleles in 1621 Scandinavian persons with MS and 1064 population-based Swedish controls and associations were replicated in 718 German persons with MS. HLA-alleles were determined by SNP imputation, sequence specific (SSP) kits and a reverse PCR sequence-specific oligonucleotide (PCR-SSO) method. An initial GWAS screen displayed a strong HLA class II region signal. The HLA-DRB1*15 haplotype was strongly negatively associated to JCV sero-status in Scandinavian MS cases (OR = 0.42, p = 7×10(-15)) and controls (OR = 0.53, p = 2×10(-5)). In contrast, the DQB1*06:03 haplotype was positively associated with JCV sero-status, in Scandinavian MS cases (OR = 1.63, p = 0.006), and controls (OR = 2.69, p = 1×10(-5)). The German dataset confirmed these findings (OR = 0.54, p = 1×10(-4) and OR = 1.58, p = 0.03 respectively for these haplotypes). HLA class II restricted immune responses, and hence CD4+ T cell immunity is pivotal for JCV infection control. Alleles within the HLA-DR1*15 haplotype are associated with a protective effect on JCV infection. Alleles within the DQB1*06:03 haplotype show an opposite association. These associations between JC virus antibody response and human leucocyte antigens supports the notion that CD4+ T cells are crucial in the immune defence to JCV and lays the

  4. Prevalence of Alloimmunization to Human Platelet Antigen Glycoproteins and Human Leucocyte Antigen Class I in β Thalassemia Major Patients in Western India.

    PubMed

    Philip, Joseph; Kumar, Sudeep; Chatterjee, T; Mallhi, R S

    2014-12-01

    Present management of β thalassemia major by regular packed red blood cell (PRBC) transfusions poses risk of alloimmunization not only to red blood cell antigens, but also to human platelet antigens (HPA) and Human leucocyte antigens class I (HLA I). However data in this context is very limited in Indian population. The aim of the study was to determine the prevalence of alloimmunization to HPA and HLA I in β thalassemia major patients who have received multiple PRBC transfusions over the years. A cross sectional study was performed at our tertiary care blood bank. β thalassemia major patients of more than 6 years of age were included who were receiving fresh, leucoreduced and irradiated PRBC units regularly with annual requirement of more than ten PRBC transfusions. A total of 9 out of 80 (11.25 %) patients were found to be alloimmunized for HPA antigens of various specificity and 24 out of 80 (30 %) developed antibodies to HLA I. The awareness of development of alloimmunization to HPA and HLA antigens in multi PRBC transfused thalassemics, despite use of leucofilters will prompt us, to look for improvement in our current PRBC preparations to minimise platelet alloimmunisation. Further studies are required to validate the findings and build the base line data in this regard. This is of importance, especially in view of providing suitable cross-matched platelets when required in future especially when considering future haematopoietic stem cell transplantation (HSCT).

  5. Performance Characteristics and Validation of Next-Generation Sequencing for Human Leucocyte Antigen Typing.

    PubMed

    Weimer, Eric T; Montgomery, Maureen; Petraroia, Rosanne; Crawford, John; Schmitz, John L

    2016-09-01

    High-resolution human leukocyte antigen (HLA) matching reduces graft-versus-host disease and improves overall patient survival after hematopoietic stem cell transplant. Sanger sequencing has been the gold standard for HLA typing since 1996. However, given the increasing number of new HLA alleles identified and the complexity of the HLA genes, clinical HLA typing by Sanger sequencing requires several rounds of additional testing to provide allele-level resolution. Although next-generation sequencing (NGS) is routinely used in molecular genetics, few clinical HLA laboratories use the technology. The performance characteristics of NGS HLA typing using TruSight HLA were determined using Sanger sequencing as the reference method. In total, 211 samples were analyzed with an overall accuracy of 99.8% (2954/2961) and 46 samples were analyzed for precision with 100% (368/368) reproducibility. Most discordant alleles were because of technical error rather than assay performance. More important, the ambiguity rate was 3.5% (103/2961). Seventy-four percentage of the ambiguities were within the DRB1 and DRB4 loci. HLA typing by NGS saves approximately $6000 per run when compared to Sanger sequencing. Thus, TruSight HLA assay enables high-throughput HLA typing with an accuracy, precision, ambiguity rate, and cost savings that should facilitate adoption of NGS technology in clinical HLA laboratories.

  6. Protective Human Leucocyte Antigen Haplotype, HLA-DRB1*01-B*14, against Chronic Chagas Disease in Bolivia

    PubMed Central

    del Puerto, Florencia; Nishizawa, Juan Eiki; Kikuchi, Mihoko; Roca, Yelin; Avilas, Cinthia; Gianella, Alberto; Lora, Javier; Velarde, Freddy Udalrico Gutierrez; Miura, Sachio; Komiya, Norihiro; Maemura, Koji; Hirayama, Kenji

    2012-01-01

    Background Chagas disease, caused by the flagellate parasite Trypanosoma cruzi affects 8–10 million people in Latin America. The mechanisms that underlie the development of complications of chronic Chagas disease, characterized primarily by pathology of the heart and digestive system, are not currently understood. To identify possible host genetic factors that may influence the clinical course of Chagas disease, Human Leucocyte Antigen (HLA) regional gene polymorphism was analyzed in patients presenting with differing clinical symptoms. Methodology Two hundred and twenty nine chronic Chagas disease patients in Santa Cruz, Bolivia, were examined by serological tests, electrocardiogram (ECG), and Barium enema colon X-ray. 31.4% of the examinees showed ECG alterations, 15.7% megacolon and 58.1% showed neither of them. A further 62 seropositive megacolon patients who had undergone colonectomy due to acute abdomen were recruited. We analyzed their HLA genetic polymorphisms (HLA-A, HLA-B, MICA, MICB, DRB1 and TNF-alpha promoter region) mainly through Sequence based and LABType SSO typing test using LUMINEX Technology. Principal Findings The frequencies of HLA-DRB1*01 and HLA-B*14:02 were significantly lower in patients suffering from megacolon as well as in those with ECG alteration and/or megacolon compared with a group of patients with indeterminate symptoms. The DRB1*0102, B*1402 and MICA*011 alleles were in strong Linkage Disequilibrium (LD), and the HLA-DRB1*01-B*14-MICA*011haplotype was associated with resistance against chronic Chagas disease. Conclusions This is the first report of HLA haplotype association with resistance to chronic Chagas disease. PMID:22448298

  7. Analysis of Class II human leucocyte antigens in Italian and Spanish systemic sclerosis

    PubMed Central

    Rueda, Blanca; Marchini, Maurizio; Santaniello, Alessandro; Simeón, Carmen P.; Fonollosa, Vicente; Caronni, Monica; Rios-Fernandez, Raquel; Carreira, Patricia; Rodriguez-Rodriguez, Luis; Moreno, Antonia; López-Nevot, Miguel A.; Escalera, Ana; González-Escribano, Maria F.; Martin, Javier; Scorza, Raffaella

    2012-01-01

    Objective. To determine the role of Class II HLAs in SSc patients from Italy and Spain and in SSc patients of Caucasian ancestry. Methods. Nine hundred and forty-four SSc patients (Italy 392 patients; Spain 452 patients) and 1320 ethnically matched healthy controls (Italy 398 patients; Spain 922 patients) were genotyped up to the fourth digit by PCR with sequence-specific oligonucleotides for HLA-DRB1, DQA1 and DQB1 loci. Patients included 390 ACA-positive and 254 anti-topo I-positive subjects. Associations between SSc or SSc-specific antibodies and HLA alleles or HLA haplotypes were sought via the chi-square test after 10 000-fold permutation testing. A meta-analysis including this study cohort and other Caucasoids samples was also conducted. Results. In both the cohorts, the strongest association was observed between the HLA-DRB1*1104 allele and SSc or anti-topo I antibodies. The HLA-DRB1*1104 -DQA1*0501 -DQB1*0301 haplotype was overrepresented in Italian [odds ratio (OR) = 2.069, 95% asymptotic CIs (CI95) 1.486, 2.881; P < 0.001] and in Spanish patients (OR = 6.707, CI95 3.974, 11.319; P < 0.001) as well as in anti-topo-positive patients: Italy (OR = 2.642, CI95 1.78, 3.924; P < 0.001) and Spain (OR = 20.625, CI95 11.536, 36.876; P < 0.001). In both the populations we also identified an additional risk allele (HLA-DQB1*03) and a protective allele (HLA-DQB1*0501) in anti-topo-positive patients. The meta-analysis showed different statistically significant associations, the most interesting being the differential association between HLA-DRB1*01 alleles and ACAs (OR = 1.724, CI95 1.482, 2.005; P < 0.001) or topo I antibodies (OR = 0.5, CI95 0.384, 0.651; P < 0.001). Conclusions. We describe multiple robust associations between SSc and HLA Class II antigens in Caucasoids that may help to understand the genetic architecture of SSc. PMID:22087014

  8. Antibodies against human platelet alloantigens and human leucocyte antigen class 1 in Saudi Arabian multiparous women and multi-transfused patients

    PubMed Central

    Al-Ouda, Sarah K.; Al-Banyan, Abdulmajeed A.; Al-Gahtani, Farjah H.; Abdel-Gader, Abdel-Galil M.; Al-Dakhil, Lateefa O.

    2015-01-01

    Objectives: To determine the frequency of alloimmunization against human platelet antigens (HPAs) and human leucocyte antigen class 1 (HLA1) in multiparous women and multi-transfused patients. Methods: This prospective study was conducted between January and August 2013, on 50 multiparous women with no history of previous blood transfusion recruited from the Obstetrics and Gynecology Clinic, and 50 patients, who received multiple platelet transfusions, recruited from the Hematology/Oncology Ward, King Khalid University Hospital, Riyadh, Saudi Arabia. Results: The frequency of alloimmunization among multiparous pregnant women was 76%, as follows: 16% against HLA1 only, 8% against HPAs only, 52% against both HPAs and HLA1 antigens. In multi-transfused patients, the rate of alloimmunization was 42% as follows: 2% against HLA1 only, 22% against HPAs only, 18% against both HPAs and HLA1 antigens. The frequency of alloimmunization increases with the number of pregnancies, but not with the number of platelet transfusions. Conclusion: Alloimmunization against HPAs and HLA1 is very common among Saudi multiparous women and multi-transfused patients, which encourages the search for the extent of the possible complications in the fetus and newborn and in multitransfused patients and how to prevent their occurrence. PMID:25987107

  9. The human leucocyte antigen-G 14-basepair polymorphism correlates with graft-versus-host disease in unrelated bone marrow transplantation for thalassaemia.

    PubMed

    La Nasa, Giorgio; Littera, Roberto; Locatelli, Franco; Lai, Sara; Alba, Francesco; Caocci, Giovanni; Lisini, Daniela; Nesci, Sonia; Vacca, Adriana; Piras, Eugenia; Bernardo, Maria Ester; Di Cesare-Merlone, Alessandra; Orrù, Sandro; Carcassi, Carlo

    2007-10-01

    The presence of the 14-bp insertion polymorphism of the human leucocyte antigen (HLA)-G gene (HLA-G) promotes immune tolerance through increased synthesis of HLA-G molecules. We investigated this polymorphism in a large cohort of 53 thalassaemia patients transplanted from an unrelated donor. Sixteen patients (30.2%) homozygous for the 14-bp deletion had a higher risk of developing acute graft-versus-host disease (aGvHD) than patients homozygous for the 14-bp insertion (-14-bp/-14-bp vs +14-bp/+14-bp: Relative Risk = 15.0; 95% confidence interval 1.59-141.24; P = 0.008). Therefore, the 14-bp polymorphism could be an important predictive factor for aGvHD following bone marrow transplantation. PMID:17897304

  10. The human leucocyte antigen DQB1*0602 allele is associated with electroencephelograph differences in individuals with obstructive sleep apnoea syndrome.

    PubMed

    Manzotte, Thais; Guindalini, Camila; Mazzotti, Diego R; Palombini, Luciana; de Souza, Altay L; Poyares, Dalva; Bittencourt, Lia R A; Tufik, Sergio

    2013-04-01

    Human leucocyte antigen (HLA) DQB1*0602 allele, a well-known genetic risk factor for narcolepsy, has been associated with sleep parameters in healthy subjects. We aimed to assess the association of this allele with daytime sleepiness and altered sleep electroencephalogram characteristics in the general population and in patients with obstructive sleep apnoea syndrome (OSAS). Eight hundred and ninety-four individuals from the Epidemiologic Study of Sleep were genotyped for the HLA DQB1*0602 allele. Full-night polysomnography was performed, and daytime sleepiness was analysed according to the Epworth Sleepiness Scale. HLA-DQB1*0602 allele-positive and -negative subjects in the general population, as well as in patients with OSAS, exhibited similar sleep parameters and levels of daytime sleepiness. However, spectral analysis showed that allele-positive individuals with OSAS exhibited higher theta power during sleep Stage 1 (P < 0.05) in occipital derivations, and lower delta power during sleep Stages 1 and 2 (P < 0.01) compared with individuals negative for the allele, even after correction for potential confounders as age, sex, body mass index and European ancestry. No significant differences in the electroencephalogram variables were found in individuals without OSAS. The data highlight the HLA-DQB1*0602 as a potential genetic factor influencing sleep physiology in individuals diagnosed with OSAS. PMID:23136848

  11. Human Leucocyte Antigen-G (HLA-G) and Its Murine Functional Homolog Qa2 in the Trypanosoma cruzi Infection

    PubMed Central

    Dias, Fabrício C.; Mendes-Junior, Celso T.; Silva, Maria C.; Tristão, Fabrine S. M.; Dellalibera-Joviliano, Renata; Soares, Edson G.; Menezes, Jean G.; Schmidt, André; Dantas, Roberto O.; Marin-Neto, José A.; Silva, João S.; Donadi, Eduardo A.

    2015-01-01

    Genetic susceptibility factors, parasite strain, and an adequate modulation of the immune system seem to be crucial for disease progression after Trypanosoma cruzi infection. HLA-G and its murine functional homolog Qa2 have well-recognized immunomodulatory properties. We evaluated the HLA-G 3′ untranslated region (3′UTR) polymorphic sites (associated with mRNA stability and target for microRNA binding) and HLA-G tissue expression (heart, colon, and esophagus) in patients presenting Chagas disease, stratified according to the major clinical variants. Further, we investigated the transcriptional levels of Qa2 and other pro- and anti-inflammatory genes in affected mouse tissues during T. cruzi experimental acute and early chronic infection induced by the CL strain. Chagas disease patients exhibited differential HLA-G 3′UTR susceptibility allele/genotype/haplotype patterns, according to the major clinical variant (digestive/cardiac/mixed/indeterminate). HLA-G constitutive expression on cardiac muscle and colonic cells was decreased in Chagasic tissues; however, no difference was observed for Chagasic and non-Chagasic esophagus tissues. The transcriptional levels of Qa2 and other anti and proinflammatory (CTLA-4, PDCD1, IL-10, INF-γ, and NOS-2) genes were induced only during the acute T. cruzi infection in BALB/c and C57BL/6 mice. We present several lines of evidence indicating the role of immunomodulatory genes and molecules in human and experimental T. cruzi infection. PMID:25688175

  12. Human leucocyte antigen-G (HLA-G) and its murine functional homolog Qa2 in the Trypanosoma cruzi Infection.

    PubMed

    Dias, Fabrício C; Mendes-Junior, Celso T; Silva, Maria C; Tristão, Fabrine S M; Dellalibera-Joviliano, Renata; Moreau, Philippe; Soares, Edson G; Menezes, Jean G; Schmidt, André; Dantas, Roberto O; Marin-Neto, José A; Silva, João S; Donadi, Eduardo A

    2015-01-01

    Genetic susceptibility factors, parasite strain, and an adequate modulation of the immune system seem to be crucial for disease progression after Trypanosoma cruzi infection. HLA-G and its murine functional homolog Qa2 have well-recognized immunomodulatory properties. We evaluated the HLA-G 3' untranslated region (3'UTR) polymorphic sites (associated with mRNA stability and target for microRNA binding) and HLA-G tissue expression (heart, colon, and esophagus) in patients presenting Chagas disease, stratified according to the major clinical variants. Further, we investigated the transcriptional levels of Qa2 and other pro- and anti-inflammatory genes in affected mouse tissues during T. cruzi experimental acute and early chronic infection induced by the CL strain. Chagas disease patients exhibited differential HLA-G 3'UTR susceptibility allele/genotype/haplotype patterns, according to the major clinical variant (digestive/cardiac/mixed/indeterminate). HLA-G constitutive expression on cardiac muscle and colonic cells was decreased in Chagasic tissues; however, no difference was observed for Chagasic and non-Chagasic esophagus tissues. The transcriptional levels of Qa2 and other anti and proinflammatory (CTLA-4, PDCD1, IL-10, INF-γ, and NOS-2) genes were induced only during the acute T. cruzi infection in BALB/c and C57BL/6 mice. We present several lines of evidence indicating the role of immunomodulatory genes and molecules in human and experimental T. cruzi infection.

  13. Effects of type II collagen epitope carbamylation and citrullination in human leucocyte antigen (HLA)-DR4(+) monozygotic twins discordant for rheumatoid arthritis.

    PubMed

    De Santis, M; Ceribelli, A; Cavaciocchi, F; Generali, E; Massarotti, M; Isailovic, N; Crotti, C; Scherer, H U; Montecucco, C; Selmi, C

    2016-09-01

    The aim of this study is to investigate the effect of the native, citrullinated or carbamylated type II human collagen T cell- and B cell-epitopes on the adaptive immune response in rheumatoid arthritis (RA). Peripheral blood T and B cells obtained from a human leucocyte D4-related (antigen DR4(-) HLA-DR4)(+) woman with early RA, her healthy monozygotic twin and an unrelated HLA-DR3(+) woman with early RA were analysed for activation (CD154/CD69), apoptosis (annexin/7-aminoactinomycin), cytokine production [interferon (IFN)γ/interleukin (IL)-17/IL-4/IL-10/IL-6] and functional phenotype (CD45Ra/CCR7) after stimulation with the collagen native T cell epitope (T261-273), the K264 carbamylated T cell epitope (carT261-273), the native B cell epitope (B359-369) or the R360 citrullinated B cell epitope (citB359-369), and the combinations of these. The T cell memory compartment was activated by T cell epitopes in both discordant DR4(+) twins, but not in the DR3(+) RA. The collagen-specific activation of CD4(+) T cells was induced with both the native and carbamylated T cell epitopes only in the RA twin. Both T cell epitopes also induced IL-17 production in the RA twin, but a greater IL-4 and IL-10 response in the healthy twin. The citrullinated B cell epitope, particularly when combined with the carbamylated T cell epitope, induced B cell activation and an increased IL-6/IL-10 ratio in the RA twin compared to a greater IL-10 production in the healthy twin. Our data suggest that circulating collagen-specific T and B cells are found in HLA-DR4(+) subjects, but only RA activated cells express co-stimulatory molecules and produce proinflammatory cytokines. Carbamylation and citrullination further modulate the activation and cytokine polarization of T and B cells. PMID:27314557

  14. Suppression of allo-human leucocyte antigen (HLA) antibodies secreted by B memory cells in vitro: intravenous immunoglobulin (IVIg) versus a monoclonal anti-HLA-E IgG that mimics HLA-I reactivities of IVIg

    PubMed Central

    Zhu, D; Ravindranath, M H; Terasaki, P I; Miyazaki, T; Pham, T; Jucaud, V

    2014-01-01

    B memory cells remain in circulation and secrete alloantibodies without antigen exposure > 20 years after alloimmunization postpartum or by transplantation. These long-lived B cells are resistant to cytostatic drugs. Therapeutically, intravenous immunoglobulin (IVIg) is administered to reduce allo-human leucocyte antigen (HLA) antibodies pre- and post-transplantation, but the mechanism of reduction remains unclear. Recently, we reported that IVIg reacts with several HLA-I alleles and the HLA reactivity of IVIg is lost after its HLA-E reactivity is adsorbed out. Therefore, we have generated an anti-HLA-E monoclonal antibody that mimics the HLA-reactivity of IVIg to investigate whether this antibody suppresses IgG secretion, as does IVIg. B cells were purified from the blood of a woman in whose blood the B memory cells remained without antigen exposure > 20 years after postpartum alloimmunization. The B cells were stimulated with cytokines using a well-defined culture system. The anti-HLA-E monoclonal antibody (mAb) significantly suppressed the allo-HLA class-II IgG produced by the B cells, and that this suppression was far superior to that by IVIg. These findings were confirmed with HLA-I antibody secreted by the immortalized B cell line, developed from the blood of another alloimmunized woman. The binding affinity of the anti-HLA-E mAb for peptide sequences shared (i.e. shared epitopes) between HLA-E and other β2-microglobulin-free HLA heavy chains (open conformers) on the cell surface of B cells may act as a ligand and signal suppression of IgG production of activated B memory cells. We propose that anti-HLA-E monoclonal antibody may also be useful to suppress allo-HLA IgG production in vivo. PMID:24611451

  15. Leucocyte migration inhibition test with two gastric antigens in pernicious anaemia and in simple atrophic gastritis.

    PubMed

    Fixa, B; Komárková, O; Nozicka, Z

    1979-02-01

    Leucocyte migration inhibition test was used for evaluation of cell-mediated immunity in patients with pernicious anaemia (PA) and simple atrophic gastritis (SAG). As antigens microsomal antigen from gastric mucosa of swine foetus and relatively pure hog intrinsic factor (IF) were used. Significant differences were found between PA and SAG with microsomal antigen, but not with IF. It was concluded, that the microsomal antigen might be more active than IF. This observation could contribute to explane the higher incidence of parietal cell antibody than that of IF antibody in PA patients.

  16. Differential regulation of toll-like receptor-2, toll-like receptor-4, CD16 and human leucocyte antigen-DR on peripheral blood monocytes during mild and severe dengue fever

    PubMed Central

    Azeredo, Elzinandes L; Neves-Souza, Patrícia C; Alvarenga, Allan R; Reis, Sônia R N I; Torrentes-Carvalho, Amanda; Zagne, Sonia-Maris O; Nogueira, Rita M R; Oliveira-Pinto, Luzia M; Kubelka, Claire F

    2010-01-01

    Dengue fever (DF), a public health problem in tropical countries, may present severe clinical manifestations as result of increased vascular permeability and coagulation disorders. Dengue virus (DENV), detected in peripheral monocytes during acute disease and in in vitro infection, leads to cytokine production, indicating that virus–target cell interactions are relevant to pathogenesis. Here we investigated the in vitro and in vivo activation of human peripheral monocytes after DENV infection. The numbers of CD14+ monocytes expressing the adhesion molecule intercellular adhesion molecule 1 (ICAM-1) were significantly increased during acute DF. A reduced number of CD14+ human leucocyte antigen (HLA)-DR+ monocytes was observed in patients with severe dengue when compared to those with mild dengue and controls; CD14+ monocytes expressing toll-like receptor (TLR)2 and TLR4 were increased in peripheral blood from dengue patients with mild disease, but in vitro DENV-2 infection up-regulated only TLR2. Increased numbers of CD14+ CD16+ activated monocytes were found after in vitro and in vivo DENV-2 infection. The CD14high CD16+ monocyte subset was significantly expanded in mild dengue, but not in severe dengue. Increased plasma levels of tumour necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and interleukin (IL)-18 in dengue patients were inversely associated with CD14high CD16+, indicating that these cells might be involved in controlling exacerbated inflammatory responses, probably by IL-10 production. We showed here, for the first time, phenotypic changes on peripheral monocytes that were characteristic of cell activation. A sequential monocyte-activation model is proposed in which DENV infection triggers TLR2/4 expression and inflammatory cytokine production, leading eventually to haemorrhagic manifestations, thrombocytopenia, coagulation disorders, plasmatic leakage and shock development, but may also produce factors that act in order to control both intense

  17. Differential regulation of toll-like receptor-2, toll-like receptor-4, CD16 and human leucocyte antigen-DR on peripheral blood monocytes during mild and severe dengue fever.

    PubMed

    Azeredo, Elzinandes L; Neves-Souza, Patrícia C; Alvarenga, Allan R; Reis, Sônia R N I; Torrentes-Carvalho, Amanda; Zagne, Sonia-Maris O; Nogueira, Rita M R; Oliveira-Pinto, Luzia M; Kubelka, Claire F

    2010-06-01

    Dengue fever (DF), a public health problem in tropical countries, may present severe clinical manifestations as result of increased vascular permeability and coagulation disorders. Dengue virus (DENV), detected in peripheral monocytes during acute disease and in in vitro infection, leads to cytokine production, indicating that virus-target cell interactions are relevant to pathogenesis. Here we investigated the in vitro and in vivo activation of human peripheral monocytes after DENV infection. The numbers of CD14(+) monocytes expressing the adhesion molecule intercellular adhesion molecule 1 (ICAM-1) were significantly increased during acute DF. A reduced number of CD14(+) human leucocyte antigen (HLA)-DR(+) monocytes was observed in patients with severe dengue when compared to those with mild dengue and controls; CD14(+) monocytes expressing toll-like receptor (TLR)2 and TLR4 were increased in peripheral blood from dengue patients with mild disease, but in vitro DENV-2 infection up-regulated only TLR2. Increased numbers of CD14(+) CD16(+) activated monocytes were found after in vitro and in vivo DENV-2 infection. The CD14(high) CD16(+) monocyte subset was significantly expanded in mild dengue, but not in severe dengue. Increased plasma levels of tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma) and interleukin (IL)-18 in dengue patients were inversely associated with CD14(high) CD16(+), indicating that these cells might be involved in controlling exacerbated inflammatory responses, probably by IL-10 production. We showed here, for the first time, phenotypic changes on peripheral monocytes that were characteristic of cell activation. A sequential monocyte-activation model is proposed in which DENV infection triggers TLR2/4 expression and inflammatory cytokine production, leading eventually to haemorrhagic manifestations, thrombocytopenia, coagulation disorders, plasmatic leakage and shock development, but may also produce factors that act in

  18. Enhancement of mite antigen-induced histamine release by deuterium oxide from leucocytes of chronic urticarial patients

    SciTech Connect

    Numata, T.; Yamamoto, S.; Yamura, T.

    1981-09-01

    The mite antigen-induced histamine release from leucocytes of chronic urticarial patients was enhanced in the presence of deuterium oxide, which stabilizes microtubules. This enhancing effect of deuterium oxide on the histamine release from leucocytes may provide a useful means for the detection of allergens in vitro in chronic urticaria.

  19. Serum antibodies to human leucocyte antigen (HLA)-E, HLA-F and HLA-G in patients with systemic lupus erythematosus (SLE) during disease flares: Clinical relevance of HLA-F autoantibodies.

    PubMed

    Jucaud, V; Ravindranath, M H; Terasaki, P I; Morales-Buenrostro, L E; Hiepe, F; Rose, T; Biesen, R

    2016-03-01

    T lymphocyte hyperactivity and progressive inflammation in systemic lupus erythematosus (SLE) patients results in over-expression of human leucocyte antigen (HLA)-Ib on the surface of lymphocytes. These are shed into the circulation upon inflammation, and may augment production of antibodies promoting pathogenicity of the disease. The objective was to evaluate the association of HLA-Ib (HLA-E, HLA-F and HLA-G) antibodies to the disease activity of SLE. The immunoglobulin (Ig)G/IgM reactivity to HLA-Ib and β2m in the sera of 69 German, 29 Mexican female SLE patients and 17 German female controls was measured by multiplex Luminex(®)-based flow cytometry. The values were expressed as mean flourescence intensity (MFI). Only the German SLE cohort was analysed in relation to the clinical disease activity. In the controls, anti-HLA-G IgG predominated over other HLA-Ib antibodies, whereas SLE patients had a preponderance of anti-HLA-F IgG over the other HLA-Ib antibodies. The disease activity index, Systemic Lupus Erythematosus Disease Activity Index (SLEDAI)-2000, was reflected only in the levels of anti-HLA-F IgG. Anti-HLA-F IgG with MFI level of 500-1999 was associated with active SLE, whereas inactive SLE revealed higher MFI (>2000). When anti-HLA-F IgG were cross-reactive with other HLA-Ib alleles, their reactivity was reflected in the levels of anti-HLA-E and -G IgG. The prevalence of HLA-F-monospecific antibodies in SLE patients was also associated with the clinical disease activity. Anti-HLA-F IgG is possibly involved in the clearance of HLA-F shed from lymphocytes and inflamed tissues to lessen the disease's severity, and thus emerges as a beneficial immune biomarker. Therefore, anti-HLA-Ib IgG should be considered as a biomarker in standard SLE diagnostics.

  20. Purification of human leucocyte DNA: proteinase K is not necessary.

    PubMed

    Douglas, A M; Georgalis, A M; Benton, L R; Canavan, K L; Atchison, B A

    1992-03-01

    A rapid nontoxic method for the purification of DNA from human leucocytes is described. Preliminary experiments which tested different methods of DNA purification indicated that digestion of proteins with proteinase K was unnecessary. This led to the development of a simple procedure involving lysis of the cells in SDS followed by extraction with 6 M NaCl. The method described overcomes the requirement for lengthy incubations in the presence of expensive proteinase K and subsequent extraction with toxic chemicals.

  1. Nitroblue tetrazolium (NBT) activity in human leucocytes after freezing.

    PubMed

    Hill, R S; Kennedy, M; Mackinder, C

    1978-01-01

    Human peripheral blood leucocytes (neutrophil-rich) were collected either with heparin or acid citrate dextrose, frozen with dimethyl sulphoxide at a controlled rate, stored in liquid nitrogen at--196 degrees C and reconstituted with a solution containing dextran. After reconstitution, 20.2% of cells (in absolute numbers 1 in 5 fresh cells) showed a strongly positive nitroblue tetrazolium (NBT) reaction. The quantitative NBT test confirmed the synthesis of formazan/10(6) reconstituted neutrophilsa s15% of the fresh capacity. A slow titration reconstitution method for cells did not improve the functional capacity of thawed leucocytes as judged by the NBT test. When comparing anticoagulants, heparin increased the post-reconstitution cell yields after freezing and increased the absolute number of reconstituted cells capable of developing a positive NBT reaction. PMID:643322

  2. Expression of functional molecules by human CD3- decidual granular leucocyte clones.

    PubMed

    Gudelj, L; Deniz, G; Rukavina, D; Johnson, P M; Christmas, S E

    1996-04-01

    Cell surface and cytoplasmic antigen expression by 35 CD3- decidual granular leucocyte (DGL) clones, derived from human endometrial tissue in the first trimester of pregnancy, has been compared with both that of fresh CD3- decidual leucocytes and that of CD3- peripheral blood natural killer (PBNK) cell clones (n = 12). The majority of DGL clones retained the antigenic phenotype of fresh cells, although CD103 (HML-1) was expressed on 50% of DGL clones but only 17% of fresh DGL. Both cytoplasmic CD3 zeta and CD3 epsilon chains were detected in > 90% of DGL clones in the absence of cell surface CD3. Cytoplasmic CD3 zeta was present in almost all fresh CD3- DGL, whereas CD3 epsilon was not. Most DGL clones did not express surface Fc gamma receptors I-III (CD64, -32 and -16, respectively) and complement receptors (CR) types 1 and 2 (CD35 and 21, respectively), but 43% expressed CR3 (CD11b/18); in contrast, all PBNK clones were CR3+. The NK cell-associated molecules Kp43 (CD94) and the p58 molecule recognized by the HP3E4 monoclonal antibody were both present on a higher proportion of CD3- PBNK (91% and 50%, respectively) than DGL clones (31% and 14%, respectively), despite expression of CD94 by > 90% of fresh CD56+ decidual leucocytes. Five of 35 CD3- DGL clones expressed cytoplasmic CD3 zeta in the absence of expression of CD2, CD16 or the p58 molecule recognized by HP3E4. These variations between CD3- DGL and PBNK cell clones in expression of functional molecules may be related to previously reported differences in major histocompatibility complex-non-restricted cytotoxic activities between these two cell types.

  3. Human leucocyte neutral proteases, with special reference to collagen metabolism.

    PubMed

    Kobayashi, S; Nagai, Y

    1978-09-01

    Three different types of neutral proteases related to collagen metabolism have been found in the granule fraction of human leucocytes from normal adults, using collagen, gelatin, and synthetic peptides as substrates. These are collagenase, an enzyme showing a potent hydrolytic activity against gelatin but little against native collagen, and one splitting the cross-links region of collagen. Their molecular weights were estimated to be about 75,000 150,000, and 25,000, respectively, by gel chromatography. The former two enzymes were inhibited by a alpha2-macroglobulin and ethylenediaminetetraacetate, but not by alpha1-proteinase inhibitor (alpha1-antitrypsin) or phenylmethylsulfonylfluoride, while the latter enzyme, associated in behavior with an enzyme hydrolyzing succinyl-(l-alanyl)3-p-nitroanilide, was inhibited by alpha1-proteinase inhibitor, alpha2-macroglobulin, and phenylmethylsulfonylfluoride, but not by ethylenediaminetetraacetate. A possible cooperative function of these enzymes in collagen catabolism is discussed.

  4. Control of leucocyte function-associated antigen-1-dependent cellular conjugation by divalent cations.

    PubMed Central

    Jackson, A M; Alexandroff, A B; Lappin, M B; Esuvaranathan, K; James, K; Chisholm, G D

    1994-01-01

    The control of integrin activation is fundamental to an understanding of the integrin-dependent cellular adhesion thought to be important for a plethora of basic cellular functions. Using a cell-cell conjugation assay the role of divalent cations in leucocyte function-associated antigen-1 (LFA-1)-dependent cellular adhesion was further investigated. The conjugation of interleukin-2 (IL-2)-activated lymphocytes to tumour cells was found to be energy dependent and required the presence of various divalent cations, removal of which decreased the level of conjugation. Increased concentrations of calcium, magnesium and manganese ions resulted in a corresponding increase in levels of conjugation. This increase in conjugation was LFA-1 dependent. Interestingly, when calcium ions were first removed from LFA-1, treatment of lymphocytes with magnesium and manganese ions gave significantly higher levels of conjugation than in the presence of calcium. Using a simple displacement study, calcium ions were shown to displace magnesium ions resulting in decreased conjugation. However, calcium ions were unable to displace manganese ions for binding to LFA-1. That manganese was exerting its effect via an LFA-1-dependent mechanism was confirmed using monoclonal antibodies to CD11a which negated the increased conjugation frequency due to manganese. PMID:7907574

  5. Aggregation of human polymorphonuclear leucocytes during phagocytosis of bacteria.

    PubMed Central

    Henricks, P A; van der Tol, M E; Verhoef, J

    1984-01-01

    The process of aggregation of human polymorphonuclear leucocytes (PMN) during the uptake of bacteria was studied. Radiolabelled S. aureus were opsonized in different sera, washed, resuspended in buffer and added to the PMN. Uptake of the bacteria and aggregation of the PMN were measured simultaneously. Maximal aggregation occurred within 6 min, when 5 X 10(6) PMN had phagocytosed 2.5 X 10(8) S. aureus. Also the effects of serum concentrations and different sera for opsonization of the bacteria on PMN aggregation were studied. Despite normal uptake, aggregation of PMN was low when bacteria were opsonized in complement-deficient sera. Furthermore when PMN were treated with pronase to inactivate complement receptors on the cell surface of the PMN, and bacteria preopsonized in immune serum were added, no change in uptake occurred, although the degree of aggregation halved compared to control PMN. So, interaction between the bacteria and the complement receptor of the PMN cell membrane is needed for triggering the process of aggregation. By using dansylcadaverin and diphenylamine to modulate lysosomal enzyme release, azide or PMN from a chronic granulomatous disease patient to study the effect of the formation of oxygen species, and theophylline, DB-cAMP or 8 Br-cAMP to increase cAMP levels, it was concluded that aggregation of PMN during phagocytosis was not dependent on oxygen metabolism, degranulation or cAMP levels of PMN. PMID:6086503

  6. Release of enzymes from human leucocytes during incubation with Neisseria gonorrhoeae

    PubMed Central

    Senff, Leah Morford; Sawyer, William D.

    1977-01-01

    The effect of Neisseria gonorrhoeae on release of enzymes from human leucocytes was determined. Supernatants from incubation mixtures containing leucocytes and gonococci were assayed for activity of the cytoplasmic enzyme, lactic acid dehydrogenase, as well as for activity of the hydrolytic enzymes, β-glucuronidase and lysozyme, which are found primarily in leucocyte granules. Thirty-minute incubation of leucocytes with pilated T1 gonococci resulted in a negligible release of lactic acid dehydrogenase and little release of β-glucuronidase even at bacteria to leucocyte ratios as high as 50 to 1. Lysozyme release, however, was significant at this ratio and at 20 to 1 but not at 5 to 1. Incubation with non-pilated T4 bacteria yielded no significant release of lactic acid dehydrogenase or β-glucuronidase, but it caused a significant release of lysozyme at bacteria to leucocyte ratios as low as 2 to 1. These results suggested that the lysozyme release might be related to the degree of phagocytic activity since, at low ratios, T4 was readily ingested but T1 was not. Consistent with this hypothesis, serum which promoted the phagocytosis of the pilated gonococci also stimulated lysozyme release at low ratios of T1 to leucocyte. Absorption of the serum with T1 abolished the opsonic effect and markedly diminished the amount of lysozyme released. PMID:414817

  7. Release of enzymes from human leucocytes during incubation with Neisseria gonorrhoeae.

    PubMed

    Senff, L M; Sawyer, W D

    1977-12-01

    The effect of Neisseria gonorrhoeae on release of enzymes from human leucocytes was determined. Supernatants from incubation mixtures containing leucocytes and gonococci were assayed for activity of the cytoplasmic enzyme, lactic acid dehydrogenase, as well as for activity of the hydrolytic enzymes, β-glucuronidase and lysozyme, which are found primarily in leucocyte granules. Thirty-minute incubation of leucocytes with pilated T1 gonococci resulted in a negligible release of lactic acid dehydrogenase and little release of β-glucuronidase even at bacteria to leucocyte ratios as high as 50 to 1. Lysozyme release, however, was significant at this ratio and at 20 to 1 but not at 5 to 1. Incubation with non-pilated T4 bacteria yielded no significant release of lactic acid dehydrogenase or β-glucuronidase, but it caused a significant release of lysozyme at bacteria to leucocyte ratios as low as 2 to 1. These results suggested that the lysozyme release might be related to the degree of phagocytic activity since, at low ratios, T4 was readily ingested but T1 was not. Consistent with this hypothesis, serum which promoted the phagocytosis of the pilated gonococci also stimulated lysozyme release at low ratios of T1 to leucocyte. Absorption of the serum with T1 abolished the opsonic effect and markedly diminished the amount of lysozyme released.

  8. Phospholipid turnover during phagocytosis in human polymorphonuclear leucocytes

    PubMed Central

    García Gil, Merche; Alonso, Fernando; Alvarez Chiva, Vicente; Sánchez Crespo, Mariano; Mato, José M.

    1982-01-01

    We have previously observed that the phagocytosis of zymosan particles coated with complement by human polymorphonuclear leucocytes is accompanied by a time- and dose-dependent inhibition of phosphatidylcholine synthesis by transmethylation [García Gil, Alonso, Sánchez Crespo & Mato (1981) Biochem. Biophys. Res. Commun. 101, 740–748]. The present studies show that phosphatidylcholine synthesis by a cholinephosphotransferase reaction is enhanced, up to 3-fold, during phagocytosis by polymorphonuclear cells. This effect was tested by both measuring the incorporation of radioactivity into phosphatidylcholine in cells labelled with [Me-14C]choline, and by assaying the activity of CDP-choline:diacylglycerol cholinephosphotransferase. The time course of CDP-choline:diacylglycerol cholinephosphotransferase activation by zymosan mirrors the inhibition of phospholipid methyltransferase activity previously reported. The extent of incorporation of radioactivity into phosphatidylcholine induced by various doses of zymosan correlates with the physiological response of the cells to this stimulus. This effect was specific for phosphatidylcholine, and phosphatidyl-ethanolamine turnover was not affected by zymosan. The purpose of this enhanced phosphatidylcholine synthesis is not to provide phospholipid molecules rich in arachidonic acid. The present studies show that about 80% of the arachidonic acid generated in response to zymosan derives from phosphatidylinositol. A transient accumulation of arachidonoyldiacylglycerol has also been observed, which indicates that a phospholipase C is responsible, at least in part, for the generation of arachidonic acid. Finally, isobutylmethylxanthine and quinacrine, inhibitors of phosphatidylinositol turnover, inhibit both arachidonic acid generation and phagocytosis, indicating a function for this pathway during this process. PMID:6181780

  9. A comparative study of two methods for the isolation of human leucocytes for DNA extraction.

    PubMed

    Lim, L H; Ton, S H; Cheong, S K

    1990-06-01

    The 'Dextran' and the 'Buffy-coat' methods for isolation of human leucocytes for DNA extraction were compared on the basis of DNA yield from the same amounts (10 ml) of blood. Human leucocytes from a total of 11 samples were isolated using both methods for each sample after which DNA was extracted. Extracted DNA samples were treated with ribonucleases and proteinase K after which the yields were quantitated by measuring absorbance at 260 nm. The 'Buffy-coat' method yielded a mean concentration of DNA of 476.7 micrograms/ml (range: 212 to 700 micrograms/ml) while the 'Dextran' method yielded 188.4 micrograms/ml (range: 64 to 340 micrograms/ml). The difference was confirmed by subjecting the extracted DNA samples to agarose gel electrophoresis.

  10. Effects of interferon-alpha (IFN-alpha) administration on leucocytes in healthy humans.

    PubMed

    Corssmit, E P; Heijligenberg, R; Hack, C E; Endert, E; Sauerwein, H P; Romijn, J A

    1997-02-01

    Plasma concentrations of IFN-alpha are increased in several inflammatory conditions. Several lines of evidence indicate that IFN-alpha has anti-inflammatory properties. To study the effects of IFN-alpha on leucocyte subsets and activation and on cytokines, we administered IFN-alpha (rhIFN-alpha2b; 5 x 10(6) U/m2) to eight healthy human subjects in a randomized controlled cross-over study and analysed changes in circulating leucocytes and parameters for neutrophil and monocyte activation. After administration of IFN-alpha, neutrophil counts increased, monocyte counts decreased transiently, whereas the number of lymphocytes, basophils and eosinophils showed a sustained decrease. IFN-alpha administration was also associated with neutrophil activation, reflected in an increase in the plasma concentrations of elastase-alpha1-antitrypsin complexes and lactoferrin. Serum neopterin, a marker for monocyte activation, was significantly increased 10 h after administration of IFN-alpha. IFN-alpha significantly increased plasma concentrations of IL-6, IL-8 and IL-10. Although IL-1 and tumour necrosis factor (TNF) remained undetectable, plasma concentrations of soluble TNF receptors p55 and p75 increased after IFN-alpha administration. We conclude that IFN-alpha induces multiple alterations in the distribution and functional properties of leucocytes. IFN-alpha exerts pro- as well as anti-inflammatory effects within the cytokine network.

  11. Phenotypic and functional analysis of human CD3- decidual leucocyte clones.

    PubMed

    Christmas, S E; Bulmer, J N; Meager, A; Johnson, P M

    1990-10-01

    CD3- leucocyte clones were generated from human first-trimester decidualized uterine endometrium in a culture system containing interleukin-2 (IL-2) and phytohaemagglutinin (PHA). All CD3- clones tested by Southern blot analysis had T-cell receptor (TcR) gamma and delta genes in germ-line configuration. Thirty-six CD3- cell clones obtained from eight decidual samples were mostly CD2+CD56+ but, unlike fresh decidual leucocytes, many were also CD16+. Morphological differences were noted between CD16+CD56+ and CD16-CD56+ clones, with the latter cells possessing granules of more variable size. All CD16+ clones expressed strong cytotoxic activity against natural killer (NK) sensitive and NK-resistant cell targets, while CD16- clones had low or negligible activity. Some CD3- clones produced high levels of interferon-gamma, tumour necrosis factor-negligible activity. Some CD3- clones produced high levels of interferon-gamma, tumour necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta) upon stimulation, but there was no relationship between specific cytokine production and cell clone phenotype or cytotoxic function. Levels of TGF-beta were generally higher than those produced by decidual CD3+ T-cell clones. Since decidual CD3- CD16- leucocytes have a low proliferative capacity in response to IL-2, and as clones with this phenotype invariably possess low NK cell activity, it is suggested that the NK cell activity of fresh decidual leucocyte populations is mediated largely by the small numbers of CD3- CD16+ cells present.

  12. A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

    PubMed Central

    Shetty, Shishir; Weston, Christopher J.; Adams, David H.; Lalor, Patricia F.

    2014-01-01

    Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this 'adhesion cascade' can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process. PMID:24686418

  13. A human model of platelet–leucocyte adhesive interactions during controlled ischaemia in patients with peripheral vascular disease

    PubMed Central

    Pasqualini, L; Pirro, M; Lombardini, R; Ciuffetti, G; Dragani, P; Mannarino, E

    2002-01-01

    Aims: In humans, little is known about the effects of platelet–leucocyte interactions on blood viscosity and microvascular perfusion. This study tested the hypotheses that (1) activation and interactions between platelets and leucocytes may have an impact on microvascular blood viscosity and perfusion in patients with stage II peripheral arterial occlusive disease, and (2) a powerful antiplatelet drug such as Clopidogrel might help to improve microvascular perfusion by reducing platelet–leucocyte activation and blood viscosity. Methods: Plasma concentrations of certain markers of leucocyte and platelet activation, in addition to low and high shear rate blood viscosity, were measured before and after a repeated exercise treadmill test. Functional parameters including maximum walking time, transcutaneous oxygen pressure, and half recovery time were also measured. Results: Blocking platelet activation only with a single dose of Clopidogrel (300 mg) did not improve microvascular blood viscosity and perfusion after repeated exercise, but a significant improvement in microvascular perfusion during controlled ischaemia and a lack of post exercise increase in low shear rate blood viscosity was achieved when both platelet and leucocyte activation were suppressed by a relatively longer treatment with Clopidogrel (four days). Conclusions: Clopidogrel, by inhibiting platelet activation and aggregation, might also block the vicious cycle of leucocyte–platelet activation, thus improving the functioning of the microcirculation. PMID:12461065

  14. Dog leucocyte antigen class II diversity and relationships among indigenous dogs of the island nations of Indonesia (Bali), Australia and New Guinea.

    PubMed

    Runstadler, J A; Angles, J M; Pedersen, N C

    2006-11-01

    The genetic polymorphism at the dog leucocyte antigen (DLA) class II loci DQA1, DQB1 and DRB1 was studied in a large genetically diverse population of feral and wild-type dogs from the large island nations of Indonesia (Bali), Australia and New Guinea (Bali street dog, dingo and New Guinea singing dog, respectively). Sequence-based typing (SBT) of the hypervariable region of DLA-DRB1, -DQA1 and -DQB1 alleles was used to determine genetic diversity. No new DQA1 alleles were recognized among the three dog populations, but five novel DLA-DRB1 and 2 novel DLA-DQB1 allele sequences were detected. Additional unknown alleles were postulated to exist in Bali street dogs, as indicated by the large percentage of individuals (15%-33%) that had indeterminate DRB1, DQA1 and DQB1 alleles by SBT. All three groups of dogs possessed alleles that were relatively uncommon in conventional purebreds. The New Guinea singing dog and dingo shared alleles that were not present in the Bali street dogs. These findings suggested that the dingo was more closely related to indigenous dogs from New Guinea. Feral dog populations, in particular large ones such as that of Bali, show genetic diversity that existed prior to phenotypic selection for breeds originating from their respective regions. This diversity needs to be identified and maintained in the face of progressive Westernization. These populations deserve further study as potential model populations for the evolution of major histocompatibility complex alleles, for the study of canine genetic diversity, for the development of dog breeds and for studies on the comigration of ancestral human and dog populations.

  15. Dog leucocyte antigen class II diversity and relationships among indigenous dogs of the island nations of Indonesia (Bali), Australia and New Guinea.

    PubMed

    Runstadler, J A; Angles, J M; Pedersen, N C

    2006-11-01

    The genetic polymorphism at the dog leucocyte antigen (DLA) class II loci DQA1, DQB1 and DRB1 was studied in a large genetically diverse population of feral and wild-type dogs from the large island nations of Indonesia (Bali), Australia and New Guinea (Bali street dog, dingo and New Guinea singing dog, respectively). Sequence-based typing (SBT) of the hypervariable region of DLA-DRB1, -DQA1 and -DQB1 alleles was used to determine genetic diversity. No new DQA1 alleles were recognized among the three dog populations, but five novel DLA-DRB1 and 2 novel DLA-DQB1 allele sequences were detected. Additional unknown alleles were postulated to exist in Bali street dogs, as indicated by the large percentage of individuals (15%-33%) that had indeterminate DRB1, DQA1 and DQB1 alleles by SBT. All three groups of dogs possessed alleles that were relatively uncommon in conventional purebreds. The New Guinea singing dog and dingo shared alleles that were not present in the Bali street dogs. These findings suggested that the dingo was more closely related to indigenous dogs from New Guinea. Feral dog populations, in particular large ones such as that of Bali, show genetic diversity that existed prior to phenotypic selection for breeds originating from their respective regions. This diversity needs to be identified and maintained in the face of progressive Westernization. These populations deserve further study as potential model populations for the evolution of major histocompatibility complex alleles, for the study of canine genetic diversity, for the development of dog breeds and for studies on the comigration of ancestral human and dog populations. PMID:17092255

  16. Simultaneous administration of 111In-human immunoglobulin and 99mTc-HMPAO labelled leucocytes in inflammatory bowel disease.

    PubMed

    Mairal, L; de Lima, P A; Martin-Comin, J; Baliellas, C; Xiol, X; Roca, M; Ricart, Y; Ramos, M

    1995-07-01

    Technetium-99m hexamethylpropylene amine oxime (HMPAO) labelled leucocytes and indium-111 polyclonal immunoglobulin (IgG) were simultaneously injected into a group of 27 patients routinely referred for the investigation of inflammatory bowel disease (IBD). Ten-minute anterior abdomen and tail on detector views were obtained at 30 min, 4 h and 24 h p.i. of both tracers. The diagnosis of IBD was obtained in all cases by endoscopy with biopsy and/or surgery. Images were blindly evaluated by two experienced observers who only knew of the clinical suspicion of IBD. IBD was confirmed in 20 patients (12 with Crohn's disease and eight with ulcerative colitis). Sensitivity, specificity and accuracy were 100%, 85% and 96% respectively for labelled leucocytes and 70%, 85% and 74% for IgG. Both IgG and leucocyte scans were normal in six out of seven patients in whom a diagnosis of IBD was excluded; the remaining patient, with ischaemic colitis, was falsely positive with both agents. As far as disease extension is concerned, the IgG study localized 27 diseased segments, whereas 49 were seen with the leucocyte study. Eighty-four segments were normal and 25 showed tracer uptake with both agents. Twenty-four were positive only with the leucocyte study and two were positive only with the IgG study. Agreement between the agents was 80.7%. These results confirm that 111In-human polyclonal scintigraphy is less sensitive than 99mTc-HMPAO scintigraphy both for the diagnosis of IBD and in the evaluation of disease extension. Nevertheless, if leucocyte labelling is not available, labelled IgG can be used only for diagnostic purposes. PMID:7498228

  17. Inhibition of PAF synthesis by stimulated human polymorphonuclear leucocytes with cloricromene, an inhibitor of phospholipase A2 activation.

    PubMed Central

    Ribaldi, E.; Mezzasoma, A. M.; Francescangeli, E.; Prosdocimi, M.; Nenci, G. G.; Goracci, G.; Gresele, P.

    1996-01-01

    1. A phospholipase A2 (PLA2) represents the key enzyme in the remodelling pathway of platelet-activating factor (PAF) synthesis in human polymorphonuclear (PMN) leucocytes. 2. PLA2 activation is also the rate-limiting step for the release of the arachidonic acid utilized for the synthesis of leukotrienes in stimulated leucocytes; however, it is unknown whether the PLA2s involved in the two biosynthetic pathways are identical. 3. Cloricromene (8-monochloro-3-beta-diethylaminoethyl-4-methyl-7-ethoxy- carbonylmethoxy coumarin) is an antithrombotic coumarin derivative which inhibits platelet and leucocyte function and suppresses arachidonic acid liberation by interfering with PLA2 activation. 4. The aim of the present study was to assess whether chloricromene inhibits PAF synthesis by stimulated human polymorphonuclear leucocytes (PMNs). 5. Cloricromene (50-500 microM) inhibited in a concentration-dependent manner the release of PAF, as measured by h.p.l.c. bioassay, from A23187-stimulated PMNs. Significant inhibition (45%) of PAF-release was obtained with 50 microM cloricromene and the IC50 was 85 microM. Mepacrine (500 microM), a non-specific PLA2 inhibitor, strikingly reduced PAF release. 6. The incorporation of [3H]-acetate into [3H]-PAF induced by serum-treated zymosan in human PMNs was also inhibited concentration-dependently by cloricromene, with an IC50 of 105 microM. Mepacrine also suppressed [3H]-acetate incorporation into [3H]-PAF. 7. Cloricromene did not affect the activities of the enzymes involved in PAF-synthesis acetyltransferase or phosphocholine transferase. 8. Our data demonstrate that cloricromene, an inhibitor of PLA2-activation in human leucocytes, reduces the synthesis of PAF by stimulated PMNs. This finding has a twofold implication: the PLA2s (or the mechanisms that regulate their activation) involved in PAF synthesis and arachidonate release in human leucocytes are either identical or else indistinguishable by their sensitivity to cloricromene

  18. Soluble mediators and cytokines produced by human CD3- leucocyte clones from decidualized endometrium.

    PubMed

    Deniz, G; Christmas, S E; Johnson, P M

    1996-01-01

    CD3- granulated leucocyte clones have been generated from human first-trimester decidualized endometrial tissue following culture in interleukin-2 (IL-2). Supernatants from both CD3- decidual granulated leucocyte (dGL) and CD3- peripheral blood natural killer (PBNK) cell clones inhibited the proliferation of choriocarcinoma cell lines. A panel of CD3- dGL clones, with or without phytohaemagglutinin stimulation, was assayed for cytokine secretion compared with CD3- PBNK clones and fresh tissue extracts. Levels of interferon-gamma, granulocyte-macrophage colony-stimulating factor (GM-CSF), tumour necrosis factor-alpha (TNF-alpha) and IL-10 produced by stimulated CD3-CD8- dGL clones were greater than those produced by stimulated CD3-CD8+ dGL clones. In contrast, CD8+ dGL clones were more effective in production of IL-6 than CD8- dGL clones. Immunoreactive transforming growth factor-beta 2 (TGF-beta 2) was undetectable in supernatants from CD3- dGL and PBNK clones. CD3- dGL clones generally produced higher levels of all cytokines than PBNK clones. Some unstimulated CD3- dGL and PBNK clones spontaneously produced these cytokines, but usually at a reduced level. Fresh extracts of first-trimester decidual tissue contained detectable GM-CSF, TNF-alpha, IL-10,IL-6 and TGF-beta 2. Cytokine production by fresh CD3- dGL and CD3- dGL clones indicates that these cells could play an important role in the regulation of placental growth.

  19. TLR21's agonists in combination with Aeromonas antigens synergistically up-regulate functional TLR21 and cytokine gene expression in yellowtail leucocytes.

    PubMed

    Reyes-Becerril, Martha; Ascencio-Valle, Felipe; Hirono, Ikuo; Kondo, Hidehiro; Jirapongpairoj, Walissara; Esteban, Maria Angeles; Alamillo, Erika; Angulo, Carlos

    2016-08-01

    The purpose of this study was to characterize the TLR21 gene from yellowtail (Seriola lalandi) and its functional activity using TLR agonist stimulation and Aeromonas antigens. The TLR21 nucleotide sequence from yellowtail was obtained using the whole-genome shotgun sequencing method and bioinformatics tools. Basal TLR21 gene expression was analyzed in several tissues. Subsequently, the gene expression of TLR21 and cytokines IL-1β and TNF-α was evaluated in TLR agonist (CpG-ODN2006, LPS, and Poly I:C) exposing head kidney leucocytes, which were then subjected to Aeromonas antigen stimulation. The yellowtail full-length cDNA sequence of SlTLR21 was 3615 bp (980 aa) showing a high degree of similarity with the counterparts of other fish species and sharing the common structural architecture of the TLR family, including LRR domains, one C-terminal LRR region, and a TIR domain. Gene expression studies revealed the constitutive expression of TLR21 mRNA in all the analyzed tissues; the highest levels were observed in spleen and head kidney where they play an important role in the fish immune system. Transcripts of TLR21 and the downstream IL-1β and TNF-α cytokine genes were most strongly up-regulated after exposure to the TLR agonists following Aeromonas antigen stimulation, suggesting they are involved in immune response. The results indicated that TLR agonists, in combination with Aeromonas antigens in head kidney leucocytes, synergistically enhance TLR21 and cytokines in yellowtail. PMID:26987525

  20. Human transfer factor in vitro. II. Augmentation of the secretion of leucocyte migration inhibitory factor (LIF) by leucocyte dialysate and by its components L-serine and glycine.

    PubMed Central

    Ashorn, R G; Räsänen, L; Marnela, K M; Krohn, K J

    1979-01-01

    The effect of human transfer factor (TF) or its components L-serine and/or glycine in tuberculin (PPD), or leucoagglutinin (LA) induced leucocyte migration inhibitory factor (LIF) secretion was studied. Augmentation of LIF secretion was seen with low concentration ( = 0.078 g/l) of TF when lymphocytes were cultured in minimum essential medium for suspension cultures (MEM-S), a culture medium lacking L-serine and glycine. High concentrations (0.3125-5.0 g/l dry weight) of TF were inhibitory in MEM-S. In RPMI 1640, a culture medium containing L-serine and glycine, TF was either inhibitory or had no effect. The combination of L-serine and glycine, at concentrations equivalent or lower than the optimum of TF, had an augmenting effect on LIF secretion identical to that of TF, but no inhibition at higher concentrations was seen. The results indicate that human TF contains components which have suppressive or augmenting effects on LIF secretion in vitro. The augmenting effect may be mainly due to L-serine and glycine and thus not related to TF's activity in vivo. PMID:385187

  1. Identification of peptides fromm foot-and-mouth disease virus structural proteins bound by class I swine leucocyte antigen (SLA) alleles, SLA-1*0401 and SLA-2*0401

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The analysis of peptide binding to porcine major histocompatibility complex (MHC) class I molecules has not been extensively performed. Critical to understanding the adaptive immune response of swine to infection is characterization of Swine Leucocyte Antigens (SLA) class I and class II peptide bind...

  2. The detection of membrane and cytoplasmic immunoglobulins in human leucocytes by immunoperoxidase staining.

    PubMed Central

    Mason, D Y; Labaume, S; Preud'homme, J L

    1977-01-01

    A sensitive immunoperoxidase technique for the detection of immunoglobulin (the peroxidase--anti-peroxidase or PAP procedure) has been applied to fixed smears of normal human white cells. IgM was detected in approximately 5% of lymphocytes from normal donors. Most positive cells showed a characteristic 'hairy' peripheral staining pattern; a similar morphological appearance was seen in samples stained for IgD. The membrane (rather than cytoplasmic) localization of this IgM was inferred from the redistribution of staining induced by preliminary incubation of cell suspensions with anti-mu antisera before smearing and staining. B cell-depleted and B cell-enriched suspensions showed, respectively, reduced and increased percentages of IgM-positive cells. IgG was detectable in approximately 25% of normal lymphoid cells. In contrast to the IgM and IgD reaction patterns, these cells commonly showed a discontinuous distribution of reactivity, often localized to the cell uropod or to small cytoplasmic vesicles. However, when cells were prepared at 0 degree C, staining tended to be diffuse. These findings suggested that the PAP procedure was detecting Fc receptor-bearing lymphoid cells which had bound serum IgG. IgG was also demonstrated in normal polymorphs and monocytes. The specificity of this reaction was confirmed by the use of immunoabsorbant-purified antibodies. The possible practical advantages of this immunoperoxidase procedure for the detection of leucocyte immunoglobulin are considered, and the relevance of the demonstration of IgG in non-lymphoid cells to recent reports of this immunoglobulin in Hodgkin's disease and malignant 'reticulum' cells is briefly discussed. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:338219

  3. Inherited human group IVA cytosolic phospholipase A2 deficiency abolishes platelet, endothelial, and leucocyte eicosanoid generation

    PubMed Central

    Kirkby, Nicholas S.; Reed, Daniel M.; Edin, Matthew L.; Rauzi, Francesca; Mataragka, Stefania; Vojnovic, Ivana; Bishop-Bailey, David; Milne, Ginger L.; Longhurst, Hilary; Zeldin, Darryl C.; Mitchell, Jane A.; Warner, Timothy D.

    2016-01-01

    Eicosanoids are important vascular regulators, but the phospholipase A2 (PLA2) isoforms supporting their production within the cardiovascular system are not fully understood. To address this, we have studied platelets, endothelial cells, and leukocytes from 2 siblings with a homozygous loss-of-function mutation in group IVA cytosolic phospholipase A2 (cPLA2α). Chromatography/mass spectrometry was used to determine levels of a broad range of eicosanoids produced by isolated vascular cells, and in plasma and urine. Eicosanoid release data were paired with studies of cellular function. Absence of cPLA2α almost abolished eicosanoid synthesis in platelets (e.g., thromboxane A2, control 20.5 ± 1.4 ng/ml vs. patient 0.1 ng/ml) and leukocytes [e.g., prostaglandin E2 (PGE2), control 21.9 ± 7.4 ng/ml vs. patient 1.9 ng/ml], and this was associated with impaired platelet activation and enhanced inflammatory responses. cPLA2α-deficient endothelial cells showed reduced, but not absent, formation of prostaglandin I2 (prostacyclin; control 956 ± 422 pg/ml vs. patient 196 pg/ml) and were primed for inflammation. In the urine, prostaglandin metabolites were selectively influenced by cPLA2α deficiency. For example, prostacyclin metabolites were strongly reduced (18.4% of control) in patients lacking cPLA2α, whereas PGE2 metabolites (77.8% of control) were similar to healthy volunteer levels. These studies constitute a definitive account, demonstrating the fundamental role of cPLA2α to eicosanoid formation and cellular responses within the human circulation.—Kirkby, N. S., Reed, D. M., Edin, M. L., Rauzi, F., Mataragka, S., Vojnovic, I., Bishop-Bailey, D., Milne, G. L., Longhurst, H., Zeldin, D. C., Mitchell, J. A., Warner, T. D. Inherited human group IVA cytosolic phospholipase A2 deficiency abolishes platelet, endothelial, and leucocyte eicosanoid generation. PMID:26183771

  4. Platelet serotonin release by human polymorphonuclear leucocytes stimulated by cotton dust bacteria

    PubMed Central

    Holt, P. G.; Holt, Barbara J.; Beijer, Lena; Rylander, R.

    1983-01-01

    The release of serotonin from platelets was examined using polymorphonuclear leucocytes (PMN) from normal volunteers. The stimulating agents emplyed were zymosan and heat-killed bacteria from Enterobacter agglomerans, which is commonly found in cotton dust. Optimal conditions for release were established. Both zymosan and E. agglomerans yielded a release of serotonin of an equal magnitude. The data are discussed in relation to the pathogenesis of respiratory disease associated with occupational exposure to cotton dust. PMID:6831770

  5. The use of reference strand-mediated conformational analysis for the study of cheetah (Acinonyx jubatus) feline leucocyte antigen class II DRB polymorphisms.

    PubMed

    Drake, G J C; Kennedy, L J; Auty, H K; Ryvar, R; Ollier, W E R; Kitchener, A C; Freeman, A R; Radford, A D

    2004-01-01

    There is now considerable evidence to suggest the cheetah (Acinonyx jubatus) has limited genetic diversity. However, the extent of this and its significance to the fitness of the cheetah population, both in the wild and captivity, is the subject of some debate. This reflects the difficulty associated with establishing a direct link between low variability at biologically significant loci and deleterious aspects of phenotype in this, and other, species. Attempts to study one such region, the feline leucocyte antigen (FLA), are hampered by a general reliance on cloning and sequencing which is expensive, labour-intensive, subject to PCR artefact and always likely to underestimate true variability. In this study we have applied reference strand-mediated conformational analysis (RSCA) to determine the FLA-DRB phenotypes of 25 cheetahs. This technique was rapid, repeatable and less prone to polymerase chain reaction (PCR)-induced sequence artefacts associated with cloning. Individual cheetahs were shown to have up to three FLA-DRB genes. A total of five alleles were identified (DRB*ha14-17 and DRB*gd01) distributed among four genotypes. Fifteen cheetahs were DRB*ha14/ha15/ha16/ha17, three were DRB*ha15/ha16/ha17, six were DRB*ha14/ha16/ha17 and one was DRB*ha14/ha15/ha16/ha17/gd01. Sequence analysis of DRB*gd01 suggested it was a recombinant of DRB*ha16 and DRB*ha17. Generation of new alleles is difficult to document, and the clear demonstration of such an event is unusual. This study confirms further the limited genetic variability of the cheetah at a biologically significant region. RSCA will facilitate large-scale studies that will be needed to correlate genetic diversity at such loci with population fitness in the cheetah and other species.

  6. Human leukocyte Antigen-DM polymorphisms in autoimmune diseases.

    PubMed

    Alvaro-Benito, Miguel; Morrison, Eliot; Wieczorek, Marek; Sticht, Jana; Freund, Christian

    2016-08-01

    Classical MHC class II (MHCII) proteins present peptides for CD4(+) T-cell surveillance and are by far the most prominent risk factor for a number of autoimmune disorders. To date, many studies have shown that this link between particular MHCII alleles and disease depends on the MHCII's particular ability to bind and present certain peptides in specific physiological contexts. However, less attention has been paid to the non-classical MHCII molecule human leucocyte antigen-DM, which catalyses peptide exchange on classical MHCII proteins acting as a peptide editor. DM function impacts the presentation of both antigenic peptides in the periphery and key self-peptides during T-cell development in the thymus. In this way, DM activity directly influences the response to pathogens, as well as mechanisms of self-tolerance acquisition. While decreased DM editing of particular MHCII proteins has been proposed to be related to autoimmune disorders, no experimental evidence for different DM catalytic properties had been reported until recently. Biochemical and structural investigations, together with new animal models of loss of DM activity, have provided an attractive foundation for identifying different catalytic efficiencies for DM allotypes. Here, we revisit the current knowledge of DM function and discuss how DM function may impart autoimmunity at the organism level.

  7. Human leukocyte Antigen-DM polymorphisms in autoimmune diseases.

    PubMed

    Alvaro-Benito, Miguel; Morrison, Eliot; Wieczorek, Marek; Sticht, Jana; Freund, Christian

    2016-08-01

    Classical MHC class II (MHCII) proteins present peptides for CD4(+) T-cell surveillance and are by far the most prominent risk factor for a number of autoimmune disorders. To date, many studies have shown that this link between particular MHCII alleles and disease depends on the MHCII's particular ability to bind and present certain peptides in specific physiological contexts. However, less attention has been paid to the non-classical MHCII molecule human leucocyte antigen-DM, which catalyses peptide exchange on classical MHCII proteins acting as a peptide editor. DM function impacts the presentation of both antigenic peptides in the periphery and key self-peptides during T-cell development in the thymus. In this way, DM activity directly influences the response to pathogens, as well as mechanisms of self-tolerance acquisition. While decreased DM editing of particular MHCII proteins has been proposed to be related to autoimmune disorders, no experimental evidence for different DM catalytic properties had been reported until recently. Biochemical and structural investigations, together with new animal models of loss of DM activity, have provided an attractive foundation for identifying different catalytic efficiencies for DM allotypes. Here, we revisit the current knowledge of DM function and discuss how DM function may impart autoimmunity at the organism level. PMID:27534821

  8. Human leukocyte Antigen-DM polymorphisms in autoimmune diseases

    PubMed Central

    Morrison, Eliot; Wieczorek, Marek; Sticht, Jana; Freund, Christian

    2016-01-01

    Classical MHC class II (MHCII) proteins present peptides for CD4+ T-cell surveillance and are by far the most prominent risk factor for a number of autoimmune disorders. To date, many studies have shown that this link between particular MHCII alleles and disease depends on the MHCII's particular ability to bind and present certain peptides in specific physiological contexts. However, less attention has been paid to the non-classical MHCII molecule human leucocyte antigen-DM, which catalyses peptide exchange on classical MHCII proteins acting as a peptide editor. DM function impacts the presentation of both antigenic peptides in the periphery and key self-peptides during T-cell development in the thymus. In this way, DM activity directly influences the response to pathogens, as well as mechanisms of self-tolerance acquisition. While decreased DM editing of particular MHCII proteins has been proposed to be related to autoimmune disorders, no experimental evidence for different DM catalytic properties had been reported until recently. Biochemical and structural investigations, together with new animal models of loss of DM activity, have provided an attractive foundation for identifying different catalytic efficiencies for DM allotypes. Here, we revisit the current knowledge of DM function and discuss how DM function may impart autoimmunity at the organism level. PMID:27534821

  9. Age-dependent alterations of Fc gamma receptor-mediated effector functions of human polymorphonuclear leucocytes.

    PubMed Central

    Fülöp, T; Fóris, G; Wórum, I; Leövey, A

    1985-01-01

    Changes in the effector functions in polymorphonuclear leucocytes (PMNL), harvested from blood of young and aged healthy subjects of both sexes, were studied. FC gamma-receptor (Fc gamma R)-mediated incorporation of IgG coated 51Cr-HRBC significantly increased in the aged male group, while the phagocytosis of pre-opsonized fungi (Saccharomyces cerevisiae and Candida albicans) was independent of both the age and sex. However, the intracellular killing capacity of neutrophils obtained from aged male subjects significantly decreased toward 51Cr-labelled c. albicans. The antibody-dependent cellular cytotoxicity (ADCC) was also impaired with ageing in both sexes. The age-dependent decrease in the effector functions of PMNL may be explained, among others, by the fact that during yeast cell incorporation the increased cAMP level does not return to the basic level in the old group. On the other hand, the cGMP level which increased in PMNL of aged subjects does not show any progressive increase as in the young subjects, but remains unchanged. The oxidative metabolism producing free radicals being necessary for the effective intracellular killing and ADCC diminished in PMNL of aged subjects of both sexes. The above findings indicate that the adaptation of cyclic nucleotide system and the oxidative burst to the cell activation becomes impaired with ageing. PMID:2994926

  10. Drug modulation of antigen-induced paw oedema in guinea-pigs: effects of lipopolysaccharide, tumour necrosis factor and leucocyte depletion.

    PubMed

    da Motta, J I; Cunha, F Q; Vargaftig, B B; Ferreira, S H

    1994-05-01

    1. In guinea-pigs previously sensitized with ovalbumin, the intra-plantar administration of the antigen induced dose-dependent and sustained oedema. An intense infiltrate of neutrophils and eosinophils was observed at the peak of the oedema (4 h). 2. Oedema induced by ovalbumin at the doses of 50 or 200 micrograms/paw was not inhibited by antihistamines (meclizine and cetirizine), a PAF antagonist (BN 50730), a cyclo-oxygenase inhibitor (indomethacin), a lipoxygenase inhibitor (MK-886), a dual type lipo- and cyclo-oxygenase inhibitor (NDGA), a bradykinin antagonist (Hoe 140) or the combination of cetirizine, MK-886, indomethacin and BN 50730. These drugs did inhibit paw oedema induced by their specific agonists or by carrageenin. These results suggest that histamine, PAF, prostaglandins, leukotrienes or bradykinin are not important in the development of immune paw oedema in guinea-pigs. 3. Dexamethasone (10 mg kg-1) inhibited oedema induced by ovalbumin (50 or 200 micrograms/paw, P < 0.05). This effect apparently does not result from inhibition of arachidonate metabolism, since indomethacin, MK-886 and NDGA were without effect. 4. Oedema induced by ovalbumin (50 or 200 micrograms/paw) was also inhibited by azelastine. This effect was not due to the anti-histaminic property of azelastine since two other potent-antihistamines, meclizine and cetirizine, were ineffective. 5. Intravenous injection of lipopolysaccharide (LPS) dose-dependently inhibited the oedema induced by ovalbumin (200 micrograms/paw). This effect could not be attributed to hypotension or leucopenia since the maximal dose applied (81 micrograms kg-1) did not induce significant changes in the blood pressure or in the white blood cell levels of the animals. It is suggested that the effect of LPS is mediated by the endogenous release of cytokines, including tumour necrosis factor (TNF alpha). Murine TNF alpha dose dependently(9-81 microg kg-1) inhibited the paw oedema induced by ovalbumin.7. The anti

  11. Gamma ray induced DNA damage in human and mouse leucocytes measured by SCGE-Pro: a software developed for automated image analysis and data processing for Comet assay.

    PubMed

    Chaubey, R C; Bhilwade, H N; Rajagopalan, R; Bannur, S V

    2001-02-20

    The studies reported in this communication had two major objectives: first to validate the in-house developed SCGE-Pro: a software developed for automated image analysis and data processing for Comet assay using human peripheral blood leucocytes exposed to radiation doses, viz. 2, 4 and 8 Gy, which are known to produce DNA/chromosome damage using alkaline Comet assay. The second objective was to investigate the effect of gamma radiation on DNA damage in mouse peripheral blood leucocytes using identical doses and experimental conditions, e.g. lyses, electrophoretic conditions and duration of electrophoresis which are known to affect tail moment (TM) and tail length (TL) of comets. Human and mouse whole blood samples were irradiated with different doses of gamma rays, e.g. 2, 4 and 8 Gy at a dose rate of 0.668Gy/min between 0 and 4 degrees C in air. After lyses, cells were electrophorased under alkaline conditions at pH 13, washed and stained with propidium iodide. Images of the cells were acquired and analyzed using in-house developed imaging software, SCGE-Pro, for Comet assay. For each comet, total fluorescence, tail fluorescence and tail length were measured. Increase in TM and TL was considered as the criteria of DNA damage. Analysis of data revealed heterogeneity in the response of leucocytes to gamma ray induced DNA damage both in human as well as in mouse. A wide variation in TM and TL was observed in control and irradiated groups of all the three donors. Data were analyzed for statistical significance using one-way ANOVA. Though a small variation in basal level of TM and TL was observed amongst human and mouse controls, the differences were not statistically significant. A dose-dependent increase in TM (P<0.001) and TL (P<0.001) was obtained at all the radiation doses (2-8 Gy) both in human and mouse leucocytes. However, there was a difference in the nature of dose response curves for human and mouse leucocytes. In human leucocytes, a linear increase in TM

  12. Colostral proteins from cows immunised with Streptococcus mutans/S. sobrinus support the phagocytosis and killing of mutans streptococci by human leucocytes.

    PubMed

    Loimaranta, V; Nuutila, J; Marnila, P; Tenovuo, J; Korhonen, H; Lilius, E M

    1999-10-01

    Passive immunisation, based on bovine colostral preparations, is an area of active research. Specific bovine antibodies inhibit the virulence factors of target pathogens but the interactions between whey preparations and human immune defence cells are not well known. Bovine colostrum inhibits the phagocytic activity of bovine leucocytes and this may reflect the biological activity of immunoglobulins in it. Therefore, this study aimed to examine the effects of bovine whey protein preparations from the colostrum of Streptococcus mutans/S. sobrinus-immunised and sham-immunised cows on binding, ingestion and killing of these bacteria by human leucocytes. Binding and ingestion of FITC-labelled bacteria were estimated by flow cytometry and leukocyte activation was measured as chemiluminescence. Killing rate was estimated by plate counting and by measuring bioluminescence from S. mutans- containing the insect luciferase gene. Colostral whey protein preparation from hyperimmunised cows activated human leucocytes by opsonising specific bacteria. Neutrophils, eosinophils and monocytes weakly phagocytosed non-opsonised bacteria and bacteria opsonised with control product. On the contrary, binding and ingestion were efficient in the presence of the preparation from immunised cows. Thus, these results show that bovine colostral whey proteins are able to support the activation of human phagocytes against pathogenic microbes and that this property is related to specific antibodies in whey preparations. These whey proteins may also be clinically useful, especially in preventing the colonisation of newly erupted teeth by mutans streptococci.

  13. Levels of expression of complement regulatory proteins CD46, CD55 and CD59 on resting and activated human peripheral blood leucocytes

    PubMed Central

    Christmas, Stephen E; de la Mata Espinosa, Claudia T; Halliday, Deborah; Buxton, Cheryl A; Cummerson, Joanne A; Johnson, Peter M

    2006-01-01

    The cell surface complement regulatory (CReg) proteins CD46, CD55 and CD59 are widely expressed on human lymphoid and non-lymphoid cells. This study aimed to compare systematically levels of CReg expression by different leucocyte subsets and to determine whether levels were increased following activation in vitro. Levels of each CReg protein were similar on freshly isolated monocytes and all major lymphocyte subsets, except that CD4+ cells expressed significantly less CD46 than CD8+ cells (P < 0·05) while the reverse was observed for CD55 (P < 0·02). CD56+ cells, predominantly natural killer cells, expressed significantly lower levels of CD59 than T cells (P < 0·02). CD45RO+ cells had higher levels of surface CD46 and CD59, but lower levels of CD55, than CD45RO– cells (P < 0·02); CD25+ cells also expressed significantly less CD55 than CD25− cells (P < 0·002). Neutrophils expressed higher levels of CD59, but lower levels of CD55, than monocytes. Following activation with phytohaemagglutinin, CD46 was up-regulated on all leucocyte subsets with the exception of CD56+ cells. Both CD55 and CD59 were also markedly up-regulated on monocytes, and CD55 expression was greater on CD8+ than CD4+ cells following activation (P < 0·02). Lipopolysaccharide treatment did not significantly alter B-cell expression of CReg proteins whereas CD55 and CD59, but not CD46, were significantly up-regulated on monocytes (P < 0·02). These observations that CReg proteins are up-regulated on certain activated leucocyte subsets indicate that levels would be increased following immune responses in vivo. This could enhance both protection against local complement activation at inflammatory sites and also the immunoregulatory properties of these leucocytes. PMID:16999828

  14. Levels of expression of complement regulatory proteins CD46, CD55 and CD59 on resting and activated human peripheral blood leucocytes.

    PubMed

    Christmas, Stephen E; de la Mata Espinosa, Claudia T; Halliday, Deborah; Buxton, Cheryl A; Cummerson, Joanne A; Johnson, Peter M

    2006-12-01

    The cell surface complement regulatory (CReg) proteins CD46, CD55 and CD59 are widely expressed on human lymphoid and non-lymphoid cells. This study aimed to compare systematically levels of CReg expression by different leucocyte subsets and to determine whether levels were increased following activation in vitro. Levels of each CReg protein were similar on freshly isolated monocytes and all major lymphocyte subsets, except that CD4(+) cells expressed significantly less CD46 than CD8(+) cells (P < 0.05) while the reverse was observed for CD55 (P < 0.02). CD56(+) cells, predominantly natural killer cells, expressed significantly lower levels of CD59 than T cells (P < 0.02). CD45RO(+) cells had higher levels of surface CD46 and CD59, but lower levels of CD55, than CD45RO(-) cells (P < 0.02); CD25(+) cells also expressed significantly less CD55 than CD25(-) cells (P < 0.002). Neutrophils expressed higher levels of CD59, but lower levels of CD55, than monocytes. Following activation with phytohaemagglutinin, CD46 was up-regulated on all leucocyte subsets with the exception of CD56(+) cells. Both CD55 and CD59 were also markedly up-regulated on monocytes, and CD55 expression was greater on CD8(+) than CD4(+) cells following activation (P < 0.02). Lipopolysaccharide treatment did not significantly alter B-cell expression of CReg proteins whereas CD55 and CD59, but not CD46, were significantly up-regulated on monocytes (P < 0.02). These observations that CReg proteins are up-regulated on certain activated leucocyte subsets indicate that levels would be increased following immune responses in vivo. This could enhance both protection against local complement activation at inflammatory sites and also the immunoregulatory properties of these leucocytes.

  15. Removal of leucocytes from whole blood and erythrocyte suspensions by filtration through cotton wool. IV. Immunization studies in rabbits.

    PubMed

    Engelfriet, C P; Diepenhorst, P; Van den Giessen, M; von Riesz, E

    1975-01-01

    Hetero-immunization experiments in rabbits were performed to evaluate the quantity of leucocyte antigens present in red cell suspensions prepared by the filtration method of DIEPENHORST et al., in which no intact leucocytes could be detected. It was found that the quantity of leucocyte antigens is smallest in blood filtered immediately after taking. More leucocyte-antigenic material seems to be present in blood that is filtered after storage and in frozen-thawed red cell suspensions. Evidence was obtained that granulocyte-specific antigens are more strongly immunogenic in the rabbit than lymphocyte-specific antigens or antigens common to both cells.

  16. Human immune response to Mycobacterium tuberculosis antigens.

    PubMed Central

    Havlir, D V; Wallis, R S; Boom, W H; Daniel, T M; Chervenak, K; Ellner, J J

    1991-01-01

    Little is known about the immunodominant or protective antigens of Mycobacterium tuberculosis in humans. Cell-mediated immunity is necessary for protection, and healthy tuberculin-positive individuals are relatively resistant to exogenous reinfection. We compared the targets of the cell-mediated immune response in healthy tuberculin-positive individuals to those of tuberculosis patients and tuberculin-negative persons. By using T-cell Western blotting (immunoblotting) of nitrocellulose-bound M. tuberculosis culture filtrate, peaks of T-cell blastogenic activity were identified in the healthy tuberculin reactors at 30, 37, 44, 57, 64, 71 and 88 kDa. Three of these fractions (30, 64, and 71 kDa) coincided with previously characterized proteins: antigen 6/alpha antigen, HSP60, and HSP70, respectively. The blastogenic responses to purified M. tuberculosis antigen 6/alpha antigen and BCG HSP60 were assessed. When cultured with purified antigen 6/alpha antigen, lymphocytes of healthy tuberculin reactors demonstrated greater [3H]thymidine incorporation than either healthy tuberculin-negative controls or tuberculous patients (8,113 +/- 1,939 delta cpm versus 645 +/- 425 delta cpm and 1,019 +/- 710 delta cpm, respectively; P less than 0.01). Healthy reactors also responded to HSP60, although to a lesser degree than antigen 6/alpha antigen (4,276 +/- 1,095 delta cpm; P less than 0.05). Partially purified HSP70 bound to nitrocellulose paper elicited a significant lymphocyte blastogenic response in two of six of the tuberculous patients but in none of the eight healthy tuberculin reactors. Lymphocytes of none of five tuberculin-negative controls responded to recombinant antigens at 14 or 19 kDa or to HSP70. Antibody reactivity generally was inversely correlated with blastogenic response: tuberculous sera had high titer antibody to M. tuberculosis culture filtrate in a range from 35 to 180 kDa. This is the first systematic evaluation of the human response to a panel of native

  17. Leucocytes isolated from simply frozen whole blood can be used in human biomonitoring for DNA damage measurement with the comet assay.

    PubMed

    Akor-Dewu, Maryam B; El Yamani, Naouale; Bilyk, Olena; Holtung, Linda; Tjelle, Torunn E; Blomhoff, Rune; Collins, Andrew R

    2014-04-01

    Preservation of human blood cells for DNA damage analysis with the comet assay conventionally involves the isolation of mononuclear cells by centrifugation, suspension in freezing medium and slow freezing to -80 °C-a laborious process. A recent publication (Al-Salmani et al. Free Rad Biol Med 2011; 51: 719-725) describes a simple method in which small volumes of whole blood are frozen to -20 or -80 °C; on subsequent thawing, the comet assay is performed, with no indication of elevated DNA strand breakage resulting from the rapid freezing. However, leucocytes in whole blood (whether fresh or frozen) are abnormally resistant to damage by H2 O2 , and so a common test of antioxidant status (resistance to strand breakage by H2 O2 ) cannot be used. We have refined this method by separating the leucocytes from the thawed blood; we find that, after three washes, the cells respond normally to H2 O2 . In addition, we have measured specific endogenous base damage (oxidized purines) in the isolated leucocytes, using the enzyme formamidopyrimidine DNA glycosylase. In a study of blood samples from 10 subjects, H2 O2 sensitivity and endogenous damage-both reflecting the antioxidant status of the cells-correlated significantly. This modified approach to sample collection and storage is particularly applicable when the available volume of blood is limited and has great potential in biomonitoring and ecogenotoxicology studies where samples are obtained in the field or at sites remote from the testing laboratory.

  18. Influence of human leucocyte antigen‐DRB1 on the susceptibility to rheumatoid arthritis and on the production of anti‐cyclic citrullinated peptide antibodies in a Portuguese population

    PubMed Central

    Ligeiro, D; Fonseca, J E; Abade, O; Abreu, I; Cruz, M; Nero, P; Cavaleiro, J; Teles, J; Trindade, H; Caetano, J M; Branco, J

    2007-01-01

    Objective To clarify the influence of the HLA‐DRB1 locus on the susceptibility to rheumatoid arthritis and the production of anti‐cyclic citrullinated peptide antibodies (anti‐CCP) in a Portuguese population. Methods: 141 patients with rheumatoid arthritis fulfilling the American College of Rheumatology 1987 revised criteria for rheumatoid arthritis were compared with 150 healthy controls. Human leucocyte antigen (HLA)‐DRB1 locus genotyping was assessed by polymerase chain reaction reverse probing assays and sequence‐specific primers. Anti‐CCP antibodies were quantified by ELISA in patients with rheumatoid arthritis. Frequencies between groups were compared by the two‐sided Fisher's exact test and considered significant if p<0.05. Results The HLA‐DRB1*04 and HLA‐DRB1*10 groups were highly associated with rheumatoid arthritis (p<0.001 and p = 0.031, respectively). High titres of anti‐CCP antibodies were largely associated with the presence of HLA‐DRB1*04/10. Conclusion The well‐recognised susceptibility alleles to rheumatoid arthritis, HLA‐DRB1*04, were associated with rheumatoid arthritis in Portuguese patients. The relatively rare DRB1*10 was also associated with rheumatoid arthritis, as was described previously in other southern European countries. Both groups were associated with high anti‐CCP titres, reinforcing its relevance to disease onset. PMID:16793843

  19. Expression of the human mucosal lymphocyte antigen, HML-1, by T cells activated with mitogen or specific antigen in vitro.

    PubMed

    Brew, R; West, D C; Burthem, J; Christmas, S E

    1995-06-01

    Expression of the human mucosal lymphocyte antigen, HML-1 (CD103), recently identified as a novel alpha E beta 7 integrin, was studied on peripheral blood lymphocytes activated with mitogen or specific antigen. HML-1 was up-regulated on PHA activated T-lymphoblasts cultured in 100IU/ml interleukin-2 (IL-2), reaching a peak of > 50% positive cells at day 7, and expression was maintained at this level throughout the 28-day culture period. Following a transient decrease in the percentage of L-selectin cells, expression of this molecule was maintained on most PHA T-lymphoblasts. Cells activated by purified protein derivative of M. tuberculosis (PPD) or in mixed lymphocyte culture also up-regulated and maintained HML-1 expression for 14 days. In contrast, in all cases the percentage of CD25+ cells rose initially but subsequently declined over the same time periods. When freshly isolated cells from tonsil, spleen, mesenteric lymph node and lung were analysed, only lung contained significant numbers (39 +/- 6%) of HML-1+ cells. In both freshly isolated and activated cell populations the great majority of HML-1+ cells co-expressed CD8 although some HML-1+ CD8- cells were also present. Production of TGF-beta 1 peaked early during T-lymphoblast and MLR cultures and was not related to induction of HML-1 expression. Immunoprecipitation studies showed that the HML-1 molecule expressed on 10-day PHA T-lymphoblasts was indistinguishable from that found on intestinal intraepithelial lymphocytes and that no alpha 4 beta 7 integrin was expressed by these cells. Although HML-1 expression is essentially restricted to mucosal leucocytes in vivo, these experiments show that it is readily induced and maintained along with co-expression of L-selectin following CD8+ T-lymphocyte activation in vitro.

  20. Changes in leucocyte migration after renal transplantation

    PubMed Central

    Smith, M. G. M.; Eddleston, A. L. W. F.; Dominguez, J. A.; Evans, D. B.; Bewick, M.; Williams, Roger

    1969-01-01

    The leucocyte migration test, an in-vitro measure of cellular immunity, has been used to follow the changes in cell-mediated hypersensitivity to kidney and histocompatibility antigens in three patients after renal transplantation. Inhibition of leucocyte migration, indicating strong sensitization to the antigens used, occurred in each patient, starting five to seven days after transplantation. Satisfactory renal function had not been established in any of the patients at this time. In one case inhibition of leucocyte migration persisted almost continuously until the 24th day and was associated with poor renal function proved histologically to be due to rejection. Treatment with increased dosage of prednisone was associated with a rapid reversion to normal of the migration index and improvement in renal function. Later, inhibition of migration occurred again, and shortly afterwards the graft ceased to function. In the other two cases the migration index became normal without alteration in immunosuppressive therapy and a satisfactory diuresis followed. It is suggested that this simple test should prove useful in the specific diagnosis of rejection and in control of immunosuppressive therapy. ImagesFig. 3Fig. 4 PMID:4899455

  1. Antigenically Modified Human Pluripotent Stem Cells Generate Antigen-Presenting Dendritic Cells

    PubMed Central

    Zeng, Jieming; Wu, Chunxiao; Wang, Shu

    2015-01-01

    Human pluripotent stem cells (hPSCs) provide a promising platform to produce dendritic cell (DC) vaccine. To streamline the production process, we investigated a unique antigen-loading strategy that suits this novel platform. Specifically, we stably modified hPSCs using tumour antigen genes in the form of a full-length tumour antigen gene or an artificial tumour antigen epitope-coding minigene. Such antigenically modified hPSCs were able to differentiate into tumour antigen-presenting DCs. Without conventional antigen-loading, DCs derived from the minigene-modified hPSCs were ready to prime a tumour antigen-specific T cell response and further expand these specific T cells in restimulation processes. These expanded tumour antigen-specific T cells were potent effectors with central memory or effector memory phenotype. Thus, we demonstrated that immunocompetent tumour antigen-loaded DCs can be directly generated from antigenically modified hPSCs. Using such strategy, we can completely eliminate the conventional antigen-loading step and significantly simplify the production of DC vaccine from hPSCs. PMID:26471005

  2. Leucocyte specific antinuclear antibodies. Preparation and fluorescence study.

    PubMed

    Lampert, I A; Evans, D J; Chesterton, C J

    1975-09-11

    Tissue and species specific anti-nuclear sera detectable by immunofluorescence were produced by immunising rabbits with D.N.A.-non-histone protein extract from human leucocytes. This is a potentially valuable method for the morphological study of differentiation.

  3. The Human Transporter Associated with Antigen Processing

    PubMed Central

    Corradi, Valentina; Singh, Gurpreet; Tieleman, D. Peter

    2012-01-01

    The human transporter associated with antigen processing (TAP) is a member of the ATP binding cassette (ABC) transporter superfamily. TAP plays an essential role in the antigen presentation pathway by translocating cytosolic peptides derived from proteasomal degradation into the endoplasmic reticulum lumen. Here, the peptides are loaded into major histocompatibility class I molecules to be in turn exposed at the cell surface for recognition by T-cells. TAP is a heterodimer formed by the association of two half-transporters, TAP1 and TAP2, with a typical ABC transporter core that consists of two nucleotide binding domains and two transmembrane domains. Despite the availability of biological data, a full understanding of the mechanism of action of TAP is limited by the absence of experimental structures of the full-length transporter. Here, we present homology models of TAP built on the crystal structures of P-glycoprotein, ABCB10, and Sav1866. The models represent the transporter in inward- and outward-facing conformations that could represent initial and final states of the transport cycle, respectively. We described conserved regions in the endoplasmic reticulum-facing loops with a role in the opening and closing of the cavity. We also identified conserved π-stacking interactions in the cytosolic part of the transmembrane domains that could explain the experimental data available for TAP1-Phe-265. Electrostatic potential calculations gave structural insights into the role of residues involved in peptide binding, such as TAP1-Val-288, TAP2-Cys-213, TAP2-Met-218. Moreover, these calculations identified additional residues potentially involved in peptide binding, in turn verified with replica exchange simulations performed on a peptide bound to the inward-facing models. PMID:22700967

  4. Killed poliovirus antigen titration in humans.

    PubMed

    Salk, J; Cohen, H; Fillastre, C; Stoeckel, P; Rey, J L; Schlumberger, M; Nicolas, A; van Steenis, G; van Wezel, A L; Triau, R; Saliou, P; Barry, L F; Moreau, J P; Mérieux, C

    1978-01-01

    To establish the antigen content of a killed poliovirus vaccine sufficiently potent to induce immunity with one or two doses and to establish a reference standard vaccine which has been tested under field conditions, a titration was carried out in infants to determine the amount of each of the three antigenic types of poliovirus vaccine required to induce seroconversion with a single dose. It has been observed that over a critical range of antigen concentration there is an essentially linear relationship between antibody response and quantity of antigen administered. More than 90 percent of the groups studied had detectable antibody after receiving single injections of 80, 8 and 64 D-antigen units of Types I, II and III, respectively. Four-fold less antigen for each of the three types was less effective. The implications of these findings for an efficient immunization procedure are discussed.

  5. Human humoral responses to antigens of Mycobacterium tuberculosis: immunodominance of high-molecular-mass antigens.

    PubMed Central

    Laal, S; Samanich, K M; Sonnenberg, M G; Zolla-Pazner, S; Phadtare, J M; Belisle, J T

    1997-01-01

    The selection of antigens of Mycobacterium tuberculosis for most studies of humoral responses in tuberculosis patients has been restricted to molecules that were either immunodominant in immunized animals or amenable to biochemical purification rather than those that were reactive with the human immune system. Delineation of antigens that elicit humoral responses during the natural course of disease progression in humans has been hindered by the presence of cross-reactive antibodies to conserved regions on ubiquitous prokaryotic antigens in sera from healthy individuals and tuberculosis patients. The levels of cross-reactive antibodies in the sera were reduced by preadsorption with Escherichia coli lysates, prior to studying their reactivity against a large panel of M. tuberculosis antigens to which the human immune system may be exposed during natural infection and disease. Thus, reactivity against pools of secreted, cellular, and cell wall-associated antigens of M. tuberculosis was assessed by an enzyme-linked immunosorbent assay (ELISA). Initial results suggested that the secreted protein preparation contained antigens most frequently recognized by the humoral responses of pulmonary tuberculosis patients. The culture filtrate proteins were subsequently size fractionated by preparative polyacrylamide gel electrophoresis, characterized by reaction with murine monoclonal antibodies to known antigens of M. tuberculosis by an ELISA, and assessed for reactivity with tuberculous and nontuberculous sera. Results show that a secreted antigen of 88 kDa elicits a strong antibody response in a high percentage of patients with pulmonary tuberculosis. This and other antigens identified on the basis of their reactivity with patient sera may prove useful for developing serodiagnosis for tuberculosis. PMID:9008280

  6. Melanocyte antigen triggers autoimmunity in human psoriasis.

    PubMed

    Arakawa, Akiko; Siewert, Katherina; Stöhr, Julia; Besgen, Petra; Kim, Song-Min; Rühl, Geraldine; Nickel, Jens; Vollmer, Sigrid; Thomas, Peter; Krebs, Stefan; Pinkert, Stefan; Spannagl, Michael; Held, Kathrin; Kammerbauer, Claudia; Besch, Robert; Dornmair, Klaus; Prinz, Jörg C

    2015-12-14

    Psoriasis vulgaris is a common T cell-mediated inflammatory skin disease with a suspected autoimmune pathogenesis. The human leukocyte antigen (HLA) class I allele, HLA-C*06:02, is the main psoriasis risk gene. Epidermal CD8(+) T cells are essential for psoriasis development. Functional implications of HLA-C*06:02 and mechanisms of lesional T cell activation in psoriasis, however, remained elusive. Here we identify melanocytes as skin-specific target cells of an HLA-C*06:02-restricted psoriatic T cell response. We found that a Vα3S1/Vβ13S1 T cell receptor (TCR), which we had reconstituted from an epidermal CD8(+) T cell clone of an HLA-C*06:02-positive psoriasis patient specifically recognizes HLA-C*06:02-positive melanocytes. Through peptide library screening, we identified ADAMTS-like protein 5 (ADAMTSL5) as an HLA-C*06:02-presented melanocytic autoantigen of the Vα3S1/Vβ13S1 TCR. Consistent with the Vα3S1/Vβ13S1-TCR reactivity, we observed numerous CD8(+) T cells in psoriasis lesions attacking melanocytes, the only epidermal cells expressing ADAMTSL5. Furthermore, ADAMTSL5 stimulation induced the psoriasis signature cytokine, IL-17A, in CD8(+) T cells from psoriasis patients only, supporting a role as psoriatic autoantigen. This unbiased analysis of a TCR obtained directly from tissue-infiltrating CD8(+) T cells reveals that in psoriasis HLA-C*06:02 directs an autoimmune response against melanocytes through autoantigen presentation. We propose that HLA-C*06:02 may predispose to psoriasis via this newly identified autoimmune pathway.

  7. Melanocyte antigen triggers autoimmunity in human psoriasis.

    PubMed

    Arakawa, Akiko; Siewert, Katherina; Stöhr, Julia; Besgen, Petra; Kim, Song-Min; Rühl, Geraldine; Nickel, Jens; Vollmer, Sigrid; Thomas, Peter; Krebs, Stefan; Pinkert, Stefan; Spannagl, Michael; Held, Kathrin; Kammerbauer, Claudia; Besch, Robert; Dornmair, Klaus; Prinz, Jörg C

    2015-12-14

    Psoriasis vulgaris is a common T cell-mediated inflammatory skin disease with a suspected autoimmune pathogenesis. The human leukocyte antigen (HLA) class I allele, HLA-C*06:02, is the main psoriasis risk gene. Epidermal CD8(+) T cells are essential for psoriasis development. Functional implications of HLA-C*06:02 and mechanisms of lesional T cell activation in psoriasis, however, remained elusive. Here we identify melanocytes as skin-specific target cells of an HLA-C*06:02-restricted psoriatic T cell response. We found that a Vα3S1/Vβ13S1 T cell receptor (TCR), which we had reconstituted from an epidermal CD8(+) T cell clone of an HLA-C*06:02-positive psoriasis patient specifically recognizes HLA-C*06:02-positive melanocytes. Through peptide library screening, we identified ADAMTS-like protein 5 (ADAMTSL5) as an HLA-C*06:02-presented melanocytic autoantigen of the Vα3S1/Vβ13S1 TCR. Consistent with the Vα3S1/Vβ13S1-TCR reactivity, we observed numerous CD8(+) T cells in psoriasis lesions attacking melanocytes, the only epidermal cells expressing ADAMTSL5. Furthermore, ADAMTSL5 stimulation induced the psoriasis signature cytokine, IL-17A, in CD8(+) T cells from psoriasis patients only, supporting a role as psoriatic autoantigen. This unbiased analysis of a TCR obtained directly from tissue-infiltrating CD8(+) T cells reveals that in psoriasis HLA-C*06:02 directs an autoimmune response against melanocytes through autoantigen presentation. We propose that HLA-C*06:02 may predispose to psoriasis via this newly identified autoimmune pathway. PMID:26621454

  8. Dihomo-gamma-linolenic acid inhibits tumour necrosis factor-alpha production by human leucocytes independently of cyclooxygenase activity.

    PubMed

    Dooper, Maaike M B W; van Riel, Boet; Graus, Yvo M F; M'Rabet, Laura

    2003-11-01

    Dietary oils (such as borage oil), which are rich in gamma-linolenic acid (GLA), have been shown to be beneficial under inflammatory conditions. Dihomo-GLA (DGLA) is synthesized directly from GLA and forms a substrate for cyclooxygenase (COX) enzymes, resulting in the synthesis of lipid mediators (eicosanoids). In the present study, the immunomodulatory effects of DGLA were investigated and compared with those of other relevant fatty acids. Freshly isolated human peripheral blood mononuclear cells (PBMC) were cultured in fatty acid (100 microm)-enriched medium for 48 hr. Subsequently, cells were stimulated with lipopolysaccharide (LPS) for 20 hr and the cytokine levels were measured, in supernatants, by enzyme-linked immunosorbent assay (ELISA). Phospholipids were analysed by gas chromatography. Fatty acids were readily taken up, metabolized and incorporated into cellular phospholipids. Compared with the other fatty acids tested, DGLA exerted pronounced modulatory effects on cytokine production. Tumour necrosis factor-alpha (TNF-alpha) and interleukin (IL)-10 levels were reduced to 60% of control levels, whereas IL-6 levels were not affected by DGLA. Kinetic studies showed that peak levels of TNF-alpha, occurring early after LPS addition, were inhibited strongly, whereas IL-10 levels were not affected until 15 hr after stimulation. Both the reduction of cytokine levels and the decrease in arachidonic acid levels in these cells, induced by DGLA, were dose dependent, suggesting a shift in eicosanoid-subtype synthesis. However, although some DGLA-derived eicosanoids similarly reduced TNF-alpha levels, the effects of DGLA were probably not mediated by COX products, as the addition of indomethacin did not alter the effects of DGLA. In conclusion, these results suggest that DGLA affects cytokine production by human PBMC independently of COX activation. PMID:14632663

  9. Human cysticercosis: antigens, antibodies and non-responders.

    PubMed Central

    Flisser, A; Woodhouse, E; Larralde, C

    1980-01-01

    Immunoelectrophoresis of sera from patients with brain cysticercosis against a crude antigenic extract from Cysticercus cellulosae indicates that nearly 50% of the patients do not make sufficient antibodies to ostensively precipitate. The other 50% of the patients who do make precipitating antibodies show a very heterogeneous response in the number of antigens they recognize as well as in the type of antigen--as classified by their electrophoretic mobilities. The most favoured, called antigen B, is recognized by 84% of positive sera and corresponds to one or a limited number of antigens isoelectric at pH 8.6. Indirect immunofluorescence with monospecific anti-human immunoglobulins, performed upon the immunoelectrophoretic preparations, reveal that all cysticercus antigens induced the synthesis of antibodies in the immunoglobulin classes in the order G greater than M greater than E greater than A greater than D. Finally, antigen H (an anodic component) seems to favour IgE relative to its ability to induce IgG. Thus, although in natural infection a good proportion of cysticercotic patients do not seem to mount an energetic antibody response against the parasite, giving rise to some speculations about immunosuppression, the fact that 50% do synthesize antibodies allows for some optimistic expectations from vaccination of humans--in view of the good results of vaccination in experimental animals mediated by IgG antibodies. A likely prospect for a human vaccine would be antigen B because it is the most frequently detected by humans, although its immunizing and toxic properties remain to be properly studied. Images FIG. 1 FIG. 3 FIG. 6 PMID:7389197

  10. Human cysticercosis: antigens, antibodies and non-responders.

    PubMed

    Flisser, A; Woodhouse, E; Larralde, C

    1980-01-01

    Immunoelectrophoresis of sera from patients with brain cysticercosis against a crude antigenic extract from Cysticercus cellulosae indicates that nearly 50% of the patients do not make sufficient antibodies to ostensively precipitate. The other 50% of the patients who do make precipitating antibodies show a very heterogeneous response in the number of antigens they recognize as well as in the type of antigen--as classified by their electrophoretic mobilities. The most favoured, called antigen B, is recognized by 84% of positive sera and corresponds to one or a limited number of antigens isoelectric at pH 8.6. Indirect immunofluorescence with monospecific anti-human immunoglobulins, performed upon the immunoelectrophoretic preparations, reveal that all cysticercus antigens induced the synthesis of antibodies in the immunoglobulin classes in the order G greater than M greater than E greater than A greater than D. Finally, antigen H (an anodic component) seems to favour IgE relative to its ability to induce IgG. Thus, although in natural infection a good proportion of cysticercotic patients do not seem to mount an energetic antibody response against the parasite, giving rise to some speculations about immunosuppression, the fact that 50% do synthesize antibodies allows for some optimistic expectations from vaccination of humans--in view of the good results of vaccination in experimental animals mediated by IgG antibodies. A likely prospect for a human vaccine would be antigen B because it is the most frequently detected by humans, although its immunizing and toxic properties remain to be properly studied.

  11. Antigenic Relationships among Human Pathogenic Orientia tsutsugamushi Isolates from Thailand

    PubMed Central

    Nawtaisong, Pruksa; Tanganuchitcharnchai, Ampai; Smith, Derek J.; Day, Nicholas P. J.; Paris, Daniel H.

    2016-01-01

    Background Scrub typhus is a common cause of undiagnosed febrile illness in certain tropical regions, but can be easily treated with antibiotics. The causative agent, Orientia tsutsugamushi, is antigenically variable which complicates diagnosis and efforts towards vaccine development. Methodology/Principal Findings This study aimed to dissect the antigenic and genetic relatedness of O. tsutsugamushi strains and investigate sero-diagnostic reactivities by titrating individual patient sera against their O. tsutsugamushi isolates (whole-cell antigen preparation), in homologous and heterologous serum-isolate pairs from the same endemic region in NE Thailand. The indirect immunofluorescence assay was used to titrate Orientia tsutsugamushi isolates and human sera, and a mathematical technique, antigenic cartography, was applied to these data to visualise the antigenic differences and cross-reactivity between strains and sera. No functional or antigen-specific analyses were performed. The antigenic variation found in clinical isolates was much less pronounced than the genetic differences found in the 56kDa type-specific antigen genes. The Karp-like sera were more broadly reactive than the Gilliam-like sera. Conclusions/Significance Antigenic cartography worked well with scrub typhus indirect immunofluorescence titres. The data from humoral responses suggest that a Karp-like strain would provide broader antibody cross-reactivity than a Gilliam-like strain. Although previous exposure to O. tsutsugamushi could not be ruled out, scrub typhus patient serum antibody responses were characterised by strong homologous, but weak heterologous antibody titres, with little evidence for cross-reactivity by Gilliam-like sera, but a broader response from some Karp-like sera. This work highlights the importance of antigenic variation in O. tsutsugamushi diagnosis and determination of new serotypes. PMID:27248711

  12. Neutrophil elastase enhances antigen presentation by upregulating human leukocyte antigen class I expression on tumor cells.

    PubMed

    Chawla, Akhil; Alatrash, Gheath; Philips, Anne V; Qiao, Na; Sukhumalchandra, Pariya; Kerros, Celine; Diaconu, Iulia; Gall, Victor; Neal, Samantha; Peters, Haley L; Clise-Dwyer, Karen; Molldrem, Jeffrey J; Mittendorf, Elizabeth A

    2016-06-01

    Neutrophil elastase (NE) is an innate immune cell-derived inflammatory mediator that we have shown increases the presentation of tumor-associated peptide antigens in breast cancer. In this study, we extend these observations to show that NE uptake has a broad effect on enhancing antigen presentation by breast cancer cells. We show that NE increases human leukocyte antigen (HLA) class I expression on the surface of breast cancer cells in a concentration and time-dependent manner. HLA class I upregulation requires internalization of enzymatically active NE. Western blots of NE-treated breast cancer cells confirm that the expression of total HLA class I as well as the antigen-processing machinery proteins TAP1, LMP2, and calnexin does not change following NE treatment. This suggests that NE does not increase the efficiency of antigen processing; rather, it mediates the upregulation of HLA class I by stabilizing and reducing membrane recycling of HLA class I molecules. Furthermore, the effects of NE extend beyond breast cancer since the uptake of NE by EBV-LCL increases the presentation of HLA class I-restricted viral peptides, as shown by their increased sensitivity to lysis by EBV-specific CD8+ T cells. Together, our results show that NE uptake increases the responsiveness of breast cancer cells to adaptive immunity by broad upregulation of membrane HLA class I and support the conclusion that the innate inflammatory mediator NE enhances tumor cell recognition and increases tumor sensitivity to the host adaptive immune response.

  13. Characterization of binding specificities of Bovine Leucocyte class I molecules: Impacts for rational epitope discovery

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The binding of peptides to classical major histocompatibility complex (MHC) class-I proteins is the single most selective step in antigen presentation. However, the peptide binding specificity of cattle MHC (bovine leucocyte antigen, BoLA) class I (BoLA-I) molecules remains poorly characterized. Her...

  14. A New Antigen Retrieval Technique for Human Brain Tissue

    PubMed Central

    Byne, William; Haroutunian, Vahram; García-Villanueva, Mercedes; Rábano, Alberto; García-Amado, María; Prensa, Lucía; Giménez-Amaya, José Manuel

    2008-01-01

    Immunohistochemical staining of tissues is a powerful tool used to delineate the presence or absence of an antigen. During the last 30 years, antigen visualization in human brain tissue has been significantly limited by the masking effect of fixatives. In the present study, we have used a new method for antigen retrieval in formalin-fixed human brain tissue and examined the effectiveness of this protocol to reveal masked antigens in tissues with both short and long formalin fixation times. This new method, which is based on the use of citraconic acid, has not been previously utilized in brain tissue although it has been employed in various other tissues such as tonsil, ovary, skin, lymph node, stomach, breast, colon, lung and thymus. Thus, we reported here a novel method to carry out immunohistochemical studies in free-floating human brain sections. Since fixation of brain tissue specimens in formaldehyde is a commonly method used in brain banks, this new antigen retrieval method could facilitate immunohistochemical studies of brains with prolonged formalin fixation times. PMID:18852880

  15. Antisera against leukaemia-associated antigens on human lymphocytes.

    PubMed Central

    Hsu, C C; Marti, G E; Mittal, K K

    1977-01-01

    Antisera were raised in rabbits against leukaemic lymphosarcoma (LSL) cells which carried surface markers of both thymus-derived T lymphocytes (T cells) and bone marrow-derived B lymphocytes (B cells). After absorption with leucocytes, erythrocytes and serum proteins from normal individuals, the antisera demonstrated significant complement-dependent cytotoxicity against leukaemic cells from patients with acute lymphoblastic leukaemia (ALL) (9/11), LSL (7/9) and chronic lymphocytic leukaemia (CLL) (9/12), with an antibody titre of 1:64 or greater. The antisera did not react with: (a) blood lymphocytes from clinically healthy individuals (0/23), patients with ono-lymphoproliferative disorders (0/8) and normal umbilical cords (0/3), (b) normal lymphocytes stimulated by pokeweed mitogen (0/7), allogeneic lymphocytes (0/3), fetuin (0/1), purified protein derivative (PPD) (0/2), and candida antigen (0/1); (C) normal marrow cells (0/3), (D) normal thymocytes (0/2) and (E) leukaemic cells from patients with acute myeloblastic (AML) (0/10) and chronic granulocytic leukaemia (CGL) (0/3). However, the antisera did react with lymphoblastoid cells from continuous B-cell lines derived from an AML patient and from a non-leukaemic individual and, to a lesser extent, with lymphocytes from patients with infectious mononucleosis. The antisera also reacted with lymphocytes from chronically infected tonsils. Cytotoxicity of the antisera against lymphoblastoid and tonsillar cells was inhibited by ALL and CLL cell-lysates; and, conversely, cytotoxicity against ALL cells was inhibited by the lymphoblastoid cell extract. In contrast, a cell lysate or extract from normal inhibited by the lymphoblastoid cell extract. In contrast, a cell lysate or extract from normal lymphocytes did not inhibit cytotoxicity toward lymphoblastoid, tonsillar or ALL cells. Cytotoxicity of the antisera was neutralized by a goat anti-rabbit IgG (GAR IgG). These results suggest that the antisera contained

  16. Synthesis of Antihemophilic Factor Antigen by Cultured Human Endothelial Cells

    PubMed Central

    Jaffe, Eric A.; Hoyer, Leon W.; Nachman, Ralph L.

    1973-01-01

    Antihemophilic factor (AHF, Factor VIII) antigen has been demonstrated in cultured human endothelial cells by immunofluorescence studies using monospecific rabbit antibody to human AHF. Control studies with cultured human smooth muscle cells and human fibroblasts were negative. By radioimmunoassay it was demonstrated that cultured human endothelial cells contain AHF antigen which is released into the culture medium. Cultured smooth muscle cells and fibroblasts did not have this property. Cultured endothelial cells incorporated radioactive amino acids into high molecular weight, AHF antigen-rich protein fractions prepared from the culture media, 7% of the radioactive amino acid counts incorporated into this material were precipitated by globulin prepared from rabbit anti-AHF whereas normal rabbit globulin precipitated only 1.5% of the counts. Although cultured endothelial cells actively synthesize AHF antigen, AHF procoagulant activity was not detected in the culture medium. Studies seeking a basis for the lack of procoagulant activity have not clarified this deficiency, but they have established that exogenous AHF procoagulant activity is not inactivated by the tissue culture system. Images PMID:4583980

  17. Antigen-specific human polyclonal antibodies from hyperimmunized cattle.

    PubMed

    Kuroiwa, Yoshimi; Kasinathan, Poothappillai; Sathiyaseelan, Thillainayagen; Jiao, Jin-an; Matsushita, Hiroaki; Sathiyaseelan, Janaki; Wu, Hua; Mellquist, Jenny; Hammitt, Melissa; Koster, Julie; Kamoda, Satoru; Tachibana, Katsumi; Ishida, Isao; Robl, James M

    2009-02-01

    Antigen-specific human polyclonal antibodies (hpAbs), produced by hyperimmunization, could be useful for treating many human diseases. However, yields from available transgenic mice and transchromosomic (Tc) cattle carrying human immunoglobulin loci are too low for therapeutic applications. We report a Tc bovine system that produces large yields of hpAbs. Tc cattle were generated by transferring a human artificial chromosome vector carrying the entire unrearranged, human immunoglobulin heavy (hIGH) and kappa-light (hIGK) chain loci to bovine fibroblasts in which two endogenous bovine IgH chain loci were inactivated. Plasma from the oldest animal contained >2 g/l of hIgG, paired with either human kappa-light chain (up to approximately 650 microg/ml, fully human) or with bovine kappa- or lambda-light chain (chimeric), with a normal hIgG subclass distribution. Hyperimmunization with anthrax protective antigen triggered a hIgG-mediated humoral immune response comprising a high proportion of antigen-specific hIgG. Purified, fully human and chimeric hIgGs were highly active in an in vitro toxin neutralization assay and protective in an in vivo mouse challenge assay.

  18. Leucocyte function in children with kwashiorkor.

    PubMed

    Rosen, E U; Geefhuysen, J; Anderson, R; Joffe, M; Rabson, A R

    1975-03-01

    A study of leucocyte response to infection, polymorphonuclear leucocyte chemotaxis and bactericidal activity, and nitroblue tetrazolium (NBT) reduction in children with kwashiorkor was undertaken and compared with a control group. The results show that total leucocyte counts were depressed in children with kwashiorkor, and lymphopenia was not infrequent. NBT reduction was normal. Abnormal polymorphonuclear leucocyte chemotaxis and bactericidal activity, though frequently found in children with kwashiorkor, was shown to be dependent on infection and not on protein depletion per se. Therefore, apart from some impairment of leucocyte mobilization in the presence of infection, the qualtiy of polymorphonuclear function, as determined by the above techniques, appears to be normal in kwashiorkor.

  19. Characterization of human sperm surface antigens with monoclonal antibodies.

    PubMed

    Wolf, D P; Sokoloski, J E; Dandekar, P; Bechtol, K B

    1983-10-01

    Monoclonal antibodies (McAb) against human ejaculated sperm were developed from mice immunized with sperm membrane preparations. A solid-phase radioimmunoassay, with dried sperm as antigen, was employed in McAb screening. The tissue and species specificity of monoclonal antibodies HS 2, 4 and 6 were evaluated after absorption of antibody preparations with heterologous sperm, human serum or seminal plasma or cells from other human organs. The sensitivity of HS 2, 4 and 6 antigens to trypsin exposure was determined: HS 4 antigen was highly sensitive while HS 2 and 6 were not. The regional distribution of McAb 4 on intact sperm cells was determined by immunofluorescence staining. HS 4 may be a sperm-coating antigen based on its presence on sperm and in seminal plasma. This possibility led to an investigation of its role in sperm capacitation. HS 4 antibody binding was reduced when capacitated sperm were compared with noncapacitated cells. HS 4 antibody, when present during capacitation and insemination, was without effect on sperm motility or fusion with zona-free hamster eggs. Trypsin removal of as much as 60% of HS 4 antigen from the cell population also did not impact on sperm function. To identify the molecular correlate of HS 4 antigen, membrane components were extracted from washed sperm with Nonidet P-40, concentrated by acetone precipitation and analyzed electrophoretically in SDS-urea on 10% polyacrylamide slab gels. Immunoassays on protein blots with peroxidase-coupled second antibody identified a single reactive species in the molecular weight range of 130,000. Multiple reactive components were detected in blot transfers of seminal plasma.

  20. Fya/Fyb antigen polymorphism in human erythrocyte Duffy antigen affects susceptibility to Plasmodium vivax malaria

    PubMed Central

    King, Christopher L.; Adams, John H.; Xianli, Jia; Grimberg, Brian T.; McHenry, Amy M.; Greenberg, Lior J.; Siddiqui, Asim; Howes, Rosalind E.; da Silva-Nunes, Monica; Ferreira, Marcelo U.; Zimmerman, Peter A.

    2011-01-01

    Plasmodium vivax (Pv) is a major cause of human malaria and is increasing in public health importance compared with falciparum malaria. Pv is unique among human malarias in that invasion of erythrocytes is almost solely dependent on the red cell's surface receptor, known as the Duffy blood-group antigen (Fy). Fy is an important minor blood-group antigen that has two immunologically distinct alleles, referred to as Fya or Fyb, resulting from a single-point mutation. This mutation occurs within the binding domain of the parasite's red cell invasion ligand. Whether this polymorphism affects susceptibility to clinical vivax malaria is unknown. Here we show that Fya, compared with Fyb, significantly diminishes binding of Pv Duffy binding protein (PvDBP) at the erythrocyte surface, and is associated with a reduced risk of clinical Pv in humans. Erythrocytes expressing Fya had 41–50% lower binding compared with Fyb cells and showed an increased ability of naturally occurring or artificially induced antibodies to block binding of PvDBP to their surface. Individuals with the Fya+b− phenotype demonstrated a 30–80% reduced risk of clinical vivax, but not falciparum malaria in a prospective cohort study in the Brazilian Amazon. The Fya+b− phenotype, predominant in Southeast Asian and many American populations, would confer a selective advantage against vivax malaria. Our results also suggest that efficacy of a PvDBP-based vaccine may differ among populations with different Fy phenotypes. PMID:22123959

  1. Identification of Goodpasture antigens in human alveolar basement membrane.

    PubMed Central

    Yoshioka, K; Iseki, T; Okada, M; Morimoto, Y; Eryu, N; Maki, S

    1988-01-01

    Goodpasture (GP) antigens, protein components reactive with human autoantibodies against glomerular basement membrane (GBM), were identified in human alveolar basement membrane (ABM) using an enzyme-linked immunoassay (ELISA), Western blotting and immunoprecipitation. All six anti-GBM antisera studied, three obtained from patients with glomerulonephritis and pulmonary haemorrhages (i.e. GP syndrome), and three from patients with glomerulonephritis alone, distinctively reacted with collagenase-digested (CD) ABM. Very cationic 22-28 kD and 40-48 kD components were detected by blot analysis combined with two-dimensional gel electrophoresis. These proteins showed some similarities to GP antigens in human GBM with respect to the monomer-dimer composition and charge distribution. Inhibition ELISA revealed that the binding of anti-GBM antisera to CDGBM decreased when they were pre-incubated with CDABM, suggesting that the anti-GBM antisera recognized the same epitope(s) on the GBM and ABM. Heterogeneity of the GP antigens in human ABM was demonstrated by blotting; monomeric antigens were absent or at low levels in the CDABM of three out of 10 normal individuals. In immunoprecipitation, anti-GBM antisera from patients with and without pulmonary haemorrhage showed different reactivities with CDABM. The former antisera precipitated both monomeric and dimeric components, but the latter did not. The observations of variation in monomer-dimer composition of ABM, and the different binding of anti-GBM antisera to it may explain why only some patients with anti-GBM nephritis have lung involvement. Images Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:2466590

  2. Characterization of membrane antigens on human cytomegalovirus-infected fibroblasts recognized by human antibodies

    SciTech Connect

    van der Voort, L.H.M.; de Leij, L.F.M.H.; The T.H.

    1989-03-01

    The antigens on the surface of human cytomegalovirus (HCMV)-infected fibroblasts which are recognized by human HCMV antibody-positive sera were characterized. Three HCMV-induced polypeptides, with apparent molecular masses of 53 to 63, 94, and 94 to 120 kilodaltons, were precipitated from /sup 125/I-surface-labeled cell extracts with different sera obtained from healthy individuals. Renal transplant recipients who were suffering from active HCMV infections recognized the same set of antigens. By the use of monoclonal antibodies, these antigens were identified as polypeptides belonging to the gcI and gcIII families of HCMV glycoproteins.

  3. Human Leukocyte Antigen Diversity: A Southern African Perspective.

    PubMed

    Tshabalala, Mqondisi; Mellet, Juanita; Pepper, Michael S

    2015-01-01

    Despite the increasingly well-documented evidence of high genetic, ethnic, and linguistic diversity amongst African populations, there is limited data on human leukocyte antigen (HLA) diversity in these populations. HLA is part of the host defense mechanism mediated through antigen presentation to effector cells of the immune system. With the high disease burden in southern Africa, HLA diversity data is increasingly important in the design of population-specific vaccines and the improvement of transplantation therapeutic interventions. This review highlights the paucity of HLA diversity data amongst southern African populations and defines a need for information of this kind. This information will support disease association studies, provide guidance in vaccine design, and improve transplantation outcomes. PMID:26347896

  4. Human Leukocyte Antigen Diversity: A Southern African Perspective

    PubMed Central

    Tshabalala, Mqondisi; Mellet, Juanita; Pepper, Michael S.

    2015-01-01

    Despite the increasingly well-documented evidence of high genetic, ethnic, and linguistic diversity amongst African populations, there is limited data on human leukocyte antigen (HLA) diversity in these populations. HLA is part of the host defense mechanism mediated through antigen presentation to effector cells of the immune system. With the high disease burden in southern Africa, HLA diversity data is increasingly important in the design of population-specific vaccines and the improvement of transplantation therapeutic interventions. This review highlights the paucity of HLA diversity data amongst southern African populations and defines a need for information of this kind. This information will support disease association studies, provide guidance in vaccine design, and improve transplantation outcomes. PMID:26347896

  5. Characterization of a human antigen specific helper factor

    SciTech Connect

    Richardson, B.

    1986-03-01

    While antigen (Ag) specific helper factors have been characterized in mice, similar molecules have not been identified in humans. To characterize human antigen specific helper molecules, an IL-2 dependent tetanus toxoid (T.T.) reactive T cell line was fused with a 6-thioguanine resistant CEM line, and hybrids selected in medium containing hypoxanthine and azaserine. Hybrids were screened by culturing the cells with /sup 35/S-Met then reacting the supernatants with T.T. or hepatitis vaccine immobilized on nitrocellulose. One hybrid, TT6BA-O, was identified which secreted a Met-containing molecule which bound T.T. but not hepatitis vaccine. Supernatants from TT6BA-O, but not the parent CEM line, when added to autologous peripheral blood mononuclear cells (PBMC's) stimulated secretion of T.T. specific antibodies (Abs). Specificity controls demonstrated that TT6BA-O supernatant did not induce antibodies to diphtheria toxoid, hepatitis vaccine or pneumococcal polysaccharide, and total immunoglobulin (lg) synthesis was minimally increased. In contrast, pokeweed mitogen stimulated significant lg synthesis as well as Ab's to pneumococcal polysaccharide and T.T. TT6BA-O supernatant induced anti-T.T.Ab's in autologous PBMC's but not PBMC's from 3 unrelated donors, suggesting that the activity of the helper factor is restricted, possibly by the MHC. The molecular weight of the helper factor was estimated at 100,000-150,000 by Sephacryl S-300 chromatography. Finally, the helper factor could be demonstrated to bind and elute from sephorose-immobilized T.T. and anti-DR antisera, but not anti-lg antisera or the T40/25 monoclonal antibody, which binds a nonpolymorphic determinant on the human T cell receptor. These results demonstrate that human Ag specific helper factors exist, bind antigen and bear class II MHC determinants.

  6. Natural micropolymorphism in human leukocyte antigens provides a basis for genetic control of antigen recognition

    SciTech Connect

    Archbold, Julia K.; Macdonald, Whitney A.; Gras, Stephanie; Ely, Lauren K.; Miles, John J.; Bell, Melissa J.; Brennan, Rebekah M.; Beddoe, Travis; Wilce, Matthew C.J.; Clements, Craig S.; Purcell, Anthony W.; McCluskey, James; Burrows, Scott R.; Rossjohn, Jamie

    2009-07-10

    Human leukocyte antigen (HLA) gene polymorphism plays a critical role in protective immunity, disease susceptibility, autoimmunity, and drug hypersensitivity, yet the basis of how HLA polymorphism influences T cell receptor (TCR) recognition is unclear. We examined how a natural micropolymorphism in HLA-B44, an important and large HLA allelic family, affected antigen recognition. T cell-mediated immunity to an Epstein-Barr virus determinant (EENLLDFVRF) is enhanced when HLA-B*4405 was the presenting allotype compared with HLA-B*4402 or HLA-B*4403, each of which differ by just one amino acid. The micropolymorphism in these HLA-B44 allotypes altered the mode of binding and dynamics of the bound viral epitope. The structure of the TCR-HLA-B*4405EENLLDFVRF complex revealed that peptide flexibility was a critical parameter in enabling preferential engagement with HLA-B*4405 in comparison to HLA-B*4402/03. Accordingly, major histocompatibility complex (MHC) polymorphism can alter the dynamics of the peptide-MHC landscape, resulting in fine-tuning of T cell responses between closely related allotypes.

  7. HLA-Modeler: Automated Homology Modeling of Human Leukocyte Antigens.

    PubMed

    Amari, Shinji; Kataoka, Ryoichi; Ikegami, Takashi; Hirayama, Noriaki

    2013-01-01

    The three-dimensional (3D) structures of human leukocyte antigen (HLA) molecules are indispensable for the studies on the functions at molecular level. We have developed a homology modeling system named HLA-modeler specialized in the HLA molecules. Segment matching algorithm is employed for modeling and the optimization of the model is carried out by use of the PFROSST force field considering the implicit solvent model. In order to efficiently construct the homology models, HLA-modeler uses a local database of the 3D structures of HLA molecules. The structure of the antigenic peptide-binding site is important for the function and the 3D structure is highly conserved between various alleles. HLA-modeler optimizes the use of this structural motif. The leave-one-out cross-validation using the crystal structures of class I and class II HLA molecules has demonstrated that the rmsds of nonhydrogen atoms of the sites between homology models and crystal structures are less than 1.0 Å in most cases. The results have indicated that the 3D structures of the antigenic peptide-binding sites can be reproduced by HLA-modeler at the level almost corresponding to the crystal structures.

  8. 76 FR 51374 - Direct Discovery of HLA Associated Influenza Epitopes Isolated From Human Cells for Vaccine and...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-08-18

    ... Isolated From Human Cells for Vaccine and Therapeutic Evaluation and Development (U01) AGENCY: Food and... processed and presented on soluble HLA (human leucocyte antigen) expressed by human cells. Initial studies... produced from influenza infected human lung cell lines. There is a growing interest in developing...

  9. Antibodies to Hepatitis B Surface Antigen Potentiate the Response of Human T Lymphocyte Clones to the Same Antigen

    NASA Astrophysics Data System (ADS)

    Celis, Esteban; Chang, Tse Wen

    1984-04-01

    Human T-helper lymphocyte clones specific for hepatitis B virus surface antigen (HBsAg) proliferate on stimulation with HBsAg in vitro. Antibodies specific for HBsAg, but no other antibodies, augment this proliferative response. In the presence of antibodies to HBsAg, the maximum response could be achieved at HBsAg concentrations that were 1 percent of those required in the absence of the antibodies. These findings suggest that antigen-specific antibodies exert regulatory controls on T cells that recognize the same antigens.

  10. Immunoanatomic distribution of cytostructural and tissue-associated antigens in the human urinary tract.

    PubMed Central

    Cordon-Cardo, C.; Finstad, C. L.; Bander, N. H.; Melamed, M. R.

    1987-01-01

    The main objective of the present study is to define the expression and/or modulation of antigenic phenotypes in cells of the normal human kidney and urothelium according to cell type. Fourteen antibodies detecting differentiation and structural antigens expressed in the human urinary tract have been used to define the immunoanatomic distribution of these antigenic systems. They include urinary tract antigens (Tamm-Horsfall glycoprotein and prostate-specific antigen), tissue-associated antigens (epithelial membrane antigen, Factor VIII antigen, and Protein S-100), and cytoskeletal antigens of the intermediate filament classes (cytokeratins, vimentin, desmin, glial fibrillary acidic protein, and neurofilaments. Immunofluorescence and immunoperoxidase analyses performed on normal human fetal and adult tissue sections have demonstrated that these antigens are expressed by different cell types and domains of the nephron. Studies correlating normal fetal and adult tissues reveal that some of the antigens appear at distinct stages of maturation, representing early and late antigenic expression events. These antibodies offer a wide range of potential applications that include studies of embryogenesis of the human urinary tract and immunopathologic analyses of neoplastic and nonneoplastic diseases of the human kidney and urothelium. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:3548401

  11. Differential stimulation by oxygen-free-radical-altered immunoglobulin G of the production of superoxide and hydrogen peroxide by human polymorphonuclear leucocytes.

    PubMed

    Kleinveld, H A; Sluiter, W; Boonman, A M; Swaak, A J; Hack, C E; Koster, J F

    1991-04-01

    1. The effect of free-radical-altered IgG (monomer and polymer u.v.-irradiated IgG), compared with that of native and heat-aggregated IgG, on the production rate of superoxide anion and hydrogen peroxide by granulocytes (polymorphonuclear leucocytes) from normal blood and granulocytes obtained from the blood and synovial fluid of patients with rheumatoid arthritis was studied. 2. Similar rates of superoxide production by granulocytes from normal blood at rest and in the presence of any form of IgG were found. In contrast, the rate of hydrogen peroxide production could be stimulated in a dose-dependent fashion by monomer or polymer u.v.-irradiated IgG. 3. The stimulatory effect of free-radical-altered IgG on the rate of hydrogen peroxide production did not occur in the presence of 2-deoxyglucose, which deprives the NADPH:O2 oxidoreductase of its substrate NADPH by inhibition of glycolysis and the pentose phosphate pathway. This points to a stimulatory effect on the direct divalent reduction of oxygen without intermediate superoxide production by this enzyme complex. 4. Granulocytes obtained from the blood and synovial fluid of patients with rheumatoid arthritis reacted differently to polymer u.v.-irradiated IgG. In the presence of this stimulus the rate of release of both superoxide and hydrogen peroxide was increased. Furthermore, these granulocytes synthesized superoxide and hydrogen peroxide at a higher rate than did granulocytes from normal blood in the presence of serum-treated zymosan but not in the presence of phorbol myristate acetate. 5. Taken together, these results indicate that the rate of superoxide and hydrogen peroxide production by the granulocyte NADPH:O2 oxidoreductase depends on the pathological condition of the donor and the type of stimulus used.

  12. Human leukocyte antigen-E alleles and expression in patients with serous ovarian cancer

    PubMed Central

    Zheng, Hui; Lu, Renquan; Xie, Suhong; Wen, Xuemei; Wang, Hongling; Gao, Xiang; Guo, Lin

    2015-01-01

    Human leukocyte antigen-E (HLA-E) is one of the most extensively studied non-classical MHC class I molecules that is almost non-polymorphic. Only two alleles (HLA-E*0101 and HLA-E*0103) are found in worldwide populations, and suggested to be functional differences between these variants. The HLA-E molecule can contribute to the escape of cancer cells from host immune surveillance. However, it is still unknown whether HLA-E gene polymorphisms might play a role in cancer immune escape. To explore the association between HLA-E alleles and the susceptibility to serous ovarian cancer (SOC), 85 primary SOC patients and 100 healthy women were enrolled. Here, we indicated that high frequency of HLA-E*0103 allele existed in SOC patients by the allele-specific quantitative real-time PCR method. The levels of HLA-E protein expression in SOC patients with the HLA-E*0103 allele were higher than those with the HLA-E*0101 allele using immunohistochemistry analysis. The cell surface expression and functional differences between the two alleles were verified by K562 cells transfected with HLA-E*0101 or HLA-E*0103 allelic heavy chains. The HLA-E*0103 allele made the transfer of the HLA-E molecule to the cell surface easier, and HLA-E/peptides complex more stable. These differences ultimately influenced the function of natural killer cells, showing that the cells transfected with HLA-E*0103 allele inhibited natural killer cells to lysis. This study reveals a novel mechanism regarding the susceptibility to SOC, which is correlated with the HLA-E*0103 allele. PMID:25711417

  13. Original antigenic sin with human bocaviruses 1-4.

    PubMed

    Li, Xuemeng; Kantola, Kalle; Hedman, Lea; Arku, Benedict; Hedman, Klaus; Söderlund-Venermo, Maria

    2015-10-01

    Human bocavirus (HBoV) 1 is a widespread parvovirus causing acute respiratory disease in young children. In contrast, HBoV2 occurs in the gastrointestinal tract and is potentially associated with gastroenteritis, whilst HBoV3 and -4 infections are less frequent and have not yet been linked with human disease. Due to HBoV1 DNA persistence in the nasopharynx, serology has been advocated as a better alternative for diagnosing acute infections. In constitutionally healthy children, we previously noted that pre-existing HBoV2 immunity in a subsequent HBoV1 infection typically resulted in low or non-existent HBoV1-specific antibody responses. A phenomenon describing such immunological events among related viruses has been known since the 1950s as 'original antigenic sin' (OAS). The aim of this study was to characterize this putative OAS phenomenon in a more controlled setting. Follow-up sera of 10 rabbit pairs, inoculated twice with HBoV1-4 virus-like particles (VLPs) or control antigens, in various combinations, were analysed with HBoV1-4 IgG enzyme immunoassays with and without depletion of heterotypic HBoV antibodies. There were no significant IgG boosts after the second inoculation in either the heterologously or the homologously HBoV-inoculated rabbits, but a clear increase in cross-reactivity was seen with time. We could, however, distinguish a distinct OAS pattern from plain cross-reactivity: half of the heterologously inoculated rabbits showed IgG patterns representative of the OAS hypothesis, in line with our prior results with naturally infected children. HBoVs are the first parvoviruses to show the possible existence of OAS. Our findings provide new information on HBoV1-4 immunity and emphasize the complexity of human bocavirus diagnosis. PMID:26224569

  14. Specificity of Heteroantisera to Human Acute Leukemia-Associated Antigens

    PubMed Central

    Baker, Michael A.; Ramachandar, K.; Taub, Robert N.

    1974-01-01

    Antisera have been raised to human leukemic blast cells from individual patients in mice rendered tolerant with cyclophosphamide to remission leukocytes from the same individual. 10 antisera were raised against acute myelogenous leukemia (AML) cells and 5 antisera were raised against acute lymphoblastic leukemia (ALL) cells. Antisera to AML cells were absorbed with ALL cells, and antisera to ALL cells were absorbed with AML cells. Unabsorbed and absorbed antisera as well as antisera raised in nontolerant mice were tested for cytotoxicity against various cells of a panel containing myeloblasts from 35 patients with AML, lymphoblasts from 7 patients with ALL, myeloblasts from 7 patients with chronic myelogenous leukemia (CML) in blast crisis, peripheral blood leukocytes from 12 patients with acute leukemia in remission and 30 nonleukemic patients, and nucleated bone marrow cells from 10 nonleukemic patients. Unabsorbed antisera to AML or ALL cells raised in tolerant mice were highly cytotoxic to leukemic blasts cells but significantly less cytotoxic to remission and control cells. Antisera to AML cells absorbed with ALL cells retained measurable cytotoxicity against AML cells but were not cytotoxic to ALL cells or control cells. Similarly, antisera to ALL cells absorbed with AML cells retained significant cytotoxicity only to ALL cells. Control antisera raised in nontolerant mice were cytotoxic to all cells tested. Although species specific, histocompatibility, differentiation, maturation, and cell cycle-associated antigens may be responsible in part for the cytotoxic activity of the unabsorbed antisera, the absorbed antisera are probably detecting antigens specific for their leukemic cell type. PMID:4140196

  15. Identification of monoclonal antibodies with specificity to alpha- or beta-chains of beta 2-integrins using peripheral blood leucocytes of normal and bovine leucocyte adhesion deficient (BLAD) cattle.

    PubMed

    Rutten, V P; Hoek, A; Müller, K E

    1996-08-01

    The reactivity of monoclonal antibodies entered in the panels of the Third Workshop on Ruminant Leucocyte Antigens with lymphocytes and monocytes of normal and beta 2-integrin deficient (Bovine Leucocyte Adhesion Deficiency: BLAD) animals was determined by flow cytometry to investigate potential specificities to alpha- or beta-chains of beta 2-integrins. From the 13 monoclonal antibodies that were entered as having specificity for CD11/CD18 antigens, ten were confirmed correct, but three had reactivity with cells from BLAD animals. We conclude that our approach provides an easy way to reliably identify the majority of beta 2-integrin specific monoclonal antibodies. PMID:8896223

  16. The antigenic similarity of human low density lipoproteins.

    PubMed

    LEVINE, L; KAUFFMAN, D L; BROWN, R K

    1955-08-01

    THE FOLLOWING HUMAN LOW DENSITY LIPOPROTEINS WERE PREPARED: beta-lipoproteins of densities greater than 1.040 (A, B,C) a beta-lipoprotein of -S(1.063) = 5 (D), a lipoprotein of -S(1.063) = 19 (E), and a lipoprotein of -S(1.063) = 70 (F). Data are presented which show the immunochemical homogeneity of the D lipoprotein rabbit-anti-D lipoprotein system. Cross-reactions between antibody to A and D lipoproteins and the above lipoproteins have been demonstrated by quantitative precipitation, quanitative complement fixation, and single and double diffusion in agar. The antigenic similarities appear to be associated with the protein portions of the molecule. The antisera produced did not differentiate the low density lipoprotein classes. PMID:13242737

  17. Thyrotropin Receptor Epitope and Human Leukocyte Antigen in Graves' Disease.

    PubMed

    Inaba, Hidefumi; De Groot, Leslie J; Akamizu, Takashi

    2016-01-01

    Graves' disease (GD) is an organ-specific autoimmune disease, and thyrotropin (TSH) receptor (TSHR) is a major autoantigen in this condition. Since the extracellular domain of human TSHR (TSHR-ECD) is shed into the circulation, TSHR-ECD is a preferentially immunogenic portion of TSHR. Both genetic factors and environmental factors contribute to development of GD. Inheritance of human leukocyte antigen (HLA) genes, especially HLA-DR3, is associated with GD. TSHR-ECD protein is endocytosed into antigen-presenting cells (APCs), and processed to TSHR-ECD peptides. These peptide epitopes bind to HLA-class II molecules, and subsequently the complex of HLA-class II and TSHR-ECD epitope is presented to CD4+ T cells. The activated CD4+ T cells secrete cytokines/chemokines that stimulate B-cells to produce TSAb, and in turn hyperthyroidism occurs. Numerous studies have been done to identify T- and B-cell epitopes in TSHR-ECD, including (1) in silico, (2) in vitro, (3) in vivo, and (4) clinical experiments. Murine models of GD and HLA-transgenic mice have played a pivotal role in elucidating the immunological mechanisms. To date, linear or conformational epitopes of TSHR-ECD, as well as the molecular structure of the epitope-binding groove in HLA-DR, were reported to be related to the pathogenesis in GD. Dysfunction of central tolerance in the thymus, or in peripheral tolerance, such as regulatory T cells, could allow development of GD. Novel treatments using TSHR antagonists or mutated TSHR peptides have been reported to be effective. We review and update the role of immunogenic TSHR epitopes and HLA in GD, and offer perspectives on TSHR epitope specific treatments. PMID:27602020

  18. Thyrotropin Receptor Epitope and Human Leukocyte Antigen in Graves’ Disease

    PubMed Central

    Inaba, Hidefumi; De Groot, Leslie J.; Akamizu, Takashi

    2016-01-01

    Graves’ disease (GD) is an organ-specific autoimmune disease, and thyrotropin (TSH) receptor (TSHR) is a major autoantigen in this condition. Since the extracellular domain of human TSHR (TSHR-ECD) is shed into the circulation, TSHR-ECD is a preferentially immunogenic portion of TSHR. Both genetic factors and environmental factors contribute to development of GD. Inheritance of human leukocyte antigen (HLA) genes, especially HLA-DR3, is associated with GD. TSHR-ECD protein is endocytosed into antigen-presenting cells (APCs), and processed to TSHR-ECD peptides. These peptide epitopes bind to HLA-class II molecules, and subsequently the complex of HLA-class II and TSHR-ECD epitope is presented to CD4+ T cells. The activated CD4+ T cells secrete cytokines/chemokines that stimulate B-cells to produce TSAb, and in turn hyperthyroidism occurs. Numerous studies have been done to identify T- and B-cell epitopes in TSHR-ECD, including (1) in silico, (2) in vitro, (3) in vivo, and (4) clinical experiments. Murine models of GD and HLA-transgenic mice have played a pivotal role in elucidating the immunological mechanisms. To date, linear or conformational epitopes of TSHR-ECD, as well as the molecular structure of the epitope-binding groove in HLA-DR, were reported to be related to the pathogenesis in GD. Dysfunction of central tolerance in the thymus, or in peripheral tolerance, such as regulatory T cells, could allow development of GD. Novel treatments using TSHR antagonists or mutated TSHR peptides have been reported to be effective. We review and update the role of immunogenic TSHR epitopes and HLA in GD, and offer perspectives on TSHR epitope specific treatments.

  19. Thyrotropin Receptor Epitope and Human Leukocyte Antigen in Graves’ Disease

    PubMed Central

    Inaba, Hidefumi; De Groot, Leslie J.; Akamizu, Takashi

    2016-01-01

    Graves’ disease (GD) is an organ-specific autoimmune disease, and thyrotropin (TSH) receptor (TSHR) is a major autoantigen in this condition. Since the extracellular domain of human TSHR (TSHR-ECD) is shed into the circulation, TSHR-ECD is a preferentially immunogenic portion of TSHR. Both genetic factors and environmental factors contribute to development of GD. Inheritance of human leukocyte antigen (HLA) genes, especially HLA-DR3, is associated with GD. TSHR-ECD protein is endocytosed into antigen-presenting cells (APCs), and processed to TSHR-ECD peptides. These peptide epitopes bind to HLA-class II molecules, and subsequently the complex of HLA-class II and TSHR-ECD epitope is presented to CD4+ T cells. The activated CD4+ T cells secrete cytokines/chemokines that stimulate B-cells to produce TSAb, and in turn hyperthyroidism occurs. Numerous studies have been done to identify T- and B-cell epitopes in TSHR-ECD, including (1) in silico, (2) in vitro, (3) in vivo, and (4) clinical experiments. Murine models of GD and HLA-transgenic mice have played a pivotal role in elucidating the immunological mechanisms. To date, linear or conformational epitopes of TSHR-ECD, as well as the molecular structure of the epitope-binding groove in HLA-DR, were reported to be related to the pathogenesis in GD. Dysfunction of central tolerance in the thymus, or in peripheral tolerance, such as regulatory T cells, could allow development of GD. Novel treatments using TSHR antagonists or mutated TSHR peptides have been reported to be effective. We review and update the role of immunogenic TSHR epitopes and HLA in GD, and offer perspectives on TSHR epitope specific treatments. PMID:27602020

  20. Mapping Antigenic Epitopes on the Human Bocavirus Capsid

    PubMed Central

    Kailasan, Shweta; Garrison, Jamie; Ilyas, Maria; Chipman, Paul; McKenna, Robert; Kantola, Kalle; Söderlund-Venermo, Maria; Kučinskaitė-Kodzė, Indrė; Žvirblienė, Aurelija

    2016-01-01

    ABSTRACT Human bocaviruses (HBoV1 to -4) are emerging pathogens associated with pneumonia and/or diarrhea in young children. Currently, there is no treatment or vaccination, so there is a need to study these pathogens to understand their disease mechanisms on a molecular and structural level for the development of control strategies. Here, we report the structures of six HBoV monoclonal antibody (MAb) fragment complexes, HBoV1-15C6, HBoV2-15C6, HBoV4-15C6, HBoV1-4C2, HBoV1-9G12, and HBoV1-12C1, determined by cryo-electron microscopy and three-dimensional image reconstruction to 18.0- to 8.5-Å resolution. Of these, the 15C6 MAb cross-reacted with HBoV1, HBoV2, and HBoV4, while the 4C2, 12C1, and 9G12 MAbs recognized only HBoV1. Pseudoatomic modeling mapped the 15C6 footprint to the capsid surface DE and HI loops, at the 5-fold axis and the depression surrounding it, respectively, which are conserved motifs in Parvoviridae. The footprints for 4C2, 12C1, and 9G12 span the surface loops that assemble portions of the 2-/5-fold wall (a raised surface feature between the 2-fold and 5-fold axes of symmetry) and the shoulder of the 3-fold protrusions. The MAb footprints, cross reactive and strain specific, coincide with regions with high and low sequence/structural identities, respectively, on the capsid surfaces of the HBoVs and identify potential regions for the development of peptide vaccines for these viruses. IMPORTANCE Human bocaviruses (HBoVs) may cause severe respiratory and gastrointestinal infections in young children. The nonenveloped parvovirus capsid carries determinants of host and tissue tropism, pathogenicity, genome packaging, assembly, and antigenicity important for virus infection. This information is currently unavailable for the HBoVs and other bocaparvoviruses. This study identifies three strain-specific antigenic epitopes on the HBoV1 capsid and a cross-reactive epitope on the HBoV1, HBoV2, and HBoV4 capsids using structures of capsid

  1. A recombinant vaccinia virus expressing human carcinoembryonic antigen (CEA).

    PubMed

    Kaufman, H; Schlom, J; Kantor, J

    1991-07-30

    Carcinoembryonic antigen (CEA) is a 180-kDa glycoprotein expressed on most gastrointestinal carcinomas. A 2.4-kb cDNA clone, containing the complete coding sequence, was isolated from a human colon tumor cell library and inserted into a vaccinia virus genome. This newly developed construct was characterized by Southern blotting, DNA hybridization studies, and polymerase chain reaction analysis. The CEA gene was stably integrated into the vaccinia virus thymidine kinase gene. The recombinant was efficiently replicated upon serial passages in cell cultures and in animals. The recombinant virus expresses on the surface of infected cells a protein product recognized by a monoclonal antibody (COL-I) directed against CEA. Immunization of mice with the vaccinia construct elicited a humoral immune response against CEA. Pilot studies also showed that administration of the recombinant CEA vaccinia construct was able to greatly reduce the growth in mice of a syngeneic murine colon adenocarcinoma which had been transduced with the human CEA gene. The use of this new recombinant CEA vaccinia construct may thus provide an approach in the specific active immunotherapy of human GI cancer and other CEA expressing carcinoma types.

  2. Human erythrocyte antigens. Regulation of expression of a novel erythrocyte surface antigen by the inhibitor Lutheran In(Lu) gene.

    PubMed Central

    Telen, M J; Eisenbarth, G S; Haynes, B F

    1983-01-01

    Our study describes a novel human erythrocyte protein antigen, the expression of which is regulated by the rare Lutheran inhibitor In(Lu) gene. We have produced a monoclonal antibody (A3D8) that bound strongly to erythrocytes from subjects with Lutheran phenotypes Lu(a+b+), Lu(a+b-), and Lu(a-b+) but bound negligibly to erythrocytes from subjects with the dominant form of Lu(a-b-) phenotype, reflecting inheritance of the In(Lu) gene. Importantly, erythrocytes from an individual with the recessive form of Lu(a-b-) phenotype (i.e., absence of the In(Lu) gene and absence of genes encoding for Lutheran antigens) showed reactivity with A3D8 antibody comparable to that seen with Lu(a+) or Lu(b+) erythrocytes. A3D8 antigen activity was also found on all leukocytes and in serum and plasma; this activity also appeared to be regulated by the In(Lu) gene in serum, plasma, and on a subset of leukocytes. Thus, we have identified a human erythrocyte protein whose expression is modified by the In(Lu) gene. This knowledge that such an antigen exists on erythrocytes and in normal plasma should allow further studies into the molecular genetics of the In(Lu) gene and into the functional and structural significance of the A3D8 antigen. PMID:6863545

  3. Increased CD11/CD18 expression on the peripheral blood leucocytes of patients with HIV disease: relationship to disease severity.

    PubMed Central

    Palmer, S; Hamblin, A S

    1993-01-01

    In HIV disease increased adhesion between leucocytes themselves and between leucocytes and endothelium may contribute to cell loss and viral spread. Using a novel method for the preparation of blood leucocytes for flow cytometry, we report increased expression of leucocyte adhesion molecules (LeuCAMs) (CD11/CD18) on peripheral blood leucocytes of patients with HIV disease compared with normal controls. Patients were divided into two groups on the basis of CD4 T lymphocyte numbers (those with > 0.5 x 10(9)/l and those with < 0.2 x 10(9)/l), and assessed for p24 antigen expression, viral load and serum tumour necrosis factor (TNF) levels as well as LeuCAM expression. Patients with < 0.2 x 10(9)/lCD4 cells had more p24 antigen and more HIV infectious virus and more serum TNF than those with > 0.5 x 10(9)/l. Whilst the percentages of only monocytes and polymorphs expressing CD11b were significantly increased in patients with the least CD4 cells, the density of LeuCAMs, expressed as mean fluorescence intensity (MFI), was significantly increased on all leucocytes, with the most significant increases being seen on patients with the fewest CD4 T cells. Our findings are consistent with leucocyte activation by a soluble factor, although we could find no correlation between levels of TNF and LeuCAM expression. The increased expression of adhesion molecules on peripheral blood leucocytes could play a role in the cellular extravasation and aggregation seen in HIV disease. PMID:8103716

  4. Human melanoma immunotherapy using tumor antigen-specific T cells generated in humanized mice

    PubMed Central

    Hu, Zheng; Xia, Jinxing; Fan, Wei; Wargo, Jennifer; Yang, Yong-Guang

    2016-01-01

    A major factor hindering the exploration of adoptive immunotherapy in preclinical settings is the limited availability of tumor-reactive human T cells. Here we developed a humanized mouse model that permits large-scale production of human T cells expressing the engineered melanoma antigen MART-1-specific TCR. Humanized mice, made by transplantation of human fetal thymic tissue and CD34+ cells virally-transduced with HLA class I-restricted melanoma antigen (MART-1)-specific TCR gene, showed efficient development of MART-1-TCR+ human T cells with predominantly CD8+ cells. Importantly, MART-1-TCR+CD8+ T cells developing in these mice were capable of mounting antigen-specific responses in vivo, as evidenced by their proliferation, phenotypic conversion and IFN-γ production following MART-1 peptide immunization. Moreover, these MART-1-TCR+CD8+ T cells mediated efficient killing of melanoma cells in an HLA/antigen-dependent manner. Adoptive transfer of in vitro expanded MART-1-TCR+CD8+ T cells induced potent antitumor responses that were further enhanced by IL-15 treatment in melanoma-bearing recipients. Finally, a short incubation of MART-1-specific T cells with rapamycin acted synergistically with IL-15, leading to significantly improved tumor-free survival in recipients with metastatic melanoma. These data demonstrate the practicality of using humanized mice to produce potentially unlimited source of tumor-specific human T cells for experimental and preclinical exploration of cancer immunotherapy. This study also suggests that pretreatment of tumor-reactive T cells with rapamycin in combination with IL-15 administration may be a novel strategy to improve the efficacy of adoptive T cell therapy. PMID:26824989

  5. Monoclonal antibody-defined human pancreatic cancer-associated antigens.

    PubMed

    Schmiegel, W H; Kalthoff, H; Arndt, R; Gieseking, J; Greten, H; Klöppel, G; Kreiker, C; Ladak, A; Lampe, V; Ulrich, S

    1985-03-01

    Three pancreatic cancer-associated antigens were characterized by use of monoclonal antibodies in immunobinding studies with various cellular and soluble target antigens, in immunoprecipitation, and in immunoperoxidase staining. C54-0 represents a tumor-associated Mr 122,000 antigen, which appears to be widely distributed on various epithelial tumors and to a lower extent on normal tissue. C1-N3 antigen exhibited a more restricted distribution, reacting with pancreatic and various gastrointestinal tract tumors as well as with chronically inflamed pancreatic tissue. The most specific antigen expression was observed for C1-P83 antigen, found on all exocrine tumors of the pancreas, but not on normal or chronically inflamed pancreatic tissue.

  6. Toxoplasma gondii recombinant antigens as tools for serodiagnosis of human toxoplasmosis: current status of studies.

    PubMed

    Holec-Gasior, Lucyna

    2013-09-01

    Toxoplasma gondii is a parasitic protozoan which is the cause of toxoplasmosis. Although human toxoplasmosis in healthy adults is usually asymptomatic, serious disease can occur in the case of congenital infections and immunocompromised individuals. Furthermore, despite the exact recognition of its etiology, it still presents a diagnostic problem. Diagnosis of toxoplasmosis is mainly based on the results of serological tests detecting anti-T. gondii-specific antibodies in the patient's serum sample. The specificities and sensitivities of serology tests depend mostly on the diagnostic antigen(s) used. Most of the commercial serological kits currently available are based on Toxoplasma lysate antigens (TLAs). In recent years, many studies showed that recombinant antigenic proteins of T. gondii may be an alternative source of antigens which are very useful for the serodiagnosis of toxoplasmosis. This article presents a review of current studies on the application and usefulness of different T. gondii recombinant antigens in serological tests for the diagnosis of human toxoplasmosis.

  7. Human platelet antigen genotyping of platelet donors in southern Brazil.

    PubMed

    Merzoni, J; Fagundes, I S; Lunardi, L W; Lindenau, J D-R; Gil, B C; Jobim, M; Dias, V G; Merzoni, L; Sekine, L; Onsten, T G H; Jobim, L F

    2015-10-01

    Human platelet antigens (HPA) are immunogenic structures that result from single nucleotide polymorphisms (SNPs) leading to single amino acid substitutions. This study sought to determine the allele and genotype frequencies of HPA-1, HPA-2, HPA-3, HPA-4, HPA-5 and HPA-15 in platelet donors from the state of Rio Grande do Sul (RS), Brazil, and compare their allele frequencies to those observed in other populations. HPA genotyping was performed by PCR-SSP method. The study sample comprised 201 platelet donors (167 Caucasians and 34 non-Caucasians). Allele 'a' was that most commonly found for HPA-1 to 5 in both groups. The HPA-15ab genotype predominated over homozygous genotypes of this system. Fisher's exact test revealed statistically significant differences for the HPA-5 system, with a greater prevalence of the HPA-5b allele in non-Caucasians. The neighbour-joining method and principal components analysis revealed genetic proximity between our Caucasian group and European populations. We conclude that the allele frequencies of HPA-1 to 5 and HPA-15 found in our Caucasian sample are similar to those reported for European populations. These findings corroborate the ethnic makeup of the population of RS. The higher frequency of the HPA-5b allele found in the non-Caucasian group of our sample suggests the possibility of allosensitization in patients who receive platelet transfusions from genetically incompatible donors.

  8. Association of human leukocyte antigen with postherpetic neuralgia in Koreans.

    PubMed

    Chung, Hye Yoon; Song, Eun Young; Yoon, Jung Ah; Suh, Dae Hun; Lee, Sang Chul; Kim, Yong Chul; Park, Myoung Hee

    2016-10-01

    Postherpetic neuralgia (PHN), the most frequent complication of varicella-zoster virus reactivation, is characterized by pain that persists for more than 3 months, often for years after healing of zoster rash. A few studies revealing the association of human leukocyte antigen (HLA) with PHN have been reported, but only in the Japanese. The aim of this study was to investigate the primary HLA locus associated with PHN susceptibility in Koreans. We compared HLA-A, -B, -C, and DRB1 genotypes of 66 PHN patients with those of 54 herpes zoster (HZ) patients without developing PHN and 235 healthy controls. Frequencies of HLA-B*13, B*44, B*15 (B75), DRB1*10:01, and DRB1*12:02 were increased, and those of HLA-C*01, C*12, and DRB1*01:01 were decreased in PHN patients compared to those in controls (each, p < 0.05). Among these alleles, only the frequency of HLA-B*44 was significantly increased in PHN patients compared to that in HZ patients and the change was due to HLA-B*44:03 (PHN vs controls, p = 0.043; PHN vs HZ, p = 0.012). The results suggest that HLA-B*44:03 or other closely linked gene of the major histocompatibility complex is associated with susceptibility to the development of PHN after HZ, but not with the onset of HZ. PMID:27457498

  9. Wild-type human p53 transactivates the human proliferating cell nuclear antigen promoter

    SciTech Connect

    Shivakumar, C.V.; Brown, D.R.; Deb, S.; Deb, S.P.

    1995-12-01

    The p53 tumor suppressor protein negatively regulates cell growth and somatic mutations in the p53 gene lead to uncontrolled cell growth and oncogenesis. This report describes research which demonstrates, using a number of different cell lines, that at low levels, wild-type p53 transactivates the human proliferating cell nuclear antigen (PCNA) promoter. When expressed at similar levels, tumor-derived p53 mutants did not transactivate the PCNA promoter. It also reports the identification of a wild-type human p53-binding site on the human PCNA promote. 84 refs., 5 figs, 3 tabs.

  10. Antigen exposure shapes the ratio between antigen-specific Tregs and conventional T cells in human peripheral blood

    PubMed Central

    Su, Laura F.; del Alcazar, Daniel; Stelekati, Erietta; Wherry, E. John; Davis, Mark M.

    2016-01-01

    The T-cell receptor (TCR) is required for maturation and function of regulatory T cells (Tregs), but the ligand specificities of Tregs outside the context of transgenic TCRs are largely unknown. Using peptide–MHC tetramers, we isolated rare specific Foxp3+ cells directly ex vivo from adult peripheral blood and defined their frequency and phenotype. We find that a proportion of circulating Tregs recognize foreign antigens and the frequency of these cells are similar to that of self-reactive Tregs in the absence of cognate infection. In contrast, the frequencies of Tregs that recognize some common microbial antigens are significantly reduced in the blood of most adults. Exposure to peripheral antigens likely has a major influence on the balance between Tregs and conventional T-cell subsets because a larger proportion of flu-specific T cells has a regulatory cell phenotype in the cord blood. Consistent with this finding, we show that lymphocytic choriomeningitis virus infection can directly modulate the ratio of virus-specific effectors and Tregs in mice. The resulting change in the balance within an antigen-specific T-cell population further correlates with the magnitude of effector response and the chronicity of infection. Taken together, our data highlight the importance of antigen specificity in the functional dynamics of the T-cell repertoire. Each specific population of CD4+ T cells in human peripheral blood contains a subset of Tregs at birth, but the balance between regulatory and effector subsets changes in response to peripheral antigen exposure and this could impact the robustness of antipathogen immunity. PMID:27681619

  11. Monoclonal antibody recognizing human melanoma-carcinoma cross-reacting oncofetal antigen epitopically associated with carcinoembryonic antigen.

    PubMed

    Liao, S K; Kwong, P C; Clarke, B J; Dent, P B; Ryan, E D; Khosravi, M J; Laferte, S; Krantz, M J

    1985-05-01

    By fusion of mouse NS1 myeloma cells with splenocytes from a BALB/c mouse immunized with human melanoma cells, an IgG1 monoclonal antibody, designated as 140.72, was produced. By the mixed hemadsorption antibody binding assay, 140.72 was shown to react with 17 of 20 melanoma cell lines and with 5 of 14 carcinoma cell lines. This antibody also reacted with 3 of 3 normal melanocyte cultures in much lower titers. It did not react with any of 35 other normal and malignant lines, including neuroblastoma, glioblastoma, sarcoma, teratoma, fibroblast, and lymphoid cell lines. Absorption with fresh melanoma and carcinoma homogenates confirmed the results of direct tests. Fetal reactivity of antibody 140.72 was determined by positive absorption with 10 of 11 tissue homogenates derived from different fetuses of 10-16 weeks' gestation. The reactivity of this antibody was completely removed by absorption with a highly purified preparation of carcinoembryonic antigen (CEA) derived from a colon carcinoma. The antigenic activity was detected in the culture medium of reactive cell lines. Immunoprecipitation analyses of melanoma and carcinoma cells indicated that the antigenic determinant recognized by antibody 140.72 is on a glycoprotein with an apparent molecular weight of 95,000-150,000 common to both serologically reactive cell types. Additionally, a 200,000-molecular-weight glycoprotein corresponding to the CEA molecule was detected only on the reactive carcinoma cells. These data confirmed previous findings obtained with polyclonal anti-CEA antisera for the existence of shared CEA-related antigenic determinants on human carcinomas and melanomas and provided additional molecular characterization of these glycoproteins. Further characterization of the molecules bearing the antigenic determinant recognized by antibody 140.72 should be performed with a view to exploring its potential in the immunodiagnosis and immunotherapy of patients with melanoma.

  12. Plasmodium vivax GPI-anchored micronemal antigen (PvGAMA) binds human erythrocytes independent of Duffy antigen status

    PubMed Central

    Cheng, Yang; Lu, Feng; Wang, Bo; Li, Jian; Han, Jin-Hee; Ito, Daisuke; Kong, Deok-Hoon; Jiang, Lubin; Wu, Jian; Ha, Kwon-Soo; Takashima, Eizo; Sattabongkot, Jetsumon; Cao, Jun; Nyunt, Myat Htut; Kyaw, Myat Phone; Desai, Sanjay A.; Miller, Louis H.; Tsuboi, Takafumi; Han, Eun-Taek

    2016-01-01

    Plasmodium vivax, a major agent of malaria in both temperate and tropical climates, has been thought to be unable to infect humans lacking the Duffy (Fy) blood group antigen because this receptor is critical for erythrocyte invasion. Recent surveys in various endemic regions, however, have reported P. vivax infections in Duffy-negative individuals, suggesting that the parasite may utilize alternative receptor-ligand pairs to complete the erythrocyte invasion. Here, we identified and characterized a novel parasite ligand, Plasmodium vivax GPI-anchored micronemal antigen (PvGAMA), that bound human erythrocytes regardless of Duffy antigen status. PvGAMA was localized at the microneme in the mature schizont-stage parasites. The antibodies against PvGAMA fragments inhibited PvGAMA binding to erythrocytes in a dose-dependent manner. The erythrocyte-specific binding activities of PvGAMA were significantly reduced by chymotrypsin treatment. Thus, PvGAMA may be an adhesion molecule for the invasion of Duffy-positive and -negative human erythrocytes. PMID:27759110

  13. Human Noroviruses' Fondness for Histo-Blood Group Antigens

    PubMed Central

    Singh, Bishal K.; Leuthold, Mila M.

    2014-01-01

    ABSTRACT Human noroviruses are the dominant cause of outbreaks of gastroenteritis around the world. Human noroviruses interact with the polymorphic human histo-blood group antigens (HBGAs), and this interaction is thought to be important for infection. Indeed, synthetic HBGAs or HBGA-expressing enteric bacteria were shown to enhance norovirus infection in B cells. A number of studies have found a possible relationship between HBGA type and norovirus susceptibility. The genogroup II, genotype 4 (GII.4) noroviruses are the dominant cluster, evolve every other year, and are thought to modify their binding interactions with different HBGA types. Here we show high-resolution X-ray crystal structures of the capsid protruding (P) domains from epidemic GII.4 variants from 2004, 2006, and 2012, cocrystallized with a panel of HBGA types (H type 2, Lewis Y, Lewis B, Lewis A, Lewis X, A type, and B type). Many of the HBGA binding interactions were found to be complex, involving capsid loop movements, alternative HBGA conformations, and HBGA rotations. We showed that a loop (residues 391 to 395) was elegantly repositioned to allow for Lewis Y binding. This loop was also slightly shifted to provide direct hydrogen- and water-mediated bonds with Lewis B. We considered that the flexible loop modulated Lewis HBGA binding. The GII.4 noroviruses have dominated outbreaks over the past decade, which may be explained by their exquisite HBGA binding mechanisms, their fondness for Lewis HBGAs, and their temporal amino acid modifications. IMPORTANCE Our data provide a comprehensive picture of GII.4 P domain and HBGA binding interactions. The exceptionally high resolutions of our X-ray crystal structures allowed us to accurately recognize novel GII.4 P domain interactions with numerous HBGA types. We showed that the GII.4 P domain-HBGA interactions involved complex binding mechanisms that were not previously observed in norovirus structural studies. Many of the GII.4 P domain

  14. Sandwich enzyme-linked immunosorbent assay for detection of excretory secretory antigens in humans with fascioliasis.

    PubMed Central

    Espino, A M; Finlay, C M

    1994-01-01

    A sandwich enzyme-linked immunosorbent assay has been developed for the detection of Fasciola hepatica excretory secretory (ES) antigens in stool specimens of infected humans. The assay uses antibodies against F. hepatica ES antigens. A monoclonal antibody (ES78, mouse immunoglobulin G2a) was used to capture ES antigens, and a rabbit polyclonal antibody, peroxidase conjugate, was used to identify ES antigens. Thirteen of 14 patients with parasitological evidence of fascioliasis had a detectable concentration of ES antigens (more than 15 ng/ml). None of the stool specimens from controls and from patients with parasites other than F. hepatica showed a positive reaction, suggesting the absence of cross-reactions in this assay. When the 14 patients were retested 2 months after treatment, all of the specimens from the 11 parasitologically cured patients were negative by the antigen detection assay while the specimens from the 3 patients with persisting F. hepatica eggs in their stools remained positive. PMID:8126178

  15. A novel tuberculosis antigen identified from human tuberculosis granulomas.

    PubMed

    Yu, Yang; Jin, Dongdong; Hu, Shizong; Zhang, Yan; Zheng, Xiaojing; Zheng, Jianhua; Liao, Mingfeng; Chen, Xinchun; Graner, Michael; Liu, Haiying; Jin, Qi

    2015-04-01

    Tuberculosis is a global infectious disease caused by Mycobacterium tuberculosis (Mtb). Although novel Mtb biomarkers from both the pathogen and host have been studied, more breakthroughs are still needed to meet different clinic requirements. In an effort to identify Mtb antigens, chaperone-peptide complexes were purified from TB infected lungs using free-solution isoelectric focusing combined with high resolution LTQ Orbitrap Velos mass spectrometry. Antigen specific cellular immune responses in vitro were then examined. Those efforts led to the identification of six Mtb peptides only identified in Tuberculosis lung samples and that were not found in the control samples. Additionally, antigen-specific IFN-γ secretion, T-cell proliferation, cytokine expression, and a cytotoxic assay were also evaluated. Among the peptides isolated, we identified a 34 amino acid peptide named PKAp belonging to a serine/threonine-protein kinase, as being able to generate Mtb-specific cellular immune responses as noted by elevated antigen-specific cytokine secretion levels, increased CD8(+) T-cell proliferation and a strong cytotoxic lymphocyte (CTL) response. Moreover, the immune stimulating abilities of PKAp were further validated in vivo, with target peptide immunized mice showing an increased cellular IFN-γ in both the lungs and spleen without causing immunopathogenesis. In conclusion, we identified novel functional Mtb antigens directly from the granulomatous lesions of Tuberculosis patients, inducing not only significant antigen-specific IFN-γ secretion but also a marked cytotoxic lymphocyte functional response. These findings indicated that PKAp has potential as a novel antigen biomarker for vaccine development. PMID:25605460

  16. Lymphoblastoid cell supernatants increase expression of C3b receptors on human polymorphonuclear leucocytes: direct binding studies with 125I-C3b.

    PubMed Central

    Berger, M; Cross, A S

    1984-01-01

    Human PMN incubated in culture supernatants of the Raji long-term human lymphoblastoid cell line showed increased rosette formation with sheep erythrocytes coated with C3b (EIgM C4b3b) but no change in rosette formation with IgG-coated erythrocytes. This suggested a specific increase in cell surface C3b receptors, which was further investigated using 125I-C3b for direct binding studies. The results confirmed that specific binding of 125I-C3b to PMN incubated in culture supernatants increased up to three- to four-fold over binding to PMN incubated in control media alone. Scatchard analysis revealed that the apparent Ka for supernatant-treated cells, 3.36 +/- 0.89 X 10(7) L/M did not differ from the Ka for cells incubated in control media, 3.76 +/- 0.75 X 10(7) L/M, suggesting an increase in a single class of C3b receptors. Kinetic studies revealed that the active factor was present within 24 hr of culture of the Raji cells, and that neutrophils incubated in culture supernatants increased their C3b receptors continuously for up to 4 hr, the longest interval tested. The effect of the culture supernatant was lost with dilution beyond eight- to 10-fold. The results suggest that culture supernatants of this long-term lymphoblastoid cell line contain soluble factors that induce increased expression of C3b receptors on PMN and may thus serve as a model for study of important physiologic effects of lymphocyte products on PMN in vivo. PMID:6230308

  17. The role of a human antigen specific T8+ cell subset in antigen presentation, helper function and contrasuppression.

    PubMed Central

    Lehner, T; Avery, J; Jones, T

    1985-01-01

    Regulation of the human immune response was studied by sequential separation of subsets of T cells, followed by assessment of their helper and suppressor functions in a series of reconstitution experiments. T8+ lymphocytes were separated by panning on streptococcal antigen (SA) coated plates into T8+ SA-adherent cells (T8+SA+) and T8+ SA-non-adherent cells (T8+SA-). The helper and suppressor functions of the T8+SA+ and T8+SA- cells, reconstituted with T4+ helper cells were then studied by a direct antibody forming cell assay. T4+ cells will not induce helper activity by 1000 ng SA alone but require the accessory function of monocytes (Mo). However, replacing Mo by T8+SA+ cells will elicit a similar helper activity by T4+ cells and SA as that induced by Mo. In addition to the antigen-specific presentation and induction of helper activity, the T8+SA+ subset displays the properties of antigen-specific contrasuppressor cells. Thus, reconstitution of T4+ cells and T8+SA- (suppressor cells) with T8+SA+ and 1000 ng SA induces helper and no suppressor activity. Substitution of Mo for the T8+SA+ cells converts the helper to a predominantly suppressor-cell function. T8+SA- cells elicit suppression with 1 ng SA in the absence of accessory cells and reconstitution with Mo, T8+SA+ or T4+ cells failed to affect the suppressor activity. Total reconstitution of the four principle subsets of T4+, T8+SA+, T8+SA- cells and Mo elicited similar antigen dose-dependent responses as those of the unseparated mononuclear cells. It seems that all four cell subsets are required for optimal immunoregulation. We suggest that the T8+SA+ can present antigen to T4+ helper cells and induce helper activity, but in addition these cells can prevent the suppressor subset of T8+ cells from inhibiting T4+ helper cells and function as contrasuppressor cells. The mechanism of these functions is not known but HLA class II antigens might play an essential role in antigen binding, presentation and

  18. Synthesis and processing in Escherichia coli of human leucocyte interferon fused with the signal sequence of Bacillus amyloliquefaciens a-amylase

    SciTech Connect

    Sorokin, A.V.; Avakov, A.S.; Bogush, V.G.; Kostrov, S.V.; Gaiga, G.Z.; Iomantas, Yu.V.; Abalakina, E.G.; Stepanov, A.I.; Strongin, A.Ya.; Kozlov, Yu.I.; Debabov, V.G.

    1985-11-01

    Earlier, the authors reported cloning of the alpha-amylase gene of B. amyloliquefaciens in B. subtilis and E. coli. Currently, the authors report results on the expression of the hybrid gene consisting of the DNA fragment coding for the leader part of B. amyloliquefaciens alpha-amylase and the structural part of the human interferon alpha-2 in E. coli cells. This gene contains an additional methionine codon at the 5'-terminal, which codes for the interferon structure (without its own signal peptide). The interferon gene was inserted into plasmid /sub p/TG 278 at the cleavage site of EcoRI. The structure of the plasmid thus obtained the signal peptide of amylase, five amino acids (Val-Gly-Glu-Phe-Met), and the structural part of the interferon. The E. coli C600 cells carrying plasmid pTGA6 were used to study interferon secretions. The interferon activity was determined radioimmunologically with the use of monoclonal anti-bodies NK2.

  19. Group-specific human granulocyte antigens on a chronic myelogenous leukemia cell line with a Philadelphia chromosome marker.

    PubMed

    Drew, S I; Terasaki, P I; Billing, R J; Bergh, O J; Minowada, J; Klein, E

    1977-05-01

    Group-specific human granulocyte antigens are serologically detectable with granulocytotoxic-positive human alloantisera on a cell line, K562, of chronic myelogenous leukemia origin which bears a Philadelphia chromosomal marker. The same cell line lacks serologically detectable HLA, B2 microglobulin, and B-lymphocyte antigens. Granulocyte antigens are important cell markers for cell lines of suspected myeloid lineage.

  20. Structural, Mechanistic, and Antigenic Characterization of the Human Astrovirus Capsid

    PubMed Central

    York, Royce L.; Yousefi, Payam A.; Bogdanoff, Walter; Haile, Sara; Tripathi, Sarvind

    2015-01-01

    ABSTRACT Human astroviruses (HAstVs) are nonenveloped, positive-sense, single-stranded RNA viruses that are a leading cause of viral gastroenteritis. HAstV particles display T=3 icosahedral symmetry formed by 180 copies of the capsid protein (CP), which undergoes proteolytic maturation to generate infectious HAstV particles. Little is known about the molecular features that govern HAstV particle assembly, maturation, infectivity, and immunogenicity. Here we report the crystal structures of the two main structural domains of the HAstV CP: the core domain at 2.60-Å resolution and the spike domain at 0.95-Å resolution. Fitting of these structures into the previously determined 25-Å-resolution electron cryomicroscopy density maps of HAstV allowed us to characterize the molecular features on the surfaces of immature and mature T=3 HAstV particles. The highly electropositive inner surface of HAstV supports a model in which interaction of the HAstV CP core with viral RNA is a driving force in T=3 HAstV particle formation. Additionally, mapping of conserved residues onto the HAstV CP core and spike domains in the context of the immature and mature HAstV particles revealed dramatic changes to the exposure of conserved residues during virus maturation. Indeed, we show that antibodies raised against mature HAstV have reactivity to both the HAstV CP core and spike domains, revealing for the first time that the CP core domain is antigenic. Together, these data provide new molecular insights into HAstV that have practical applications for the development of vaccines and antiviral therapies. IMPORTANCE Astroviruses are a leading cause of viral diarrhea in young children, immunocompromised individuals, and the elderly. Despite the prevalence of astroviruses, little is known at the molecular level about how the astrovirus particle assembles and is converted into an infectious, mature virus. In this paper, we describe the high-resolution structures of the two main astrovirus

  1. CARbodies: Human Antibodies Against Cell Surface Tumor Antigens Selected From Repertoires Displayed on T Cell Chimeric Antigen Receptors

    PubMed Central

    Alonso-Camino, Vanesa; Sánchez-Martín, David; Compte, Marta; Nuñez-Prado, Natalia; Diaz, Rosa M; Vile, Richard; Alvarez-Vallina, Luis

    2013-01-01

    A human single-chain variable fragment (scFv) antibody library was expressed on the surface of human T cells after transduction with lentiviral vectors (LVs). The repertoire was fused to a first-generation T cell receptor ζ (TCRζ)-based chimeric antigen receptor (CAR). We used this library to isolate antibodies termed CARbodies that recognize antigens expressed on the tumor cell surface in a proof-of-principle system. After three rounds of activation-selection there was a clear repertoire restriction, with the emergence dominant clones. The CARbodies were purified from bacterial cultures as soluble and active proteins. Furthermore, to validate its potential application for adoptive cell therapy, human T cells were transduced with a LV encoding a second-generation costimulatory CAR (CARv2) bearing the selected CARbodies. Transduced human primary T cells expressed significant levels of the CARbodies-based CARv2 fusion protein on the cell surface, and importantly could be specifically activated, after stimulation with tumor cells. This approach is a promising tool for the generation of antibodies fully adapted to the display format (CAR) and the selection context (cell synapse), which could extend the scope of current adoptive cell therapy strategies with CAR-redirected T cells. PMID:23695536

  2. An antigenic study of human plasma cells in normal tissue and in myeloma: identification of a novel plasma cell associated antigen.

    PubMed Central

    Nathan, P D; Walker, L; Hardie, D; Richardson, P; Khan, M; Johnson, G D; Ling, N R

    1986-01-01

    A mouse monoclonal antibody named BU11 which detects an antigen strongly expressed on human plasma cells is described. The antibody stains plasma cells in tonsil sections, fresh and cultured plasmacytoid cells from the bone marrow of patients with multiple myeloma and cells of the plasmacytoid cell line RPMI 8226 used as the immunogen. In vitro studies of pokeweed mitogen (PWM) stimulated peripheral blood B cells and Epstein-Barr virus (EBV) stimulated tonsil B cells show that the antigen is present mainly on cells coexpressing the OKT10 antigen and containing cytoplasmic immunoglobulin (cIg). The BU11 antigen is expressed weakly on some normal B cells and is not present on T cells, monocytes or granulocytes. The antigen is of molecular weight 58kD under reducing conditions and is biochemically distinct from previously described plasma cell antigens. Images Fig. 4 PMID:3024883

  3. Common antigenic determinants on human melanoma, glioma, neuroblastoma, and sarcoma cells defined with monoclonal antibodies.

    PubMed

    Seeger, R C; Rosenblatt, H M; Imai, K; Ferrone, S

    1981-07-01

    Antigenic determinants that are common to melanomas, gliomas, neuroblastomas, and sarcomas but that are minimally or not detectably expressed by adult tissues were defined with monoclonal antibodies. Quantitative absorption of monoclonal antibody (Ab 165) with adult tissues followed by testing on antigen-positive UCLA-SO-M14 melanoma cells did not demonstrate antigenic determinant (Ag 165) in brain, lung, liver, kidney, intestine, adrenal, and muscle, Absorption of Ab 376 demonstrated Ag 376 in adult lung but minimal or no antigen in other tissues. Both antigens were associated with a variety of fetal tissues. Assessment of 28 human tumor cell lines with the 131I-staphylococcal Protein A-binding test demonstrated that Ab 165 reacted strongly with melanomas and gliomas and weakly with sarcomas. Ab 376 reacted strongly with melanomas, gliomas, neuroblastomas, and sarcomas. Neither of these antibodies reacted appreciably with carcinoma or teratoma cell lines. Absorption of Ab 165 and Ab 376 with noncultured tumors demonstrated that melanomas, sarcomas, and neuroblastomas can have greater quantities of these antigens in vivo than do normal adult tissues. Qualitative and quantitative antigenic heterogeneity within positive classes of tumors was demonstrated for both cultured and noncultured tumors. The differences in antigen expression in vivo between normal and neoplastic cells suggest potential value for these antibodies in immunodiagnosis and possibly immunotherapy.

  4. Immune profiling with a Salmonella Typhi antigen microarray identifies new diagnostic biomarkers of human typhoid.

    PubMed

    Liang, Li; Juarez, Silvia; Nga, Tran Vu Thieu; Dunstan, Sarah; Nakajima-Sasaki, Rie; Davies, D Huw; McSorley, Stephen; Baker, Stephen; Felgner, Philip L

    2013-01-01

    Current serological diagnostic assays for typhoid fever are based on detecting antibodies against Salmonella LPS or flagellum, resulting in a high false-positive rate. Here we used a protein microarray containing 2,724 Salmonella enterica serovar Typhi antigens (>63% of proteome) and identified antibodies against 16 IgG antigens and 77 IgM antigens that were differentially reactive among acute typhoid patients and healthy controls. The IgG target antigens produced a sensitivity of 97% and specificity of 80%, whereas the IgM target antigens produced 97% and 91% sensitivity and specificity, respectively. Our analyses indicated certain features such as membrane association, secretion, and protein expression were significant enriching features of the reactive antigens. About 72% of the serodiagnostic antigens were within the top 25% of the ranked antigen list using a Naïve bayes classifier. These data provide an important resource for improved diagnostics, therapeutics and vaccine development against an important human pathogen. PMID:23304434

  5. Antigenic Patterns and Evolution of the Human Influenza A (H1N1) Virus.

    PubMed

    Liu, Mi; Zhao, Xiang; Hua, Sha; Du, Xiangjun; Peng, Yousong; Li, Xiyan; Lan, Yu; Wang, Dayan; Wu, Aiping; Shu, Yuelong; Jiang, Taijiao

    2015-09-28

    The influenza A (H1N1) virus causes seasonal epidemics that result in severe illnesses and deaths almost every year. A deep understanding of the antigenic patterns and evolution of human influenza A (H1N1) virus is extremely important for its effective surveillance and prevention. Through development of antigenicity inference method for human influenza A (H1N1), named PREDAC-H1, we systematically mapped the antigenic patterns and evolution of the human influenza A (H1N1) virus. Eight dominant antigenic clusters have been inferred for seasonal H1N1 viruses since 1977, which demonstrated sequential replacements over time with a similar pattern in Asia, Europe and North America. Among them, six clusters emerged first in Asia. As for China, three of the eight antigenic clusters were detected in South China earlier than in North China, indicating the leading role of South China in H1N1 transmission. The comprehensive view of the antigenic evolution of human influenza A (H1N1) virus can help formulate better strategy for its prevention and control.

  6. Flow cytometric determination of the frequency and heterogeneity of expression of human melanoma-associated antigens.

    PubMed

    Berd, D; Herlyn, M; Koprowski, H; Mastrangelo, M J

    1989-12-01

    We used flow cytometry to measure the expression of human melanoma antigens on cell suspensions dissociated from metastatic masses. The objective was to study the heterogeneity between tumor samples from different patients and between different tumors excised from a single patient. Fifty-three metastases excised from 34 melanoma patients were analyzed with a panel of nine murine monoclonal antibodies (MOABs). Melanoma cells were stained by an indirect fluorescent method and analyzed on a Coulter EPICS C flow cytometer after gating to exclude tumor-infiltrating leukocytes and dead cells. The most consistently and most strongly expressed antigen was the high-molecular-weight proteoglycan (detected by the MOAB 9.2.27), which was expressed on 95% of the melanoma specimens and by a high proportion of cells within each specimen (mean +/- SE, 79.2 +/- 5.5). However, strong expression of this antigen was limited to melanoma cells that had been dissociated mechanically and was markedly diminished by exposure to collagenase. Culture of collagenase-dissociated tumor cells for 24 to 48 h resulted in reexpression of the antigen. The expression of other melanoma-associated antigens was not affected by collagenase treatment, but for these antigens there was more variability between cells from an individual tumor and between tumors from different patients. The percentage of enzyme-dissociated tumors considered positive for MOAB binding (defined as at least 10% of cells positive) and the mean +/- SE of the percentage of positive cells within a tumor were as follows: MOAB ME-9-61 (antigen, p97) = 84% + (41.2 +/- 5.4%); MOAB ME-20.4 (antigen, nerve growth factor receptor) = 40% + (18.7 +/- 5.1%); MOAB ME-24 (antigen, ganglioside GD3) = 84% + (50.8 +/- 4.8%); MOAB ME-311 (antigen, ganglioside 9-O-acetyl-GD3) = 76% + (42.5 +/- 5.1%); MOAB ME-361 (antigen, mainly ganglioside GD2) = 3% + (1.9 +/- 0.8%); MOAB 3F8 (antigen, ganglioside GD2) = 36% (10.5 +/- 3.8%); MOAB 14G2a (antigen

  7. Comparison of Z and R3 antigen expression and of genes encoding other antigenic markers in invasive human and bovine Streptococcus agalactiae strains from Norway.

    PubMed

    Maeland, Johan A; Radtke, Andreas

    2013-12-27

    Streptococcus agalactiae (GBS) may cause a variety of infectious diseases in humans caused by human GBS and mastitis in cattle caused by bovine GBS. Over the last few years molecular testing has provided evidence that human and bovine GBS have evolved along diverse phylogenetic lines. In the present study 173 invasive human GBS strains and 52 invasive bovine strains were tested for altogether 18 strain-variable and surface-localized antigenic markers including all 10 capsular polysaccharides (CPS) and proteins including Cβ, the alpha-like proteins, R3 and the recently described Z1 and Z2 antigens. PCR was used to detect encoding genes and antibody-based methods to detect expression of antigens. Thirteen of the 18 markers were detected in isolates of both strain categories. Seven of the ten CPS antigens were detected in both groups with types III and V predominating in the human GBS strains, types IV and V in the bovine isolates. Z1, Z2 and/or R3 expression and the genes encoding Cβ, Cα, Alp1, Alp2/3 or R4 (Rib) were detected in both groups. Protein antigen-CPS associations well known for human strains were essentially the same in the bovine isolates. The results show that in spite of evolution along different lines, human and bovine GBS share a variety of surface-exposed antigenic markers, substantiating close relationship between the two GBS subpopulations. PMID:24120184

  8. Inhibition of leucocytic lysosomal enzymes by glycosaminoglycans in vitro.

    PubMed Central

    Avila, J L; Convit, J

    1975-01-01

    1. A lysosomal fraction was separated by density-gradient centrifugation from a highly purified human polymorphonuclear leucocyte suspension. 2. Some 23 different lysosomal enzymes were assayed for activity in the presence of various concentrations of glycosaminoglycans. 3. The 21 acid hydrolases assayed were strongly inhibited to different degrees by low (0-12 mmol/l) concentrations of glycosaminoglycans in a pH-dependent manner. Thus inhibitions were stronger below pH4.5, with activity returning to control values at about pH5.0. 4. On a molar basis, the inhibitory activity for the several glycosaminoglycans studied was: heparin greater than chondroitin sulphate greater than hyaluronic acid. 5. Once the glycosaminoglycan-acid hydrolase complex was formed, it was partially dissociated by slight elevations in the pH of the incubation medium, by increasing the ionic strength of the incubation medium, or by adding several cationic proteins (e.g. histone, protamine). 6. As leucocytic lysosomes contain large amounts of chondroitin sulphate, and have a strongly acid intragranular pH, we suggest that glycosaminoglycans may modify lysosomal function through the formation of complexes with lysosomal enzymes, by inhibiting the digestive activity of the acid hydrolases when the intralysosomal pH is below their pI. PMID:2162

  9. Immunohistochemical demonstration of specific antigens in the human brain fixed in zinc-ethanol-formaldehyde.

    PubMed

    Korzhevskii, D E; Sukhorukova, E G; Kirik, O V; Grigorev, I P

    2015-08-05

    Tissue fixation is critical for immunohistochemistry. Recently, we developed a zinc-ethanol-formalin fixative (ZEF), and the present study was aimed to assess the applicability of the ZEF for the human brain histology and immunohistochemistry and to evaluate the detectability of different antigens in the human brain fixed with ZEF. In total, 11 antigens were tested, including NeuN, neuron-specific enolase, GFAP, Iba-1, calbindin, calretinin, choline acetyltransferase, glutamic acid decarboxylase (GAD65), tyrosine hydroxylase, synaptophysin, and α-tubulin. The obtained data show that: i) the ZEF has potential for use in general histological practice, where detailed characterization of human brain morphology is needed; ii) the antigens tested are well-preserved in the human brain specimens fixed in the ZEF.

  10. Immunohistochemical Demonstration of Specific Antigens in the Human Brain Fixed in Zinc-ethanol-Formaldehyde

    PubMed Central

    Korzhevskii, D.E.; Sukhorukova, E.G.; Kirik, O.V.; Grigorev, I.P.

    2015-01-01

    Tissue fixation is critical for immunohistochemistry. Recently, we developed a zinc-ethanol-formalin fixative (ZEF), and the present study was aimed to assess the applicability of the ZEF for the human brain histology and immunohistochemistry and to evaluate the detectability of different antigens in the human brain fixed with ZEF. In total, 11 antigens were tested, including NeuN, neuron-specific enolase, GFAP, Iba-1, calbindin, calretinin, choline acetyltransferase, glutamic acid decarboxylase (GAD65), tyrosine hydroxylase, synaptophysin, and α-tubulin. The obtained data show that: i) the ZEF has potential for use in general histological practice, where detailed characterization of human brain morphology is needed; ii) the antigens tested are well-preserved in the human brain specimens fixed in the ZEF. PMID:26428887

  11. Immunohistochemical demonstration of specific antigens in the human brain fixed in zinc-ethanol-formaldehyde.

    PubMed

    Korzhevskii, D E; Sukhorukova, E G; Kirik, O V; Grigorev, I P

    2015-01-01

    Tissue fixation is critical for immunohistochemistry. Recently, we developed a zinc-ethanol-formalin fixative (ZEF), and the present study was aimed to assess the applicability of the ZEF for the human brain histology and immunohistochemistry and to evaluate the detectability of different antigens in the human brain fixed with ZEF. In total, 11 antigens were tested, including NeuN, neuron-specific enolase, GFAP, Iba-1, calbindin, calretinin, choline acetyltransferase, glutamic acid decarboxylase (GAD65), tyrosine hydroxylase, synaptophysin, and α-tubulin. The obtained data show that: i) the ZEF has potential for use in general histological practice, where detailed characterization of human brain morphology is needed; ii) the antigens tested are well-preserved in the human brain specimens fixed in the ZEF. PMID:26428887

  12. Position 156 influences the peptide repertoire and tapasin dependency of human leukocyte antigen B*44 allotypes

    PubMed Central

    Badrinath, Soumya; Saunders, Philippa; Huyton, Trevor; Aufderbeck, Susanne; Hiller, Oliver; Blasczyk, Rainer; Bade-Doeding, Christina

    2012-01-01

    Background Polymorphic differences between donor and recipient human leukocyte antigen class I molecules can result in graft-versus-host disease due to distinct peptide presentation. As part of the peptide-loading complex, tapasin plays an important role in selecting peptides from the pool of potential ligands. Class I polymorphisms can significantly alter the tapasin-mediated interaction with the peptide-loading complex and although most class I allotypes are highly dependent upon tapasin, some are able to load peptides independently of tapasin. Several human leukocyte antigen B*44 allotypes differ exclusively at position 156 (B*44:02156Asp, 44:03156Leu, 44:28156Arg, 44:35156Glu). From these alleles, only the high tapasin-dependency of human leukocyte antigen B*44:02 has been reported. Design and Methods We investigated the influence of position 156 polymorphisms on both the requirement of tapasin for efficient surface expression of each allotype and their peptide features. Genes encoding human leukocyte antigen B*44 variants bearing all possible substitutions at position 156 were lentivirally transduced into human leukocyte antigen class I-negative LCL 721.221 cells and the tapasin-deficient cell line LCL 721.220. Results Exclusively human leukocyte antigen B*44:28156Arg was expressed on the surface of tapasin-deficient cells, suggesting that the remaining B*44/156 variants are highly tapasin-dependent. Our computational analysis suggests that the tapasin-independence of human leukocyte antigen B*44:28156Arg is a result of stabilization of the peptide binding region and generation of a more peptide receptive state. Sequencing of peptides eluted from human leukocyte antigen B*44 molecules by liquid chromatography-electrospray ionization-mass spectrometry (LTQ-Orbitrap) demonstrated that both B*44:02 and B*44:28 share the same overall peptide motif and a certain percentage of their individual peptide repertoires in the presence and/or absence of tapasin

  13. Human leukocyte antigen-G polymorphism influences the age of onset and autoantibody status in rheumatoid arthritis.

    PubMed

    Mariaselvam, C M; Chaaben, A B; Salah, S; Charron, D; Krishnamoorthy, R; Tamouza, R; Negi, V S

    2015-03-01

    The study was conducted to investigate the frequency of three gene polymorphisms in the 3'-untranslated region (3'-UTR) of human leucocyte antigen-G (HLA-G) gene in south Indian patients with rheumatoid arthritis (RA) and analyze their influence on disease susceptibility, phenotype and treatment response. HLA-G 14 bp insertion (Ins)/deletion (del) (rs66554220), HLA-G +3142G>C (rs1063320) and +3187A>G (rs9380142) polymorphism was analyzed in 221 RA patients and 200 healthy controls. Frequency of HLA-G genotypes or alleles did not differ between patients and controls. Analysis based on rheumatoid factor (RF) status revealed that the frequency of allele 'A' (rs9380142) was significantly higher in RF-positive than in RF-negative patients [84% vs 74%, Yates-corrected P value (Pc) = 0.04, odds ratio (OR) = 1.8, 95% confidence interval (CI) = 1.0-3.2]. A similar difference was maintained in RF-positive female patients than their RF-negative counterparts (83% vs 71%, Pc = 0.02, OR = 1.9, 95% CI = 1.0 to 3.4) and between RF-positive and RF-negative young onset RA (YORA) patients (84% vs 73%, Pc = 0.03, OR = 1.9, 95% CI = 1.0-3.2), suggesting that rs9380142 polymorphism influenced RF status. The 14 bp Ins allele of rs66554220 was significantly more prevalent in RF-positive YORA than in RF-positive late onset RA (LORA) patients (51% vs 25%, P = 0.03, OR = 3.1, 95% CI = 1.1-9.8). Frequency of the four major haplotypes [InsGA (48%), DelGA (22%), DelCG (18%), DelCA (9.7%)] observed did not differ between cases and controls. HLA-G does not appear to be a risk factor for development of RA in south Indian Tamils but may act as a genetic modifier of clinical phenotype in terms of autoantibody production, gender preference and age at disease onset.

  14. Effects of 1 alpha,25-dihydroxyvitamin D3 and cytokines on the expression of MHC antigens, complement receptors and other antigens on human blood monocytes and U937 cells: role in cell differentiation, activation and phagocytosis.

    PubMed Central

    Spittler, A; Willheim, M; Leutmezer, F; Ohler, R; Krugluger, W; Reissner, C; Lucas, T; Brodowicz, T; Roth, E; Boltz-Nitulescu, G

    1997-01-01

    The effect of calcitriol/1 alpha,25-dihydroxyvitamin D3, alone and in combination with cytokines, on the expression of various antigens (Ag) on human peripheral blood monocytes and U937 cells was studied by flow cytometry. Both constitutive and interferon-gamma (IFN-gamma), interleukin-4 (IL-4), IL-6 and tumour necrosis factor-alpha (TNF-alpha)-induced human leucocyte antigen (HLA)-DR, HLA-DP and HLA-DQ Ag expression on monocytes was significantly down-regulated by calcitriol, IL-10 and transforming growth factor-beta (TGF-beta). The effects of calcitriol were concentration dependent and reached maximal inhibitory levels after 3-5 days. Modulation of HLA-DR by calcitriol and IFN-gamma at the protein level correlated with the amount of mRNA specific for the HLA-DR alpha-chain, as judged by Northern blot analysis. The basal as well as IL-4, IL-6, IFN-gamma, TNF-alpha and TGF-beta-driven levels of HLA-ABC Ag were significantly diminished by calcitriol. On U937 cells calcitriol markedly induced CD11a and CD11b expression and weakly up-regulated CD11c whereas on monocytes, constitutive CD11a, CD11b and CD11c expression was significantly down-regulated by calcitriol. The expression of CD14 Ag was strongly induced on U937 cells but only modestly on monocytes. Both the basal level of CD71 and IL-4, IFN-gamma or TNF-alpha-driven expression was diminished on calcitriol-treated U937 cells. In addition, calcitriol suppressed the expression of CD71 Ag on monocytes. The ability of monocytes to phagocytize opsonized Escherichia coli was diminished by calcitriol. Our results demonstrate that calcitriol, alone or in combination with cytokines, modulates expression of MHC, CD11b, CD11c, CD14 and CD71 Ag on both monocytes and U937 cells, and impairs the phagocytic property of monocytes. Images Figure 2 PMID:9135559

  15. Human T cell activation. III. Induction of an early activation antigen, EA 1 by TPA, mitogens and antigens

    SciTech Connect

    Hara, T.; Jung, L.K.L.; FU, S.M.

    1986-03-01

    With human T cells activated for 12 hours by 12-o-tetradecanoyl phorbol-13-acetate (TPA) as immunogen, an IgG/sub 2a/ monoclonal antibody, mAb Ea 1, has been generated to a 60KD phosphorylated protein with 32KD and 28KD subunits. The antigen, Ea 1, is readily detected on 60% of isolated thymocytes by indirect immunofluorescence. A low level of Ea 1 expression is detectable on 2-6% of blood lymphocytes. Isolated T cells have been induced to express Ea 1 by TPA, mitogens and anitgens. TPA activated T cells express Ea 1 as early as 1 hour after activation. By 4 hours, greater than 95% of the T cells stain with mAb Ea 1. About 50% of the PHA or Con A activated T cells express Ea 1 with a similar kinetics. Ea 1 expression proceeds that of IL-2 receptor in these activation processes. T cells activated by soluble antigens (tetanus toxoid and PPD) and alloantigens in MLR also express Ea 1 after a long incubation. About 20% of the T cells stain for Ea 1 at day 6. Ea 1 expression is not limited to activated T cells. B cells activated by TPA or anti-IgM Ab plus B cell growth factor express Ea 1. The kinetics of Ea 1 expression is slower and the staining is less intense. Repeated attempts to detect Ea 1 on resting and activated monocytes and granulocytes have not been successful. Ea 1 expression is due to de novo synthesis for its induction is blocked by cycloheximide and actinomycin D. Ea 1 is the earliest activation antigen detectable to-date.

  16. Flow cytometry analyses of phagocytic and respiratory burst activities and cytochemical characterization of leucocytes isolated from wrasse (Labrus bergylta A.).

    PubMed

    Haugland, Gyri T; Rønneseth, Anita; Wergeland, Heidrun I

    2014-07-01

    We have isolated leucocytes from peripheral blood (PBL), head kidney (HKL) and spleen (SL) of wrasse (Labrus bergylta A.) and studied the innate immune responses phagocytosis and respiratory burst using flow cytometry. Further, we have characterized the phenotypic properties of the leucocytes by cytochemical staining. We could differentiate between several subsets of leucocytes; lymphocytes, monocytes/macrophages, neutrophils, eosinophils, basophils and small leucocytes that might be precursor or immature cells. One striking observation was the eosinophils which were present among HKL, PBL and SL. The neutrophils had rounded, bean shaped or bi-lobed nuclei and resembled neutrophils in Atlantic cod (Gadus morhua L.) and lumpsucker (Cyclopterus lumpus L.), but were different from the polymorphonucleated neutrophils in Atlantic salmon (Salmo salar L.) and humans. Basophils were observed, but they were rare. Phagocytosis and respiratory burst activities were detected among different cell types. Highest phagocytic activity was observed among monocytes/macrophages and small leucocytes. Several different subtypes had ability to perform an oxygen-dependent degradation of microbes, measured as respiratory burst activity. Knowledge of the basic properties of wrasse's leucocytes and innate immunology can benefit further studies on its adaptive immune responses.

  17. Fy(a)/Fy(b) antigen polymorphism in human erythrocyte Duffy antigen affects susceptibility to Plasmodium vivax malaria.

    PubMed

    King, Christopher L; Adams, John H; Xianli, Jia; Grimberg, Brian T; McHenry, Amy M; Greenberg, Lior J; Siddiqui, Asim; Howes, Rosalind E; da Silva-Nunes, Monica; Ferreira, Marcelo U; Zimmerman, Peter A

    2011-12-13

    Plasmodium vivax (Pv) is a major cause of human malaria and is increasing in public health importance compared with falciparum malaria. Pv is unique among human malarias in that invasion of erythrocytes is almost solely dependent on the red cell's surface receptor, known as the Duffy blood-group antigen (Fy). Fy is an important minor blood-group antigen that has two immunologically distinct alleles, referred to as Fy(a) or Fy(b), resulting from a single-point mutation. This mutation occurs within the binding domain of the parasite's red cell invasion ligand. Whether this polymorphism affects susceptibility to clinical vivax malaria is unknown. Here we show that Fy(a), compared with Fy(b), significantly diminishes binding of Pv Duffy binding protein (PvDBP) at the erythrocyte surface, and is associated with a reduced risk of clinical Pv in humans. Erythrocytes expressing Fy(a) had 41-50% lower binding compared with Fy(b) cells and showed an increased ability of naturally occurring or artificially induced antibodies to block binding of PvDBP to their surface. Individuals with the Fy(a+b-) phenotype demonstrated a 30-80% reduced risk of clinical vivax, but not falciparum malaria in a prospective cohort study in the Brazilian Amazon. The Fy(a+b-) phenotype, predominant in Southeast Asian and many American populations, would confer a selective advantage against vivax malaria. Our results also suggest that efficacy of a PvDBP-based vaccine may differ among populations with different Fy phenotypes.

  18. Use of MAG1 recombinant antigen for diagnosis of Toxoplasma gondii infection in humans.

    PubMed

    Holec, Lucyna; Hiszczyńska-Sawicka, Elzbieta; Gasior, Artur; Brillowska-Dabrowska, Anna; Kur, Józef

    2007-03-01

    This paper describes the cloning, purification, and serological applications of matrix antigen MAG1 of Toxoplasma gondii. The expression system used allows the production of a large amount of T. gondii recombinant protein, which was assessed for its potential use in an enzyme-linked immunosorbent assay (ELISA) for detection of T. gondii infection in humans. Serum samples from 117 patients with different stages of infection, along with 10 serum samples from seronegative patients obtained for routine diagnostic tests, were used. The results were compared with those of an ELISA that uses a native T. gondii antigen extract. The MAG1 antigen detected antibodies more frequently from the acute stage (97.3%) than from the chronic stage (7.5%) of toxoplasmosis. Hence, this antigen may be used as a tool for detection of T. gondii immunoglobulin G antibodies in persons with acute toxoplasmosis.

  19. Expression of Human Herpesvirus-6 Antigens in Benign and Malignant Lymphoproliferative Diseases

    PubMed Central

    Luppi, Mario; Barozzi, Patrizia; Garber, Richard; Maiorana, Antonio; Bonacorsi, Goretta; Artusi, Tullio; Trovato, Raffaella; Marasca, Roberto; Torelli, Giuseppe

    1998-01-01

    Immunohistochemistry was used to look for the expression of human herpesvirus-6 (HHV-6) antigens in a well characterized series of benign, atypical, and malignant lymphoid lesions, which tested positive for the presence of HHV-6 DNA. A panel of specific antibodies against HHV-6 antigens, characteristic either of the early (p41) or late (p101K, gp106, and gp116) phases of the viral cycle, was applied to the lymphoid tissues from 15 non-Hodgkin’s lymphomas, 14 Hodgkin’s disease cases, 5 angioimmunoblastic lymphadenopathies with dysproteinemia, 14 reactive lymphadenopathies, and 2 cases of sinus histiocytosis with massive lymphadenopathy (Rosai-Dorfman disease). In lymphomatous tissues, the expression of late antigens was documented only in reactive cells, and mainly in plasma cells. Of interest, the expression of the early p41 antigen was detected in the so-called “mummified” Reed-Sternberg cells, in two Hodgkin’s disease cases. In reactive lymphadenopathies, the HHV-6 late antigen-expressing cells were plasma cells, histiocytes, and rare granulocytes distributed in interfollicular areas. In both cases of Rosai-Dorfman disease, the p101K showed an intense staining in follicular dendritic cells of germinal centers, whereas the gp106 exhibited an intense cytoplasmic reaction in the abnormal histiocytes, which represent the histological hallmark of the disease. The expression of HHV-6 antigens is tightly controlled in lymphoid tissues. The lack of HHV-6 antigen expression in neoplastic cells and the limited expression in degenerating Reed-Sternberg cells argue against a major pathogenetic role of the virus in human lymphomagenesis. The detection of a rather unique pattern of viral late antigen expression in Rosai-Dorfman disease suggests a possible pathogenetic involvement of HHV-6 in some cases of this rare lymphoproliferative disorder. PMID:9736030

  20. Regulation of human peripheral blood monocyte DR antigen expression in vitro by lymphokines and recombinant interferons.

    PubMed Central

    Sztein, M B; Steeg, P S; Johnson, H M; Oppenheim, J J

    1984-01-01

    The in vitro regulation of adult human monocyte DR antigen expression was studied. Normally about 75% of freshly obtained human peripheral blood monocytes express DR antigens as determined by anti-DR and complement-mediated cytotoxicity assays. DR expression on monocytes in unfractionated peripheral blood mononuclear cell cultures persisted to variable degrees for up to 5 d of incubation. However, when the mononuclear cells were thoroughly depleted of nonadherent cells, cultured monocytes consistently exhibited progressively decreased DR expression over 2-5 d of incubation. Readdition of nonadherent cells to the adherent cell population prevented or delayed this decrease in monocyte DR antigen expression. Thus, monocyte DR expression diminished markedly during in vitro incubation; however, the presence of nonadherent cells somehow interfered with this process. In other experiments, peripheral adherent monocytes, which had been cultured for 2-3 d to reduce their DR expression, could be induced to reexpress DR antigens after 2 d of incubation with unpurified lymphokine-containing culture supernatants, recombinant human interferon-alpha, or recombinant human gamma interferon (IFN-gamma). The reinduction of DR expression on human monocytes by lymphokines was abrogated by an antiserum produced to the synthetic N-terminal amino acids of human IFN-gamma, indicating that IFN-gamma is the active mediator in the lymphokine-containing preparations. Monocytes cultured with lymphokines or recombinant interferons also could initiate a significantly greater mixed lymphocyte response than control monocytes. Thus, IFN-gamma-containing lymphokines and recombinant interferons are required to induce human monocyte DR expression and accessory cell capacity in vitro, since in their absence monocytes become DR antigen-deficient. Finally, incubation of unfractionated human mononuclear cells with anti-human IFN-gamma also promoted the loss of monocyte DR expression. These findings suggest

  1. A new MIC1-MAG1 recombinant chimeric antigen can be used instead of the Toxoplasma gondii lysate antigen in serodiagnosis of human toxoplasmosis.

    PubMed

    Holec-Gąsior, Lucyna; Ferra, Bartłomiej; Drapała, Dorota; Lautenbach, Dariusz; Kur, Józef

    2012-01-01

    This study presents an evaluation of the MIC1 (microneme protein 1)-MAG1 (matrix antigen 1) Toxoplasma gondii recombinant chimeric antigen for the serodiagnosis of human toxoplasmosis for the first time. The recombinant MIC1-MAG1 antigen was obtained as a fusion protein containing His tags at the N- and C-terminal ends using an Escherichia coli expression system. After purification by metal affinity chromatography, the chimeric protein was tested for usefulness in an enzyme-linked immunosorbent assay (ELISA) for the detection of anti-T. gondii immunoglobulin G (IgG). One hundred ten sera from patients at different stages of infection and 40 sera from seronegative patients were examined. The results obtained for the MIC1-MAG1 chimeric antigen were compared with those of IgG ELISAs using a Toxoplasma lysate antigen (TLA), a combination of recombinant antigens (rMIC1ex2-rMAG1) and single recombinant proteins (rMIC1ex2 and rMAG1). The sensitivity of the IgG ELISA calculated from all of the positive serum samples was similar for the MIC1-MAG1 chimeric antigen (90.8%) and the TLA (91.8%), whereas the sensitivities of the other antigenic samples used were definitely lower, at 69.1% for the mixture of antigens, 75.5% for the rMIC1ex2, and 60% for rMAG1. This study demonstrates that the MIC1-MAG1 recombinant chimeric antigen can be used instead of the TLA in the serodiagnosis of human toxoplasmosis.

  2. CD1 antigen presentation and autoreactivity in the pregnant human uterus

    PubMed Central

    Guerin, Leigh; Wu, Vernon; Houser, Brandy; Tilburgs, Tamara; de Jong, Annemieke; Moody, D. Branch; Strominger, Jack L.

    2015-01-01

    Problem CD11cHI human decidual macrophages express several isoforms of CD1 molecules. Their expression pattern and function required investigation. Method of Study CD11cHI macrophages were isolated from decidua. Expression of CD1 isoforms and their ability to present lipid antigens to T cells was studied. Results CD1a, CD1c, and CD1d were all expressed on CD11cHI dMϕ, a pattern differing from those previously observed. Exposure of peripheral monocytes and dendritic cells to lipid isolates from decidua led to increased surface CD1a levels only. The CD1a and CD1c on dMϕ were able to present the appropriate lipid antigens to lipid antigen-specific T cells. Finally, autoreactivity of decidual T cells to CD1a was observed. Conclusion The unique pattern of expression of CD1 isoforms on CD11cHI dMϕ is consistent with organ-specific roles of CD1 in human T cell responses. dMϕ are able to present lipid antigens to both peripheral and decidual T cells and are major antigen presenting cells in human de-cidua. PMID:25739697

  3. Sero-epidemiological value of some hydatid cyst antigen in diagnosis of human cystic echinococcosis.

    PubMed

    Hassanain, Mohey Abdelhafez; Shaapan, Raafat Mohamed; Khalil, Fathia Abdelrazik M

    2016-03-01

    Cystic echinococcosis (CE) is a severe zoonotic disease which affects both human and animals. The disease has a considerable economic and social impact, because it has numerous complications leading to important disabilities and even death. CE is a widespread chronic endemic helminthic disease caused by infection with metacestodes of tapeworm Echinococcus granulosus. This study was conducted to diagnosis human CE by hydatid cyst antigens from camels and sheep. Hydatid fluid and protoscoleces crude antigens corresponding to camel and sheep were resolute by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and the protein bands of different antigens were exposed to infected patients serum CE through western blot (WB) assay. The camel hydatid fluid antigen revealed five polypeptide bands of 18-98.8 kDa by SDS-PAGE while sheep hydatid fluid antigen revealed four polypeptide bands of 20-100 kDa. Immune reactive bands were obtained through WB ranged from 25 to 125 kDa. The study showed prominent immune reactive bands of 92, 52.2 and 35.7 kDa which may helpful in diagnosis of human CE. PMID:27065597

  4. Induction of human complement activation without cytolysis by mouse monoclonal antibodies to human leukocyte antigens.

    PubMed

    Sugita, K; Majdic, O; Stockinger, H; Holter, W; Burger, R; Knapp, W

    1987-04-01

    Ten monoclonal antibodies to human leukocyte subsets that had previously been shown to lyse their respective target cells in the presence of rabbit serum as complement source were evaluated for their cytolytic capacity with human complement. Four of the ten were lytic with human complement. All were of IgM type. Antibodies were also evaluated for their capacity to induce C3 binding to target cells. With this method we could demonstrate that, indeed, 3 of the 6 noncytolytic antibodies had the capacity to initiate the human complement activation process and to induce C3 binding. Two of these 3 antibodies were of IgM class (VIT3 and VIM13), one of IgG3 (562). From the practical point of view the most interesting of these 3 antibodies is the nonmitogenic anti-CD3 pan-T cell antibody VIT3. Therefore, this antibody was analyzed in more detail. VIT3 antibody concentrations needed to induce detectable C3 binding to human T cells are very low (down to 1 ng VIT3/ml). Human serum as complement source can also be considerably (100X) diluted before C3 binding becomes undetectable. Activation of C3 is a prerequesite for VIT3-induced C3 binding, and bound C3 seems to lack the C3a fragment. Bound C3, in contrast to the quickly occuring antigenic modulation of the CD3 complex and the simultaneous disappearance of the antibody coat, remains expressed also after prolonged incubation at 37 degrees C. C3 fragments bound to T cells after activation with VIT3 are also recognized by cells bearing C3 receptors of types CR1 and CR2. PMID:3576673

  5. Evolutionary origin and human-specific expansion of a cancer/testis antigen gene family.

    PubMed

    Zhang, Qu; Su, Bing

    2014-09-01

    Cancer/testis (CT) antigens are encoded by germline genes and are aberrantly expressed in a number of human cancers. Interestingly, CT antigens are frequently involved in gene families that are highly expressed in germ cells. Here, we presented an evolutionary analysis of the CTAGE (cutaneous T-cell-lymphoma-associated antigen) gene family to delineate its molecular history and functional significance during primate evolution. Comparisons among human, chimpanzee, gorilla, orangutan, macaque, marmoset, and other mammals show a rapid and primate specific expansion of CTAGE family, which starts with an ancestral retroposition in the haplorhini ancestor. Subsequent DNA-based duplications lead to the prosperity of single-exon CTAGE copies in catarrhines, especially in humans. Positive selection was identified on the single-exon copies in comparison with functional constraint on the multiexon copies. Further sequence analysis suggests that the newly derived CTAGE genes may obtain regulatory elements from long terminal repeats. Our result indicates the dynamic evolution of primate genomes, and the recent expansion of this CT antigen family in humans may confer advantageous phenotypic traits during early human evolution.

  6. Antigenic Relatedness of Norovirus GII.4 Variants Determined by Human Challenge Sera

    PubMed Central

    Dai, Ying-Chun; Zhang, Xu-Fu; Xia, Ming; Tan, Ming; Quigley, Christina; Lei, Wen; Fang, Hao; Zhong, Weiming; Lee, Bonita; Pang, Xiaoli; Nie, Jun; Jiang, Xi

    2015-01-01

    The GII.4 noroviruses (NoVs) are a single genotype that is responsible for over 50% of NoV gastroenteritis epidemics worldwide. However, GII.4 NoVs have been found to undergo antigenic drifts, likely selected by host herd immunity, which raises an issue for vaccine strategies against NoVs. We previously characterized GII.4 NoV antigenic variations and found significant levels of antigenic relatedness among different GII.4 variants. Further characterization of the genetic and antigenic relatedness of recent GII.4 variants (2008b and 2010 cluster) was performed in this study. The amino acid sequences of the receptor binding interfaces were highly conserved among all GII.4 variants from the past two decades. Using serum samples from patients enrolled in a GII.4 virus challenge study, significant cross-reactivity between major GII.4 variants from 1998 to 2012 was observed using enzyme-linked immunosorbent assays and HBGA receptor blocking assays. The overall abilities of GII.4 NoVs to bind to the A/B/H HBGAs were maintained while their binding affinities to individual ABH antigens varied. These results highlight the importance of human HBGAs in NoV evolution and how conserved antigenic types impact vaccine development against GII.4 variants. PMID:25915764

  7. Human immunodeficiency virus (HIV) antigen testing to detect HIV infection in female sex workers in Singapore.

    PubMed

    Chan, R K; Ali, K; Thoe, S Y

    1995-07-01

    Human immunodeficiency virus (HIV) infection is characterised by seroconversion after a ¿window¿ period of 2 to 3 months. After this period antibodies are usually detectable by screening tests (enzyme immunoassay or particle agglutination) confirmed by Western blot analysis. We studied 1000 newly enrolled female sex workers who had not been previously tested for HIV to assess the usefulness of HIV antigen testing to improve the efficacy of HIV infection detection. Blood was taken at enrollment for HIV antigen and HIV antibody testing. The Abbott HIVAG-1 test was used to detect antigen; antibody detection was by the Abbott recombinant HIV-1/HIV-2 3rd generation enzyme immunoassay (EIA) test, the Fujirebio Serodia-HIV particle agglutination (PA) test for screening, and the Diagnostic Biotechnology HIV Blot 2.2 Western blot (WB) test for antibody confirmation. Of the 1000 samples, 26 were positive for HIV antibody testing (26/26 for EIA, 25/25 for PA, 26/26 for WB), giving a prevalence rate of 2.6%, Of these 26 seropositive samples 1 was positive on HIV antigen testing. There were no samples which were antigen-positive and antibody-negative. HIV antigen testing does not add to increased efficacy of HIV detection among female sex workers in Singapore.

  8. Human peripheral blood monocytes display surface antigens recognized by monoclonal antinuclear antibodies

    SciTech Connect

    Holers, V.M.; Kotzin, B.L.

    1985-09-01

    The authors used monoclonal anti-nuclear autoantibodies and indirect immunofluorescence to examine normal human peripheral blood mononuclear leukocytes for the presence of cell surface nuclear antigens. Only one monoclonal anti-histone antibody (MH-2) was found to bind to freshly isolated PBL, staining approximately 10% of large cells. However, after cells were placed into culture for 16-24 h, a high percentage (up to 60%) of large-sized cells were recognized by an anti-DNA (BWD-1) and several different antihistone monoclonal antibodies (BWH-1, MH-1, and MH-2). These antibodies recognize separate antigenic determinants on chromatin and histones extracted from chromatin. The histone antigen-positive cells were viable, and the monoclonal antibodies could be shown to be binding to the cell surface and not to the nucleus. Using monoclonal antibodies specific for monocytes and T cells, and complement-mediated cytotoxicity, the cells bearing histone antigens were shown to be primarily monocytes. The appearance of histone and DNA antigen-positive cells was nearly completely inhibited by the addition of low concentrations of cycloheximide at initiation of the cultures. In contrast, little effect on the percentage of positive cells was detected if cells were exposed to high doses of gamma irradiation before culture. These data further support the existence of cell surface nuclear antigens on selected cell subsets, which may provide insight into the immunopathogenesis of systemic lupus erythematosus and related autoimmune diseases.

  9. Serological speciation of human schistosome infections by ELISA with a panel of three antigens

    PubMed Central

    Turner, P; Lalloo, K; Bligh, J; Armstrong, M; Whitty, C J M; Doenhoff, M J; Chiodini, P L

    2004-01-01

    Aims: To find out whether serology can reliably speciate human schistosomiasis using a simple enzyme linked immunosorbent assay (ELISA) technique. Methods: Stored sera from 66 patients with microscopically confirmed schistosomiasis were subjected to ELISA using a panel of three antigens, namely: unfractionated Schistosoma mansoni soluble egg antigen (SEA); CEF6, a cationic fraction of SEA; and crude S margrebowiei egg antigen, prepared from an animal schistosome closely related to S haematobium. Results: The optical densities (ODs) obtained using CEF6 as antigen were significantly higher in sera from S mansoni infected patients than in sera from S haematobium infected patients (median OD, 0.810 v 0.595). Using S margrebowiei egg antigen, the optical densities were significantly higher in S haematobium sera than in S mansoni sera (median OD, 0.794 v 0.544). There was no significant difference in optical densities between S mansoni and S haematobium sera using SEA (median OD, 0.725 v 0.737). The ratio of ODs (CEF6 to S margrebowiei egg antigen) was calculated: a ratio of >1 indicated S mansoni infection (sensitivity, 88%) and a ratio of <1 indicated S haematobium infection (sensitivity, 84%). The odds ratio for S haematobium having an OD ratio of <1 was 36.8 (95% confidence interval, 7.0 to 194). Conclusions: The identity of the infecting species of schistosome can be determined using the panel of antigens described. SEA should be used to screen serum samples, and the CEF6 : S margrebowiei egg antigen ELISA optical density ratio can be used where serological speciation is required. PMID:15509682

  10. Polymorphic Expression of a Human Superficial Bladder Tumor Antigen Defined by Mouse Monoclonal Antibodies

    NASA Astrophysics Data System (ADS)

    Fradet, Yves; Islam, Nazrul; Boucher, Lucie; Parent-Vaugeois, Carmen; Tardif, Marc

    1987-10-01

    Three mouse monoclonal antibodies (mAbs), which define a highly restricted antigen, were obtained by simultaneous immunizations with superficial papillary bladder tumor cells and mouse polyclonal serum against normal urothelium. The antigen was detected by the avidin/biotin/peroxidase method in 30/44 superficial bladder tumors (68%) but in only 4/27 infiltrating urothelial cancers (with much less intensity). No normal adult or fetal tissues tested expressed the antigen, including normal urothelium from 40 individuals, 13 of whom had a bladder tumor positive for the antigen. Only 1 of 45 nonbladder tumors showed some reactivity with one of the three mAbs. Serological tests on a large panel of human cancer cell lines and normal cultured cells were negative. The antigen is highly stable and well preserved on paraffin-embedded tissues. Electrophoretic transfer blot experiments with fresh tumor extracts showed that all three mAbs react with a determinant on a component of 300,000 Mr (pI 9.5) and 62,000 Mr (pI 6.5). The antigen shows polymorphic expression at the cellular level on tissue sections and also at a molecular level on immunoblots where the two bands are differentially detected on extracts of a series of tumors but are not visualized on normal urothelium extracts. The characteristics of this antigenic system suggest that it may provide some insights about the biology of bladder cancer. Specific detection of the antigen on 70% of superficial bladder tumors with normal cytology may be useful for their diagnosis and follow-up.

  11. Structural Basis for Distinct Binding Properties of the Human Galectins to Thomsen-Friedenreich Antigen

    PubMed Central

    Bian, Cheng-Feng; Zhang, Ying; Sun, Hui; Li, De-Feng; Wang, Da-Cheng

    2011-01-01

    The Thomsen-Friedenreich (TF or T) antigen, Galβ1-3GalNAcα1-O-Ser/Thr, is the core 1 structure of O-linked mucin type glycans appearing in tumor-associated glycosylation. The TF antigen occurs in about 90% of human cancer cells and is a potential ligand for the human endogenous galectins. It has been reported that human galectin-1 (Gal-1) and galectin-3 (Gal-3) can perform their cancer-related functions via specifically recognizing TF antigen. However, the detailed binding properties have not been clarified and structurally characterized. In this work, first we identified the distinct TF-binding abilities of Gal-1 and Gal-3. The affinity to TF antigen for Gal-3 is two orders of magnitude higher than that for Gal-1. The structures of Gal-3 carbohydrate recognition domain (CRD) complexed with TF antigen and derivatives, TFN and GM1, were then determined. These structures show a unique Glu-water-Arg-water motif-based mode as previously observed in the mushroom galectin AAL. The observation demonstrates that this recognition mode is commonly adopted by TF-binding galectins, either as endogenous or exogenous ones. The detailed structural comparisons between Gal-1 and Gal-3 CRD and mutagenesis experiments reveal that a pentad residue motif (51AHGDA55) at the loop (g1-L4) connecting β-strands 4 and 5 of Gal-1 produces a serious steric hindrance for TF binding. This motif is the main structural basis for Gal-1 with the low affinity to TF antigen. These findings provide the intrinsic structural elements for regulating the TF-binding activity of Gal-1 in some special conditions and also show certain target and approach for mediating some tumor-related bioactivities of human galectins. PMID:21949831

  12. Regulation of leucocyte homeostasis in the circulation.

    PubMed

    Scheiermann, Christoph; Frenette, Paul S; Hidalgo, Andrés

    2015-08-01

    The functions of blood cells extend well beyond the immune functions of leucocytes or the respiratory and hemostatic functions of erythrocytes and platelets. Seen as a whole, the bloodstream is in charge of nurturing and protecting all organs by carrying a mixture of cell populations in transit from one organ to another. To optimize these functions, evolution has provided blood and the vascular system that carries it with various mechanisms that ensure the appropriate influx and egress of cells into and from the circulation where and when needed. How this homeostatic control of blood is achieved has been the object of study for over a century, and although the major mechanisms that govern it are now fairly well understood, several new concepts and mediators have recently emerged that emphasize the dynamism of this liquid tissue. Here we review old and new concepts that relate to the maintenance and regulation of leucocyte homeostasis in blood and briefly discuss the mechanisms for platelets and red blood cells.

  13. Deglycosylation of Toxocara excretory-secretory antigens improves the specificity of the serodiagnosis for human toxocariasis.

    PubMed

    Roldán, W H; Elefant, G R; Ferreira, A W

    2015-11-01

    Serodiagnosis of human toxocariasis is difficult in tropical areas where other helminthiasis are endemic. Many studies have shown that glycans from helminths may be the responsible for cross-reactions in the immunoassays. In this study, we have evaluated the deglycosylation of the Toxocara canis excretory-secretory (TES) antigens for the detection of IgG antibodies using a panel of 228 serum samples (58 patients with toxocariasis, 75 patients with other helminth infections and 95 healthy individuals) by ELISA and Western blot assays. Our results showed that the deglycosylation of TES antigens resulted in a single fraction of 26 kDa (dTES) and was able to detect IgG antibodies with a sensitivity and specificity of 100% in both above-mentioned assays. The rate of cross-reactions, observed in ELISA with TES (13·3%), was significantly reduced (5·3%) when the dTES antigens were used. Likewise, the cross-reactivity observed with the fractions of 32, 55 and 70 kDa of the TES antigens was totally eliminated when the dTES were used in the Western blot. All these results showed that the deglycosylation of the TES antigens really improves the specificity of the serodiagnosis of human toxocariasis in endemic areas for helminth infections. PMID:26315805

  14. A human centromere antigen (CENP-B) interacts with a short specific sequence in alphoid DNA, a human centromeric satellite

    PubMed Central

    1989-01-01

    We report the interaction between a human centromere antigen and an alphoid DNA, a human centromeric satellite DNA, which consists of 170- bp repeating units. A cloned alphoid DNA fragment incubated with a HeLa cell nuclear extract is selectively immunoprecipitated by the anticentromere sera from scleroderma patients. Immunoprecipitation of the DNA made by primer extension defines the 17-bp segment on the alphoid DNA that is required for formation of DNA-antigen complex. On the other hand, when proteins bound to the biotinylated alphoid DNA carrying the 17-bp motif are recovered by streptavidin agarose and immunoblotted, the 80-kD centromere antigen (CENP-B) is detected. DNA binding experiments for proteins immunoprecipitated with anticentromere serum, separated by gel electrophoresis, and transferred to a membrane strongly suggest that the 80-kD antigen specifically binds to the DNA fragment with the 17-bp motif. The 17-bp motif is termed the "CENP-B box." Alphoid monomers with the CENP-B box are found in all the known alphoid subclasses, with varying frequencies, except the one derived from the Y chromosome so far cloned. These results imply that the interaction of the 80-kD centromere antigen with the CENP-B box in the alphoid repeats may play some crucial role in the formation of specified structure and/or function of human centromere. PMID:2808515

  15. Antigenic properties of pleuropneumonia-like organisms from tissue cell cultures and the human genital area.

    PubMed

    BAILEY, J S; CLARK, H W; FELTS, W R; FOWLER, R C; BROWN, T M

    1961-10-01

    Bailey, Jack S. (George Washington University, Washington, D. C.), Harold W. Clark, William R. Felts, Richard C. Fowler, and Thomas McP. Brown. Antigenic properties of pleuropneumonia-like organisms from tissue cell cultures and the human genital area. J. Bacteriol. 82:542-547. 1961.-Antigens were prepared from several tissue culture and human genital strains of pleuropneumonia-like organisms (PPLO) by a method utilizing continuous agitation of the incubating cultures. Antisera were produced in rabbits by intravenous injection of suspensions of these organisms standardized turbidimetrically. The antigenic properties of the selected strains were compared by agglutination techniques supplemented by a test based upon the inhibition of growth of PPLO by specific antisera.The majority of tissue culture strains of PPLO studied, including contaminants from several HeLa cell lines, appeared to be antigenically similar to the human type 1 strains. However, one strain (Sp-1) from a HeLa cell line was found to be related to the human type 2 PPLO.

  16. Viral Engineering of Chimeric Antigen Receptor Expression on Murine and Human T Lymphocytes.

    PubMed

    Hammill, Joanne A; Afsahi, Arya; Bramson, Jonathan L; Helsen, Christopher W

    2016-01-01

    The adoptive transfer of a bolus of tumor-specific T lymphocytes into cancer patients is a promising therapeutic strategy. In one approach, tumor specificity is conferred upon T cells via engineering expression of exogenous receptors, such as chimeric antigen receptors (CARs). Here, we describe the generation and production of both murine and human CAR-engineered T lymphocytes using retroviruses. PMID:27581020

  17. The natural antibody repertoire of sharks and humans recognizes the potential universe of antigens.

    PubMed

    Adelman, Miranda K; Schluter, Samuel F; Marchalonis, John J

    2004-02-01

    In ancestral sharks, a rapid emergence in the evolution of the immune system occurred, giving jawed-vertebrates the necessary components for the combinatorial immune response (CIR). To compare the natural antibody (NAb) repertoires of the most divergent vertebrates with the capacity to produce antibodies, we isolated NAbs to the same set of antigens by affinity chromatography from two species of Carcharhine sharks and from human polyclonal IgG and IgM antibody preparations. The activities of the affinity-purified anti-T-cell receptor (anti-TCR) NAbs were compared with those of monoclonal anti-TCR NAbs that were generated from a systemic lupus erythematosus patient. We report that sharks and humans, representing the evolutionary extremes of vertebrate species sharing the CIR, have NAbs to human TCRs, Igs, the human senescent cell antigen, and to numerous retroviral antigens, indicating that essential features of the combinatorial repertoire and the capacity to recognize the potential universe of antigens is shared among all jawed-vertebrates.

  18. Leucocyte responses to fighting in the adult male bandicoot rat.

    PubMed

    Ghosh, P R; Sahu, A; Maiti, B R

    1983-01-01

    The effect of fighting stress on blood leucocyte count was studied in the adult male bandicoot rat. Exposure to fighting stress for 3 h induced neutrophilia, eosinopenia, lymphopenia and monocytopenia. The changes were more significant in the subordinate rat than in the dominant animal. It is suggested that leucocyte responses to fighting are perhaps mediated by the adrenal gland in these animals.

  19. Phagocytosis and leucocyte enzymes in protein–calorie malnutrition

    PubMed Central

    Selvaraj, Ratnam J.; Bhat, K. Seetharam

    1972-01-01

    1. Enzymes pertinent to bactericidal activities of leucocytes were assayed in children suffering from protein–calorie malnutrition. 2. Leucocytes obtained from malnourished and control children contained similar activities for glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Granule-bound NADPH oxidase activity was low in leucocytes isolated from malnourished patients and failed to show the phagocytic stimulation which is normally seen in control leucocytes. Further, leucocytes obtained from malnourished patients did not release the acid phosphatase from lysosomes during phagocytosis, unlike those from controls. 3. Treatment of the malnourishment with a diet high in calories and protein resulted in significant increase in the activities of glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and NADPH oxidase and in releasing the acid phosphatase from the lysosomes into the supernatant fraction during phagocytosis. 4. The significance of these enzyme changes are discussed in relation to the increased susceptibility of these patients to infection. PMID:4403728

  20. Inhibition of leucocyte elastase by heparin and its derivatives.

    PubMed Central

    Redini, F; Tixier, J M; Petitou, M; Choay, J; Robert, L; Hornebeck, W

    1988-01-01

    Leucocyte proteinases, e.g. leucocyte elastase and cathepsin G, are inhibited by heparin. The activities of pig pancreatic and Pseudomonas aeruginosa elastases are unaffected by this polysaccharide. Heparin derivatives of known Mr and degree of sulphation were isolated. The inhibition of leucocyte elastase by these oligosaccharides can be classified as tight-binding hyperbolic non-competitive. Ki values ranged from 40 nM to 100 microM and were found to be inversely correlated with the chain length of the oligosaccharides. Desulphated compounds lacked inhibitory potential towards leucocyte elastase. Over-O-sulphated di- and tetra-saccharides are more potent inhibitors than their over-N-sulphated counterparts. It is proposed that the therapeutic use of heparin and its derivatives could be extended to disease states such as emphysema and rheumatoid arthritis, where the role of leucocyte elastase has been clearly established. PMID:3415672

  1. Human carcinoembryonic antigen and biliary glycoprotein can serve as mouse hepatitis virus receptors.

    PubMed Central

    Chen, D S; Asanaka, M; Chen, F S; Shively, J E; Lai, M M

    1997-01-01

    Receptors for murine coronavirus mouse hepatitis virus (MHV) are members of the murine carcinoembryonic antigen (CEA) gene family. Since MHV can also infect primates and cause central nervous system lesions (G. F. Cabirac et al., Microb. Pathog. 16:349-357, 1994; R. S. Murray et al., Virology 188:274-284, 1992), we examined whether human CEA-related molecules can be used by MHV as potential receptors. Transfection of plasmids expressing human carcinoembryonic antigen (hCEA) and human biliary glycoprotein into COS-7 cells, which lack a functional MHV receptor, conferred susceptibility to two MHV strains, A59 and MHV-2. Domain exchange experiments between human and murine CEA-related molecules identified the immunoglobulin-like loop I of hCEA as the region conferring the virus-binding specificity. This finding expands the potential MHV receptors to primate species. PMID:8995701

  2. Human kallikrein 3 (prostate specific antigen) and human kallikrein 5 expression in salivary gland tumors.

    PubMed

    Darling, M R; Tsai, S; Jackson-Boeters, L; Daley, T D; Diamandis, E P

    2006-01-01

    The human kallikrein 5 protein (hK5) is expressed in many normal tissues, most notably in skin, breast, salivary gland and esophagus. It has also been shown to be a potential biomarker for breast, ovarian and testicular cancer. Human kallikrein 3 (hK3; prostate-specific antigen) is the most useful marker for adenocarcinoma of the prostate gland. The aim of this study was to determine whether hK3 and hK5 are expressed in salivary gland tissues and salivary gland tumors (both benign and malignant), in order to compare normal with tumor tissues. Pleomorphic adenomas, adenoid cystic carcinomas, polymorphous low-grade adenocarcinomas, acinic cell carcinomas, mucoepidermoid carcinomas and adenocarcinomas not otherwise specified of both minor and major salivary glands were examined. The results of this study indicate that most salivary gland tumors do not show high levels of expression of hK5. Staining was most prominent in keratinizing epithelia in pleomorphic adenomas. hK3 is not expressed in salivary gland tumors.

  3. Human tumour antigens defined by cytotoxicity and proliferative responses of cultured lymphoid cells

    NASA Astrophysics Data System (ADS)

    Vose, Brent M.; Bonnard, Guy D.

    1982-03-01

    The long-term goal of many laboratories has been to develop cellular reagents having specific reactivity against human tumour cells. Such immune cells should prove useful for defining the antigenicity of human malignancies and may have important therapeutic potential, as has been clearly shown in some animal models1. Here we describe methods of initiating continued lymphocyte cultures (CLC) having specific anti-tumour reactivity using conditioned media containing interleukin-2 (IL-2).

  4. Antigen-induced generation of lyso-phospholipids in human airways

    PubMed Central

    1996-01-01

    mass, (14 kD) human synovial PLA2 and dithiothreitol. Acetyl hydrolase activity also markedly increased in BALF obtained after antigen challenge. This study indicates that high levels of lyso-PLs are present in airways of allergic subjects challenged with antigen and provides evidence for two distinct mechanisms that could induce lyso-PL formation. Future studies will be necessary to determine the ramifications of these high levels of lyso- phospholipids on airway function. PMID:8642333

  5. Serological survey of normal humans for natural antibody to cell surface antigens of melanoma.

    PubMed Central

    Houghton, A N; Taormina, M C; Ikeda, H; Watanabe, T; Oettgen, H F; Old, L J

    1980-01-01

    Sera of 106 normal adult men were tested for antibodies reacting with cell surface antigens of three established lines of cultured malignant melanoma. Positive reactions with a protein A assay for IgG antibodies were extremely rare (1-2%). The frequency of positive reactions with assays for IgM antibodies was higher: 5-15% in immune adherence assays and 55-82% in anti-C3 mixed hemadsorption assays. After low-titered sera and sera reacting with fetal calf serum components, conventional alloantigens, and widely distributed class 3 antigens were excluded, sera from seven individuals (one with IgG antibody and six with IgM antibodies) were selected for detailed analysis. The serum containing the IgG antibody came from a healthy 65-year-old Caucasian man; titers of antibody in his serum ranged from < 1/10 to 1/40,000 in tests with different melanoma cell lines. This IgG antibody identifies a differentiation antigen of melanocytes, provisionally designated Mel 1, that distinguishes two classes of melanomas: 22 melanoma cell lines typed Mel 1+ and 17 types Mel 1-. Mel 1 is expressed by fetal fibroblasts but not adult fibroblasts and can be found on a proportion of cultured epithelial cancer cell lines (5 out of 23) but not on glioma or B-cell lines. The melanoma antigens detected by the naturally occurring IgM antibodies are serologically unrelated to Mel 1 but, like Mel 1, appear to be differentiation antigens that distinguish subsets of melanoma. These IgM antibodies detect antigens that are identical or closely related to the AH antigen, a melanoma surface antigen that was initially defined by autologous antibody in a patient with melanoma. In view of the immunogenicity of both Mel 1 and the AH antigens in humans and their occurrence on more than 50% of melanomas, it remains to be seen whether antibody to these antigens can be elicited by specific vaccination of seronegative melanoma patients and whether this will have an influence on the clinical course of the disease

  6. Cell-surface antigens of melanoma recognized by human monoclonal antibodies.

    PubMed Central

    Yamaguchi, H; Furukawa, K; Fortunato, S R; Livingston, P O; Lloyd, K O; Oettgen, H F; Old, L J

    1987-01-01

    Human monoclonal antibodies (mAbs) were derived from lymph node lymphocytes and peripheral blood lymphocytes (PBL) from patients with melanoma. Four methods for generating human mAbs were compared: fusion with human [LICR-LON-HMy-2 (LICR-2)] or mouse (NS-1) cells; transformation by Epstein-Barr virus (EBV); and EBV transformation followed by NS-1 fusion. NS-1 fusion with lymph node lymphocytes resulted in a higher number of growing hybrids than LICR-2 fusion. Virtually no hybrids were obtained from NS-1 or LICR-2 fusions with PBL. EBV transformed lymphocytes from lymph node and peripheral blood with equal efficiency, and the yield of proliferating cultures for antibody screening was more than 10- to 30-fold greater than that obtained by fusion techniques. However, once antibody-producing cultures had been identified, stability and clonability of EBV-transformed cells were poorer than that of NS-1 hybrid cells. To combine the strengths of both methods, cultures of EBV-transformed cells were fused with NS-1; and hybrid clones were isolated that showed vigorous growth, clonability, and stable antibody secretion. Detailed specificity analysis of the mAbs produced by six of these clones indicated detection of a class 1 (unique) melanoma antigen, a class 3 melanoma antigen, and four ganglioside antigens (GD3, GM3, and two other, as yet uncharacterized, heterophile antigens). Images PMID:3031684

  7. Antibody recognition of an immunogenic influenza hemagglutinin-human leukocyte antigen class II complex

    PubMed Central

    1991-01-01

    The A/Japan/57 influenza hemagglutin (HA) peptide HA 128-145, when bound by human histocompatibility leukocyte antigen-DRw11 cells, is recognized by the human CD4+ T cell clone V1. A rabbit antiserum has been raised against HA 128-145 which recognizes not only the free peptide, but also the HA 128-145/DRw11 complex on a solid matrix, in solution, or on the surface of viable cells. The detection of these complexes on viable cells was shown to be class II specific, DRw11 restricted, and commensurate with the level of DRw11 expression. The identity of DRw11 as the cell surface molecule binding HA 128-145 was confirmed by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and tryptic peptide mapping. Using this antiserum HA 128-145/DRw11 complexes could be detected on the cell surface as soon as 30 min after the peptide was added, and increased up to 24 h. Dissociation kinetics showed these complexes were long-lived, with a half-life of approximately 14 h. This anti-HA peptide antiserum represents the first direct means of studying antigenic peptide-human leukocyte antigen class II complexes on the surface of living cells without the addition of a non-amino acid moiety to the peptide. The properties of this antiserum thus provide the potential to study naturally processed antigenic peptides as well as the mechanism of processing itself in a physiologically relevant system. PMID:2056278

  8. Transient induction of a nuclear antigen unrelated to Epstein-Barr nuclear antigen in cells of two human B-lymphoma lines converted by Epstein-Barr virus.

    PubMed

    Fresen, K O; zur Hausen, H

    1977-01-01

    Infection of cells of the Epstein-Barr virus (EBV)-negative human B-lymphoma lines BJAB and Ramos with EBV preparations from P3HR-1 or B 95-8 cells converted these cells to EBV genome carriers expressing Epstein-Barr nuclear antigen (EBNA) in almost 100% of these cells. Induction of these cells as well as of clones from P3HR-1 EBV-converted BJAB cells with iododeoxyuridine, aminopterin, and hypoxanthine resulted in the appearance of a nuclear antigen in about 1-6% of the cells 1-4 days after induction. The antigen is different from known EBV-induced antigens like EBNA, viral capsid antigen (VCA) or the D- and R-subspecificities of the early antigen (EA) complex. It is demonstrated by indirect immunofluorescence and inactivated after acetone fixation. The antigen was not detectable after induction of uninfected BJAB and Ramos cells nor has it been found in noninduced or induced P3HR-1 and Raji cells. Thus, it appears that EBV-infection mediates the expression of this antigen, for which the name TINA (transiently induced nuclear antigen) is suggested. Sera reacting against TINA generally contained high antibody titers against EBV-induced EA. Only a limited number of highly EA-reactive sera, however, were also positive for TINA. Among 200 sera tested thus far, TINA reactivity was most frequently observed in sera of patients with nasopharyngeal carcinoma (7 out of 28), in sera of the only two patients with immunoblastoma tested and occasionally in sera from patients with Hodgkin's disease and chronic lymphatic leukemia. Among 70 sera from nontumor patients, TINA reactivity was observed three times: two patients suffered from "chronic" infectious mononucleosis, the other revealed persistent splenomegaly. PMID:189313

  9. CTLA4 mediates antigen-specific apoptosis of human T cells.

    PubMed Central

    Gribben, J G; Freeman, G J; Boussiotis, V A; Rennert, P; Jellis, C L; Greenfield, E; Barber, M; Restivo, V A; Ke, X; Gray, G S

    1995-01-01

    The regulation of T cell-mediated immune responses requires a balance between amplification and generation of effector function and subsequent selective termination by clonal deletion. Although apoptosis of previously activated T cells can be induced by signaling of the tumor necrosis factor receptor family, these molecules do not appear to regulate T-cell clonal deletion in an antigen-specific fashion. We demonstrate that cross-linking of the inducible T-cell surface molecule CTLA4 can mediate apoptosis of previously activated human T lymphocytes. This function appears to be antigen-restricted, since a concomitant signal T-cell receptor signal is required. Regulation of this pathway may provide a novel therapeutic strategy to delete antigen-specific activated T cells. Images Fig. 3 PMID:7846057

  10. Correlated analysis of cellular DNA, membrane antigens and light scatter of human lymphoid cells

    SciTech Connect

    Braylan, R.C.; Benson, N.A.; Nourse, V.; Kruth, H.S.

    1982-03-01

    Flow cytometric correlated analysis of membrane antigens, DNA, and light scatter was performed on human lymphoid cells using fluorescein (FITC)-conjugated antibodies to label B- and T-cell antigens and propidium iodide (PI) to stain DNA after ethanol fixation and RNase treatment. A FACS II flow cytometer was modified to obtain digitized measurements of two color fluorescence and light scatter emissions, simultaneously. Software was written to allow single parameter analysis or correlated analysis of any two of the three parameters acquired. Ethanol fixation preserved FITC surface labeling for at least 15 weeks, but produced marked changes in light scatter. No changes in FITC distributions were observed after RNase treatment and PI staining, and the presence of FITC labeling did not affect DNA distributions. Within heterogeneous cell populations, the DNA distribution of cell subpopulations identified by a membrane antigen was clearly demonstrated.

  11. Immunoregulatory adherent cells in human tuberculosis: radiation-sensitive antigen-specific suppression by monocytes

    SciTech Connect

    Kleinhenz, M.E.; Ellner, J.J.

    1985-07-01

    In human tuberculosis, adherent mononuclear cells (AMC) selectively depress in vitro responses to the mycobacterial antigen tuberculin purified protein derivative (PPD). The phenotype of this antigen-specific adherent suppressor cell was characterized by examining the functional activity of adherent cells after selective depletion of sheep erythrocyte-rosetting T cells or OKM1-reactive monocytes. Adherent cell suppression was studied in the (/sup 3/H)thymidine-incorporation microculture assay by using T cells rigorously depleted of T cells with surface receptors for the Fc portion of IgG (T gamma cells) as antigen-responsive cells. PPD-induced (/sup 3/H)thymidine incorporation by these non gamma T cells was uniformly reduced (mean, 42% +/- 10% (SD)) when autologous AMC were added to non gamma T cells at a ratio of 1:2. Antigen-specific suppression by AMC was not altered by depletion of sheep erythrocyte-rosetting T cells or treatment with indomethacin. However, AMC treated with OKM1 and complement or gamma irradiation (1,500 rads) no longer suppressed tuberculin responses in vitro. These studies identify the antigen-specific adherent suppressor cell in tuberculosis as an OKM1-reactive, non-erythrocyte-rosetting monocyte. The radiosensitivity of this monocyte immunoregulatory function may facilitate its further definition.

  12. Internalization and re-expression of antigens of human melanoma cells following exposure to monoclonal antibody

    SciTech Connect

    Wang, B.S.; Lumanglas, A.L.; Silva, J.; Ruszala-Mallon, V.; Durr, F.E.

    1987-04-15

    Modulation of the surface membrane of human Sk-Mel-28 melanoma cells by monoclonal antibody (MoAb) 96.5 recognizing p97 determinants was examined using direct radioimmunoassay and indirect fluorescent antibody-staining techniques. It was determined that the majority of /sup 111/In-labeled antibody that remained associated with cells after a 24-hr incubation at 37 degrees C had been internalized because MoAb 96.5 was no longer visible on the cell surface. A second treatment of these cells with the same antibody 24 hr later not only increased the cell-associated radioactivity, reflecting an increase of total antibody bound, but also rendered these cells membrane immunofluorescent again, indicating the re-expression of surface antigens. Autoradiographs of the electrophoretically analyzed membrane components of Sk-Mel-28 cells further demonstrated the appearance of newly synthesized 97-kDa proteins that were immunoprecipitable with MoAb 96.5. Taken together, the present findings suggest that p97 antigens undergo endocytosis in Sk-Mel-28 cells following exposure to MoAb 96.5. However, the same antigens were regenerated and expressed on the cell surface within a period of 24 hr. The re-expression of tumor cell surface antigen following initial internalization of the MoAb-antigen complex may have implications for diagnosis and therapy.

  13. Development and Characterization of a New Antipeptide Monoclonal Antibody Directed to Human CD20 Antigen.

    PubMed

    Habibi-Anbouhi, Mahdi; Azadmanesh, Kayhan; Behdani, Mahdi; Hajizadeh-Saffar, Ensiyeh; Vahabpour, Rouhollah; Shokrgozar, Mohammad Ali

    2015-09-01

    The rapid expansion of immunotherapeutic approaches for treatment of various diseases, including cancers, has been greatly facilitated by the invention of new generation of antibodies. Clinical studies have indicated that anti-CD20 mAb-based therapies represent an effective treatment for various diseases with overexpression of CD20 on their cell surface, such as non-Hodgkin's lymphoma, hemolytic anemia, as well as autoimmune diseases like rheumatoid arthritis. Technically, due to a short extra membrane domain, the recombinant CD20 protein is a difficult antigen to raise immune responses. In search for new monoclonal antibodies, the authors used an antigenic polypeptide, which yielded numbers of new binders that may lead to production of anti-CD20 antibodies, with improved diagnostic or clinical attributes. Mice were immunized with extra membrane loop of human CD20 (exCD20) polypeptide. The exCD20 antigen showed a desired immune response and was able to develop a monoclonal antibody, 3B4C10, which reacted well with peptide antigen as well as native antigen on the surface of Raji B-cell line. The antibody 3B4C10 with a balanced K(on) and K(off) may be applicable in the construction of affinity columns or beads for isolation and purification of CD20-positive cells and cancer stem cells. PMID:26352927

  14. Human macrophages can express the Hodgkin's cell-associated antigen Ki-1 (CD30).

    PubMed Central

    Andreesen, R.; Brugger, W.; Löhr, G. W.; Bross, K. J.

    1989-01-01

    The normal precursor of the neoplastic cell in Hodgkin's lymphoma is still unknown. Previous reports on the expression of the Hodgkin's cell-associated antigen Ki-1, CD30, on normal cells have been limited to activated lymphocytes. This study demonstrates, however, that cells of the macrophage lineage also are able to express the Ki-1 antigen. The Ki-1 antigen is absent from normal blood monocytes but expressed on up to 85% of macrophage-type cells developed during subsequent in vitro differentiation on Teflon membranes. Unlike other maturation-associated antigens, Ki-1 is found only at late stages of the macrophage primary cultures. Its expression can be enhanced by human interferon-gamma in a fashion similar to that of HLA-DR molecules. In addition, freshly explanted tumor cells from three patients with histopathologic and clinical features consistent with the diagnosis of true histiocytic lymphoma or malignant histiocytosis as well as the permanent cell line SU-DHL-1 could be demonstrated to express the Ki-1 antigen. The phenotype of histiocytic malignancy was further evaluated to be HLA-DR+MAX.26+CD25+-EMA+OKT9+Ki-1+. The results could indicate either that Hodgkin's lymphoma may arise not only from the lymphocyte but also from the macrophage lineage or may emphasize a macrophage involvement in the pathogenesis of this disease. Images Figure 3 PMID:2536522

  15. Detection of Goodpasture antigen in fractions prepared from collagenase digests of human glomerular basement membrane.

    PubMed Central

    Fish, A J; Lockwood, M C; Wong, M; Price, R G

    1984-01-01

    Preparations of human glomerular basement membrane (GBM) were digested with collagenase, and a Goodpasture (GP) antigen rich pool from gel filtration column runs was identified by antibody inhibition radioimmunoassay. The components of the GP antigen pool were separated on polyacrylamide gels, and transferred to nitrocellulose sheets by the 'western' blotting technique. The blots were separately reacted with thirteen GP sera as primary antibody, followed by peroxidase labelled goat anti-human IgG and revealed 45-50K (two bands) and 25-28K (one-three bands) components. No corresponding reactivity was observed using convalescent GP sera or other control sera (normal human serum, rapidly progressive glomerulonephritis with or without pulmonary haemorrhage, and lupus erythematosus) as primary antibody. Images Fig. 3 PMID:6319059

  16. Tracing antigen signatures in the human IgE repertoire

    PubMed Central

    Marth, Katharina; Novatchkova, Maria; Focke-Tejkl, Margarete; Jenisch, Stefan; Jäger, Siegfried; Kabelitz, Dieter; Valenta, Rudolf

    2010-01-01

    Allergen recognition by IgE antibodies is a key event in allergic inflammation. In this study, the IgE IGHV repertoires of individuals with allergy to the major birch pollen allergen, Bet v 1, were analyzed over a four years period of allergen exposure by RT-PCR and sequencing of cDNA. Approximately half of the IgE transcripts represented non-redundant sequences, which belonged to seventeen different IGHV genes. Most variable regions contained somatic mutations but also non-mutated sequences were identified. There was no evidence for relevant increases of somatic mutations over time of allergen exposure. Highly similar IgE variable regions were found after four years of allergen exposure in the same and in genetically non-related individuals. Our results indicate that allergens select and shape a limited number of similar IgE variable regions in the human IgE repertoire. PMID:20573403

  17. S-antigen. Identification of human T-cell lymphocyte proliferation sites.

    PubMed

    Vrabec, T R; Reber, R N; Magargal, L E; Donoso, L A

    1990-10-01

    Immune responses to normal retinal proteins, including S-antigen, have been demonstrated in patients with a variety of retinal disorders, as well as in those who have received panretinal laser photocoagulation. T-cell lymphocytes (T cells) have been implicated in the pathogenesis of several ocular inflammatory diseases of possible autoimmune etiology. We used synthetic peptides that correspond to the amino acid sequence of S-antigen in lymphocyte proliferation assays to identify specific sites in the molecule recognized by human T cells. Ten patients with type II diabetes were studied before and after initial panretinal laser photocoagulation for proliferative diabetic retinopathy. T-cell responses, expressed as a stimulation index, to S-antigen and peptides were negative in all patients before treatment. Three weeks after panretinal laser photocoagulation, eight of 10 assays were positive (stimulation index greater than 2; P less than .01) when lymphocytes were stimulated with peptide BSA(273-292); six of nine were positive (P less than .01) with peptide BSA(303-332); and six of six were positive (P less than .001) with peptide BSA(343-362). Our study identifies several specific sites in S-antigen that elicit human immune responses. The implications of these findings with regard to the pathogenesis and treatment of autoimmune uveitis are discussed. PMID:2222280

  18. Uncommon structural motifs dominate the antigen binding site in human autoantibodies reactive with basement membrane collagen.

    PubMed

    Foster, Mary H; Buckley, Elizabeth S; Chen, Benny J; Hwang, Kwan-Ki; Clark, Amy G

    2016-08-01

    Autoantibodies mediate organ destruction in multiple autoimmune diseases, yet their origins in patients remain poorly understood. To probe the genetic origins and structure of disease-associated autoantibodies, we engrafted immunodeficient mice with human CD34+ hematopoietic stem cells and immunized with the non-collagenous-1 (NC1) domain of the alpha3 chain of type IV collagen. This antigen is expressed in lungs and kidneys and is targeted by autoantibodies in anti-glomerular basement membrane (GBM) nephritis and Goodpasture syndrome (GPS), prototypic human organ-specific autoimmune diseases. Using Epstein Barr virus transformation and cell fusion, six human anti-alpha3(IV)NC1 collagen monoclonal autoantibodies (mAb) were recovered, including subsets reactive with human kidney and with epitopes recognized by patients' IgG. Sequence analysis reveals a long to exceptionally long heavy chain complementarity determining region3 (HCDR3), the major site of antigen binding, in all six mAb. Mean HCDR3 length is 25.5 amino acids (range 20-36), generated from inherently long DH and JH genes and extended regions of non-templated N-nucleotides. Long HCDR3 are suited to forming noncontiguous antigen contacts and to binding recessed, immunologically silent epitopes hidden from conventional antibodies, as seen with self-antigen crossreactive broadly neutralizing anti-HIV Ig (bnAb). The anti-alpha3(IV)NC1 collagen mAb also show preferential use of unmutated variable region genes that are enriched among human chronic lymphocytic leukemia antibodies that share features with natural polyreactive Ig. Our findings suggest unexpected relationships between pathogenic anti-collagen Ig, bnAb, and autoreactive Ig associated with malignancy, all of which arise from B cells expressing unconventional structural elements that may require transient escape from tolerance for successful expansion. PMID:27450516

  19. Experimental studies of a vaccine formulation of recombinant human VEGF antigen with aluminum phosphate.

    PubMed

    Pérez Sánchez, Lincidio; Morera Díaz, Yanelys; Bequet-Romero, Mónica; Ramses Hernández, Gerardo; Rodríguez, Yadira; Castro Velazco, Jorge; Puente Pérez, Pedro; Ayala Avila, Marta; Gavilondo, Jorge V

    2015-01-01

    CIGB-247 is a cancer vaccine that is a formulation of a recombinant protein antigen representative of the human vascular endothelial growth factor (VEGF) with a bacterially-derived adjuvant (VSSP). The vaccine has shown an excellent safety profile in mice, rats, rabbits, not-human primates and in recent clinical trials in cancer patients. Response to the vaccine is characterized by specific antibody titers that neutralize VEGF/VEGFR2 binding and a cytotoxic tumor-specific response. To expand our present anti-VEGF active immunotherapy strategies, we have now studied in mice and non-human primates the effects of vaccination with a formulation of our recombinant VEGF antigen and aluminum phosphate adjuvant (hereafter denominated CIGB-247-A). Administered bi-weekly, CIGB-247-A produces high titers of anti-VEGF IgG blocking antibodies in 2 mice strains. Particularly in BALB/c, the treatment impaired subcutaneous F3II mammary tumor growth and reduced the number of spontaneous lung macro metastases, increasing animals' survival. Spleen cells from specifically immunized mice directly killed F3II tumor cells in vitro. CIGB-247-A also showed to be immunogenic in non-human primates, which developed anti-VEGF blocking antibodies and the ability for specific direct cell cytotoxic responses, all without impairing the healing of deep skin wounds or other side effect. Our results support consideration of aluminum phosphate as a suitable adjuvant for the development of new vaccine formulations using VEGF as antigen.

  20. Human peripheral blood monocytes display surface antigens recognized by monoclonal antinuclear antibodies.

    PubMed Central

    Holers, V M; Kotzin, B L

    1985-01-01

    We used monoclonal anti-nuclear autoantibodies and indirect immunofluorescence to examine normal human peripheral blood mononuclear leukocytes for the presence of cell surface nuclear antigens. Only one monoclonal anti-histone antibody (MH-2) was found to bind to freshly isolated PBL, staining approximately 10% of large cells. However, after cells were placed into culture for 16-24 h, a high percentage (up to 60%) of large-sized cells were recognized by an anti-DNA (BWD-1) and several different antihistone monoclonal antibodies (BWH-1, MH-1, and MH-2). These antibodies recognize separate antigenic determinants on chromatin and histones extracted from chromatin. None of the monoclonal autoantibodies appeared to bind to a significant percentage of cells of relatively small cell size, either before or after culture. The histone antigen-positive cells were viable, and the monoclonal antibodies could be shown to be binding to the cell surface and not to the nucleus. Further experiments, including those using aggregated Ig to block antibody binding, strongly indicated that anti-histone antibody binding was not Fc receptor mediated. Using monoclonal antibodies specific for monocytes and T cells, and complement-mediated cytotoxicity, the cells bearing histone antigens were shown to be primarily monocytes. The appearance of histone and DNA antigen-positive cells was nearly completely inhibited by the addition of low concentrations (0.25 micrograms/ml) of cycloheximide at initiation of the cultures. In contrast, little effect on the percentage of positive cells was detected if cells were exposed to high doses of gamma irradiation before culture. These data further support the existence of cell surface nuclear antigens on selected cell subsets, which may provide insight into the immunopathogenesis of systemic lupus erythematosus and related autoimmune diseases. Images PMID:3876357

  1. Chromosomal localization of the gene for human B-cell antigen CD40

    SciTech Connect

    Ramesh, N.; Geha, R. ); Ramesh, V.; Gusella, J.F. )

    1993-05-01

    CD40 is a surface glycoprotein expressed on all human B lymphocytes and plays an important role in B-cell development, growth, and differentiation. Anti-CD40 monoclonal antibodies cause isotype switching in B cells treated with IL-4. CD40 is a member of a family of proteins that include low-affinity nerve growth factor receptor, TNF receptor, and the antigen Fas. The ligand for CD40 had been recently identified and had been assigned to the X chromosome. Using a panel of human-rodent somatic cell hybrids, the authors now show that CD40 maps to human chromosome 20.

  2. Human thymic epithelial primary cells produce exosomes carrying tissue-restricted antigens.

    PubMed

    Skogberg, Gabriel; Lundberg, Vanja; Berglund, Martin; Gudmundsdottir, Judith; Telemo, Esbjörn; Lindgren, Susanne; Ekwall, Olov

    2015-09-01

    Exosomes are nano-sized vesicles released by cells into the extracellular space and have been shown to be present in thymic tissue both in mice and in humans. The source of thymic exosomes is however still an enigma and hence it is not known whether thymic epithelial cells (TECs) are able to produce exosomes. In this work, we have cultured human TECs and isolated exosomes. These exosomes carry tissue-restricted antigens (TRAs), for example, myelin basic protein and desmoglein 3. The presence of TRAs indicates a possible role for thymic epithelium-derived exosomes in the selection process of thymocytes. The key contribution of these exosomes could be to disseminate self-antigens from the thymic epithelia, thus making them more accessible to the pool of maturing thymocytes. This would increase the coverage of TRAs within the thymus, and facilitate the process of positive and negative selection.

  3. Recognition of Major Histocompatibility Complex Antigens on Cultured Human Biliary Epithelial Cells by Alloreactive Lymphocytes

    PubMed Central

    Saidman, Susan L.; Duquesnoy, Rene J.; Zeevi, Adriana; Fung, John J.; Starzl, Thomas E.; Demetris, A. Jake

    2010-01-01

    We have developed an in vitro system to study the interactions between biliary epithelium and lymphocytes using cultured human biliary epithelial cells. No class II antigens were detected by immunoperoxidase staining of the normal biliary epithelial cells, but alloactivated lymphocyte culture supernatants were able to induce class II expression. The activity of the supernatants was blocked with an anti-γ-interferon monoclonal antibody. In addition, recombinant human γ-interferon alone induced the expression of class II antigens and increased the intensity of class I staining of cultured biliary epithelial cells. Biliary epithelial cell–induced proliferation of alloreactive T lymphocytes demonstrated that the major histocompatibility complex molecules carry functional lymphocyte-activating determinants. The recognition of major histocompatibility complex determinants was confirmed by monoclonal antibody–blocking studies and by stimulation of an alloreactive T-cell clone. However, the biliary epithelial cells were much less potent stimulators than arterial endothelial cells tested in the same assay system. PMID:1704868

  4. Reactivity of human Lyme borreliosis sera with a 39-kilodalton antigen specific to Borrelia burgdorferi.

    PubMed Central

    Simpson, W J; Schrumpf, M E; Schwan, T G

    1990-01-01

    Borrelia burgdorferi is the causative agent of Lyme borreliosis, a spirochetal illness with a variety of acute clinical manifestations that may lead to debilitating neurological and arthritic complications. Diagnosis is difficult because symptoms mimic a variety of unrelated clinical conditions, spirochetes cannot always be isolated from infected patients, and current serological tests are frequently inconclusive because of the presence of cross-reacting non-B. burgdorferi antibodies. To identify antigens specific to B. burgdorferi that could be used in the serodiagnosis of Lyme borreliosis, we screened a Borrelia DNA expression library in Escherichia coli for antigens reactive with human Lyme borreliosis sera. One clone carried a 6.3-kilobase EcoRI chromosomal fragment (pSPR33), which encoded two species-specific antigens with molecular masses of 28 (P28) and 39 (P39) kilodaltons (kDa). These two antigens were immunologically distinct from OspA, OspB, and the 41-kDa flagellin. Ninety-four serum specimens from patients having Lyme borreliosis were tested for reactivity with P39. All of 33 the serum specimens with immunofluorescence assay titers of greater than or equal to 1:256, 13 of 17 serum specimens with titers of 1:128, and 14 of 44 serum specimens with titers of less than or equal to 1:64 reacted with P39. Notably, many sera reactive to P39 did not appear to react with the 41-kDa flagellin. Therefore, antibody to P39 could be mistaken for antibody to the 41-kDa flagellin in tests of human sera by Western blot (immunoblot). Twenty-five control serum specimens, which included sera from syphilitic, relapsing fever, and amyotrophic lateral sclerosis patients as well as from 10 normal individuals, did not react to P39. Our data suggest that P39 may be a useful antigen for the serological confirmation of Lyme borreliosis. Images PMID:2380361

  5. Restricted expression of LW antigen on subsets of human B and T lymphocytes.

    PubMed

    Oliveira, O L; Thomas, D B; Lomas, C G; Tippett, P

    1984-01-01

    NIM-M8 is a monoclonal IgM antibody, specific for the LWab antigen as shown by its reaction with red cells of all donors except those lacking LWa, LWb and LWab. Indirect immunofluorescent staining and cell sorter analyses have shown that LWab is present on a subpopulation of human lymphocytes. Cell fractionation studies indicate that subsets of both B and T cells express LWab and it may, therefore, provide a further marker for heterogeneity in these lymphocyte populations. PMID:6443217

  6. In vitro interactions between Neoparamoeba spp. and salmonid leucocytes; The effect of parasite sonicate on anterior kidney leucocyte function

    USGS Publications Warehouse

    Gross, K.; Alcorn, S.; Murray, A.; Morrison, R.; Nowak, B.

    2006-01-01

    Sonicated Neoparamoeba spp. (Nspp) did not affect the in vitro respiratory burst response of leucocytes isolated from Atlantic salmon Salmo salar, rainbow trout Oncorhynchus mykiss and chinook salmon Oncorhynchus tshawytscha anterior kidneys (P > 0.05). Atlantic salmon and chinook salmon leucocytes pre-incubated with the parasites, however, responded to phorbol myristate acetate (PMA) stimulation with a greater response compared to cells incubated with PMA on its own (P < 0.05). Sonicated Nspp was not chemo-attractive for anterior kidney leucocytes isolated from all three fish species. ?? 2006 The Fisheries Society of the British Isles.

  7. Antigen-Specific Acquired Immunity in Human Brucellosis: Implications for Diagnosis, Prognosis, and Vaccine Development

    PubMed Central

    Cannella, Anthony P.; Tsolis, Renee M.; Liang, Li; Felgner, Philip L.; Saito, Mayuko; Sette, Alessandro; Gotuzzo, Eduardo; Vinetz, Joseph M.

    2012-01-01

    Brucella spp., are Gram negative bacteria that cause disease by growing within monocyte/macrophage lineage cells. Clinical manifestations of brucellosis are immune mediated, not due to bacterial virulence factors. Acquired immunity to brucellosis has been studied through observations of naturally infected hosts (cattle, goats), mouse models (mice), and human infection. Even though Brucella spp. are known for producing mechanisms that evade the immune system, cell-mediated immune responses drive the clinical manifestations of human disease after exposure to Brucella species, as high antibody responses are not associated with protective immunity. The precise mechanisms by which cell-mediated immune responses confer protection or lead to disease manifestations remain undefined. Descriptive studies of immune responses in human brucellosis show that TH1 (interferon-γ-producing T cells) are associated with dominant immune responses, findings consistent with animal studies. Whether these T cell responses are protective, or determine the different clinical responses associated with brucellosis is unknown, especially with regard to undulant fever manifestations, relapsing disease, or are associated with responses to distinct sets of Brucella spp. antigens are unknown. Few data regarding T cell responses in terms of specific recognition of Brucella spp. protein antigens and peptidic epitopes, either by CD4+ or CD8+ T cells, have been identified in human brucellosis patients. Additionally because current attenuated Brucella vaccines used in animals cause human disease, there is a true need for a recombinant protein subunit vaccine for human brucellosis, as well as for improved diagnostics in terms of prognosis and identification of unusual forms of brucellosis. This review will focus on current understandings of antigen-specific immune responses induced Brucella peptidic epitopes that has promise for yielding new insights into vaccine and diagnostics development, and for

  8. Stability of free and complexed human immunodeficiency virus type 1 antigen at 4 degrees C and at room temperature.

    PubMed Central

    Griffith, B P; Garner, R B; Chacko, T M

    1995-01-01

    Free and immune-complex-dissociated (ICD) human immunodeficiency virus type 1 (HIV-1) antigenemias in serum specimens stored at room temperature (RT) and 4 degrees C for 1 to 35 days were evaluated. At all time points examined, there was no significant loss in detectable levels of ICD HIV-1 antigen at either RT or 4 degrees C. Free HIV-1 antigen was not stable in serum samples stored at RT for more than 2 days but was stable in samples stored at 4 degrees C for up to 4 days. Loss of free antigen occurred more rapidly in samples with high antigen content at baseline. Use of the ICD antigen assay allowed accurate quantitation of antigen in samples stored at RT or 4 degrees C for as long as 1 month. PMID:7615753

  9. Optimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins.

    PubMed

    Zhong, Nan; Loppnau, Peter; Seitova, Alma; Ravichandran, Mani; Fenner, Maria; Jain, Harshika; Bhattacharya, Anandi; Hutchinson, Ashley; Paduch, Marcin; Lu, Vincent; Olszewski, Michal; Kossiakoff, Anthony A; Dowdell, Evan; Koide, Akiko; Koide, Shohei; Huang, Haiming; Nadeem, Vincent; Sidhu, Sachdev S; Greenblatt, Jack F; Marcon, Edyta; Arrowsmith, Cheryl H; Edwards, Aled M; Gräslund, Susanne

    2015-01-01

    We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in vivo biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 in vivo biotinylated antigen domains. High-affinity Fabs (<20nM) were obtained for 196. We constructed and optimized a new expression vector to produce in vivo biotinylated Fabs in E. coli. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. In summary, we have optimized each step of the pipeline to produce recombinant antibodies, significantly increasing both efficiency and yield, and also showed that these Fabs and IgGs can be generally useful for chromatin immunoprecipitation (ChIP) protocols. PMID:26437229

  10. Excretory-secretory antigenic components of Paragonimus heterotremus recognized by infected human sera.

    PubMed Central

    Maleewong, W; Wongkham, C; Intapan, P; Pariyanonda, S; Morakote, N

    1992-01-01

    Antigenic components of Paragonimus heterotremus metabolic products were revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis of sera from patients with P. heterotremus infection, from patients with other illnesses, and from healthy adults. By SDS-PAGE, it was found that the metabolic products comprised more than eight major polypeptides. Immunoblot analysis revealed 11 components which were strongly recognized by paragonimiasis antisera. These antigenic components had molecular masses ranging from less than 12.3 kDa to 144 kDa. One antigenic band of 31.5 kDa was found to give a consistent reaction with paragonimiasis antisera (97% sensitivity). Of the other patient sera, only sera from patients with Fasciola sp. infection reacted with antigenic bands of 56, 38, and 18.5 kDa. The present findings suggest that the 31.5-kDa component is sensitive and specific for the diagnosis of human P. heterotremus paragonimiasis. Images PMID:1500515

  11. Optimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins

    PubMed Central

    Zhong, Nan; Loppnau, Peter; Seitova, Alma; Ravichandran, Mani; Fenner, Maria; Jain, Harshika; Bhattacharya, Anandi; Hutchinson, Ashley; Paduch, Marcin; Lu, Vincent; Olszewski, Michal; Kossiakoff, Anthony A.; Dowdell, Evan; Koide, Akiko; Koide, Shohei; Huang, Haiming; Nadeem, Vincent; Sidhu, Sachdev S.; Greenblatt, Jack F.; Marcon, Edyta; Arrowsmith, Cheryl H.; Edwards, Aled M.; Gräslund, Susanne

    2015-01-01

    We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in vivo biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 in vivo biotinylated antigen domains. High-affinity Fabs (<20nM) were obtained for 196. We constructed and optimized a new expression vector to produce in vivo biotinylated Fabs in E. coli. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. In summary, we have optimized each step of the pipeline to produce recombinant antibodies, significantly increasing both efficiency and yield, and also showed that these Fabs and IgGs can be generally useful for chromatin immunoprecipitation (ChIP) protocols. PMID:26437229

  12. Alphoid satellite DNA is tightly associated with centromere antigens in human chromosomes throughout the cell cycle

    SciTech Connect

    Masumoto, Hiroshi; Sugimoto, Kenji; Okazaki, Tuneko )

    1989-03-01

    In this study, the authors have examined a DNA element specific to the centromere domain of human chromosomes. Purified HeLa chromosomes were digested with the restriction enzyme Sau3AI and fractionated by sedimentation through a sucrose gradient. Fractions showing antigenicity to anticentromere (kinetochore) serum obtained from a scleroderma CREST patient were used to construct a DNA library. From this library they found one clone which has specifically hybridized to the centromere domain of metaphase chromosomes using a biotinylated probe DNA and FITC-conjugated avidin. The clone contained a stretch of alphoid DNA dimer. To determine precisely the relative location of the alphoid DNA stretch and the centromere antigen, a method was developed to carry out in situ hybridization of DNA and indirect immunofluorescent staining of antigen on the same cell preparation. Using this method, they have found perfect overlapping of the alphoid DNA sites with the centromere antigen in both metaphase chromosomes and nuclei at various stages in the cell cycle. They have also observed this exact correlation at the attachment sites of artificially extended sister chromatids. These results suggest the possibility that alphoid DNA repeats are a key component of kinetochore structure.

  13. Surface antigens of melanoma and melanocytes. Specificity of induction of Ia antigens by human gamma-interferon

    PubMed Central

    1984-01-01

    IFN-gamma is known to induce expression of Ia antigens on a variety of cell types. In the present study, this activity of IFN-gamma has been analyzed with a panel of 36 melanoma cell lines, normal melanocytes, and 97 cell lines representing a range of other differentiation lineages. 55% of the melanoma cell lines express Ia antigens in a constitutive manner without IFN-gamma induction. Of the 16 Ia-melanoma lines, 13 could be induced to express Ia antigens by IFN-gamma, whereas three were noninducible. Melanocytes, which do not normally express Ia antigens, are converted to Ia expression by IFN-gamma. Ia antigens expressed constitutively or after IFN-gamma induction were identified with antibodies detecting monomorphic and allomorphic products of DR and DC loci. IFN-gamma appeared to be unique in its ability to induce Ia expression on melanoma and melanocytes; 14 other agents (including IFN-alpha and IFN-beta) known to influence growth or differentiation did not have Ia-inducing activity. Equally striking is the restriction of antigenic changes following IFN-gamma induction to HLA-associated products; of the 38 systems of cell surface antigens examined, only HLA- A,B,C, beta 2m, and Ia antigens were affected. A variety of other Ia- cell types were shown to be Ia-inducible by IFN-gamma; these included established lines of breast, colon, pancreas, bladder, kidney, ovary, and brain cancers, and cultures of normal fibroblasts, kidney epithelia, and epidermal keratinocytes. In contrast, three tumor types, teratocarcinoma, choriocarcinoma, and neuroblastoma, were not inducible for Ia expression, even though IFN-gamma could induce expression of HLA- A,B,C products. The broad representation of Ia antigens on most somatic cell types expressed either constitutively or after IFN-gamma can be viewed in an immunological context (antigen presentation/immune regulatory signals) or could indicate that Ia products have functions other than those related to immune reactions. PMID

  14. Characteristics of the transport of ascorbic acid into leucocytes

    SciTech Connect

    Raghoebar, M.; Huisman, J.A.M.; van den Berg, W.B.; van Ginneken, C.A.M.

    1987-02-02

    The degree and the mode of association of (/sup 14/C)-ascorbic acid with leucocytes are examined. The degree of association of ascorbic acid with polymorphonuclear leucocytes (1-3 %) is dependent on cell type, extracellular concentration of ascorbic acid, incubation temperature, intactness of the cells and the extracellular pH. All experiments are performed according to strict protocols as these compounds are labile in aqueous solutions. Further it is noticed that in all experiments an outward gradient of leucocyte endogenic ascorbic acid exists. The results suggest that the association process comprises at least one saturable pathway. The activation of polymorphonuclear leucocytes by phorbol myristate acetate increases the accumulation of ascorbic acid threefold. 30 references, 7 figures, 3 tables.

  15. Generation of a human anti-idiotypic antibody that mimics the GD2 antigen.

    PubMed

    Saleh, M N; Stapleton, J D; Khazaeli, M B; LoBuglio, A F

    1993-09-15

    In a phase 1 trial, patients with metastatic melanoma received the anti-GD2 murine mAb 14G2a. All patients developed human anti-14G2a antibodies including anti-Id antibodies. Peripheral blood MNCs from one such patient were fused with the murine myeloma cell line Ag8. Four human anti-14G2a secreting hybridomas were generated and the mAb product of one of the hybridomas was characterized. The human mAb 4B5 (hu-IgG, lambda) binds to the variable region of murine 14G2a (anti-Id). The 4B5 binds to the antigen-combining site of 14G2a and inhibits its binding to GD2 expressing Mel-21 cells. Rabbits were immunized with the human anti-Id 4B5. Sera from the immunized rabbits demonstrated anti-4B5 antibodies and anti-Mel-21 and anti-GD2 reactivity. Furthermore, rabbit sera competitively inhibited binding of 14G2a to Mel-21 cells. Rabbits immunized with 4B5 developed a DTH response when challenged with 4B5 antibody and Mel-21 cells. These studies demonstrate that the human anti-Id 4B5 mimics the GD2 antigen and is capable of eliciting both a humoral and cellular anti-GD2 immune response. This antibody could be potentially used as a human anti-Id vaccine in patients with malignant melanoma. PMID:8376782

  16. Taenia saginata metacestode antigenic fractions without affinity to concanavalin A are an important source of specific antigens for the diagnosis of human neurocysticercosis.

    PubMed

    Oliveira, Heliana B; Machado, Gleyce A; Mineo, José R; Costa-Cruz, Julia M

    2010-04-01

    Taenia saginata metacestode antigens have been constituted a useful alternative antigen for neurocysticercosis (NC) serodiagnosis, particularly due to an increasing difficulty to obtain Taenia solium homologous antigen. Cross-reactivity with Echinococcus granulosus infection occurs in homologous and heterologous antigens and could be avoided by using different purified methods. The present study evaluated antigen fractions obtained from saline extracts of T. saginata metacestodes purified by affinity chromatography with jacalin or concanavalin A (ConA) lectins to detect IgG antibodies by enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis to diagnose human NC. Serum samples were collected from 142 individuals: 40 of them were diagnosed with NC, 62 presented Taenia sp. and other parasites, and 40 were apparently healthy individuals. The jacalin- and ConA-unbound fractions demonstrated sensitivity and specificity higher than those of bound fractions. Among unbound fractions, ConA demonstrated statistically higher sensitivity and specificity by ELISA (90% and 93.1%, respectively). By immunoblot assay, the 64- to 68-kDa component from the ConA-unbound fraction showed 100% sensitivity and specificity, making this component suitable for use as a specific antigen for diagnosis of NC. To our knowledge, this is the first report showing the relevance of using the unbound ConA fraction of T. saginata metacestodes to diagnose NC. In conclusion, the results obtained herein clearly demonstrate that antigenic fractions without affinity to ConA, obtained from T. saginata metacestodes, are an important source of specific peptides and are efficient in the diagnosis of NC when tested by immunoblot assay.

  17. Primary in vitro generation of cytotoxic cells specific for human minor histocompatibility antigens between HLA-identical siblings.

    PubMed

    Tekolf, W A; Shaw, S

    1984-04-01

    A limiting dilution culture system was developed for the primary in vitro detection of human minor histocompatibility antigens by cytotoxic T lymphocytes (CTL). CTL were generated in primary in vitro culture between two HLA-identical sibling pairs and propagated as stable CTL lines. Population and family studies indicate that these CTL lines recognize minor histocompatibility antigens in an HLA-restricted manner. The antigen recognized by one CTL line is detected on six (out of 37) HLA-B7-positive donors but not on 32 HLA-B7-negative donors. The cytotoxicity of this CTL line is mediated by T3+, T8+ effector cells. The antigen detected by this CTL population is different from all known human minor histocompatibility antigens. The data of this study, like those in the mouse system, suggest that a suppressor cell is diluted out in a limiting dilution culture, which allows the activation of the CTL precursors.

  18. Specific and cross-reacting antigens of Staphylococcus aureus of human and canine origins.

    PubMed

    Live, I

    1985-01-01

    Biotype -specificity of Staphylococcus aureus of human and canine origins has been found to be associated with thermolabile agglutinogens represented in S. aureus strains 17 and 61218, respectively. Both strains also have exhibited a common thermostable antigen. On that basis, absorbed antisera have been developed for the differentiation of S. aureus of the two biotypes. In the present study, still another thermostable agglutinogen was established, shared by strain 17 and some S. aureus strains of canine origin, as represented by S. aureus strain 887. These findings led to modification and enhanced specificity of the serological method of distinguishing S. aureus of the human biotype from S. aureus of the canine biotype. PMID:2578480

  19. Detection of antigens specific for B-lymphoid cultured cell lines with human alloantisera.

    PubMed

    Mann, D L; Abelson, L; Harris, S; Amos, D B

    1975-07-01

    Human sera were tested for cytotoxicity to pairs of long-term tissue-cultured cell lines. Each pair had been derived from the same individual and one of the pairs possessed the characteristics of either "T" or "B" cells. The alloantisera used were HL-A-typing reagents or sera obtained from Amish multiparas. Selected cytotoxicity was found against the B-cell lines by direct testing. Cytotoxicity was abolished by absorption with B-cell line but not by absorption with the T-cell lines. The results suggest that a group of allotypic antigens may be expressed exculsively on human B cells. PMID:1080182

  20. Detection of a human intracisternal A-type retroviral particle antigenically related to HIV

    NASA Technical Reports Server (NTRS)

    Garry, R. F.; Fermin, C. D.; Hart, D. J.; Alexander, S. S.; Donehower, L. A.; Luo-Zhang, H.

    1990-01-01

    Sjogren's syndrome is an autoimmune disease that is characterized by dryness of the mouth and eyes. The loss of salivary and lacrimal gland function is accompanied by lymphocytic infiltration. Because similar symptoms and glandular pathology are observed in certain persons infected with human immunodeficiency virus (HIV), a search was initiated for a possible retroviral etiology in this syndrome. A human intracisternal A-type retroviral particle that is antigenically related to HIV was detected in lymphoblastoid cells exposed to homogenates of salivary tissue from patients with Sjogren's syndrome. Comparison of this retroviral particle to HIV indicates that they are distinguishable by several ultrastructural, physical, and enzymatic criteria.

  1. Antigenic relationship between the animal and human pathogen Pythium insidiosum and nonpathogenic Pythium species.

    PubMed Central

    Mendoza, L; Kaufman, L; Standard, P

    1987-01-01

    Identification of the newly named pathogenic oomycete Pythium insidiosum and its differentiation from other Pythium species by morphologic criteria alone can be difficult and time-consuming. Antigenic analysis by fluorescent-antibody and immunodiffusion precipitin techniques demonstrated that the P. insidiosum isolates that cause pythiosis in dogs, horses, and humans are identical and that they were distinguishable from other Pythium species by these means. The immunologic data agreed with the morphologic data. This indicated that the animal and human isolates belonged to a single species, P. insidiosum. Fluorescent-antibody and immunodiffusion reagents were developed for the specific identification of P. insidiosum. PMID:3121666

  2. Avian leucocyte counting using the hemocytometer

    USGS Publications Warehouse

    Dein, F.J.; Wilson, A.; Fischer, D.; Langenberg, P.

    1994-01-01

    Automated methods for counting leucocytes in avian blood are not available because of the presence of nucleated erythrocytes and thrombocytes. Therefore, total white blood cell counts are performed by hand using a hemocytometer. The Natt and Herrick and the Unopette methods are the most common stain and diluent preparations for this procedure. Replicate hemocytometer counts using these two methods were performed on blood from four birds of different species. Cells present in each square of the hemocytometer were counted. Counting cells in the corner, side, or center hemocytometer squares produced statistically equivalent results; counting four squares per chamber provided a result similar to that obtained by counting nine squares; and the Unopette method was more precise for hemocytometer counting than was the Natt and Herrick method. The Unopette method is easier to learn and perform but is an indirect process, utilizing the differential count from a stained smear. The Natt and Herrick method is a direct total count, but cell identification is more difficult.

  3. Single antigen flow beads for identification of human leukocyte antigen antibody specificities in hypersensitized patients with chronic renal failure

    PubMed Central

    Kılıçaslan-Ayna, Tülay; Özkızılcık-Koçyiğit, Aslı; Güleç, Derya; Pirim, İbrahim

    2016-01-01

    Aims of this study Aims of this study were to identify class I and class II antibodies in highly sensitized patients by flow cytometry single antigen bead (FC-SAB) assay and to evaluate according to donor HLA type in order to increase their kidney transplantation chance. Material and methods We analyzed 60 hypersensitive patients of 351 individuals, who applied to our laboratory for PRA test in November 2013-December 2014. Flow cytometric PRA screening and single antigen bead commercial kits were used for these analyses. Results In our study group, 19 (31.7%) of these patients were male while 41 (68.3%) patients were female. The most common acceptable antigens were A*02 (10.11%), HLA-A*23 (10.11%), HLA-B*38 (8.79%) and HLA-DRB1*03 (7.83%) in hypersensitive patients. The highest antibody reactivity on SAB was observed against HLA-A*25, HLA-B*45, HLA-DRB1*04 and HLA-DRB1*08 antigens. Conclusions The determination of these acceptable and unacceptable antigens may increase their transplantation chance. Pre-transplant HLA antibody identifications provide prognostic information with respect to the determination of patients who are at increased risk of graft loss. PMID:27095928

  4. Generation of Large Numbers of Antigen-Expressing Human Dendritic Cells Using CD14-ML Technology.

    PubMed

    Imamura, Yuya; Haruta, Miwa; Tomita, Yusuke; Matsumura, Keiko; Ikeda, Tokunori; Yuno, Akira; Hirayama, Masatoshi; Nakayama, Hideki; Mizuta, Hiroshi; Nishimura, Yasuharu; Senju, Satoru

    2016-01-01

    We previously reported a method to expand human monocytes through lentivirus-mediated introduction of cMYC and BMI1, and we named the monocyte-derived proliferating cells, CD14-ML. CD14-ML differentiated into functional DC (CD14-ML-DC) upon addition of IL-4, resulting in the generation of a large number of DC. One drawback of this method was the extensive donor-dependent variation in proliferation efficiency. In the current study, we found that introduction of BCL2 or LYL1 along with cMYC and BMI1 was beneficial. Using the improved method, we obtained CD14-ML from all samples, regardless of whether the donors were healthy individuals or cancer patients. In vitro stimulation of peripheral blood T cells with CD14-ML-DC that were loaded with cancer antigen-derived peptides led to the establishment of CD4+ and CD8+ T cell lines that recognized the peptides. Since CD14-ML was propagated for more than 1 month, we could readily conduct genetic modification experiments. To generate CD14-ML-DC that expressed antigenic proteins, we introduced lentiviral antigen-expression vectors and subjected the cells to 2 weeks of culture for drug-selection and expansion. The resulting antigen-expressing CD14-ML-DC successfully induced CD8+ T cell lines that were reactive to CMVpp65 or MART1/MelanA, suggesting an application in vaccination therapy. Thus, this improved method enables the generation of a sufficient number of DC for vaccination therapy from a small amount of peripheral blood from cancer patients. Information on T cell epitopes is not necessary in vaccination with cancer antigen-expressing CD14-ML-DC; therefore, all patients, irrespective of HLA type, will benefit from anti-cancer therapy based on this technology. PMID:27050553

  5. Molecular detection of tumor-associated antigens shared by human cutaneous melanomas and gliomas.

    PubMed Central

    Chi, D. D.; Merchant, R. E.; Rand, R.; Conrad, A. J.; Garrison, D.; Turner, R.; Morton, D. L.; Hoon, D. S.

    1997-01-01

    Both melanocytes and glial cells are derived embryologically from the neural ectoderm. Their malignant transformed counterparts, melanoma and glioma cells, respectively, may share common antigens. Numerous tumor-associated antigens have been identified in melanomas but only a few a gliomas. Using an established reverse transcriptase polymerase chain reaction plus Southern blot assay, we compared the mRNA expression of melanoma-associated antigens (MAAs) of melanomas to brain tumors primarily derived from glial cells. The MAAs studied included tyrosinase (Tyr), tyrosinase-related protein-1 and -2 (TRP-1 and TRP-2), gp100, human melanoma antigen-encoding genes 1 and 3 (MAGE-1 and MAGE-3), and melanotransferrin (p97). Glioblastoma multiforme (n = 21), anaplastic astrocytoma (n = 3), ependymoma (n = 2), meningioma (n = 3), oligodendroglioma (n = 1), and melanoma (n = 12) tumor specimens were assayed for MAA mRNA expression. Glioblastoma multiforme, astrocytoma, and melanoma cell lines were also assayed. We observed that individual MAA mRNAs were expressed in these brain tumors and cell lines at varying frequencies. The melanogenesis-pathway-related MAAs Tyr, TRP-1, TRP-2, and gp100 mRNAs were also expressed at different levels in normal brain tissues but at a much lower frequency than in glioblastoma multiforme and melanoma. MAGE-1 and MAGE-3 mRNA were expressed in different types of tumor specimens and cell lines but never in normal brain tissue. Tumor antigen p97 was expressed in all types of tumors and also in normal brain tissues. These studies demonstrate that melanomas and primary brain tumors express common MAAs and could be exploited in patients with malignant glioma by active specific immunotherapy against these common MAAs. Images Figure 1 Figure 2 Figure 3 PMID:9176405

  6. Glomerular Autoimmune Multicomponents of Human Lupus Nephritis In Vivo (2): Planted Antigens

    PubMed Central

    Bruschi, Maurizio; Galetti, Maricla; Sinico, Renato Alberto; Moroni, Gabriella; Bonanni, Alice; Radice, Antonella; Tincani, Angela; Pratesi, Federico; Migliorini, Paola; Murtas, Corrado; Franceschini, Franco; Trezzi, Barbara; Brunini, Francesca; Gatti, Rita; Tardanico, Regina; Barbano, Giancarlo; Piaggio, Giorgio; Messa, Piergiorgio; Ravani, Pietro; Scolari, Francesco; Candiano, Giovanni; Martini, Alberto; Allegri, Landino

    2015-01-01

    Glomerular planted antigens (histones, DNA, and C1q) are potential targets of autoimmunity in lupus nephritis (LN). However, the characterization of these antigens in human glomeruli in vivo remains inconsistent. We eluted glomerular autoantibodies recognizing planted antigens from laser-microdissected renal biopsy samples of 20 patients with LN. Prevalent antibody isotypes were defined, levels were determined, and glomerular colocalization was investigated. Renal and circulating antibodies were matched, and serum levels were compared in 104 patients with LN, 84 patients with SLE without LN, and 50 patients with rheumatoid arthritis (RA). Autoantibodies against podocyte antigens (anti–α-enolase/antiannexin AI) were also investigated. IgG2 autoantibodies against DNA, histones (H2A, H3, and H4), and C1q were detected in 50%, 55%, and 70% of biopsy samples, respectively. Anti-DNA IgG3 was the unique non-IgG2 anti-DNA deposit, and anti-C1q IgG4 was mainly detected in subepithelial membranous deposits. Anti-H3, anti-DNA, and anti-C1q IgG2 autoantibodies were also prevalent in LN serum, which also contained IgG3 against the antigen panel and anti-C1q IgG4. Serum and glomerular levels of autoantibodies were not strictly associated. High serum levels of all autoantibodies detected, including anti–α-enolase and antiannexin AI, identified LN versus SLE and RA. Anti-H3 and anti–α-enolase IgG2 levels had the most remarkable increase in LN serum and represented a discriminating feature of LN in principal component analysis. The highest levels of these two autoantibodies were also associated with proteinuria>3.5 g/24 hours and creatinine>1.2 mg/dl. Our findings suggest that timely autoantibody characterization might allow outcome prediction and targeted therapies for patients with nephritis. PMID:25398787

  7. Diagnostic significance of soluble human leukocyte antigen-G for gastric cancer.

    PubMed

    Pan, Ying-Qiu; Ruan, Yan-Yun; Peng, Jin-Bang; Han, Qiu-Yue; Zhang, Xia; Lin, Aifen; Yan, Wei-Hua

    2016-04-01

    Human leukocyte antigen-G (HLA-G) is a novel tumor marker. Increased level of soluble HLA-G (sHLA-G) in various tumor types has been reported. However, the potential diagnostic value of sHLA-G with other tumor markers in gastric cancer (GC) diagnosis is yet to be explored. In this study, plasma level of sHLA-G was measured in 81 GC patients, 53 benign gastric disease patients and 77 normal controls by ELISA. The serum levels of alpha fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen 125 (CA125), cancer antigen 19-9 (CA19-9) and cancer antigen 72-4 (CA72-4) were also determined. Data showed that plasma level of sHLA-G in GC was dramatically increased compared with normal controls and benign gastric disease patients (both p<0.001). The AUC for sHLA-G was 0.730 (p<0.001), superior to serum AFP, CEA, CA125, CA19-9 and CA72-4. After evaluating three cut-offs of sHLA-G, we concluded sHLA-G (cut-off at 128U/ml) plus CA125 in two-biomarker panel test and CA125 plus CA199 plus sHLA-G or CA125 plus CA724 plus sHLA-G in three-biomarker panel test were better choices for GC discrimination. Our findings indicated that sHLA-G was a potential biomarker for GC diagnosis and the combination of sHLA-G with CA125, CA19-9 and CA72-4 can improve the clinical screening and diagnosis for GC.

  8. Antigen-specific activation and cytokine-facilitated expansion of naive, human CD8+ T cells

    PubMed Central

    Wölfl, Matthias; Greenberg, Philip D

    2014-01-01

    Antigen-specific priming of human, naïve T-cells has been difficult to assess. Due to the low initial frequency in the naïve cell pool of specific T-cell precursors, such an analysis has been obscured by the requirements for repeated stimulations and prolonged culture time. In this protocol, we describe how to rapidly evaluate antigen-specific priming of CD8+ -cells following a single stimulation. The assay provides reference conditions, which result in the expansion of a significant population of antigen-specific T-cells from the naïve repertoire. Various conditions and modifications during the priming process (e.g. testing new cytokines, costimulators, etc.) can now be directly compared to the reference conditions. Factors relevant to achieving effective priming include the dendritic cell preparation, the T-cell preparation, the cell ratio at the time of priming, the serum source used for the experiment, and the timing of addition and concentration of the cytokines used for expansion. This protocol is relevant for human immunology, vaccine biology and drug development. PMID:24675735

  9. Involvement of fertilization antigen (FA-1) in involuntary immunoinfertility in humans.

    PubMed Central

    Naz, R K

    1987-01-01

    Sera from immunoinfertile patients (n = 32) and fertile controls (n = 20) were analyzed for cross-reaction with a purified and characterized sperm-specific glycoprotein, the fertilization antigen (FA-1), employing an enzyme-linked immunosorbent assay. The immunoinfertile sera demonstrated a strong reaction with FA-1 when compared with fertile control sera. There was no correlation between the reaction of sera with FA-1 and the titers obtained through the sperm agglutination technique and the sperm immobilization technique. Immunoinfertile sera showed binding with the protein bands in the regions corresponding to FA-1 on Western blots involving sodium deoxycholate-solubilized human sperm. Antigens isolated with immunoaffinity chromatography involving immunoinfertile sera also demonstrated antigen bands corresponding to FA-1 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Of the seven immunoinfertile couples, three that had antibodies to FA-1 in the male as well as female partners demonstrated a block of fertilization (IVF) due to antibodies bound on the sperm surface. The anti-FA-1 antibody activity was detected in serum as well as in follicular fluid and seminal plasma. Immunoinfertile sera that showed an inhibition of human sperm penetration of zona-free hamster ova showed a significant (P less than 0.001) increase in penetration rates after absorption with FA-1. These results indicate that sera from immunoinfertile patients had antibodies reacting with FA-1, and these antibodies are involved in the fertilization process. Images PMID:3316276

  10. Allopurinol reduces antigen-specific and polyclonal activation of human T cells

    PubMed Central

    Pérez-Mazliah, Damián; Albareda, María C.; Alvarez, María G.; Lococo, Bruno; Bertocchi, Graciela L.; Petti, Marcos; Viotti, Rodolfo J.; Laucella, Susana A.

    2012-01-01

    Allopurinol is the most popular commercially available xanthine oxidase inhibitor and it is widely used for treatment of symptomatic hyperuricaemia, or gout. Although, several anti-inflammatory actions of allopurinol have been demonstrated in vivo and in vitro, there have been few studies on the action of allopurinol on T cells. In the current study, we have assessed the effect of allopurinol on antigen-specific and mitogen-driven activation and cytokine production in human T cells. Allopurinol markedly decreased the frequency of IFN-γ and IL-2-producing T cells, either after polyclonal or antigen-specific stimulation with Herpes Simplex virus 1, Influenza (Flu) virus, tetanus toxoid and Trypanosoma cruzi-derived antigens. Allopurinol attenuated CD69 upregulation after CD3 and CD28 engagement and significantly reduced the levels of spontaneous and mitogen-induced intracellular reactive oxygen species in T cells. The diminished T cell activation and cytokine production in the presence of allopurinol support a direct action of allopurinol on human T cells, offering a potential pharmacological tool for the management of cell-mediated inflammatory diseases. PMID:23049532

  11. Characterization of human platelet glycoprotein antigens giving rise to individual immunoprecipitates in crossed-immunoelectrophoresis

    SciTech Connect

    Kunicki, T.J.; Nurden, A.T.; Pidard, D.; Russell, N.R.; Caen, J.P.

    1981-12-01

    Washed human platelets were labeled with 125I by the lactoperoxidase-catalyzed method and solubilized in 1% Triton X-100. The soluble proteins were analyzed by crossed-immunoelectrophoresis in 1% agarose, employing a rabbit antibody raised against whole human platelets. Analysis of autoradiograms developed from dried agarose gels led to the establishment of a normal reference pattern that was consistent for platelets obtained from more than 50 normal individuals. Six platelet membrane glycoprotein antigens contained in four distinguishable precipitates were identified. Each identification was based on direct sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of 125I-antigens contained in individually excised precipitates. These platelet antigens include major membrane glycoproteins previously designated la, lb, lla, llb, llla, and lllb. Glycoproteins llb and llla were shown to be contained in a single immunoprecipitate, while glycoproteins la and lla were routinely detected in a single different immunoprecipitate. Analysis of soluble proteins from platelets of five patients with Glanzmann's thrombasthenia demonstrated either a complete absence or a marked reduction of only one radiolabeled precipitate, that containing membrane glycoproteins llb and llla. Platelet samples from two patients with Bernard-Soulier syndrome were devoid of a different precipitate, that containing membrane glycoprotein lb.

  12. Monoclonal antibodies directed against human Rh antigens in tests with the red cells of nonhuman primates.

    PubMed

    Socha, W W; Ruffie, J

    1990-01-01

    Monoclonal antibodies against Rh related antigens on human red cells often crossreact with the red cells of the highest subhuman primate species. Depending on specificity of antibody, the species tested, and technique used, these reactions can be either species-specific or type specific. In tests with chimpanzee red cells, some of the latter type reactions have specificities related to the R antigen of the R-C-E-F blood group system of chimpanzee; specificities of some others seem to be unrelated to any known chimpanzee blood groups. Monoclonal anti-D reagents that give uniformly positive reactions with human D-positive (common and rare types) red cells, display wide individual differences in tests with chimpanzee blood. This indicates that there are minute structural variations of antibody molecules from one monoclonal anti-D antibodies apparently have no bearing on recognition of the D combining site on the human red cells, but come into play when in contact with chimpanzee rbcs. Some of the monoclonal antibodies directed against Rh and LW molecules are distinguished by unusually strong reactions with the red cells of the Old World monkeys (macaques and baboons), which is in contrast with negative or weak reactions of the same antibodies with the red cells of anthropoid apes and human bloods. One may recall, that polyclonal anti-Rh sera do not react with the blood of rhesus monkeys, the phenomenon that was the source of controversy surrounding the discovery of the rhesus factor of the human blood.

  13. Alloantibody Generation and Effector Function Following Sensitization to Human Leukocyte Antigen

    PubMed Central

    Hickey, Michelle J.; Valenzuela, Nicole M.; Reed, Elaine F.

    2016-01-01

    Allorecognition is the activation of the adaptive immune system to foreign human leukocyte antigen (HLA) resulting in the generation of alloantibodies. Due to a high polymorphism, foreign HLA is recognized by the immune system following transplant, transfusion, or pregnancy resulting in the formation of the germinal center and the generation of long-lived alloantibody-producing memory B cells. Alloantibodies recognize antigenic epitopes displayed by the HLA molecule on the transplanted allograft and contribute to graft damage through multiple mechanisms, including (1) activation of the complement cascade resulting in the formation of the MAC complex and inflammatory anaphylatoxins, (2) transduction of intracellular signals leading to cytoskeletal rearrangement, growth, and proliferation of graft vasculature, and (3) immune cell infiltration into the allograft via FcγR interactions with the FC portion of the antibody. This review focuses on the generation of HLA alloantibody, routes of sensitization, alloantibody specificity, and mechanisms of antibody-mediated graft damage. PMID:26870045

  14. Development of Lateral Flow Immunoassay for Antigen Detection in Human Angiostrongylus cantonensis Infection

    PubMed Central

    Chen, Mu-Xin; Chen, Jia-Xu; Chen, Shao-Hong; Huang, Da-Na; Ai, Lin; Zhang, Ren-Li

    2016-01-01

    Angiostrongyliasis is difficult to be diagnosed for the reason that no ideal method can be used. Serologic tests require specific equipment and are not always available in poverty-stricken zone and are time-consuming. A lateral flow immunoassay (LFIA) may be useful for angiostrongyliasis control. We established a LFIA for the diagnosis of angiostrongyliasis based on 2 monoclonal antibodies (mAbs) against antigens of Angiostrongylus cantonensis adults. The sensitivity and specificity were 91.1% and 100% in LFIA, while those of commercial ELISA kit was 97.8% and 86.3%, respectively. Youden index was 0.91 in LFIA and 0.84 in commercial ELISA kit. LFIA showed detection limit of 1 ng/ml of A. cantonensis ES antigens. This LFIA was simple, rapid, highly sensitive and specific, which opened an alternative approach for the diagnosis of human angiostrongyliasis. PMID:27417097

  15. Antigenicity and Diagnostic Potential of Vaccine Candidates in Human Chagas Disease

    PubMed Central

    Gupta, Shivali; Wan, Xianxiu; Zago, Maria P.; Martinez Sellers, Valena C.; Silva, Trevor S.; Assiah, Dadjah; Dhiman, Monisha; Nuñez, Sonia; Petersen, John R.; Vázquez-Chagoyán, Juan C.; Estrada-Franco, Jose G.; Garg, Nisha Jain

    2013-01-01

    Background Chagas disease, caused by Trypanosoma cruzi, is endemic in Latin America and an emerging infectious disease in the US and Europe. We have shown TcG1, TcG2, and TcG4 antigens elicit protective immunity to T. cruzi in mice and dogs. Herein, we investigated antigenicity of the recombinant proteins in humans to determine their potential utility for the development of next generation diagnostics for screening of T. cruzi infection and Chagas disease. Methods and Results Sera samples from inhabitants of the endemic areas of Argentina-Bolivia and Mexico-Guatemala were analyzed in 1st-phase for anti-T. cruzi antibody response by traditional serology tests; and in 2nd-phase for antibody response to the recombinant antigens (individually or mixed) by an ELISA. We noted similar antibody response to candidate antigens in sera samples from inhabitants of Argentina and Mexico (n = 175). The IgG antibodies to TcG1, TcG2, and TcG4 (individually) and TcGmix were present in 62–71%, 65–78% and 72–82%, and 89–93% of the subjects, respectively, identified to be seropositive by traditional serology. Recombinant TcG1- (93.6%), TcG2- (96%), TcG4- (94.6%) and TcGmix- (98%) based ELISA exhibited significantly higher specificity compared to that noted for T. cruzi trypomastigote-based ELISA (77.8%) in diagnosing T. cruzi-infection and avoiding cross-reactivity to Leishmania spp. No significant correlation was noted in the sera levels of antibody response and clinical severity of Chagas disease in seropositive subjects. Conclusions Three candidate antigens were recognized by antibody response in chagasic patients from two distinct study sites and expressed in diverse strains of the circulating parasites. A multiplex ELISA detecting antibody response to three antigens was highly sensitive and specific in diagnosing T. cruzi infection in humans, suggesting that a diagnostic kit based on TcG1, TcG2 and TcG4 recombinant proteins will be useful in diverse situations. PMID

  16. The presence of HLA-DR antigens on lactating human breast epithelium and milk fat globule membranes.

    PubMed Central

    Newman, R A; Ormerod, M G; Greaves, M F

    1980-01-01

    HLA-DR antigens have been demonstrated on the secretory epithelia of lactating breast using rabbit anti-p28,33 ('Ia-like') and mouse monoclonal anti-HLA-DR 'framework'. Normal non-lactating breast, benign or malignant tumours, epithelial cells from normal breast or isolated from milk and a presumptive breast carcinoma cell line (MCF-7) were all HLA-DR-negative. HLA-DR, HLA (ABC 'framework') and beta 2-microglobulin determinants were also demonstrated on the surface of milk fat globules (MFG) which were unreactive with monoclonal antibodies to thymus cells or leucocytes. A monoclonal antibody detecting allelic HLA-DR determinants (HLA-DRw 1,2,6) was positive on 40% of MFG samples tested, positive reactions being concordant, when tested, with blood B lymphocytes. Antisera raised against MFG membranes also contain anti-HLA-DR activity. Whether the breast epithelial cells synthesize HLA-DR molecules or acquire these passively from mononuclear cells which infiltrate during lactation is not yet resolved. Images Fig. 1 PMID:7002399

  17. Biochemical and functional characterization of the leucocyte tyrosine phosphatase CD45 (CD45RO, 180 kD) from human neutrophils. In vivo upregulation of CD45RO plasma membrane expression on patients undergoing haemodialysis.

    PubMed Central

    Pulido, R; Alvarez, V; Mollinedo, F; Sánchez-Madrid, F

    1992-01-01

    The biochemical and functional characterization, and the regulation of plasma membrane expression of the leucocyte tyrosine phosphatase CD45, have been investigated in neutrophils from healthy donors and patients undergoing haemodialysis. CD45 proteins of 180 kD and 130-150 kD were precipitated from neutrophils from both healthy subjects and haemodialysed patients. Prolonged storing, as well as trypsin treatment of samples containing the 180-kD CD45 protein, generated the 130-150-kD polypeptides. The 130-150-kD CD45 polypeptides carried extracellular CD45 epitopes, including the sialic acid-related UCHL1 epitope (CD45RO). Furthermore, these trypsin-generated CD45 polypeptides did not possess phosphatase activity, which could be detected on the 180-kD protein. A remarkable quantitative increase of cell surface expression of the neutrophil CD45 components was detected both after in vitro neutrophil activation and after dialysis treatment with neutropenic membranes. The CD45 biochemical pattern did not qualitatively change upon either in vitro or in vivo dialysis-induced neutrophil activation. The upregulated expression of CD45 on neutrophils from dialysed patients correlated with the neutropenic effect induced by the different dialyser membranes. Maximal upregulation of CD45 expression was observed after 15 min of dialysis with neutropenic membranes, and normal expression levels were restored after 1 h. By contrast, increase of CD45 plasma membrane expression induced in vitro by treatment of normal neutrophils with the degranulatory agents fMLP or Ca2+ ionophore was maintained. These results demonstrate that neutrophil cell surface expression of the 180-kD CD45 protein is upregulated during the in vivo haemodialysis process, and suggest that a proteolytic activity could regulate the enzymatic activity of CD45 by degranulation of its cytoplasmic phosphatase domains. Images Fig. 1 Fig. 2 Fig. 3 PMID:1370931

  18. Human monoclonal antibody to a neuroectodermal tumor antigen (OFA-I-2).

    PubMed

    Katano, M; Sidell, N; Irie, R F

    1983-01-01

    Human IgM kappa monoclonal antibody to human tumors of neuroectodermal origin was produced in the spent medium of an Epstein-Barr virus-transformed B-lymphoblastoid cell line, L72. Chemically, the antigen was identified as ganglioside GD2 [Gal NAc beta 1----4 (Neu Ac alpha 2----8 Neu Ac alpha 2---3) Gal beta 1----4 Glc----ceramide]. Twenty-seven mg of pure human IgM were obtained from 10 liters of L72 spent medium using salt and hypotonic precipitation, ultracentrifugation, and Sephacryl-S 300 superfine gel filtration. The monoclonal origin of the antibody was determined by agarose isoelectrofocusing. This human monoclonal antibody may be a particularly useful reagent for immunotherapy trials in cancer patients.

  19. Structural Constraints on Human Norovirus Binding to Histo-Blood Group Antigens.

    PubMed

    Singh, Bishal K; Leuthold, Mila M; Hansman, Grant S

    2016-01-01

    Human norovirus interacts with the polymorphic human histo-blood group antigens (HBGAs), and this interaction is thought to be important for infection. The genogroup II genotype 4 (GII.4) noroviruses are the dominant cluster, evolve every other year, and are thought to modify their binding interactions with different HBGA types. Most human noroviruses bind HBGAs, while some strains were found to have minimal or no HBGA interactions. Here, we explain some possible structural constraints for several noroviruses that were found to bind poorly to HBGAs by using X-ray crystallography. We showed that one aspartic acid was flexible or positioned away from the fucose moiety of the HBGAs and this likely hindered binding, although other fucose-interacting residues were perfectly oriented. Interestingly, a neighboring loop also appeared to influence the loop hosting the aspartic acid. These new findings might explain why some human noroviruses bound HBGAs poorly, although further studies are required. PMID:27303720

  20. Engineering antigen-specific T cells from genetically modified human hematopoietic stem cells in immunodeficient mice.

    PubMed

    Kitchen, Scott G; Bennett, Michael; Galić, Zoran; Kim, Joanne; Xu, Qing; Young, Alan; Lieberman, Alexis; Joseph, Aviva; Goldstein, Harris; Ng, Hwee; Yang, Otto; Zack, Jerome A

    2009-01-01

    There is a desperate need for effective therapies to fight chronic viral infections. The immune response is normally fastidious at controlling the majority of viral infections and a therapeutic strategy aimed at reestablishing immune control represents a potentially powerful approach towards treating persistent viral infections. We examined the potential of genetically programming human hematopoietic stem cells to generate mature CD8+ cytotoxic T lymphocytes that express a molecularly cloned, "transgenic" human anti-HIV T cell receptor (TCR). Anti-HIV TCR transduction of human hematopoietic stem cells directed the maturation of a large population of polyfunctional, HIV-specific CD8+ cells capable of recognizing and killing viral antigen-presenting cells. Thus, through this proof-of-concept we propose that genetic engineering of human hematopoietic stem cells will allow the tailoring of effector T cell responses to fight HIV infection or other diseases that are characterized by the loss of immune control.

  1. Approach for Identifying Human Leukocyte Antigen (HLA)-DR Bound Peptides from Scarce Clinical Samples.

    PubMed

    Heyder, Tina; Kohler, Maxie; Tarasova, Nataliya K; Haag, Sabrina; Rutishauser, Dorothea; Rivera, Natalia V; Sandin, Charlotta; Mia, Sohel; Malmström, Vivianne; Wheelock, Åsa M; Wahlström, Jan; Holmdahl, Rikard; Eklund, Anders; Zubarev, Roman A; Grunewald, Johan; Ytterberg, A Jimmy

    2016-09-01

    Immune-mediated diseases strongly associating with human leukocyte antigen (HLA) alleles are likely linked to specific antigens. These antigens are presented to T cells in the form of peptides bound to HLA molecules on antigen presenting cells, e.g. dendritic cells, macrophages or B cells. The identification of HLA-DR-bound peptides presents a valuable tool to investigate the human immunopeptidome. The lung is likely a key player in the activation of potentially auto-aggressive T cells prior to entering target tissues and inducing autoimmune disease. This makes the lung of exceptional interest and presents an ideal paradigm to study the human immunopeptidome and to identify antigenic peptides.Our previous investigation of HLA-DR peptide presentation in the lung required high numbers of cells (800 × 10(6) bronchoalveolar lavage (BAL) cells). Because BAL from healthy nonsmokers typically contains 10-15 × 10(6) cells, there is a need for a highly sensitive approach to study immunopeptides in the lungs of individual patients and controls.In this work, we analyzed the HLA-DR immunopeptidome in the lung by an optimized methodology to identify HLA-DR-bound peptides from low cell numbers. We used an Epstein-Barr Virus (EBV) immortalized B cell line and bronchoalveolar lavage (BAL) cells obtained from patients with sarcoidosis, an inflammatory T cell driven disease mainly occurring in the lung. Specifically, membrane complexes were isolated prior to immunoprecipitation, eluted peptides were identified by nanoLC-MS/MS and processed using the in-house developed ClusterMHCII software. With the optimized procedure we were able to identify peptides from 10 × 10(6) cells, which on average correspond to 10.9 peptides/million cells in EBV-B cells and 9.4 peptides/million cells in BAL cells. This work presents an optimized approach designed to identify HLA-DR-bound peptides from low numbers of cells, enabling the investigation of the BAL immunopeptidome from individual patients

  2. Phagocytosis, germination and killing of Bacillus subtilis spores presenting heterologous antigens in human macrophages.

    PubMed

    Ceragioli, Mara; Cangiano, Giuseppina; Esin, Semih; Ghelardi, Emilia; Ricca, Ezio; Senesi, Sonia

    2009-02-01

    Bacillus subtilis is a Gram-positive spore-bearing bacterium long used as a probiotic product and more recently regarded as an attractive vehicle for delivering heterologous antigens to be used for mucosal vaccination. This report describes the in vitro interaction between human macrophages and B. subtilis spores displaying the tetanus toxin fragment C or the B subunit of the heat-labile toxin of Escherichia coli on their surface in comparison to spores of the parental strain. Recombinant and parental B. subtilis spores were similarly internalized by human macrophages, at a frequency lower than 2.5%. Inside macrophages, nearly all spores germinated and were killed within 6 h. Using germination-defective spores and inhibiting spore germination inside macrophages, evidence was produced that only germinated spores were killed by human macrophages and that intracellular spore germination was mediated by an alanine-dependent pathway. The germinated spores were killed by macrophages before any round of cell duplication, as estimated by fluorescence microscopy analysis of macrophages infected with spores carrying the gfp gene fused to abrB, a B. subtilis gene shown here to be expressed at the transition between outgrowth and vegetative growth. Monitoring of macrophage infection never revealed cytotoxic effects being exerted by B. subtilis spores. These in vitro data support the hypothesis that B. subtilis spores may potentially be used as a suitable and safe vehicle for administering heterologous antigens to humans.

  3. Leucocyte phagocytosis during the luteal phase in bitches.

    PubMed

    Holst, Bodil Ström; Gustavsson, Malin Hagberg; Lilliehöök, Inger; Morrison, David; Johannisson, Anders

    2013-05-15

    Pyometra is a disease that affects a large proportion of intact bitches, and typically is seen during the latter half of dioestrus. Several factors contribute to the development of pyometra, including genetic factors, an infectious component (most often Escherichia coli), and hormonal factors. Hormones may act directly on the endometrium, and also affect the immune system. In dogs, the phagocytic ability has been shown to decrease with age, and ovarian hormones have also been shown to affect immune resistance. The aim of the present study was to examine whether phagocytosis by canine leucocytes varies significantly during the luteal phase. Eight bitches were followed by repeated blood sampling. Samples were taken at the calculated optimal day for mating (Day 1), and thereafter on days 8, 15 and 22 (early luteal phase) and 29, 43, 57 and 71 (late luteal phase). Blood was collected from the cephalic vein into EDTA tubes for leucocyte counts and heparinised tubes for testing of phagocytosis and oxidative burst using commercial kits and flow cytometry. The cell activity of the phagocyting leucocytes, expressed as mean fluorescence activity, MFI, was significantly lower during late luteal phase than during early luteal phase. The proportion of leucocytes that was induced to phagocyte did not differ significantly. The percentage of cells stimulated by E. coli to oxidative burst was significantly lower during late luteal phase. Their activity did not differ between the two periods. The number of cells stimulated to oxidative burst by a low stimulus was too low to evaluate, and leucocytes stimulated with the high stimulus did not vary in oxidative burst between the two periods. The changes in phagocytic activity and in the number of leucocytes that showed oxidative burst were not associated with any change in the proportion of different leucocytes. The decreased phagocytic capacity possibly contributes to the higher incidence of diseases such as pyometra during the latter

  4. An Integrated Peptide-Antigen Microarray on Plasmonic Gold Films for Sensitive Human Antibody Profiling

    PubMed Central

    Price, Jordan V.; Tabakman, Scott M.; Li, Yanguang; Gong, Ming; Hong, Guosong; Feng, Ju; Utz, Paul J.; Dai, Hongjie

    2013-01-01

    High-throughput screening for interactions of peptides with a variety of antibody targets could greatly facilitate proteomic analysis for epitope mapping, enzyme profiling, drug discovery and biomarker identification. Peptide microarrays are suited for such undertaking because of their high-throughput capability. However, existing peptide microarrays lack the sensitivity needed for detecting low abundance proteins or low affinity peptide-protein interactions. This work presents a new peptide microarray platform constructed on nanostructured plasmonic gold substrates capable of metal enhanced NIR fluorescence enhancement (NIR-FE) by hundreds of folds for screening peptide-antibody interactions with ultrahigh sensitivity. Further, an integrated histone peptide and whole antigen array is developed on the same plasmonic gold chip for profiling human antibodies in the sera of systemic lupus erythematosus (SLE) patients, revealing that collectively a panel of biomarkers against unmodified and post-translationally modified histone peptides and several whole antigens allow more accurate differentiation of SLE patients from healthy individuals than profiling biomarkers against peptides or whole antigens alone. PMID:23923050

  5. Peptide-dependent Conformational Fluctuation Determines the Stability of the Human Leukocyte Antigen Class I Complex*

    PubMed Central

    Yanaka, Saeko; Ueno, Takamasa; Shi, Yi; Qi, Jianxun; Gao, George F.; Tsumoto, Kouhei; Sugase, Kenji

    2014-01-01

    In immune-mediated control of pathogens, human leukocyte antigen (HLA) class I presents various antigenic peptides to CD8+ T-cells. Long-lived peptide presentation is important for efficient antigen-specific T-cell activation. Presentation time depends on the peptide sequence and the stability of the peptide-HLA complex (pHLA). However, the determinant of peptide-dependent pHLA stability remains elusive. Here, to reveal the pHLA stabilization mechanism, we examined the crystal structures of an HLA class I allomorph in complex with HIV-derived peptides and evaluated site-specific conformational fluctuations using NMR. Although the crystal structures of various pHLAs were almost identical independent of the peptides, fluctuation analyses identified a peptide-dependent minor state that would be more tightly packed toward the peptide. The minor population correlated well with the thermostability and cell surface presentation of pHLA, indicating that this newly identified minor state is important for stabilizing the pHLA and facilitating T-cell recognition. PMID:25028510

  6. Study of human leukocyte antigen-cw in Egyptian patients with vitiligo.

    PubMed

    Hassab El Naby, Hussein M; Alnaggar, Mohamed R; Abdelhamid, Mahmoud F; Alsaid, Khadiga; Al Shawadfy, Eslam M; Elsaie, Mohamed L

    2015-04-01

    Human leukocyte antigen (HLA) antigens vary considerably in different racial groups, and an analysis of results from several geographical locations suggests that vitiligo appears to be associated with different HLA antigens in different groups. The aim of this work was to assess the association of HLA-Cw with vitiligo in the Egyptian population. Forty unrelated patients with nonsegmental vitiligo and 20 matched controls were selected. A polymerase chain reaction sequence specific primer (PCR-SSP) method was used to determine HLA DNA typing. There was a statistically significant difference in the association of HLA-Cw6 with vitiligo in the 2 studied groups. A comparatively increased number of patients showed HLA-Cw2 and HLA-Cw7 (13.64%). However, there were no statistically significant differences. To the best of our knowledge, this is the first molecular study of HLA typing in Egyptian patients with vitiligo. Our findings are in agreement with earlier studies that reported statistically increased frequencies for allele of HLA-Cw6 in Northern Italian, Kuwaiti, Chinese Han, and Saudi populations (45.45%, P<.05).

  7. Keyhole limpet haemocyanin – a model antigen for human immunotoxicological studies

    PubMed Central

    Swaminathan, Ashwin; Lucas, Robyn M; Dear, Keith; McMichael, Anthony J

    2014-01-01

    Immunization with a T-cell dependent antigen has been promoted as a reliable and sensitive tool for assessing the influence of putative immunotoxic exposures or agents on immune function. Keyhole limpet haemocyanin (KLH) is a very large, copper-containing protein molecule derived from the haemolymph of the inedible mollusc, Megathura crenulata. KLH is a highly immunogenic T-cell dependent antigen that is used increasingly in immunotoxicological studies, particularly in those involving animals. This report systematically reviews the human clinical studies that have used trans-cutaneous KLH immunization for assessment of the influence of various physiological and disease states and exposures on immune function over the last 20 years (1994–2013). These studies varied in their immunization protocols, formulation of KLH, dose, site and route of administration and immunoassay platforms developed to assess KLH-specific responses. KLH immunization has been well tolerated with only mild to moderate adverse effects reported. Though very promising as a model antigen candidate in immunotoxicology research, more work on standardizing immunization and immunoassay protocols is required. PMID:24833186

  8. Comparative efficacy of antigen and antibody detection tests for human trichinellosis

    SciTech Connect

    Ivanoska, D.; Cuperlovic, K.; Gamble, H.R.; Murrell, K.D.

    1989-02-01

    Sera collected from patients with suspected or confirmed exposure to Trichinella spiralis were tested for circulating parasite antigens and antiparasite antibodies. Using an immunoradiometric assay, excretory--secretory antigens from muscle-stage larvae of T. spiralis were detected in the sera of 47% of 62 patients with clinical trichinellosis and 13% of 39 patients without clinical signs but suspected of exposure to infected meat. In comparison, antibodies were detected using an indirect immunofluorescent test in the circulation of 100% of the 62 patients with clinical trichinellosis and 46% of the 39 patients with suspected exposure. The presence of antibodies specific to excretory-secretory products of T. spiralis muscle larvae was confirmed in the majority of the samples tested by a monoclonal antibody-based competitive inhibition assay. These results indicate that antibody detection is a more sensitive diagnostic method for human trichinellosis, but that antigen detection might be a useful confirmatory test because it is a direct demonstration of parasite products in the circulation.

  9. Antigenic assessment of a recombinant human CD90 protein expressed in prokaryotic expression system.

    PubMed

    Yousefi-Rad, Narges; Shokrgozar, Mohammad Ali; Behdani, Mahdi; Moradi-Kalbolandi, Shima; Motamedi-Rad, Mahdieh; Habibi-Anbouhi, Mahdi

    2015-12-01

    Cluster of Differentiation 90 (CD90, Thy-1) has been proposed as one of the most important biomarkers in several cancer cells including cancer stem cells (CSCs). CD90 is considered as a potential normal stem cell and CSCs biomarker and also has been identified in lung cancer stem cells, hepatocellular carcinoma cells and high-grade gliomas. Using eukaryotic host systems involves complex procedures and frequently results in low protein yields. The expression of recombinant proteins in Escherichia coli is comparatively easier than eukaryotic host cells. The potential of large scale production of recombinant protein has made this system an economic production platform. In this study we expressed the extra-membrane domain of human CD90 (exCD90) antigen (Gln15-Cys130) in E. coli expression host cells. The epitope integrity of purified recombinant antigen was confirmed by antibody-antigen interaction using 5E10 anti-CD90 monoclonal antibody and binding study through ELISA and florescent staining of CD90(+) cells in a flow cytometry experiment. PMID:26297626

  10. Visualization of antigen-specific human cytotoxic T lymphocytes labeled with superparamagnetic iron-oxide particles.

    PubMed

    Beer, Ambros J; Holzapfel, Konstantin; Neudorfer, Juliana; Piontek, Guido; Settles, Marcus; Krönig, Holger; Peschel, Christian; Schlegel, Jürgen; Rummeny, Ernst J; Bernhard, Helga

    2008-06-01

    New technologies are needed to characterize the migration and survival of antigen-specific T cells in vivo. In this study, we developed a novel technique for the labeling of human cytotoxic T lymphocytes with superparamagnetic iron-oxide particles and the subsequent depiction with a conventional 1.5-T magnetic resonance scanner. Antigen-specific CD8(+) T lymphocytes were labeled with ferucarbotran by lipofection. The uptake of ferucarbotran was confirmed by immunofluorescence microscopy using a dextran-specific antibody, and the intracellular enrichment of iron was measured by atomic absorption spectrometry. The imaging of T cells was performed by magnetic resonance on day 0, 2, 7 and 14 after the labeling procedure. On day 0 and 2 post labeling, a pronounced shortening of T2*-relaxation times was observed, which diminished after 7 days and was not detectable anymore after 14 days, probably due to the retained mitotic activity of the labeled T cells. Of importance, the antigen-specific cytolytic activity of the T cells was preserved following ferucarbotran labeling. Efficient ferucarbotran labeling of functionally active T lymphocytes and their detection by magnetic resonance imaging allows the in vivo monitoring of T cells and, subsequently, will impact the further development of T cell-based therapies. PMID:18286290

  11. Antigenic assessment of a recombinant human CD90 protein expressed in prokaryotic expression system.

    PubMed

    Yousefi-Rad, Narges; Shokrgozar, Mohammad Ali; Behdani, Mahdi; Moradi-Kalbolandi, Shima; Motamedi-Rad, Mahdieh; Habibi-Anbouhi, Mahdi

    2015-12-01

    Cluster of Differentiation 90 (CD90, Thy-1) has been proposed as one of the most important biomarkers in several cancer cells including cancer stem cells (CSCs). CD90 is considered as a potential normal stem cell and CSCs biomarker and also has been identified in lung cancer stem cells, hepatocellular carcinoma cells and high-grade gliomas. Using eukaryotic host systems involves complex procedures and frequently results in low protein yields. The expression of recombinant proteins in Escherichia coli is comparatively easier than eukaryotic host cells. The potential of large scale production of recombinant protein has made this system an economic production platform. In this study we expressed the extra-membrane domain of human CD90 (exCD90) antigen (Gln15-Cys130) in E. coli expression host cells. The epitope integrity of purified recombinant antigen was confirmed by antibody-antigen interaction using 5E10 anti-CD90 monoclonal antibody and binding study through ELISA and florescent staining of CD90(+) cells in a flow cytometry experiment.

  12. Identification of sVSG117 as an Immunodiagnostic Antigen and Evaluation of a Dual-Antigen Lateral Flow Test for the Diagnosis of Human African Trypanosomiasis

    PubMed Central

    Sullivan, Lauren; Fleming, Jennifer; Sastry, Lalitha; Mehlert, Angela; Wall, Steven J.; Ferguson, Michael A. J.

    2014-01-01

    Background The diagnosis of human African trypanosomiasis (HAT) caused by Trypanosoma brucei gambiense relies mainly on the Card Agglutination Test for Trypanosomiasis (CATT). There is no immunodiagnostic for HAT caused by T. b. rhodesiense. Our principle aim was to develop a prototype lateral flow test that might be an improvement on CATT. Methodology/Principle Findings Pools of infection and control sera were screened against four different soluble form variant surface glycoproteins (sVSGs) by ELISA and one, sVSG117, showed particularly strong immunoreactivity to pooled infection sera. Using individual sera, sVSG117 was shown to be able to discriminate between T. b. gambiense infection and control sera by both ELISA and lateral flow test. The sVSG117 antigen was subsequently used with a previously described recombinant diagnostic antigen, rISG65, to create a dual-antigen lateral flow test prototype. The latter was used blind in a virtual field trial of 431 randomized infection and control sera from the WHO HAT Specimen Biobank. Conclusion/Significance In the virtual field trial, using two positive antigen bands as the criterion for infection, the sVSG117 and rISG65 dual-antigen lateral flow test prototype showed a sensitivity of 97.3% (95% CI: 93.3 to 99.2) and a specificity of 83.3% (95% CI: 76.4 to 88.9) for the detection of T. b. gambiense infections. The device was not as good for detecting T. b. rhodesiense infections using two positive antigen bands as the criterion for infection, with a sensitivity of 58.9% (95% CI: 44.9 to 71.9) and specificity of 97.3% (95% CI: 90.7 to 99.7). However, using one or both positive antigen band(s) as the criterion for T. b. rhodesiense infection improved the sensitivity to 83.9% (95% CI: 71.7 to 92.4) with a specificity of 85.3% (95% CI: 75.3 to 92.4). These results encourage further development of the dual-antigen device for clinical use. PMID:25033401

  13. Antigen-bound C3b and C4b enhance antigen-presenting cell function in activation of human T-cell clones.

    PubMed

    Arvieux, J; Yssel, H; Colomb, M G

    1988-10-01

    The effect of complement fragments C3b and C4b, on the triggering of antigen-specific human T-cell clones by Epstein-Barr virus-transformed human lymphoblastoid B cells (LCL) when these fragments are covalently coupled to the antigen tetanus toxin (TT) is described. TT was chemically cross-linked to purified C3b [(TT-C3b)n], C4b [(TT-C4b)n] or bovine serum albumin [(TT-BSA)n] as a control. T-cell activation was quantified by tritiated thymidine incorporation and 51Cr release. (TT-C3b)n and (TT-C4b)n induced proliferative responses comparable to (TT-BSA)n but at 18-25 and 4-6 lower concentrations, respectively. This enhancing effect required the covalent cross-linking of the complement fragments to the antigen and involved intracellular processing of the latter by LCL. Antigen presentation was similarly enhanced when measuring the cytotoxic activity of a helper T-cell clone against LCL previously pulsed with (TT-C3b)n or (TT-C4b)n compared with (TT-BSA)n. Binding studies, carried out on LCL using TT radiolabelled with 125I before cross-linking, indicated that (TT-C3b)n and (TT-C4b)n gave three- to four-fold more binding than (TT-BSA)n. Addition of antibodies against CR1 and CR2 or proteolytic removal of these complement receptors with trypsin inhibited by about 60% the enhancing effect of TT-bound C3b and C4b in both binding and functional assays. These results indicate that binding of C3b or C4b to antigen enhances antigen-specific proliferative and cytotoxic responses of T cells by targeting opsonized antigen onto complement receptors CR1 and CR2 of LCL. The putative significance of these findings in terms of regulation of immune responses by complement is discussed.

  14. Antigen-bound C3b and C4b enhance antigen-presenting cell function in activation of human T-cell clones.

    PubMed

    Arvieux, J; Yssel, H; Colomb, M G

    1988-10-01

    The effect of complement fragments C3b and C4b, on the triggering of antigen-specific human T-cell clones by Epstein-Barr virus-transformed human lymphoblastoid B cells (LCL) when these fragments are covalently coupled to the antigen tetanus toxin (TT) is described. TT was chemically cross-linked to purified C3b [(TT-C3b)n], C4b [(TT-C4b)n] or bovine serum albumin [(TT-BSA)n] as a control. T-cell activation was quantified by tritiated thymidine incorporation and 51Cr release. (TT-C3b)n and (TT-C4b)n induced proliferative responses comparable to (TT-BSA)n but at 18-25 and 4-6 lower concentrations, respectively. This enhancing effect required the covalent cross-linking of the complement fragments to the antigen and involved intracellular processing of the latter by LCL. Antigen presentation was similarly enhanced when measuring the cytotoxic activity of a helper T-cell clone against LCL previously pulsed with (TT-C3b)n or (TT-C4b)n compared with (TT-BSA)n. Binding studies, carried out on LCL using TT radiolabelled with 125I before cross-linking, indicated that (TT-C3b)n and (TT-C4b)n gave three- to four-fold more binding than (TT-BSA)n. Addition of antibodies against CR1 and CR2 or proteolytic removal of these complement receptors with trypsin inhibited by about 60% the enhancing effect of TT-bound C3b and C4b in both binding and functional assays. These results indicate that binding of C3b or C4b to antigen enhances antigen-specific proliferative and cytotoxic responses of T cells by targeting opsonized antigen onto complement receptors CR1 and CR2 of LCL. The putative significance of these findings in terms of regulation of immune responses by complement is discussed. PMID:2973431

  15. Allele-Independent Turnover of Human Leukocyte Antigen (HLA) Class Ia Molecules

    PubMed Central

    Prevosto, Claudia; Usmani, M. Farooq; McDonald, Sarah; Gumienny, Aleksandra M.; Key, Tim; Goodman, Reyna S.; Gaston, J. S. Hill; Deery, Michael J.; Busch, Robert

    2016-01-01

    Major histocompatibility complex class I (MHCI) glycoproteins present cytosolic peptides to CD8+ T cells and regulate NK cell activity. Their heavy chains (HC) are expressed from up to three MHC gene loci (human leukocyte antigen [HLA]-A, -B, and -C in humans), whose extensive polymorphism maps predominantly to the antigen-binding groove, diversifying the bound peptide repertoire. Codominant expression of MHCI alleles is thus functionally critical, but how it is regulated is not fully understood. Here, we have examined the effect of polymorphism on the turnover rates of MHCI molecules in cell lines with functional MHCI peptide loading pathways and in monocyte-derived dendritic cells (MoDCs). Proteins were labeled biosynthetically with heavy water (2H2O), folded MHCI molecules immunoprecipitated, and tryptic digests analysed by mass spectrometry. MHCI-derived peptides were assigned to specific alleles and isotypes, and turnover rates quantified by 2H incorporation, after correcting for cell growth. MHCI turnover half-lives ranged from undetectable to a few hours, depending on cell type, activation state, donor, and MHCI isotype. However, in all settings, the turnover half-lives of alleles of the same isotype were similar. Thus, MHCI protein turnover rates appear to be allele-independent in normal human cells. We propose that this is an important feature enabling the normal function and codominant expression of MHCI alleles. PMID:27529174

  16. Domain specificity of the human antibody response to Bacillus anthracis protective antigen.

    PubMed

    Reason, Donald C; Ullal, Anuska; Liberato, Justine; Sun, Jinying; Keitel, Wendy; Zhou, Jianhui

    2008-07-29

    Protective antigen (PA) is the cell surface recognition moiety of the Bacillus anthracis A-B toxin system, and the active immunogenic component in the currently licensed human anthrax vaccine (BioThrax, or AVA). The serum antibody response to the PA protein is polyclonal and complex both in terms of the antibody combining sites utilized to bind PA and the PA-associated epitopes recognized. We have cloned, sequenced, and expressed a large panel of PA-specific human monoclonal antibodies from seven AVA-immunized donors. Dot blots, Western blots, and radiolabeled antigen capture assays employing both proteolytic fragments of PA and engineered PA sub-domain fusion proteins were used to determine the region (domain) of the PA monomer to which each of the cloned human antibodies bound. The domain specificity of the isolated monoclonals was highly biased towards the amino-terminal 20kDa fragment of PA (PA(20)), with the majority (62%) of independently arising antibody clones reacting with determinants located on this PA fragment. A similar bias in domain specificity was also demonstrated in the serum response of AVA-vaccinated donors. Since PA(20) is cleaved from the remainder of the monomer rapidly following cell surface binding and has no known role in the intoxication process, the immunodominance of PA(20)-associated epitopes may directly affect the efficacy of PA-based anthrax vaccines.

  17. Allele-Independent Turnover of Human Leukocyte Antigen (HLA) Class Ia Molecules.

    PubMed

    Prevosto, Claudia; Usmani, M Farooq; McDonald, Sarah; Gumienny, Aleksandra M; Key, Tim; Goodman, Reyna S; Gaston, J S Hill; Deery, Michael J; Busch, Robert

    2016-01-01

    Major histocompatibility complex class I (MHCI) glycoproteins present cytosolic peptides to CD8+ T cells and regulate NK cell activity. Their heavy chains (HC) are expressed from up to three MHC gene loci (human leukocyte antigen [HLA]-A, -B, and -C in humans), whose extensive polymorphism maps predominantly to the antigen-binding groove, diversifying the bound peptide repertoire. Codominant expression of MHCI alleles is thus functionally critical, but how it is regulated is not fully understood. Here, we have examined the effect of polymorphism on the turnover rates of MHCI molecules in cell lines with functional MHCI peptide loading pathways and in monocyte-derived dendritic cells (MoDCs). Proteins were labeled biosynthetically with heavy water (2H2O), folded MHCI molecules immunoprecipitated, and tryptic digests analysed by mass spectrometry. MHCI-derived peptides were assigned to specific alleles and isotypes, and turnover rates quantified by 2H incorporation, after correcting for cell growth. MHCI turnover half-lives ranged from undetectable to a few hours, depending on cell type, activation state, donor, and MHCI isotype. However, in all settings, the turnover half-lives of alleles of the same isotype were similar. Thus, MHCI protein turnover rates appear to be allele-independent in normal human cells. We propose that this is an important feature enabling the normal function and codominant expression of MHCI alleles. PMID:27529174

  18. Detection and monitoring of human bocavirus 1 infection by a new rapid antigen test

    PubMed Central

    Bruning, A.H.L.; Susi, P.; Toivola, H.; Christensen, A.; Söderlund-Venermo, M.; Hedman, K.; Aatola, H.; Zvirbliene, A.; Koskinen, J.O.

    2016-01-01

    Clinically relevant diagnosis of human bocavirus 1 (HBoV1) is challenging, as the virus is frequently detected in asymptomatic patients, and cofindings with other respiratory viruses are common. The clinical value of current diagnostic methods, such as PCR, is therefore low, and alternative diagnostic strategies are needed. We describe for the first time the use of an antigen detection assay for the rapid identification of HBoV1 in a paediatric patient with respiratory tract infection symptoms. We estimate the duration of active HBoV1 infection to be 6 days. PMID:27014463

  19. Detection and monitoring of human bocavirus 1 infection by a new rapid antigen test.

    PubMed

    Bruning, A H L; Susi, P; Toivola, H; Christensen, A; Söderlund-Venermo, M; Hedman, K; Aatola, H; Zvirbliene, A; Koskinen, J O

    2016-05-01

    Clinically relevant diagnosis of human bocavirus 1 (HBoV1) is challenging, as the virus is frequently detected in asymptomatic patients, and cofindings with other respiratory viruses are common. The clinical value of current diagnostic methods, such as PCR, is therefore low, and alternative diagnostic strategies are needed. We describe for the first time the use of an antigen detection assay for the rapid identification of HBoV1 in a paediatric patient with respiratory tract infection symptoms. We estimate the duration of active HBoV1 infection to be 6 days. PMID:27014463

  20. Immunohistochemical demonstration of human papilloma virus (HPV) antigens in oral squamous cell lesions.

    PubMed

    Syrjänen, K J; Pyrhönen, S; Syrjänen, S M; Lamberg, M A

    1983-06-01

    Six oral squamous cell tumours classified as focal epithelial hyperplasia (FEH), Condyloma acuminatum (CA) and squamous cell papilloma (SQP) were subjected to indirect immunoperoxidase staining with anti-human papillomavirus (anti-HPV) antiserum to demonstrate the possible presence of HPV antigens in these lesions. The results are discussed in the light of the observations on HPV-lesions elsewhere in the body (in uterine cervix), and a suggestion is made to adopt the name condyloma for all those tumours where HPV aetiology can be established by ultrastructural or immunohistochemical means.

  1. Reverse vaccinology 2.0: Human immunology instructs vaccine antigen design

    PubMed Central

    Bottomley, Matthew J.; D’Oro, Ugo; Finco, Oretta; De Gregorio, Ennio

    2016-01-01

    Traditionally, vaccines have been developed by cultivating infectious agents and isolating the inactivated whole pathogen or some of its purified components. 20 years ago, reverse vaccinology enabled vaccine discovery and design based on information deriving from the sequence of microbial genomes rather than via the growth of pathogens. Today, the high throughput discovery of protective human antibodies, sequencing of the B cell repertoire, and the increasing structural characterization of protective antigens and epitopes provide the molecular and mechanistic understanding to drive the discovery of novel vaccines that were previously impossible. We are entering a “reverse vaccinology 2.0” era. PMID:27022144

  2. Human lymphocyte antigen CD38 catalyzes the production of cyclic ADP-ribose.

    PubMed

    Summerhill, R J; Jackson, D G; Galione, A

    1993-12-01

    The human lymphocyte antigen CD38 has been shown to share sequence homology with ADP-ribosyl cyclase, the enzyme that catalyzes the conversion of NAD+ to cyclic ADP-ribose (cADPR), a potent Ca(2+)-mobilizing agent. In this study COS1 cells from African Green Monkey kidney were transiently transfected with CD38 cDNA, inducing expression of authentic CD38 on the cell surface. We demonstrate that CD38 expressed in this manner can convert NAD+ to cADPR in the extracellular medium as assessed by Ca2+ release from sea-urchin egg microsomes. PMID:8253202

  3. Appropriate clinical use of human leukocyte antigen typing for coeliac disease: an Australasian perspective.

    PubMed

    Tye-Din, J A; Cameron, D J S; Daveson, A J; Day, A S; Dellsperger, P; Hogan, C; Newnham, E D; Shepherd, S J; Steele, R H; Wienholt, L; Varney, M D

    2015-04-01

    The past decade has seen human leukocyte antigen (HLA) typing emerge as a remarkably popular test for the diagnostic work-up of coeliac disease with high patient acceptance. Although limited in its positive predictive value for coeliac disease, the strong disease association with specific HLA genes imparts exceptional negative predictive value to HLA typing, enabling a negative result to exclude coeliac disease confidently. In response to mounting evidence that the clinical use and interpretation of HLA typing often deviates from best practice, this article outlines an evidence-based approach to guide clinically appropriate use of HLA typing, and establishes a reporting template for pathology providers to improve communication of results.

  4. Demonstration of cross-reactivity between bacterial antigens and class I human leukocyte antigens by using monoclonal antibodies to Shigella flexneri.

    PubMed Central

    Williams, K M; Raybourne, R B

    1990-01-01

    Bacterial envelope proteins which share immunodeterminants with the human leukocyte antigen (HLA) class I histocompatibility antigen HLA-B27 may invoke spondyloarthritic disease through the process of molecular mimicry in patients expressing this phenotype. Monoclonal antibodies generated by the immunization of BALB/c mice with envelope proteins of Shigella flexneri type 2a were tested for reactivity against cultured lymphoblastoid cell lines of defined HLA phenotype. As measured by flow microfluorometry, four immunoglobulin M monoclonal antibodies reacted preferentially with HLA-B27-positive lymphocytes (HOM-2, MM) as compared with a B27-loss mutant line (1065) or cells lacking major histocompatibility complex class I antigen (Daudi, K562). Monoclonal antibodies also reacted with mouse EL-4 cells transfected with and expressing the HLA-B7 gene. Western immunoblot analysis of isolated enterobacterial envelopes demonstrated that the reactive epitope was present on bacterial proteins with an apparent relative molecular mass of 36 and 19 kilodaltons. The structural basis for the cross-reactivity of bacterial antigen and HLA-B27 appeared to reside in the portion of the HLA molecule that is responsible for allotypic specificity (amino acids 63 through 83), since monoclonal antibodies were positive by enzyme-linked immunosorbent assay with synthetic polypeptides corresponding to this segment. Images PMID:2187807

  5. Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

    PubMed Central

    Schütz, Christian; Fleck, Martin; Schneck, Jonathan P.; Oelke, Mathias

    2014-01-01

    Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion. PMID:25145915

  6. Killer artificial antigen presenting cells (KaAPC) for efficient in vitro depletion of human antigen-specific T cells.

    PubMed

    Schütz, Christian; Fleck, Martin; Schneck, Jonathan P; Oelke, Mathias

    2014-01-01

    Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion. PMID:25145915

  7. Identification of antigen-specific human monoclonal antibodies using high-throughput sequencing of the antibody repertoire.

    PubMed

    Liu, Ju; Li, Ruihua; Liu, Kun; Li, Liangliang; Zai, Xiaodong; Chi, Xiangyang; Fu, Ling; Xu, Junjie; Chen, Wei

    2016-04-22

    High-throughput sequencing of the antibody repertoire provides a large number of antibody variable region sequences that can be used to generate human monoclonal antibodies. However, current screening methods for identifying antigen-specific antibodies are inefficient. In the present study, we developed an antibody clone screening strategy based on clone dynamics and relative frequency, and used it to identify antigen-specific human monoclonal antibodies. Enzyme-linked immunosorbent assay showed that at least 52% of putative positive immunoglobulin heavy chains composed antigen-specific antibodies. Combining information on dynamics and relative frequency improved identification of positive clones and elimination of negative clones. and increase the credibility of putative positive clones. Therefore the screening strategy could simplify the subsequent experimental screening and may facilitate the generation of antigen-specific antibodies.

  8. Human Immune Response Varies by the Degree of Relative Cryptococcal Antigen Shedding.

    PubMed

    Boulware, David R; von Hohenberg, Maximilian; Rolfes, Melissa A; Bahr, Nathan C; Rhein, Joshua; Akampurira, Andrew; Williams, Darlisha A; Taseera, Kabanda; Schutz, Charlotte; McDonald, Tami; Muzoora, Conrad; Meintjes, Graeme; Meya, David B; Nielsen, Kirsten; Huppler Hullsiek, Katherine

    2016-01-01

    Background.  Cerebrospinal fluid (CSF) cryptococcal glucuronoxylomannan antigen (CrAg) titers generally correlate with quantitative fungal culture burden; however, correlation is not precise. Some patients have higher CrAg titers with lower fungal burdens and vice versa. We hypothesized that the relative discordancy between CrAg titer and quantitative culture burden reflects the relative degree of CrAg shedding by Cryptococcus neoformans and is associated with human immune responses. Methods.  One hundred ninety human immunodeficiency virus-infected individuals with cryptococcal meningitis were enrolled in Uganda and South Africa. We compared initial CSF CrAg titers relative to their CSF quantitative cultures to determine low (n = 58), intermediate (n = 68), or high (n = 64) CrAg shedders. We compared cytokines measured by Luminex multiplex assay on cryopreserved CSF and 10-week mortality across shedding groups using linear and logistic regression and distribution of genotypes by multilocus sequence typing. Results.  The relative degree of CrAg shedding was positively associated with increasing CSF levels of the following: interleukin (IL)-6, IL-7, IL-8, and tumor necrosis factor-α (each P < 0.01), which are all secreted by antigen-presenting cells and negatively associated with vascular endothelial growth factor (P = .01). In addition, IL-5, IL-13, granulocyte colony-stimulating factor, and macrophage chemotactic protein were decreased in low-CrAg shedders compared with intermediate shedders (each P ≤ .01). Type 1 T-helper cells (Th1) cytokine responses and 10-week mortality did not differ between the shedding groups. Cryptococcal genotypes were equally distributed across shedding groups. Conclusions.  Discordancy between CrAg shedding and expected shedding based on quantitative fungal burden is associated with detectable immunologic differences in CSF, primarily among secreted cytokines and chemokines produced by antigen-presenting cells and Th2. PMID

  9. Antigen nature and complexity influence human antibody light chain usage and specificity.

    PubMed

    Smith, Kenneth; Shah, Hemangi; Muther, Jennifer J; Duke, Angie L; Haley, Kathleen; James, Judith A

    2016-05-27

    Human antibodies consist of a heavy chain and one of two possible light chains, kappa (κ) or lambda (λ). Here we tested how these two possible light chains influence the overall antibody response to polysaccharide and protein antigens by measuring light chain usage in human monoclonal antibodies from antibody secreting cells obtained following vaccination with Pneumovax23. Remarkably, we found that individuals displayed restricted light chain usage to certain serotypes and that lambda antibodies have different specificities and modes of cross-reactivity than kappa antibodies. Thus, at both the monoclonal (7 kappa, no lambda) and serum levels (145μg/mL kappa, 2.82μg/mL lambda), antibodies to cell wall polysaccharide were nearly always kappa. The pneumococcal reference serum 007sp was analyzed for light chain usage to 12 pneumococcal serotypes for which it is well characterized. Similar to results at the monoclonal level, certain serotypes tended to favor one of the light chains (14 and 19A, lambda; 6A and 23F, kappa). We also explored differences in light chain usage at the serum level to a variety of antigens. We examined serum antibodies to diphtheria toxin mutant CRM197 and Epstein-Barr virus protein EBNA-1. These responses tended to be kappa dominant (average kappa-to-lambda ratios of 4.52 and 9.72 respectively). Responses to the influenza vaccine were more balanced with kappa-to-lambda ratio averages having slight strain variations: seasonal H1N1, 1.1; H3N2, 0.96; B, 0.91. We conclude that antigens with limited epitopes tend to produce antibodies with restricted light chain usage and that in most individuals, antibodies with lambda light chains have specificities different and complementary to kappa-containing antibodies.

  10. Antigen nature and complexity influence human antibody light chain usage and specificity.

    PubMed

    Smith, Kenneth; Shah, Hemangi; Muther, Jennifer J; Duke, Angie L; Haley, Kathleen; James, Judith A

    2016-05-27

    Human antibodies consist of a heavy chain and one of two possible light chains, kappa (κ) or lambda (λ). Here we tested how these two possible light chains influence the overall antibody response to polysaccharide and protein antigens by measuring light chain usage in human monoclonal antibodies from antibody secreting cells obtained following vaccination with Pneumovax23. Remarkably, we found that individuals displayed restricted light chain usage to certain serotypes and that lambda antibodies have different specificities and modes of cross-reactivity than kappa antibodies. Thus, at both the monoclonal (7 kappa, no lambda) and serum levels (145μg/mL kappa, 2.82μg/mL lambda), antibodies to cell wall polysaccharide were nearly always kappa. The pneumococcal reference serum 007sp was analyzed for light chain usage to 12 pneumococcal serotypes for which it is well characterized. Similar to results at the monoclonal level, certain serotypes tended to favor one of the light chains (14 and 19A, lambda; 6A and 23F, kappa). We also explored differences in light chain usage at the serum level to a variety of antigens. We examined serum antibodies to diphtheria toxin mutant CRM197 and Epstein-Barr virus protein EBNA-1. These responses tended to be kappa dominant (average kappa-to-lambda ratios of 4.52 and 9.72 respectively). Responses to the influenza vaccine were more balanced with kappa-to-lambda ratio averages having slight strain variations: seasonal H1N1, 1.1; H3N2, 0.96; B, 0.91. We conclude that antigens with limited epitopes tend to produce antibodies with restricted light chain usage and that in most individuals, antibodies with lambda light chains have specificities different and complementary to kappa-containing antibodies. PMID:27113164

  11. Progressive graft fibrosis and donor-specific human leukocyte antigen antibodies in pediatric late liver allografts.

    PubMed

    Miyagawa-Hayashino, Aya; Yoshizawa, Atushi; Uchida, Yoichiro; Egawa, Hiroto; Yurugi, Kimiko; Masuda, Satohiro; Minamiguchi, Sachiko; Maekawa, Taira; Uemoto, Shinji; Haga, Hironori

    2012-11-01

    The role of donor-specific anti-human leukocyte antigen antibodies (DSAs) that develop late after living donor liver transplantation is unknown. Seventy-nine pediatric recipients who had good graft function and underwent protocol liver biopsy more than 5 years after transplantation (median = 11 years, range = 5-20 years) were reviewed. DSAs were determined with the Luminex single-antigen bead assay at the time of the last biopsy, and complement component 4d (C4d) immunostaining was assessed at the times of the last biopsy and the previous biopsy. The donor specificity of antibodies could be identified in 67 patients: DSAs were detected in 32 patients (48%), and they were usually against human leukocyte antigen class II (30 cases) but were rarely against class I (2 cases). These patients had a higher frequency of bridging fibrosis or cirrhosis (28/32 or 88%) than DSA-negative patients (6/35 or 17%, P < 0.001). Fibrosis was likely to be centrilobular-based. DSA-positive patients, in comparison with DSA-negative patients, had higher frequencies of diffuse/focal endothelial C4d staining (P < 0.001) and mild/indeterminate acute rejection [15/32 (47%) versus 5/35 (14%), P = 0.004]. Four DSA-negative patients were off immunosuppression, whereas no patients in the DSA-positive group were (P = 0.048). In conclusion, the high prevalence of graft fibrosis and anti-class II DSAs in late protocol biopsy samples suggests that humoral alloreactivity may contribute to the process of unexplained graft fibrosis late after liver transplantation.

  12. Intra-Blood-Brain Barrier Synthesis of Human Immunodeficiency Virus Antigen and Antibody in Humans and Chimpanzees

    NASA Astrophysics Data System (ADS)

    Goudsmit, Jaap; Epstein, Leon G.; Paul, Deborah A.; van der Helm, Hayo J.; Dawson, George J.; Asher, David M.; Yanagihara, Richard; Wolff, Axel V.; Gibbs, Clarence J.; Carleton Gajdusek, D.

    1987-06-01

    The presence of human immunodeficiency virus (HIV) antigens in cerebrospinal fluid (CSF) was associated with progressive encephalopathy in adult and pediatric patients with acquired immunodeficiency syndrome (AIDS). HIV antigen was detected in CSF from 6 of 7 AIDS patients with progressive encephalopathy. By contrast, HIV antigen, whether free or complexed, was detected in CSF from only 1 of 18 HIV antibody seropositive patients without progressive encephalopathy and from 0 of 8 experimentally infected chimpanzees without clinical signs. Intra-blood-brain barrier synthesis of HIV-specific antibody was demonstrated in the majority of patients with AIDS (9/12) or at risk for AIDS (8/13) as well as in the experimentally infected chimpanzees, indicating HIV-specific B-cell reactivity in the brain without apparent neurological signs. In 6 of 11 patients with HIV infection, antibodies synthesized in the central nervous system were directed against HIV envelope proteins. Active viral expression appears to be necessary for both the immunodeficiency and progressive encephalopathy associated with HIV infection.

  13. Human natural immunoglobulin M antibodies induce apoptosis of human neuroblastoma cells by binding to a Mr 260,000 antigen.

    PubMed

    David, K; Ollert, M W; Vollmert, C; Heiligtag, S; Eickhoff, B; Erttmann, R; Bredehorst, R; Vogel, C W

    1999-08-01

    Sera of healthy humans contain natural cytotoxic IgM antibodies that specifically recognize a Mr 260,000 antigen (NB-p260) on the surface of human neuroblastoma (NB) cells. Here we demonstrate that anti-NB IgM antibodies prepared from different healthy individuals induce, in all human NB cell lines analyzed thus far, typical morphological and biochemical features of apoptosis including nuclear fragmentation, chromatin condensation, and DNA fragmentation. Both the binding of human anti-NB IgM to NB cells and the induction of apoptosis could be inhibited by preincubation of NB cells with murine IgG raised against purified NB-p260. Furthermore, preincubation of human anti-NB IgM with purified NB-p260 immobilized onto a solid support abolished its ability to induce apoptosis in NB cells. Natural human anti-NB IgM failed to bind to and induce apoptosis in control tumor cell lines that lack expression of NB-p260. The anti-NB IgM-induced apoptotic response was also observed in vivo in xenografted human NB tumors. After a single i.v. injection of anti-NB IgM into nude rats bearing solid NB xenografts, many areas of pyknotic cells with fragmented nuclei were observed that stained positive using the terminal dUTP nick end labeling method. In conclusion, the data demonstrate that natural anti-NB IgM antibodies in the sera of healthy individuals are potent mediators of apoptotic cell death of NB cells both in vitro and in vivo. The NB-p260 antigen was identified as the apoptosis-inducing receptor for anti-NB IgM. Whereas natural anti-NB IgM and NB-p260 may be useful tools for immunotherapy of NB, their biological significance remains to be determined.

  14. Human monoclonal antibody against Rh(D) antigen: partial characterization of the Rh(D) polypeptide from human erythrocytes.

    PubMed

    Bloy, C; Blanchard, D; Lambin, P; Goossens, D; Rouger, P; Salmon, C; Cartron, J P

    1987-05-01

    A human monoclonal anti-Rh(D) antibody produced by an Epstein-Barr virus (EBV)-transformed B-cell line (IgG1(lambda), clone H2D5D2) has been purified on protein A-Sepharose column and used for binding studies and immune precipitation of the blood group rhesus (Rh) antigens. Scatchard plot analyses show that the 125I-labeled antibody (iodo-gen procedure), binds to 1.09 X 10(5), 0.43 X 10(5), and 0.32 X 10(5) antigen sites on each D--/D--, R2R2 and R1R1 RBC, respectively, with an association constant of approximately 0.6 X 10(8) mol/L-1. Immune precipitation studies indicate also that the Rh(D) antigen of the Rh(D)-positive RBCs is carried by a 29 kd polypeptide as deduced from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). No material could be precipitated from Rh(D)-negative or Rhnull RBCs. These results indicate that the monoclonal and the polyclonal human anti-Rh(D) behave similarly. A sample (Blo., presumed genotype R2r or R0R2) showing an increased number of antigen sites (0.76 X 10(5)/cell) and a high binding constant (5.7 X 10(8) mol/L-1) was used, as well as D--/D-- RBCs, for further purification of the 29-kd component. Extraction by Triton X-100 (0.1% to 5%) of the immune complexes formed between the membrane-bound Rh(D) antigens and the monoclonal antibody as well as a direct quantitative estimate of the 29-kd component, suggest that the Rh(D) polypeptide is loosely bound to the skeleton, since less than or equal to 80% can be solubilized from the membrane. In similar conditions, glycophorin A showed a slight association with the Triton-insoluble residue, whereas glycophorin B was easily and completely extracted. In contrast, both the minor RBC sialoglycoproteins, glycophorin C and glycoprotein gamma, remained predominantly bound to the membrane skeleton. The purified Rh(D) polypeptide obtained from Blo. and D--/D-- RBCs by immunoprecipitation and preparative gel electrophoresis was homogenous as judged by SDS-PAGE. Amino acid

  15. Human monoclonal antibody against Rh(D) antigen: partial characterization of the Rh(D) polypeptide from human erythrocytes.

    PubMed

    Bloy, C; Blanchard, D; Lambin, P; Goossens, D; Rouger, P; Salmon, C; Cartron, J P

    1987-05-01

    A human monoclonal anti-Rh(D) antibody produced by an Epstein-Barr virus (EBV)-transformed B-cell line (IgG1(lambda), clone H2D5D2) has been purified on protein A-Sepharose column and used for binding studies and immune precipitation of the blood group rhesus (Rh) antigens. Scatchard plot analyses show that the 125I-labeled antibody (iodo-gen procedure), binds to 1.09 X 10(5), 0.43 X 10(5), and 0.32 X 10(5) antigen sites on each D--/D--, R2R2 and R1R1 RBC, respectively, with an association constant of approximately 0.6 X 10(8) mol/L-1. Immune precipitation studies indicate also that the Rh(D) antigen of the Rh(D)-positive RBCs is carried by a 29 kd polypeptide as deduced from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). No material could be precipitated from Rh(D)-negative or Rhnull RBCs. These results indicate that the monoclonal and the polyclonal human anti-Rh(D) behave similarly. A sample (Blo., presumed genotype R2r or R0R2) showing an increased number of antigen sites (0.76 X 10(5)/cell) and a high binding constant (5.7 X 10(8) mol/L-1) was used, as well as D--/D-- RBCs, for further purification of the 29-kd component. Extraction by Triton X-100 (0.1% to 5%) of the immune complexes formed between the membrane-bound Rh(D) antigens and the monoclonal antibody as well as a direct quantitative estimate of the 29-kd component, suggest that the Rh(D) polypeptide is loosely bound to the skeleton, since less than or equal to 80% can be solubilized from the membrane. In similar conditions, glycophorin A showed a slight association with the Triton-insoluble residue, whereas glycophorin B was easily and completely extracted. In contrast, both the minor RBC sialoglycoproteins, glycophorin C and glycoprotein gamma, remained predominantly bound to the membrane skeleton. The purified Rh(D) polypeptide obtained from Blo. and D--/D-- RBCs by immunoprecipitation and preparative gel electrophoresis was homogenous as judged by SDS-PAGE. Amino acid

  16. CD Nomenclature 2015: Human Leukocyte Differentiation Antigen Workshops as a Driving Force in Immunology.

    PubMed

    Engel, Pablo; Boumsell, Laurence; Balderas, Robert; Bensussan, Armand; Gattei, Valter; Horejsi, Vaclav; Jin, Bo-Quan; Malavasi, Fabio; Mortari, Frank; Schwartz-Albiez, Reinhard; Stockinger, Hannes; van Zelm, Menno C; Zola, Heddy; Clark, Georgina

    2015-11-15

    CD (cluster of differentiation) Ags are cell surface molecules expressed on leukocytes and other cells relevant for the immune system. CD nomenclature has been universally adopted by the scientific community and is officially approved by the International Union of Immunological Societies and sanctioned by the World Health Organization. It provides a unified designation system for mAbs, as well as for the cell surface molecules that they recognize. This nomenclature was established by the Human Leukocyte Differentiation Antigens Workshops. In addition to defining the CD nomenclature, these workshops have been instrumental in identifying and determining the expression and function of cell surface molecules. Over the past 30 y, the data generated by the 10 Human Leukocyte Differentiation Antigens Workshops have led to the characterization and formal designation of more than 400 molecules. CD molecules are commonly used as cell markers, allowing the identification and isolation of leukocyte populations, subsets, and differentiation stages. mAbs against these molecules have proven to be essential for biomedical research and diagnosis, as well as in biotechnology. More recently, they have been recognized as invaluable tools for the treatment of several malignancies and autoimmune diseases. In this article, we describe how the CD nomenclature was established, present the official updated list of CD molecules, and provide a rationale for their usefulness in the 21st century. PMID:26546687

  17. Association between human leukocyte antigen-DR and demylinating Guillain-Barré syndrome

    PubMed Central

    Hasan, Zaki N.; Zalzala, Haider H.; Mohammedsalih, Hyam R.; Mahdi, Batool M.; Abid, Laheeb A.; Shakir, Zena N.; Fadhel, Maithem J.

    2014-01-01

    Objective: To find an association between human leukocyte antigen (HLA) class II DRB1, DRB3, DRB4, and DRB5 alleles frequencies in a sample of Iraqi patients with Guillain-Barré syndrome (GBS) and compare with a healthy control group. Methods: We performed a cross-sectional study consisting of 30 Iraqi Arab patients with GBS attending the Neurological Department in the Neuroscience Hospital, Baghdad, Iraq between September 2012 and June 2013. The control group comprised 42 apparently healthy volunteers. Human leukocyte antigen genotyping for HLA DRB1, DRB3, DRB4, and DRB5 was performed using the polymerase chain reaction-sequence-specific primers method. The allele frequencies were compared across both groups. Major histocompatibility complex (MHC)-class II HLA-DR genotyping and serotyping were performed by software analysis. Results: We found increased frequencies of HLA genotype DRB1*03:01 (p=0.0009), DRB1*07:01 (p=0.0015), and DRB4*01:01 (p<0.0001) in patients with GBS compared with healthy controls. The HLA DR6 was increased in the control group (p<0.0001). Conclusions: Our results suggest an association between HLA-DRB1*03:01, DRB1*07:01, DRB4*01:01, and HLA DR3, DR7 and a susceptibility to GBS. PMID:25274590

  18. IMMUNODIAGNOSIS OF HUMAN STRONGYLOIDIASIS: USE OF SIX DIFFERENT ANTIGENIC FRACTIONS FROM Strongyloides venezuelensis PARASITIC FEMALES

    PubMed Central

    CORRAL, Marcelo Andreetta; de PAULA, Fabiana Martins; GOTTARDI, Maiara; MEISEL, Dirce Mary Correia Lima; CASTILHO, Vera Lucia Pagliusi; GONÇALVES, Elenice Messias do Nascimento; CHIEFFI, Pedro Paulo; GRYSCHEK, Ronaldo Cesar Borges

    2015-01-01

    SUMMARY The aim of this study was to evaluate six different antigenic fractions from Strongyloides venezuelensis parasitic females for the immunodiagnosis of human strongyloidiasis. Soluble and membrane fractions from S. venezuelensis parasitic females were prepared in phosphate-buffered saline (SSF and SMF, respectively), Tris-HCl (TSF and TMF, respectively), and an alkaline buffer (ASF and AMF, respectively). Serum samples obtained from patients with strongyloidiasis or, other parasitic diseases, and healthy individuals were analyzed by enzyme-linked immunosorbent assay (ELISA). Soluble fractions SSF, TSF, and ASF showed 85.0%, 75.0%, and 80.0% sensitivity and 93.1%, 93.1%, and 87.5% specificity, respectively. Membrane fractions SMF, TMF, and AMF showed 80.0%, 75.0%, and 85.0% sensitivity, and 95.8%, 90.3%, and 91.7% specificity, respectively. In conclusion, the present results suggest that the fractions obtained from parasitic females, especially the SSF and SMF, could be used as alternative antigen sources in the serodiagnosis of human strongyloidiasis. PMID:26603231

  19. Different human vaccine adjuvants promote distinct antigen-independent immunological signatures tailored to different pathogens

    PubMed Central

    Knudsen, Niels Peter H.; Olsen, Anja; Buonsanti, Cecilia; Follmann, Frank; Zhang, Yuan; Coler, Rhea N.; Fox, Christopher B.; Meinke, Andreas; D´Oro, Ugo; Casini, Daniele; Bonci, Alessandra; Billeskov, Rolf; De Gregorio, Ennio; Rappuoli, Rino; Harandi, Ali M.; Andersen, Peter; Agger, Else Marie

    2016-01-01

    The majority of vaccine candidates in clinical development are highly purified proteins and peptides relying on adjuvants to enhance and/or direct immune responses. Despite the acknowledged need for novel adjuvants, there are still very few adjuvants in licensed human vaccines. A vast number of adjuvants have been tested pre-clinically using different experimental conditions, rendering it impossible to directly compare their activity. We performed a head-to-head comparison of five different adjuvants Alum, MF59®, GLA-SE, IC31® and CAF01 in mice and combined these with antigens from M. tuberculosis, influenza, and chlamydia to test immune-profiles and efficacy in infection models using standardized protocols. Regardless of antigen, each adjuvant had a unique immunological signature suggesting that the adjuvants have potential for different disease targets. Alum increased antibody titers; MF59® induced strong antibody and IL-5 responses; GLA-SE induced antibodies and Th1; CAF01 showed a mixed Th1/Th17 profile and IC31® induced strong Th1 responses. MF59® and GLA-SE were strong inducers of influenza HI titers while CAF01, GLA-SE and IC31® enhanced protection to TB and chlamydia. Importantly, this is the first extensive attempt to categorize clinical-grade adjuvants based on their immune profiles and protective efficacy to inform a rational development of next generation vaccines for human use. PMID:26791076

  20. Chromogranin A is a T Cell Antigen in Human Type 1 Diabetes

    PubMed Central

    Gottlieb, Peter A; Delong, Thomas; Baker, Rocky L.; Fitzgerald-Miller, Lisa; Wagner, Rebecca; Cook, Gabrielle; Rewers, Marian R.; Michels, Aaron; Haskins, Kathryn

    2013-01-01

    Chromogranin A (ChgA) is a beta cell secretory granule protein and a peptide of ChgA, WE14, was recently identified as a ligand for diabetogenic CD4 T cell clones derived from the NOD mouse. In this study we compared responses of human CD4 T cells from recent onset type 1 diabetic (T1D) and control subjects to WE14 and to an enzymatically modified version of this peptide. T cell responders to antigens were detected in PBMCs from study subjects by an indirect CD4 ELISPOT assay for IFN-γ. T1D patients (n=27) were recent onset patients within one year of diagnosis, typed for HLA-DQ8. Controls (n=31) were either 1st degree relatives with no antibodies or from the HLA-matched general population cohort of DAISY/TEDDY. A second cohort of patients (n=11) and control subjects (n=11) was tested at lower peptide concentrations. We found that WE14 is recognized by T cells from diabetic subjects vs. controls in a dose dependent manner. Treatment of WE14 with transglutaminase increased reactivity to the peptide in some patients. This work suggests that ChgA is an important target antigen in human T1D subjects and that post-translational modification may play a role in its reactivity and relationship to disease. PMID:24239002

  1. Different human vaccine adjuvants promote distinct antigen-independent immunological signatures tailored to different pathogens.

    PubMed

    Knudsen, Niels Peter H; Olsen, Anja; Buonsanti, Cecilia; Follmann, Frank; Zhang, Yuan; Coler, Rhea N; Fox, Christopher B; Meinke, Andreas; D'Oro, Ugo; Casini, Daniele; Bonci, Alessandra; Billeskov, Rolf; De Gregorio, Ennio; Rappuoli, Rino; Harandi, Ali M; Andersen, Peter; Agger, Else Marie

    2016-01-01

    The majority of vaccine candidates in clinical development are highly purified proteins and peptides relying on adjuvants to enhance and/or direct immune responses. Despite the acknowledged need for novel adjuvants, there are still very few adjuvants in licensed human vaccines. A vast number of adjuvants have been tested pre-clinically using different experimental conditions, rendering it impossible to directly compare their activity. We performed a head-to-head comparison of five different adjuvants Alum, MF59®, GLA-SE, IC31® and CAF01 in mice and combined these with antigens from M. tuberculosis, influenza, and chlamydia to test immune-profiles and efficacy in infection models using standardized protocols. Regardless of antigen, each adjuvant had a unique immunological signature suggesting that the adjuvants have potential for different disease targets. Alum increased antibody titers; MF59® induced strong antibody and IL-5 responses; GLA-SE induced antibodies and Th1; CAF01 showed a mixed Th1/Th17 profile and IC31® induced strong Th1 responses. MF59® and GLA-SE were strong inducers of influenza HI titers while CAF01, GLA-SE and IC31® enhanced protection to TB and chlamydia. Importantly, this is the first extensive attempt to categorize clinical-grade adjuvants based on their immune profiles and protective efficacy to inform a rational development of next generation vaccines for human use. PMID:26791076

  2. Paratope diversity in the human antibody response to Bacillus anthracis protective antigen.

    PubMed

    Zhou, Jianhui; Ullal, Anuska; Liberato, Justine; Sun, Jinying; Keitel, Wendy; Reason, Donald C

    2008-01-01

    The active component of the licensed human anthrax vaccine (BioThrax, or AVA) is a Bacillus anthracis toxin known as protective antigen (PA). Second generation anthrax vaccines currently under development are also based on a recombinant form of PA. Since the current and future anthrax vaccines are based on this toxin, it is important that the immunobiology of this protein in vaccinated humans be understood in detail. We have isolated and analyzed the PA-specific antibody repertoire from an AVA-vaccinated individual. When examined at the clonal level, we find an antibody response that is complex in terms of the combinatorial elements and immunoglobulin variable genes employed. All PA-specific antibodies had undergone somatic hypermutation and class switch recombination, both signs of affinity maturation. Although the antigenic epitopes recognized by the response were distributed throughout the PA monomer, the majority of antibodies arising in this individual following vaccination recognize determinants located on the amino-terminal (PA20) sub-domain of the molecule. This latter finding may have implications for the rational design of future PA-based anthrax vaccines.

  3. Induction of the Epstein-Barr virus latent membrane protein 2 antigen-specific cytotoxic T lymphocytes using human leukocyte antigen tetramer-based artificial antigen-presenting cells.

    PubMed

    Lu, Xiao-Ling; Liang, Zhi-Hui; Zhang, Cai-E; Lu, Sheng-Jun; Weng, Xiu-Fang; Wu, Xiong-Wen

    2006-03-01

    Cytotoxic T lymphocytes (CTLs) specific for the Epstein-Barr virus (EBV) latent membrane protein 2 (LMP2) antigen are important reagents for the treatment of some EBV-associated malignancies, such as EBV-positive Hodgkin's disease and nasopharyngeal carcinoma. However, the therapeutic amount of CTLs is often hampered by the limited supply of antigen-presenting cells. To address this issue, an artificial antigen-presenting cell (aAPC) was made by coating a human leukocyte antigen (HLA)-pLMP2 tetrameric complex, anti-CD28 antibody and CD54 molecule to a cell-sized latex bead, which provided the dual signals required for T cell activation. By co-culture of the HLA-A2-LMP2 bearing aAPC and peripheral blood mononuclear cells from HLA-A2 positive healthy donors, LMP2 antigen-specific CTLs were induced and expanded in vitro. The specificity of the aAPC-induced CTLs was demonstrated by both HLA-A2-LMP2 tetramer staining and cytotoxicity against HLA-A2-LMP2 bearing T2 cell, the cytotoxicity was inhibited by the anti-HLA class I antibody (W6/32). These results showed that LMP2 antigen-specific CTLs could be induced and expanded in vitro by the HLA-A2-LMP2-bearing aAPC. Thus, aAPCs coated with an HLA-pLMP2 complex, anti-CD28 and CD54 might be promising tools for the enrichment of LMP2-specific CTLs for adoptive immunotherapy. PMID:16518539

  4. Artificial antigen-presenting cells transduced with telomerase efficiently expand epitope-specific, human leukocyte antigen-restricted cytotoxic T cells.

    PubMed

    Dupont, Jakob; Latouche, Jean-Baptiste; Ma, Chia; Sadelain, Michel

    2005-06-15

    Human telomerase reverse transcriptase (hTERT) is overexpressed in most human tumors, making it a potential target for cancer immunotherapy. hTERT-derived CTL epitopes have been identified previously, including p865 (RLVDDFLLV) and p540 (ILAKFLHWL), which are restricted by the human leukocyte antigen (HLA) class I A*0201 allele. However, it remains a major challenge to efficiently and consistently expand hTERT-specific CTLs from donor peripheral blood T lymphocytes. To bypass the need for generating conventional antigen-presenting cells (APC) on an autologous basis, we investigated the potential ability of fibroblast-derived artificial APCs (AAPC) to activate and expand HLA-A*0201-restricted CTLs. We show here that AAPCs stably expressing HLA-A*0201, human beta(2)-microglobulin, B7.1, intercellular adhesion molecule-1, and LFA-3, together with either p540 and p865 minigenes or the full-length hTERT, effectively stimulate tumoricidal, hTERT-specific CTLs. hTERT-expressing AAPCs stimulated both p540 and p865 CTLs as shown by peptide-specific cytolysis and tetramer staining, indicating that hTERT is processed by the AAPCs and that the two peptides are presented as codominant epitopes. The level of cytotoxic activity against a panel of tumors comprising hematologic and epithelial malignancies varied, correlating overall with the level of HLA-A2 and hTERT expression by the target cell. Starting from 100 mL blood, approximately 100 million hTERT-specific CTLs could be generated over the course of five sequential stimulations, representing an expansion of approximately 1 x 10(5). Our data show that AAPCs process hTERT antigen and efficiently stimulate hTERT-specific CTLs from human peripheral blood T lymphocytes and suggest that sufficient expansion could be achieved to be clinically useful for adoptive cell therapy.

  5. Non-pathogenic bacteria elicit a differential cytokine response by intestinal epithelial cell/leucocyte co-cultures

    PubMed Central

    Haller, D; Bode, C; Hammes, W; Pfeifer, A; Schiffrin, E; Blum, S

    2000-01-01

    BACKGROUND AND AIM—Intestinal epithelial cells (IEC) are thought to participate in the mucosal defence against bacteria and in the regulation of mucosal tissue homeostasis. Reactivity of IEC to bacterial signals may depend on interactions with immunocompetent cells. To address the question of whether non-pathogenic bacteria modify the immune response of the intestinal epithelium, we co-cultivated enterocyte-like CaCO-2 cells with human blood leucocytes in separate compartments of transwell cultures.
METHODS—CaCO-2/PBMC co-cultures were stimulated with non-pathogenic bacteria and enteropathogenic Escherichia coli. Expression of tumour necrosis factor alpha (TNF-α), interleukin (IL)-1β, IL-8, monocyte chemoattracting protein 1 (MCP-1), and IL-10 was studied by enzyme linked immunosorbent assays (cytokine secretion) and by semiquantitative reverse transcription-polymerase chain reaction.
RESULTS—Challenge of CaCO-2 cells with non-pathogenic E coli and Lactobacillus sakei induced expression of IL-8, MCP-1, IL-1β, and TNF-α mRNA in the presence of underlying leucocytes. Leucocyte sensitised CaCO-2 cells produced TNF-α and IL-1β whereas IL-10 was exclusively secreted by human peripheral blood mononuclear cells. CaCO-2 cells alone remained hyporesponsive to the bacterial challenge. Lactobacillus johnsonii, an intestinal isolate, showed reduced potential to induce proinflammatory cytokines but increased transforming growth factor beta mRNA in leucocyte sensitised CaCO-2 cells. TNF-α was identified as one of the early mediators involved in cellular cross talk. In the presence of leucocytes, discriminative activation of CaCO-2 cells was observed between enteropathogenic E coli and non-pathogenic bacteria.
CONCLUSION—The differential recognition of non-pathogenic bacteria by CaCO-2 cells required the presence of underlying leucocytes. These results strengthen the hypothesis that bacterial signalling at the mucosal surface is dependent on a network of

  6. [Pharmacogenetic research in the association between human leukocyte antigen and adverse drug reactions].

    PubMed

    Xiong, Yan; Zhang, Wei; Chen, Xiaoping

    2014-07-01

    With the rapid development of pharmacogenetics, more and more studies have shown evidence in the association between polymorphisms at the human leukocyte antigen (HLA) loci and severe adverse drug reactions (SADRs). Several HLA-B alleles proved to be associated with SADRs for drugs such as carbamazepine, allopurinol, lamotrigine, and flucloxacillin. The USA Food and Drug Administration (FDA) has even recommended routine screening for HLA-B allele before the use of abacavir and carbamazepine. With the completion of human genome project and the Hapmap project, several new pharmacogenetics approaches such as genome-wide association study (GWAS) have emerged. These newly developed methods will undoubtedly accelerate the identification and clinical utilization of the pharmacogenetic biomakers. In addition, the immunogenetic mechanisms by which the HLA alleles cause SADRs are explored at the cellular and molecular level. This review focuses on the recent progresses in HLA alleles and ADRs regarding both the clinical translation and modern pharmacogenetic methods. PMID:25080918

  7. A human monoclonal antibody to high-frequency red cell antigen Jra.

    PubMed

    Miyazaki, T; Kwon, K W; Yamamoto, K; Tone, Y; Ihara, H; Kato, T; Ikeda, H; Sekiguchi, S

    1994-01-01

    A human-mouse heterohybridoma (HMR0921) secreting human monoclonal IgG3, lambda antibody was produced from peripheral blood lymphocytes of a healthy blood donor with serum antibody to Jra, by EBV transformation and hybridization with mouse myeloma cell line P3X63Ag8.653. The reactivity of HMR0921 antibody was assessed by antiglobulin test with a panel of red cells including 14 different rare blood types. Only Jr(a-) red cells were negative. The strict specificity of this antibody to Jra antigen was further confirmed by absorption test with fluorescence flow cytometry. On screening of 28,744 blood donor samples by HMR0921 antibody, we detected 19 agglutination-negative samples, which were confirmed as Jr(a-) by conventional anti-Jra antisera. Therefore, our HMR0921 antibody is extremely useful for detecting rare Jr(a-) blood.

  8. Histocompatibility antigens in a population based silicosis series.

    PubMed Central

    Kreiss, K; Danilovs, J A; Newman, L S

    1989-01-01

    Individual susceptibility to silicosis is suggested by the lack of a uniform dose response relation and by the presence of immunological epiphenomena, such as increased antibody levels and associated diseases that reflect altered immune regulation. Human leucocyte antigens (HLA) are linked with immune response capability and might indicate a possible genetic susceptibility to silicosis. Forty nine silicotic subjects were identified from chest radiographs in a population based study in Leadville, Colorado. They were interviewed for symptoms and occupational history and gave a blood specimen for HLA-A, -B, -DR, and -DQ typing and for antinuclear antibody, immune complexes, immunoglobulins, and rheumatoid factor. Silicotic subjects had twice the prevalence of B44 (45%) of the reference population and had triple the prevalence of A29 (20%), both of which were statistically significant when corrected for the number of comparisons made. No perturbations in D-region antigen frequencies were detected. B44-positive subjects were older at diagnosis and had less dyspnoea than other subjects. A29-positive subjects were more likely to have abnormal levels of IgA and had higher levels of immune complexes. This study is the first to find significant HLA antigen excesses among a series of silicotic cases and extends earlier reported hypotheses that were based on groups of antigens of which B44 and A29 are components. PMID:2818968

  9. MUC-1 Tumor Antigen Agonist Epitopes for Enhancing T-cell Responses to Human Tumors | NCI Technology Transfer Center | TTC

    Cancer.gov

    Scientists at NIH have identified 7 new agonist epitopes of the MUC-1 tumor associated antigen. Compared to their native epitope counterparts, peptides reflecting these agonist epitopes have been shown to enhance the generation of human tumor cells, which in turn have a greater ability to kill human tumor cells endogenously expressing the native MUC-1 epitope.

  10. Somatic cell genetic analysis of human cell surface antigens 5.1H11 and F35/9 (gp45).

    PubMed

    Rettig, W J; Triche, T J; Bander, N H

    1990-01-01

    Serologic analysis of rodent-human somatic cell hybrids has permitted the assignment of loci coding for cell surface differentiation antigens 5.1H11 (gene symbol MSK39) and F35/9 (MSK40) to human chromosomes 11q13-qter and 22, respectively. Both antigens are expressed in hybrids constructed with antigen-positive human cells and certain hybrids constructed with antigen-negative human cells, indicating that the coding genes are not irreversibly silenced in human nonexpressor cells. Antigens 5.1H11 and F35/9, and at least six additional cell surface antigens encoded by chromosomes 11 and 22, are expressed on human Ewing sarcoma and peripheral neuroepithelioma cells, providing selectable markers for isolating and characterizing the specific t(11;22)(q24;q12) marker chromosomes of these tumors in interspecies hybrids.

  11. Hepatitis B surface antigen-specific cell-mediated immune responses in human chronic hepatitis B surface antigen carriers.

    PubMed Central

    Beutner, K R; Tiku, M L; Ogra, P L

    1978-01-01

    The presence of hepatitis B surface antigen (HBsAg) and antibody (anti-HBs), hepatitis B e antigen (HBeAg) and antibody (anti-HBe), the nature of T-cell function, and specific cell-mediated immunity to HBsAg were determined and evaluated serially in groups of subjects with chronic HBsAg carrier states and in seronegative controls. The techniques of in vitro lymphocyte transformation, spontaneous rosette formation, radioimmunoassay, reverse passive hemagglutination, passive hemagglutination, rheophoresis, and liver function tests were employed for these studies. For the lymphocyte transformation assay, multiple concentrations of phytohemagglutinin and purified HBsAg were used as stimulants. Cell-mediated immunity to HBsAg was detectable in 50% of the chronic HBsAg carriers (responders) at one or more concentrations of HBsAg. The remaining carriers (nonresponders) and controls failed to manifest HBsAg-specific lymphocyte transformation activity. The profile of the responders was characterized by elevated serum glutamic pyruvic transaminase levels, the presence of anti-HBe, high HBsAg titers, and the conspicuous absence of HBeAg in the serum. The nonresponders were characterized by normal serum glutamic pyruvic transaminase levels, the presence of HBeAg and anti-HBe, and lower HBsAg titers. These observations demonstrate the presence of specific cell-mediated immunity to HBsAg in chronic HBsAg carriers who manifest biochemical evidence of liver disease. PMID:80380

  12. Human immunodeficiency virus type 1 Nef does not alter T-cell sensitivity to antigen-specific stimulation.

    PubMed Central

    Page, K A; van Schooten, W C; Feinberg, M B

    1997-01-01

    We have developed an in vitro model to study the influence that human immunodeficiency virus type 1 (HIV-1) may have on the ability of T cells to respond to antigenic challenge. We have examined consequences of HIV-1 gene expression on T-cell activation in antigen-dependent T cells that have stably integrated copies of replication-defective proviral HIV-1. Virus production by HIV-infected, antigen-dependent T cells was induced in response to antigenic stimulation and then decreased as infected cells returned to a state of quiescence. Contrary to the predictions of models proposing that Nef alters signal transduction pathways in T lymphocytes and thereby alters cellular activation, Nef expression in antigen-dependent T-cell clones did not influence their proliferative responses to low or intermediate concentrations of antigen and did not affect other measures of T-cell activation, such as induction of interleukin 2 receptor alpha-chain expression and cytokine production. In addition, we found no evidence for alteration of T-cell responsiveness to antigen by the gag, pol, vif, tat, or rev gene of HIV-1. PMID:9094653

  13. The efficacy of serum carcinoembryonic antigen (CEA), cancer antigen 125 (CA125), carbohydrate antigen 19-9 (CA19-9), carbohydrate antigen 15-3 (CA15-3), alpha-fetoprotein (AFP) and human chorionic gonadotropin (hCG) levels in determining the malignancy of solitary pulmonary nodules.

    PubMed

    Bekci, T T; Senol, T; Maden, E

    2009-01-01

    We investigated the utility of the tumour markers carcinoembryonic antigen (CEA), cancer antigen 125 (CA125), carbohydrate antigen 19-9 (CA19-9), carbohydrate antigen 15-3 (CA15-3), alpha-fetoprotein (AFP) and human chorionic gonadotropin (hCG) for the differential diagnosis of benign and malignant solitary pulmonary nodules in 42 hospitalized patients. Routine medical history and physical examination of each patient was performed and each patient also had a chest X-ray and a thoracic computed tomography scan. The following diagnostic procedures were also undertaken: bronchoscopy, transthoracic needle aspiration biopsy, sputum cytology and culture, analysis of sputum acid-fast bacilli and thoracotomy. Measurement of serum levels of tumour antigens by Immulite 2000 radioimmunoassay found that three tumour markers, CEA, CA125 and CA15-3, could be used in the diagnosis of malignant solitary pulmonary nodules. More research is now required involving a larger group of patients.

  14. Histo-Blood Group Antigen-Like Substances of Human Enteric Bacteria as Specific Adsorbents for Human Noroviruses

    PubMed Central

    Miura, Takayuki; Suenaga, Atsushi; Yoshimura, Takeshi; Fuzawa, Miyu; Nakagomi, Toyoko; Nakagomi, Osamu; Okabe, Satoshi

    2013-01-01

    Histo-blood group antigens (HBGAs) have been suggested to be receptors or coreceptors for human noroviruses (HuNoVs) expressed on the intestinal epithelium. We isolated an enteric bacterium strain (SENG-6), closely related to Enterobacter cloacae, bearing HBGA-like substances from a fecal sample of a healthy individual by using a biopanning technique with anti-HBGA antibodies. The binding capacities of four genotypes of norovirus-like particles (NoVLPs) to Enterobacter sp. SENG-6 cells were confirmed by enzyme-linked immunosorbent assay (ELISA). Transmission electron microscopy demonstrated that NoVLPs bound mainly to extracellular polymeric substances (EPS) of Enterobacter sp. SENG-6, where the HBGA-like substances were localized. EPS that contained HBGA-like substances extracted from Enterobacter sp. SENG-6 was shown by enzyme-linked immunosorbent assay (ELISA) to be capable of binding to NoVLPs of a GI.1 wild-type strain (8fIIa) and a GII.6 strain that can recognize A antigen but not to an NoVLP GI.1 mutant strain (W375A) that loses the ability to bind to A antigen. Enzymatic cleavage of terminal N-acetyl-galactosamine residues in the bacterial EPS weakened bacterial EPS binding to the GI.1 wild-type strain (8fIIa). These results indicate that A-like substances in the bacterial EPS play a key role in binding to NoVLPs. Since the specific binding of HuNoVs to HBGA-positive enteric bacteria is likely to affect the transmission and infection processes of HuNoVs in their hosts and in the environment, further studies of human enteric bacteria and their binding capacity to HuNoVs will provide a new scientific platform for understanding interactions between two types of microbes that were previously regarded as biologically unrelated. PMID:23804639

  15. An Integrated Multiomics Approach to Identify Candidate Antigens for Serodiagnosis of Human Onchocerciasis.

    PubMed

    McNulty, Samantha N; Rosa, Bruce A; Fischer, Peter U; Rumsey, Jeanne M; Erdmann-Gilmore, Petra; Curtis, Kurt C; Specht, Sabine; Townsend, R Reid; Weil, Gary J; Mitreva, Makedonka

    2015-12-01

    Improved diagnostic methods are needed to support ongoing efforts to eliminate onchocerciasis (river blindness). This study used an integrated approach to identify adult female Onchocerca volvulus antigens that can be explored for developing serodiagnostic tests. The first step was to develop a detailed multi-omics database of all O. volvulus proteins deduced from the genome, gene transcription data for different stages of the parasite including eight individual female worms (providing gene expression information for 94.8% of all protein coding genes), and the adult female worm proteome (detecting 2126 proteins). Next, female worm proteins were purified with IgG antibodies from onchocerciasis patients and identified using LC-MS with a high-resolution hybrid quadrupole-time-of-flight mass spectrometer. A total of 241 immunoreactive proteins were identified among those bound by IgG from infected individuals but not IgG from uninfected controls. These included most of the major diagnostic antigens described over the past 25 years plus many new candidates. Proteins of interest were prioritized for further study based on a lack of conservation with orthologs in the human host and other helminthes, their expression pattern across the life cycle, and their consistent expression among individual female worms. Based on these criteria, we selected 33 proteins that should be carried forward for testing as serodiagnostic antigens to supplement existing diagnostic tools. These candidates, together with the extensive pan-omics dataset generated in this study are available to the community (http://nematode.net) to facilitate basic and translational research on onchocerciasis. PMID:26472727

  16. Human Antibodies to a Mr 155,000 Plasmodium falciparum Antigen Efficiently Inhibit Merozoite Invasion

    NASA Astrophysics Data System (ADS)

    Wahlin, Birgitta; Wahlgren, Mats; Perlmann, Hedvig; Berzins, Klavs; Bjorkman, Anders; Patarroyo, Manuel E.; Perlmann, Peter

    1984-12-01

    IgG from a donor clinically immune to Plasmodium falciparum malaria strongly inhibited reinvasion in vitro of human erythrocytes by the parasite. When added to monolayers of glutaraldehyde-fixed and air-dried erythrocytes infected with the parasite, this IgG also displayed a characteristic immunofluorescence restricted to the surface of infected erythrocytes. Elution of the IgG adsorbed to such monolayers gave an antibody fraction that was 40 times more efficient in the reinvasion inhibition assay (50% inhibition titer, <1 μ g/ml) than the original IgG preparation. The major antibody in this eluate was directed against a parasite-derived antigen of Mr 155,000 (Pf 155) deposited by the parasite in the erythrocyte membrane in the course of invasion. A detailed study of IgG fractions from 11 donors with acute P. falciparum malaria or clinical immunity revealed the existence of an excellent correlation between their capacities to stain the surface of infected erythrocytes, their titers in reinvasion inhibition, and the presence of antibodies to Pf 155 as detected by immunoblotting. No such correlations were seen when the IgG fractions were analyzed for immunofluorescence of intracellular parasites or for the presence of antibodies to other parasite antigens as detected by immunoprecipitation of [35S]methionine-labeled and NaDodSO4/PAGE-separated parasite extracts. The results suggest that Pf 155 has an important role in the process of erythrocyte infection and that host antibodies to this antigen may efficiently interfere with this process.

  17. A human endogenous retroviral sequence encoding an antigen recognized on melanoma by cytolytic T lymphocytes.

    PubMed

    Schiavetti, Francesca; Thonnard, Joëlle; Colau, Didier; Boon, Thierry; Coulie, Pierre G

    2002-10-01

    We have identified a gene encoding an antigen recognized by cytolytic T lymphocytes on the autologous tumor cells of a melanoma patient, AVL3. The gene shows homologies with members of the HERV-K family of human endogenous retroviruses, and it was provisionally named HERV-K-MEL. It contains many mutations that disrupt the open reading frames coding for all of the viral proteins. The HERV-K-MEL gene is not expressed in normal tissues with the exception of testis and some skin samples. It is expressed in most samples of cutaneous and ocular melanoma. It is also expressed in a majority of naevi and in a minority of carcinomas and sarcomas. The antigenic peptide, presented by HLA-A2 molecules, is encoded by a very short open reading frame present in the env region of a spliced HERV-K-MEL transcript. Anti-HERV.A2 CTLp could not be detected in the blood of three individuals without cancer but were present at a frequency of 3 x 10(-5) among blood CD8 T cells in patient AVL3 and 6 x 10(-7) in another HLA-A2 melanoma patient whose tumor expressed HERV-K-MEL. Anti-HERV.A2 CTL clones derived from each patient lysed melanoma cells. Analysis of T-cell receptor beta chain sequences indicated that the anti-HERV.A2 CTL population was oligoclonal in patient AVL3 and probably monoclonal in the other patient. These results suggest that HERV-K-MEL is a source of antigens that are targeted by CTLs in melanoma patients and could therefore be used for vaccination.

  18. An Integrated Multiomics Approach to Identify Candidate Antigens for Serodiagnosis of Human Onchocerciasis*

    PubMed Central

    McNulty, Samantha N.; Rosa, Bruce A.; Fischer, Peter U.; Rumsey, Jeanne M.; Erdmann-Gilmore, Petra; Curtis, Kurt C.; Specht, Sabine; Townsend, R. Reid; Weil, Gary J.; Mitreva, Makedonka

    2015-01-01

    Improved diagnostic methods are needed to support ongoing efforts to eliminate onchocerciasis (river blindness). This study used an integrated approach to identify adult female Onchocerca volvulus antigens that can be explored for developing serodiagnostic tests. The first step was to develop a detailed multi-omics database of all O. volvulus proteins deduced from the genome, gene transcription data for different stages of the parasite including eight individual female worms (providing gene expression information for 94.8% of all protein coding genes), and the adult female worm proteome (detecting 2126 proteins). Next, female worm proteins were purified with IgG antibodies from onchocerciasis patients and identified using LC-MS with a high-resolution hybrid quadrupole-time-of-flight mass spectrometer. A total of 241 immunoreactive proteins were identified among those bound by IgG from infected individuals but not IgG from uninfected controls. These included most of the major diagnostic antigens described over the past 25 years plus many new candidates. Proteins of interest were prioritized for further study based on a lack of conservation with orthologs in the human host and other helminthes, their expression pattern across the life cycle, and their consistent expression among individual female worms. Based on these criteria, we selected 33 proteins that should be carried forward for testing as serodiagnostic antigens to supplement existing diagnostic tools. These candidates, together with the extensive pan-omics dataset generated in this study are available to the community (http://nematode.net) to facilitate basic and translational research on onchocerciasis. PMID:26472727

  19. A study of human leukocyte D locus related antigens in Graves' disease.

    PubMed

    Farid, N R; Sampson, L; Noel, E P; Barnard, J M; Mandeville, R; Larsen, B; Marshall, W H; Carter, N D

    1979-01-01

    An association between Graves' disease and the human leukocyte antigen (HLA) system has previously been reported. The disease was more strongly associated with the HLA D locus antigen Dw3 than with HLA B8. Products of the HLA D locus are determined by the interaction of test cells with standard typing lymphocytes, a technically difficult procedure. Recently, it has been possible to type serologically for D locus related (DRw) specificities on peripheral bone marrow-derived (B) lymphocytes. Blood B lymphocytes from 50 unrelated controls and 41 patients with Graves' disease were typed for seven HLA DRw specificities. 28 patients with Graves' disease (68%) were positive for DRw3, in contrast to 14 controls (28%); whereas only 21 patients (50%) were HLA B8 positive, compared with 13 (26%) controls. Thus, positivity for DRw3 afforded a relative risk for Graves' disease of 5.5, whereas that for HLA B8 amounted to 3.0. Additionally, a family with multiple cases of Graves' disease in which the disease was previously shown to be inherited with the haplotype, was linked to DRw2, which suggests that the susceptibility to the disease was inherited in association with that antigen. Two HLA B/glyoxalase recombination events were observed in this family; in both instances HLA DRw followed HLA B. This study thus demonstrates that the disease susceptibility gene for Graves' disease is in strong linkage disequilibrium with DRw3; however, it may be associated with other DRw specificities and inherited within family units in association with them.

  20. Epidermal surface antigen (MS17S1) is highly conserved between mouse and human

    SciTech Connect

    Cho, Y.J.; Chema, D.; Cho, M.

    1995-05-20

    A mouse monoclonal antibody ECS-1 raised to human keratinocytes detects a 35-kDa epidermal surface antigen (ESA) and causes keratinocyte dissociation in vitro. ECS-1 stains skin of 16-day mouse embryo and 8- to 9-week human fetus. Mouse Esa cDNA encodes a 379-amino-acid protein that is 99.2% identical to the human, differing at only 3 amino acids. The gene (M17S1) was mapped to mouse chromosome 11, highlighting the conserved linkage synteny existing between human chromosome 17 and mouse chromosome 11. Although the nude locus has been mapped to the same region of chromosome 11, no abnormalities in protein, mRNA, or cDNA or genomic sequences were detected in nude mice. However, both nude and control mice were found to have a second Esa mRNA transcript that conserves amino acid sequence and molecular weight. The mouse and human 5{prime} and 3{prime} untranslated sequences are conserved. Similar RNA folding patterns of the 5{prime} untranslated region are predicted despite a 91-bp insertion in the mouse. These data suggest that both the function and the regulation of ESA protein are of importance and that Esa (M17S1) is not the nude locus gene. 42 refs., 7 figs., 3 tabs.

  1. Genetic Mechanism of Human Neutrophil Antigen 2 Deficiency and Expression Variations

    PubMed Central

    Li, Yunfang; Mair, David C.; Schuller, Randy M.; Li, Ling; Wu, Jianming

    2015-01-01

    Human neutrophil antigen 2 (HNA-2) deficiency is a common phenotype as 3–5% humans do not express HNA-2. HNA-2 is coded by CD177 gene that associates with human myeloproliferative disorders. HNA-2 deficient individuals are prone to produce HNA-2 alloantibodies that cause a number of disorders including transfusion-related acute lung injury and immune neutropenia. In addition, the percentages of HNA-2 positive neutrophils vary significantly among individuals and HNA-2 expression variations play a role in human diseases such as myelodysplastic syndrome, chronic myelogenous leukemia, and gastric cancer. The underlying genetic mechanism of HNA-2 deficiency and expression variations has remained a mystery. In this study, we identified a novel CD177 nonsense single nucleotide polymorphism (SNP 829A>T) that creates a stop codon within the CD177 coding region. We found that all 829TT homozygous individuals were HNA-2 deficient. In addition, the SNP 829A>T genotypes were significantly associated with the percentage of HNA-2 positive neutrophils. Transfection experiments confirmed that HNA-2 expression was absent on cells expressing the CD177 SNP 829T allele. Our data clearly demonstrate that the CD177 SNP 829A>T is the primary genetic determinant for HNA-2 deficiency and expression variations. The mechanistic delineation of HNA-2 genetics will enable the development of genetic tests for diagnosis and prognosis of HNA-2-related human diseases. PMID:26024230

  2. The human E48 antigen, highly homologous to the murine Ly-6 antigen ThB, is a GPI-anchored molecule apparently involved in keratinocyte cell-cell adhesion

    PubMed Central

    1995-01-01

    The E48 antigen, a putative human homologue of the 20-kD protein present in desmosomal preparations of bovine muzzle, and formerly called desmoglein III (dg4), is a promising target antigen for antibody- based therapy of squamous cell carcinoma in man. To anticipate the effect of high antibody dose treatment, and to evaluate the possible biological involvement of the antigen in carcinogenesis, we set out to molecularly characterize the antigen. A cDNA clone encoding the E48 antigen was isolated by expression cloning in COS cells. Sequence analysis revealed that the clone contained an open reading frame of 128 amino acids, encoding a core protein of 13,286 kD. Database searching showed that the E48 antigen has a high level of sequence similarity with the mouse ThB antigen, a member of the Ly-6 antigen family. Phosphatidylinositol-specific (PI-specific) phospholipase-C treatment indicated that the E48 antigen is glycosylphosphatidylinositol-anchored (GPI-anchored) to the plasma membrane. The gene encoding the E48 antigen is a single copy gene, located on human chromosome 8 in the 8q24-qter region. The expression of the gene is confined to keratinocytes and squamous tumor cells. The putative mouse homologue, the ThB antigen, originally identified as an antigen on cells of the lymphocyte lineage, was shown to be highly expressed in squamous mouse epithelia. Moreover, the ThB expression level is in keratinocytes, in contrast to that in lymphocytes, not mouse strain related. Transfection of mouse SV40-polyoma transformed mouse NIH/3T3 cells with the E48 cDNA confirmed that the antigen is likely to be involved in cell-cell adhesion. PMID:7790363

  3. Identifying protective Streptococcus pyogenes vaccine antigens recognized by both B and T cells in human adults and children

    PubMed Central

    Mortensen, Rasmus; Nissen, Thomas Nørrelykke; Fredslund, Sine; Rosenkrands, Ida; Christensen, Jan Pravsgaard; Andersen, Peter; Dietrich, Jes

    2016-01-01

    No commercial vaccine exists against Group A streptococci (GAS; Streptococcus pyogenes) and only little is known about anti-GAS protective immunity. In our effort to discover new protective vaccine candidates, we selected 21 antigens based on an in silico evaluation. These were all well-conserved among different GAS strains, upregulated in host-pathogen interaction studies, and predicted to be extracellular or associated with the surface of the bacteria. The antigens were tested for both antibody recognition and T cell responses in human adults and children. The antigenicity of a selected group of antigens was further validated using a high-density peptide array technology that also identified the linear epitopes. Based on immunological recognition, four targets were selected and tested for protective capabilities in an experimental GAS infection model in mice. Shown for the first time, three of these targets (spy0469, spy1228 and spy1801) conferred significant protection whereas one (spy1643) did not. PMID:26911649

  4. Crystal structure of human prostate-specific antigen in a sandwich antibody complex.

    PubMed

    Stura, Enrico A; Muller, Bruno H; Bossus, Marc; Michel, Sandrine; Jolivet-Reynaud, Colette; Ducancel, Frédéric

    2011-12-01

    Human prostate-specific antigen (PSA or human kallikrein-related peptidase 3) present in small quantities in the sera of healthy men becomes elevated in prostate cancer (PCa) and other prostate disorders. The ability to identify the free PSA fraction associated with PCa could increase the reliability of the PSA diagnostic test. Here we present the crystal structure of human PSA from seminal fluid in a sandwich complex with two monoclonal antibodies (mAbs). MAb 5D5A5 captures total PSA with exceptionally high affinity, and mAb 5D3D11 selectively discriminates between free PSA subforms that are more abundant in sera from patients with PCa. Although the antigen is not of seric origin, several insights into cancer diagnosis can be discerned from this complex. MAb 5D3D11 recognizes a PSA conformation different from that previously reported. Interacting with the kallikrein loop, the PSA N-linked glycan attached to asparagine 61 is an uncommonly complex sialated triantennary chain. O-linked glycosylation is observed at threonine 125. The description of how PSA subforms in prostatic fluid can be discriminated using pairs of antibodies is a first step in the design of new strategies that are capable of real discrimination among PSA subforms, which will lead to the formulation of more reliable diagnostic tests. In a companion article [Muller, B. H., Savatier, A., L'Hostis, G., Costa, N., Bossus, M., Michel, S., et al. (2011). In vitro affinity maturation of an anti-PSA antibody for prostate cancer diagnostic assay. J. Mol. Biol.], we describe engineering efforts to improve the affinity of mAb 5D3D11, a first step towards such goal. PMID:22037582

  5. Preparation and characterization of human monoclonal antibodies directed against the hepatitis B virus surface antigen.

    PubMed

    Colucci, G; Kohtz, D S; Waksal, S D

    1986-06-01

    The hepatitis B surface antigen (HBsAg) is highly immunogenic and induces an antibody response which is protective in vivo against hepatitis B virus (HBV) infection. Human monoclonal antibodies specific for HBsAg were produced, which could have potential therapeutic applications. Lymphocytes obtained from a vaccinated donor were stimulated in vitro and fused with the human myeloma cell line GM 4672, and eight hybridomas were obtained. Three of these clones, which reacted in an ELISA against the HBsAg vaccine, were expanded, subcloned and further analyzed. The subclones E7C2, C4C10, and D5B2 were able to bind to different HBsAg preparations, which express various subtypes, and recognized the major HBsAg peptides in Western blot analysis. Cross-inhibition experiments showed that E7C2, C4C10 and D5B2 are directed against the same epitope and have an affinity constant ranging from 5 X 10(7) to 3.3 X 10(9) M. Furthermore, these antibodies stained the surface and cytoplasm of the HBsAg-secreting cell lines PLC/PRF/5 and 4.10. The production of immunoglobulins varies from 0.3 to 1.3 micrograms/ml/10(6) and has remained stable over a period of 8 months. These human monoclonal antibodies, which appear to be directed against an antigenic determinant common to all HBsAg subtypes, could be useful in the study of HBV-related liver diseases as well as in their diagnosis and experimental therapy.

  6. Experimental radioimmunotherapy of a xenografted human colonic tumor (GW-39) producing carcinoembryonic antigen

    SciTech Connect

    Goldenberg, D.M.; Gaffar, S.A.; Bennett, S.J.; Beach, J.L.

    1981-11-01

    Experiments were undertaken to evaluate the antitumor effects of 131I-labeled goat antibody immunoglobulin G prepared against carcinoembryonic antigen in hamsters bearing the carcinoembryonic antigen-producing GW-39 human colonic carcinoma. At a single injection of 1 mCi 131I and higher, a marked growth inhibition of GW-39 tumors, as well as a considerable increase in the survival time of the tumor-bearing hamsters, could be achieved. At a dose of 1 mCi, the radioactive affinity-purified antibody appeared to be superior to radioactive normal goat immunoglobulin G in influencing tumor growth and survival time, but no significant difference could be seen at the higher dose of 2 mCi given. Radiobiological calculations indicated that the tumors received, at up to 20 days after therapy, 1325 rads for the specific antibody and only 411 rads for the normal immunoglobulin G preparation. These findings encourage the further evaluation of antibodies to tumor markers for isotopic cancer therapy.

  7. Neisserial Heparin Binding Antigen (NHBA) Contributes to the Adhesion of Neisseria meningitidis to Human Epithelial Cells

    PubMed Central

    Vacca, Irene; Del Tordello, Elena; Gasperini, Gianmarco; Pezzicoli, Alfredo; Di Fede, Martina; Rossi Paccani, Silvia; Marchi, Sara; Mubaiwa, Tsisti D.; Hartley-Tassell, Lauren E.; Jennings, Michael P.; Seib, Kate L.; Masignani, Vega; Pizza, Mariagrazia; Serruto, Davide; Aricò, Beatrice; Delany, Isabel

    2016-01-01

    Neisserial Heparin Binding Antigen (NHBA) is a surface-exposed lipoprotein ubiquitously expressed by Neisseria meningitidis strains and an antigen of the Bexsero® vaccine. NHBA binds heparin through a conserved Arg-rich region that is the target of two proteases, the meningococcal NalP and human lactoferrin (hLf). In this work, in vitro studies showed that recombinant NHBA protein was able to bind epithelial cells and mutations of the Arg-rich tract abrogated this binding. All N-terminal and C-terminal fragments generated by NalP or hLf cleavage, regardless of the presence or absence of the Arg-rich region, did not bind to cells, indicating that a correct positioning of the Arg-rich region within the full length protein is crucial. Moreover, binding was abolished when cells were treated with heparinase III, suggesting that this interaction is mediated by heparan sulfate proteoglycans (HSPGs). N. meningitidis nhba knockout strains showed a significant reduction in adhesion to epithelial cells with respect to isogenic wild-type strains and adhesion of the wild-type strain was inhibited by anti-NHBA antibodies in a dose-dependent manner. Overall, the results demonstrate that NHBA contributes to meningococcal adhesion to epithelial cells through binding to HSPGs and suggest a possible role of anti-Bexsero® antibodies in the prevention of colonization. PMID:27780200

  8. Human leukocyte antigen (HLA)-E and HLA-F expression in gastric cancer.

    PubMed

    Ishigami, Sumiya; Arigami, Takaaki; Okumura, Hiroshi; Uchikado, Yasuto; Kita, Yoshiaki; Kurahara, Hiroshi; Maemura, Kosei; Kijima, Yuko; Ishihara, Yuka; Sasaki, Ken; Uenosono, Yoshikazu; Natsugoe, Shoji

    2015-04-01

    Human leukocyte antigen (HLA)-E and HLA-F are classified as non-classical HLA class Ib antigens. Ectopic HLA-E and HLA-F expression was recently detected in cancer cells; however, the clinical implication of their expression remains unknown. A total of 209 patients with gastric cancer were enrolled in this study. Immunohistochemistry was used to evaluate the expression of HLA-E and HLA-F in gastric cancer specimens. HLA-E and HLA-F expression were seen in the cell membrane. HLA-E and HLA-F expression significantly correlated with depth of invasion, nodal involvement, lymphatic invasion, and venous invasion. No significant correlation between HLA-E and HLA-F expression was found (p<0.05, r=0.24). The five-year survival rate of the HLA-E-positive group and HLA-F-positive group were significantly poorer than that of their respective negative groups. Combination of HLA-E and HLA-F made the p-value smaller than single analysis (p<0.009). This is the first report detailing a clinical implication of HLA-E and HLA-F expression simultaneously in gastric cancer. We identified that the HLA-E and HLA-F in gastric cancer independently affected clinical factors, including postoperative outcome. For HLA-E- or HLA-F-positive gastric cancer, we should settle on a treatment strategy that reinforces the host immune response.

  9. Antigenic and genomic diversity of human rotavirus VP4 in two consecutive epidemic seasons in Mexico.

    PubMed

    Padilla-Noriega, L; Méndez-Toss, M; Menchaca, G; Contreras, J F; Romero-Guido, P; Puerto, F I; Guiscafré, H; Mota, F; Herrera, I; Cedillo, R; Muñoz, O; Calva, J; Guerrero, M L; Coulson, B S; Greenberg, H B; López, S; Arias, C F

    1998-06-01

    In the present investigation we characterized the antigenic diversity of the VP4 and VP7 proteins in 309 and 261 human rotavirus strains isolated during two consecutive epidemic seasons, respectively, in three different regions of Mexico. G3 was found to be the prevalent VP7 serotype during the first year, being superseded by serotype G1 strains during the second season. To antigenically characterize the VP4 protein of the strains isolated, we used five neutralizing monoclonal antibodies (MAbs) which showed specificity for VP4 serotypes P1A, P1B, and P2 in earlier studies. Eight different patterns of reactivity with these MAbs were found, and the prevalence of three of these patterns varied from one season to the next. The P genotype of a subset of 52 samples was determined by PCR. Among the strains characterized as genotype P[4] and P[8] there were three and five different VP4 MAb reactivity patterns, respectively, indicating that the diversity of neutralization epitopes in VP4 is greater than that previously appreciated by the genomic typing methods.

  10. IgA detection in human neurocysticercosis using different preparations of heterologous antigen.

    PubMed

    da S Ribeiro, Vanessa; Manhani, Marianna N; Costa-Cruz, Julia M

    2010-06-01

    Neurocysticercosis (NC) is the most important neurological disease of parasitic origin in humans. IgA and IgG detection in serum from neurocysticercosis patients was tested using some antigenic preparations of total saline extract from Taenia saginata: detergent (D) and aqueous (A) phases extracted with Triton X-114 and the jacalin bound (JBF) and unbound fractions (JUF) obtained by affinity chromatography using jacalin column. Samples were obtained from 45 patients with definitive NC, who were subdivided into active-NC group and inactive-NC group; 35 patients with other parasitoses; and 30 apparently healthy individuals. Sensitivity and specificity were calculated. Specificity to detect IgA and IgG for D phase, respectively, were 89.8% and 86.9% and for IgG detection 91.3% and 76.8% when using D phase and JUF, respectively. D phase and JBF proved to be specific and efficient and could be efficiently utilized as an alternative antigen for IgA detection in NC, with comparable results with IgG.

  11. Localization of human immunodeficiency virus antigens in infected cells by scanning/transmission-immunogold techniques

    SciTech Connect

    Herrera, M.I.; Santa Maria, I.; de Andres, R.; Najera, R.

    1988-01-01

    An application of high resolution scanning/transmission electron microscopy (STEM) and gold-labelling techniques for the rapid detection of human immunodeficiency virus (HIV) in infected cells has been developed. Experimental in vitro studies for detecting two HIV structural proteins, gp41 and p17, were performed following an indirect labeling procedure that uses monoclonal anti-p17 and anti-gp41 antibodies as primary antibodies and 40 nm gold-linked goat antimouse IgG as secondary antibodies. The cells were then studied by STEM in the scanning mode. Unambiguous localization of the viral antigens was possible by combining the three-dimensional image provided by the secondary electron image and the atomic number-dependent backscattered electron image for the identification of the gold marker. This technique combines both the morphological information and the rapid procedures of scanning electron microscopy with the precise and sensitive antigen detection provided by the use of STEM and immunological methods. The preliminary results of its application to the study of peripheral blood mononuclear cells from four anti-HIV-seropositive patients showing the presence of specific labeling in all of them suggest that it might prove useful for early detection of HIV infection before seroconversion, as well as for quantitative studies.

  12. Studies on thyroid cell surface antigens using cultured human thyroid cells.

    PubMed Central

    Fenzi, G F; Bartalena, L; Chiovato, L; Marcocci, C; Rotella, C M; Zonefrati, R; Toccafondi, R; Pinchera, A

    1982-01-01

    Human thyroid cells in primary culture were used for studies of thyroid cell surface antibodies in patients with thyroid autoimmune disorders. Radioiodinated IgG preparations containing thyroid microsomal antibody (TMAb), thyroid stimulating antibody (TSAb) and/or thyroglobulin antibody (TgAb) were tested for binding to thyroid cells. Binding was observed with radioiodinated IgG from patients with Graves' disease, Hashimoto's thyroiditis and idiopathic myxoedema containing TMAb, irrespective of the presence of TSAb and TgAb, while negative results were obtained with normal IgG. A dose-dependent inhibition of binding to thyroid cells was produced by the addition of the corresponding unlabelled IgG preparations. Evidence for tissue specificity was provided by the absence of binding to human skin fibroblasts used as controls. Preabsorption with human thyroid microsomes completely abolished the binding to thyroid cells of a radioiodinated TMAb positive IgG preparation, while only incomplete removal of the reactivity to thyroid microsomes was produced by preabsorption with thyroid cells. These data suggest that some but not all microsomal antigenic determinants are expressed on the thyroid cell surface. Binding to thyroid cells was also observed with purified TgAb, indicating that thyroglobulin antigenic determinants are present on the surface of thyroid cells. No evidence of binding was obtained with a TSAb positive Graves' IgG preparation with undetectable TMAb and TgAb. Unlabelled IgG preparations containing TMAb from patients with either Hashimoto's thyroiditis or idiopathic myxoedema were shown to inhibit the binding to thyroid cells of radioiodinated TMAb positive Graves' IgG and vice versa. These data indicate that antibodies present in these thyroid autoimmune disorders share common thyroid cell surface antigens. However, the binding of radioiodinated IgG from a patient with idiopathic myxoedema was only partially inhibited by Graves' or Hashimoto's Ig

  13. Identification of a Neutralizing Epitope within Antigenic Domain 5 of Glycoprotein B of Human Cytomegalovirus

    PubMed Central

    Wiegers, Anna-Katharina; Sticht, Heinrich; Winkler, Thomas H.; Britt, William J.

    2014-01-01

    ABSTRACT Human cytomegalovirus (HCMV) is an important, ubiquitous pathogen that causes severe clinical disease in immunocompromised individuals, such as organ transplant recipients and infants infected in utero. The envelope glycoprotein B (gB) of HCMV is a major antigen for the induction of virus-neutralizing antibodies. We have begun to define target structures within gB that are recognized by virus-neutralizing antibodies. Antigenic domain 5 (AD-5) of gB has been identified as an important target for neutralizing antibodies in studies using human monoclonal antibodies (MAbs). Anti-AD-5 MAbs share a target site on gB, despite originating from different, healthy, HCMV-infected donors. Mutational analysis of AD-5 identified tyrosine 280 in combination with other surface-exposed residues (the YNND epitope) as critical for antibody binding. The YNND epitope is strictly conserved among different HCMV strains. Recombinant viruses carrying YNND mutations in AD-5 were resistant to virus-neutralizing MAbs. Competition enzyme-linked immunosorbent assays (ELISAs) with human HCMV-convalescent-phase sera from unselected donors confirmed the conserved antibody response for the YNND epitope in HCMV-infected individuals and, because a significant fraction of the gB AD-5 response was directed against the YNND epitope, further argued that this epitope is a major target of anti-AD-5 antibody responses. In addition, affinity-purified polyclonal anti-AD-5 antibodies prepared from individual sera showed reactivity to AD-5 and neutralization activity toward gB mutant viruses that were similar to those of AD-5-specific MAbs. Taken together, our data indicate that the YNND epitope represents an important target for anti-gB antibody responses as well as for anti-AD-5 virus-neutralizing antibodies. IMPORTANCE HCMV is a major global health concern, and a vaccine to prevent HCMV disease is a widely recognized medical need. Glycoprotein B of HCMV is an important target for neutralizing

  14. DCAF4, a novel gene associated with leucocyte telomere length

    PubMed Central

    Mangino, Massimo; Christiansen, Lene; Stone, Rivka; Hunt, Steven C; Horvath, Kent; Eisenberg, Dan T A; Kimura, Masayuki; Petersen, Inge; Kark, Jeremy D; Herbig, Utz; Reiner, Alex P; Benetos, Athanase; Codd, Veryan; Nyholt, Dale R; Sinnreich, Ronit; Christensen, Kaare; Nassar, Hisham; Hwang, Shih-Jen; Levy, Daniel; Bataille, Veronique; Fitzpatrick, Annette L; Chen, Wei; Berenson, Gerald S; Samani, Nilesh J; Martin, Nicholas G; Tishkoff, Sarah; Schork, Nicholas J; Kyvik, Kirsten Ohm; Dalgård, Christine; Spector, Timothy D; Aviv, Abraham

    2015-01-01

    Background Leucocyte telomere length (LTL), which is fashioned by multiple genes, has been linked to a host of human diseases, including sporadic melanoma. A number of genes associated with LTL have already been identified through genome-wide association studies. The main aim of this study was to establish whether DCAF4 (DDB1 and CUL4-associated factor 4) is associated with LTL. In addition, using ingenuity pathway analysis (IPA), we examined whether LTL-associated genes in the general population might partially explain the inherently longer LTL in patients with sporadic melanoma, the risk for which is increased with ultraviolet radiation (UVR). Results Genome-wide association (GWA) meta-analysis and de novo genotyping of 20 022 individuals revealed a novel association (p=6.4×10−10) between LTL and rs2535913, which lies within DCAF4. Notably, eQTL analysis showed that rs2535913 is associated with decline in DCAF4 expressions in both lymphoblastoid cells and sun-exposed skin (p=4.1×10−3 and 2×10−3, respectively). Moreover, IPA revealed that LTL-associated genes, derived from GWA meta-analysis (N=9190), are over-represented among genes engaged in melanoma pathways. Meeting increasingly stringent p value thresholds (p<0.05, <0.01, <0.005, <0.001) in the LTL-GWA meta-analysis, these genes were jointly over-represented for melanoma at p values ranging from 1.97×10−169 to 3.42×10−24. Conclusions We uncovered a new locus associated with LTL in the general population. We also provided preliminary findings that suggest a link of LTL through genetic mechanisms with UVR and melanoma in the general population. PMID:25624462

  15. Discordance at human leukocyte antigen-DRB3 and protection from human immunodeficiency virus type 1 transmission.

    PubMed

    Hader, Shannon L; Hodge, Thomas W; Buchacz, Kate A; Bray, Robert A; Padian, Nancy S; Rausa, Alfio; Slaviniski, Sally A; Holmberg, Scott D

    2002-06-15

    Host human leukocyte antigens (HLAs) integrated into the human immunodeficiency virus (HIV) type 1 envelope could theoretically determine, as in tissue transplants, whether HIV-1 is "rejected" by exposed susceptible persons, preventing transmission. HLA discordance (mismatch) was examined among 45 heterosexual partner pairs in which at least 1 partner was HIV-1 infected and exposure or transmission between partners had occurred. Immunologic discordance at class II HLA-DRB3 (present in the HIV donor partner but absent in the recipient partner) was associated with lack of transmission of HIV-1. Eight (35%) of 23 partner pairs in which HIV-1 transmission did not occur were immunologically discordant at HLA-DRB3, compared with 0 of 11 partner pairs in which HIV-1 transmission did occur (P=.027). Further investigation of the roles of class II HLAs in HIV-1 transmission and as possible components of HIV-1 vaccines should be pursued. PMID:12085318

  16. Comparison of Effects of Running and Playing Exercises on Differential Leucocyte Count in Young Elite Athletes

    ERIC Educational Resources Information Center

    Cenikli, Abdullah

    2016-01-01

    The aims of the present research are to test the effects of running and playing exercises on leucocyte and differential leucocyte accounts, and to test the possible differences between running and playing exercises in terms of leucocyte accounts. They were thirty two male young soccer players. Participants arrived at the laboratory after a 12-hour…

  17. Prior human leukocyte antigen-allosensitization and left ventricular assist device type affect degree of post-implantation human leukocyte antigen-allosensitization.

    PubMed

    Drakos, Stavros G; Kfoury, Abdallah G; Kotter, John R; Reid, Bruce B; Clayson, Stephen E; Selzman, Craig H; Stehlik, Josef; Fisher, Patrick W; Merida, Mario; Eckels, David D; Brunisholz, Kim; Horne, Benjamin D; Stoker, Sandi; Li, Dean Y; Renlund, Dale G

    2009-08-01

    Left ventricular assist device (LVAD) implantation before heart transplantation has been associated with formation of antibodies directed against human leukocyte antigens (HLA), often referred to as sensitization. This study investigated whether prior sensitization or LVAD type affected the degree of post-implantation sensitization. The records of consecutive HeartMate (HM) I and HM II LVAD patients were reviewed. Panel reactive antibody (PRA) was assessed before LVAD implantation and biweekly thereafter. Sensitization was defined as PRA > 10%, and high-degree sensitization was defined as PRA > 90%. An HM LVAD was implanted in 64 patients, and 11 received a HM II LVAD as a bridge to transplant. Ten HM I patients (16%) were sensitized before LVAD implantation (HM I-S), and 54 (84%) were not (HM I-Non-S). Nine HM I-S patients (90%) became highly sensitized (PRA > 90%) compared with 9 HM I-Non-S patients (16.7%; p < 0.001). The PRA remained elevated (> 90%) in 8 of the 9 (88.9%) highly sensitized HM I-S patients vs 5 of the 9 (55.6%) HM I-Non-S highly sensitized patients. The PRA levels in the rest of the HM I-S highly sensitized patients declined from 93% +/- 4% to 55% +/- 15% (p = 0.01). Among the 11 HM II patients, 1 (9%) was sensitized before LVAD implantation (PRA, 40%) and the PRA moderately increased to 80%. No other HM II patient became sensitized after implantation. Thus, 1 of 11 (9%) HM II patients became sensitized compared with 29 of 64 (45%) HM I patients (p = 0.04). Pre-sensitized patients are at higher risk for becoming and remaining highly HLA-allosensitized after LVAD implantation. The HeartMate II LVAD appears to cause less sensitization than HeartMate I.

  18. Antigenic Maps of Influenza A(H3N2) Produced With Human Antisera Obtained After Primary Infection

    PubMed Central

    Fonville, Judith M.; Fraaij, Pieter L. A.; de Mutsert, Gerrie; Wilks, Samuel H.; van Beek, Ruud; Fouchier, Ron A. M.; Rimmelzwaan, Guus F.

    2016-01-01

    Background Antigenic characterization of influenza viruses is typically based on hemagglutination inhibition (HI) assay data for viral isolates tested against strain-specific postinfection ferret antisera. Here, similar virus characterizations were performed using serological data from humans with primary influenza A(H3N2) infection. Methods We screened sera collected between 1995 and 2011 from children between 9 and 24 months of age for influenza virus antibodies, performed HI tests for the positive sera against 23 influenza viruses isolated between 1989 and 2011, and measured HI titers of antisera against influenza A(H3N2) from 24 ferrets against the same panel of viruses. Results Of the 17 positive human sera, 6 had a high response, showing HI patterns that would be expected from primary infection antisera, while 11 sera had lower, more dispersed patterns of reactivity that are not easily explained. The antigenic map based on the high-response human HI data was similar to the map created using ferret data. Conclusions Although the overall structure of the ferret and human antigenic maps is similar, local differences in virus positions indicate that the human and ferret immune system might see antigenic properties of viruses differently. Further studies are needed to establish the degree of similarity between serological patterns in ferret and human data. PMID:26142433

  19. Production of leucocyte migration inhibitory factor by neonatal lymphocytes.

    PubMed

    Wolf, R L; Lomnitzer, R; Rabson, A R

    1976-06-19

    Lymphocytes (mononuclear cells) from cord blood of 10 normal placentas and from 10 normal adults were assessed for production of leucocyte migration inhibiting factor (LIF) after phytohaemagglutinin (PHA) stimulation, as a measure of cell-mediated immunity. Mononuclear cells from both adult and cord blood produced adequate quantities of LIF, indicating that neonatal lymphocytes have the ability to manufacture normal amounts of lymphokines.

  20. Human Leukocyte Antigen Class II Alleles Influence Levels of Antibodies to the Plasmodium falciparum Asexual-Stage Apical Membrane Antigen 1 but Not to Merozoite Surface Antigen 2 and Merozoite Surface Protein 1

    PubMed Central

    Johnson, Armead H.; Leke, Rose G. F.; Mendell, Nancy R.; Shon, Dewon; Suh, Young Ju; Bomba-Nkolo, Dennis; Tchinda, Viviane; Kouontchou, Samuel; Thuita, Lucy W.; van der Wel, Anne Marie; Thomas, Alan; Stowers, Anthony; Saul, Allan; Zhou, Ainong; Taylor, Diane W.; Quakyi, Isabella A.

    2004-01-01

    The apical membrane antigen 1 (AMA1), merozoite surface antigen 2 (MSA2), and merozoite surface protein 1 (MSP1) are asexual-stage proteins currently being evaluated for inclusion in a vaccine for Plasmodium falciparum. Accordingly, it is important to understand factors that control antibody responses to these antigens. Antibody levels in plasma from residents of Etoa, Cameroon, between the ages of 5 and 70 years, were determined using recombinant AMA1, MSA2, and the N-terminal region of MSP1 (MSP1-190L). In addition, antibody responses to four variants of the C-terminal region of MSP1 (MSP119) were assessed. Results showed that all individuals produced antibodies to AMA1, MSA2, and MSP1-190L; however, a proportion of individuals never produced antibodies to the MSP119 variants, although the percentage of nonresponders decreased with age. The influence of age and human leukocyte antigen (HLA)-DRB1/DQB1 alleles on antibody levels was evaluated using two-way analysis of variance. Age was correlated with levels of antibodies to AMA1 and MSP119 but not with levels of antibodies to MSA2 and MSP1-190L. No association was found between a single HLA allele and levels of antibodies to MSA2, MSP1-190L, or any of the MSP119 variants. However, individuals positive for DRB1*1201 had higher levels of antibodies to the variant of recombinant AMA1 tested than did individuals of all other HLA types. Since the effect was seen across all age groups, HLA influenced the level but not the rate of antibody acquisition. This association for AMA1, combined with the previously reported association between HLA class II alleles and levels of antibodies to rhoptry-associated protein 1 (RAP1) and RAP2, indicates that HLA influences the levels of antibodies to three of the five vaccine candidate antigens that we have evaluated. PMID:15102786

  1. Human Skin Cells That Express Stage-Specific Embryonic Antigen 3 Associate with Dermal Tissue Regeneration

    PubMed Central

    Vega Crespo, Agustin; Awe, Jason P.; Reijo Pera, Renee

    2012-01-01

    Abstract Stage-specific embryonic antigen 3 (SSEA3) is a glycosphingolipid that has previously been used to identify cells with stem cell-like, multipotent, and pluripotent characteristics. A rare subpopulation of SSEA3-expressing cells exists in the dermis of adult human skin. These SSEA3-expressing cells undergo a significant increase in cell number in response to injury, suggesting a possible role in regeneration. These SSEA3-expressing regeneration-associated (SERA) cells were derived through primary cell culture, purified by fluorescence-activated cell sorting (FACS), and characterized. Longer in vitro culture of the primary skin cells led to lower SSEA3 expression stability after FACS-based purification, suggesting that the current culture conditions may need to be optimized to permit the large-scale expansion of SERA cells. The SERA cells demonstrated a global transcriptional state that was most similar to bone marrow- and fat-derived mesenchymal stem cells (MSCs), and the highest expressing SSEA3-expressing cells co-expressed CD105 (clone 35). However, while a rare population of MSCs was observed in primary human skin cell cultures that could differentiate into adipocytes, osteoblasts, or chondrocytes, SERA cells did not possess this differentiation capacity, suggesting that there are at least two different rare subpopulations in adult human skin primary cultures. The identification, efficient purification, and large-scale expansion of these rare subpopulations (SERA cells and MSCs) from heterogeneous adult human skin primary cell cultures may have applications for future patient-specific cellular therapies. PMID:23514702

  2. Biodegradable nanoparticle mediated antigen delivery to human cord blood derived dendritic cells for induction of primary T cell responses.

    PubMed

    Diwan, Manish; Elamanchili, Praveen; Lane, Helena; Gainer, Anita; Samuel, John

    2003-01-01

    Dendritic cells (DCs) in the peripheral tissues act as sentinels of the immune system. They detect and capture pathogens entering the body and present their antigens to T cells to trigger responses directed towards elimination of the pathogen. The induction of peripheral tolerance against self and certain foreign antigens is also believed to be mediated through DCs. The outcome of any immune response is largely controlled by the microenvironment of antigen capture, processing and presentation by DCs. The "context" of antigen delivery to DCs will directly influence the microenvironment of antigen presentation and hence the regulation of immune responses. We report here preliminary investigations describing the formulation of a pharmaceutically acceptable, biodegradable, and strategic nanoparticulate delivery system, and its application for efficient antigen loading of DCs to achieve antigen specific T cell activation. "Pathogen-mimicking" nanoparticles capable of interacting with DCs were fabricated by incorporating monophosphoryl lipid A (MPLA; toll-like receptor (TLR) 4 ligand) or CpG ODN (seq #2006; TLR9 ligand) in biodegradable copolymer, poly(D,L,-lactic-co-glycolic acid) (PLGA). The uptake of PLGA nanoparticles by human umbilical cord blood derived DCs (DCs propagated from CD34 progenitors) was conclusively demonstrated by scanning electron microscopy (SEM), fluorescence activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM). Cell phenotype at day 12 of cultures was determined as immature DC using specific cell surface markers, i.e. CD11c (approximately 90%), MHC-II (approximately 70%), CD86 (approximately 20%), CD83 (approximately 5%), CD80 (approximately 40%), CD40 (approximately 40%), and CCR7 (approximately 5%). Tetanus toxoid (TT), a model antigen, was encapsulated in nanoparticles along with an immunomodulator, i.e. either MPLA or CpG ODN. DCs pulsed with various antigen formulations were co-cultured with autologous naïve T cells at

  3. Current approaches to measuring human islet-antigen specific T cell function in type 1 diabetes

    PubMed Central

    Mannering, S I; Wong, F S; Durinovic-Belló, I; Brooks-Worrell, B; Tree, T I; Cilio, C M; Schloot, N C; Mallone, R

    2010-01-01

    Type 1 diabetes (T1D) is an autoimmune disease caused by the T cell-mediated destruction of the pancreatic insulin-producing beta cells. Currently there are no widely accepted and standardized assays available to analyse the function of autoreactive T cells involved in T1D. The development of such an assay would greatly aid efforts to understand the pathogenesis of T1D and is also urgently required to guide the development of antigen-based therapies intended to prevent, or cure, T1D. Here we describe some of the assays used currently to detect autoreactive T cells in human blood and review critically their strengths and weaknesses. The challenges and future prospects for the T cell assays are discussed. PMID:20846160

  4. Human leukocyte antigen (HLA) DR4. Positive association with rapidly progressing periodontitis.

    PubMed

    Katz, J; Goultschin, J; Benoliel, R; Brautbar, C

    1987-09-01

    The relationship between human leukocyte antigens (HLA) determinants and periodontitis has been examined by several authors without showing any particular pattern. However, no study has investigated the HLA-D determinants, which are generally associated with immune disorders, and rapidly progressive periodontitis (RPP). The HLA profile of 10 RPP patients was compared with that of a healthy control population (n = 120). Although no significant difference was found for HLA-A, HLA-B, and HLA-C, HLA-DR4 of the HLA-D group was found in 80% of patients but only in 38.3% of controls. A high frequency of HLA-DR4 has been reported in rheumatoid arthritis (RA) patients. This finding may be significant in light of previous reports highlighting similarities between RA and periodontal disease. PMID:3498813

  5. Molecular cloning of a human histocompatibility antigen cDNA fragment.

    PubMed Central

    Ploegh, H L; Orr, H T; Strominger, J L

    1980-01-01

    A clone (pHLA-1) containing HLA-specific cDNA was constructed by reverse transcription of partially purified HLA mRNA from the human lymphoblastoid cell line LKT. The identity of pHLA-1 was established by its ability to hybridize to HLA heavy chain mRNA and by nucleotide sequence analysis. The pHLA-1 cDNA insert (approximately 525 base pairs) corresponds to the COOH-terminal 46 amino acids of an HLA-A, -B, or -C antigen (15 residues from the hydrophobic region and the remainder from the COOH-termial hydrophilic region), together with a portion of the 3' untranslated region of the mRNA. Images PMID:6934534

  6. In vitro antigen-specific induction of IL-22 in human subjects that resolved HCV infection

    PubMed Central

    Cusick, Matthew F; Libbey, Jane E; Fujinami, Robert S; Eckels, David D

    2012-01-01

    Aims To determine if in vitro production of IL-22 and IL-17 correlated with resolution of HCV infection. Materials & methods Human peripheral blood cells isolated from a well-defined cohort of resolved and chronic HCV-infected subjects were used to measure HCV-, influenza- and mitogen-activated T-cell proliferation. In addition, IL-22 and IL-17 production was measured via ELISAs and flow cytometry. Results Resolved HCV subjects had a significantly higher T-cell proliferative response to recombinant NS3 protein compared with chronic HCV subjects. Resolved subjects had a dose-dependent IL-22 response to recombinant NS3 compared with chronic HCV subjects. Conclusion IL-22 production is associated with antigen-specific induction of CD4 + T cells in individuals that resolved HCV infection, suggesting a potential role for IL-22 in HCV clearance. PMID:23185211

  7. Buffered Plate Antigen Test as a Screening Test for Diagnosis of Human Brucellosis

    PubMed Central

    Lucero, Nidia E.; Bolpe, Jorge E.

    1998-01-01

    Brucellosis in Argentina is currently investigated in bank donor blood by the standard plate agglutination test (PAT). This study evaluated the buffered plate antigen test (BPA), now used to screen for bovine brucellosis, as a screen for human disease. Of 57 sera from patients with culture-confirmed brucellosis, 100% were detected with the BPA. Of 142 sera positive by rose bengal (RB) and complement fixation (CF), from patients with clinical evidence of brucellosis, the BPA detected 100%. Of 307 sera from a nonsymptomatic population that were RB and CF negative, the BPA detected 99.67% of the negative sera. The data indicate that the BPA is satisfactory compared to the other agglutination tests employed. It is an inexpensive and practical screening test and reduces the nonspecific reactions detected by the PAT. PMID:9574720

  8. Physiological Level Production of Antigen-Specific Human Immunoglobulin in Cloned Transchromosomic Cattle

    PubMed Central

    Wu, Hua; Jiao, Jin-An; Kasinathan, Poothappillai; Sullivan, Eddie J.; Wang, Zhongde; Kuroiwa, Yoshimi

    2013-01-01

    Therapeutic human polyclonal antibodies (hpAbs) derived from pooled plasma from human donors are Food and Drug Administration approved biologics used in the treatment of a variety of human diseases. Powered by the natural diversity of immune response, hpAbs are effective in treating diseases caused by complex or quickly-evolving antigens such as viruses. We previously showed that transchromosomic (Tc) cattle carrying a human artificial chromosome (HAC) comprising the entire unrearranged human immunoglobulin heavy-chain (hIGH) and kappa-chain (hIGK) germline loci (named as κHAC) are capable of producing functional hpAbs when both of the bovine immunoglobulin mu heavy-chains, bIGHM and bIGHML1, are homozygously inactivated (double knockouts or DKO). However, B lymphocyte development in these Tc cattle is compromised, and the overall production of hpAbs is low. Here, we report the construction of an improved HAC, designated as cKSL-HACΔ, by incorporating all of the human immunoglobulin germline loci into the HAC. Furthermore, for avoiding the possible human-bovine interspecies incompatibility between the human immunoglobulin mu chain protein (hIgM) and bovine transmembrane α and β immunoglobulins (bIgα and bIgβ) in the pre-B cell receptor (pre-BCR) complex, we partially replaced (bovinized) the hIgM constant domain with the counterpart of bovine IgM (bIgM) that is involved in the interaction between bIgM and bIgα/Igβ; human IgM bovinization would also improve the functionality of hIgM in supporting B cell activation and proliferation. We also report the successful production of DKO Tc cattle carrying the cKSL-HACΔ (cKSL-HACΔ/DKO), the dramatic improvement of B cell development in these cattle and the high level production of hpAbs (as measured for the human IgG isotype) in the plasma. We further demonstrate that, upon immunization by tumor immunogens, high titer tumor immunogen-specific human IgG (hIgG) can be produced from such Tc cattle. PMID:24205120

  9. Physiological level production of antigen-specific human immunoglobulin in cloned transchromosomic cattle.

    PubMed

    Sano, Akiko; Matsushita, Hiroaki; Wu, Hua; Jiao, Jin-An; Kasinathan, Poothappillai; Sullivan, Eddie J; Wang, Zhongde; Kuroiwa, Yoshimi

    2013-01-01

    Therapeutic human polyclonal antibodies (hpAbs) derived from pooled plasma from human donors are Food and Drug Administration approved biologics used in the treatment of a variety of human diseases. Powered by the natural diversity of immune response, hpAbs are effective in treating diseases caused by complex or quickly-evolving antigens such as viruses. We previously showed that transchromosomic (Tc) cattle carrying a human artificial chromosome (HAC) comprising the entire unrearranged human immunoglobulin heavy-chain (hIGH) and kappa-chain (hIGK) germline loci (named as κHAC) are capable of producing functional hpAbs when both of the bovine immunoglobulin mu heavy-chains, bIGHM and bIGHML1, are homozygously inactivated (double knockouts or DKO). However, B lymphocyte development in these Tc cattle is compromised, and the overall production of hpAbs is low. Here, we report the construction of an improved HAC, designated as cKSL-HACΔ, by incorporating all of the human immunoglobulin germline loci into the HAC. Furthermore, for avoiding the possible human-bovine interspecies incompatibility between the human immunoglobulin mu chain protein (hIgM) and bovine transmembrane α and β immunoglobulins (bIgα and bIgβ) in the pre-B cell receptor (pre-BCR) complex, we partially replaced (bovinized) the hIgM constant domain with the counterpart of bovine IgM (bIgM) that is involved in the interaction between bIgM and bIgα/Igβ; human IgM bovinization would also improve the functionality of hIgM in supporting B cell activation and proliferation. We also report the successful production of DKO Tc cattle carrying the cKSL-HACΔ (cKSL-HACΔ/DKO), the dramatic improvement of B cell development in these cattle and the high level production of hpAbs (as measured for the human IgG isotype) in the plasma. We further demonstrate that, upon immunization by tumor immunogens, high titer tumor immunogen-specific human IgG (hIgG) can be produced from such Tc cattle.

  10. Antigens of human trophoblasts: A working hypothesis for their role in normal and abnormal pregnancies

    PubMed Central

    Faulk, W. Page; Temple, Anne; Lovins, R. E.; Smith, Nancy

    1978-01-01

    This report describes the preparation and characterization of antisera to human trophoblast membranes. Rabbit antisera were raised to trophoblast microvilli prepared by differential ultracentrifugation. Antibodies to serum proteins were removed by solid-phase immunoabsorption with normal human serum, and indirect immunofluorescence experiments with cryostat sections of human placentas showed that the absorbed anti-trophoblast sera reacted with trophoblasts as well as with stromal cells and endothelium of chorionic villi. The antisera also produced membrane fluorescence when studied on viable lymphocytes and certain human cell lines. These anti-trophoblast sera were also lymphocytotoxic, and this reaction was abolished by prior absorption of the antisera with leukocytes. The leukocyte-absorbed anti-trophoblast sera retained their ability to react with trophoblasts and certain human cell lines, but no longer reacted with lymphocytes or placental stromal cells and endothelium. Two categories of trophoblast membrane antigens are thus defined: one present on trophoblasts and certain human cells lines (tentatively designated TA1), and the other on trophoblasts and lymphocytes, villous fibroblasts, and endothelium (tentatively designated TA2). A working hypothesis is proposed stating that normal pregnancy involves the generation of anti-TA2 subsequent to blastocyst implantation and entrance of trophoblasts into the maternal circulation. This involves a mechanism similar to allogeneic cell stimulation and results in antibodies that block either the recognition or cytotoxicity of TA1. Failure to mount this response allows TA1 recognition and trophoblast immunopathology. Experimental and clinical studies in support of this working hypothesis, particularly involving abortion and toxemia, are cited from published reports. Images PMID:273921

  11. Antigenic variation of the human influenza A (H3N2) virus during the 2014-2015 winter season.

    PubMed

    Hua, Sha; Li, XiYan; Liu, Mi; Cheng, YanHui; Peng, YouSong; Huang, WeiJuan; Tan, MinJu; Wei, HeJiang; Guo, JunFeng; Wang, DaYan; Wu, AiPing; Shu, YueLong; Jiang, TaiJiao

    2015-09-01

    The human influenza A (H3N2) virus dominated the 2014-2015 winter season in many countries and caused massive morbidity and mortality because of its antigenic variation. So far, very little is known about the antigenic patterns of the recent H3N2 virus. By systematically mapping the antigenic relationships of H3N2 strains isolated since 2010, we discovered that two groups with obvious antigenic divergence, named SW13 (A/Switzerland/9715293/2013-like strains) and HK14 (A/Hong Kong/5738/2014-like strains), co-circulated during the 2014-2015 winter season. HK14 group co-circulated with SW13 in Europe and the United States during this season, while there were few strains of HK14 in mainland China, where SW13 has dominated since 2012. Furthermore, we found that substitutions near the receptor-binding site on hemagglutinin played an important role in the antigenic variation of both the groups. These findings provide a comprehensive understanding of the recent antigenic evolution of H3N2 virus and will aid in the selection of vaccine strains.

  12. Antigenic variation of the human influenza A (H3N2) virus during the 2014-2015 winter season.

    PubMed

    Hua, Sha; Li, XiYan; Liu, Mi; Cheng, YanHui; Peng, YouSong; Huang, WeiJuan; Tan, MinJu; Wei, HeJiang; Guo, JunFeng; Wang, DaYan; Wu, AiPing; Shu, YueLong; Jiang, TaiJiao

    2015-09-01

    The human influenza A (H3N2) virus dominated the 2014-2015 winter season in many countries and caused massive morbidity and mortality because of its antigenic variation. So far, very little is known about the antigenic patterns of the recent H3N2 virus. By systematically mapping the antigenic relationships of H3N2 strains isolated since 2010, we discovered that two groups with obvious antigenic divergence, named SW13 (A/Switzerland/9715293/2013-like strains) and HK14 (A/Hong Kong/5738/2014-like strains), co-circulated during the 2014-2015 winter season. HK14 group co-circulated with SW13 in Europe and the United States during this season, while there were few strains of HK14 in mainland China, where SW13 has dominated since 2012. Furthermore, we found that substitutions near the receptor-binding site on hemagglutinin played an important role in the antigenic variation of both the groups. These findings provide a comprehensive understanding of the recent antigenic evolution of H3N2 virus and will aid in the selection of vaccine strains. PMID:26219513

  13. Heterophile antibodies in human transplantation

    PubMed Central

    Rapaport, F. T.; Kano, K.; Milgrom, F.

    1968-01-01

    Sensitization of human recipients with transplantation antigens (leucocytes, skin, or kidney allografts) has resulted in the appearance of serum hemagglutinins directed against sheep, guinea pig, and rat erythrocytes. Such hemagglutinins have been identified as IgG and IgM antibodies. Their appearance was not related to AB0 erythrocyte group incompatibility between donors and recipients, and the antibodies were not of the Forssman or Paul-Bunnel type. The antibody responses appeared to be primarily directed against antigen(s) present on rat erythrocytes, but shared to varying extents by other species. The peak antibody titers occurred in association with allograft rejection. In this regard, they may be of interest as a possible early warning system for the diagnosis and prompt management of rejection crises in clinical organ transplantation. Images PMID:4866325

  14. Defining the HLA class I-associated viral antigen repertoire from HIV-1-infected human cells.

    PubMed

    Ternette, Nicola; Yang, Hongbing; Partridge, Thomas; Llano, Anuska; Cedeño, Samandhy; Fischer, Roman; Charles, Philip D; Dudek, Nadine L; Mothe, Beatriz; Crespo, Manuel; Fischer, William M; Korber, Bette T M; Nielsen, Morten; Borrow, Persephone; Purcell, Anthony W; Brander, Christian; Dorrell, Lucy; Kessler, Benedikt M; Hanke, Tomáš

    2016-01-01

    Recognition and eradication of infected cells by cytotoxic T lymphocytes is a key defense mechanism against intracellular pathogens. High-throughput definition of HLA class I-associated immunopeptidomes by mass spectrometry is an increasingly important analytical tool to advance our understanding of the induction of T-cell responses against pathogens such as HIV-1. We utilized a liquid chromatography tandem mass spectrometry workflow including de novo-assisted database searching to define the HLA class I-associated immunopeptidome of HIV-1-infected human cells. We here report for the first time the identification of 75 HIV-1-derived peptides bound to HLA class I complexes that were purified directly from HIV-1-infected human primary CD4(+) T cells and the C8166 human T-cell line. Importantly, one-third of eluted HIV-1 peptides had not been previously known to be presented by HLA class I. Over 82% of the identified sequences originated from viral protein regions for which T-cell responses have previously been reported but for which the precise HLA class I-binding sequences have not yet been defined. These results validate and expand the current knowledge of virus-specific antigenic peptide presentation during HIV-1 infection and provide novel targets for T-cell vaccine development. PMID:26467324

  15. Evaluation of liver fluke recombinant cathepsin B-1 protease as a serodiagnostic antigen for human opisthorchiasis.

    PubMed

    Sripa, Jittiyawadee; Brindley, Paul J; Sripa, Banchob; Loukas, Alex; Kaewkes, Sasithorn; Laha, Thewarach

    2012-03-01

    A cathepsin B-like cysteine protease belonging to family C1 is abundantly expressed in the transcriptome and proteome of the carcinogenic liver fluke of humans, Opisthorchis viverrini. This enzyme is present in excretory/secretory (ES) products released by parasites cultured in vitro. This study evaluated the performance of recombinant O. viverrini cathepsin B1 (rOv-CB-1) as an antigen for immunodiagnosis of opisthorchiasis. The full length Ov-CB-1 cDNA was cloned and recombinant protein was produced in catalytically active form in Pichia pastoris. The recombinant Ov-CB-1 (rOv-CB-1) was affinity purified via nickel-NTA chromatography and tested in enzyme-linked immunosorbent assays (ELISA) with human sera from an opisthorchiasis endemic area. Sera from egg-positive O. viverrini infections produced a strong IgG antibody response to rOv-CB-1 both in ELISA and immunoblot analysis. The sensitivity and specificity of the ELISA test was 67% and 81%, respectively. These findings support the feasibility of using recombinant Ov-CB-1 in ELISA for the serodiagnosis of human opisthorchiasis. PMID:21704728

  16. Human leukocyte antigen-G in the male reproductive system and in seminal plasma.

    PubMed

    Larsen, Margit Hørup; Bzorek, Michael; Pass, Malene B; Larsen, Lise Grupe; Nielsen, Mette Weidinger; Svendsen, Signe Goul; Lindhard, Anette; Hviid, Thomas Vauvert F

    2011-12-01

    One of the non-classical human leukocyte antigen (HLA) class Ib proteins, HLA-G, is believed to exert important immunoregulatory functions, especially during pregnancy. The presence of HLA protein in paternal seminal fluid has been suggested to have an influence on the risk of developing pre-eclampsia. We have investigated whether HLA-G protein is present in human seminal plasma and in different tissue samples of the male reproductive system. Western blot technique and a soluble HLA-G (sHLA-G) assay were used to detect sHLA-G in human seminal plasma samples. Immunohistochemical staining was performed on paraffin-embedded tissue samples. We detected sHLA-G protein in seminal plasma, and HLA-G expression in normal testis and in epididymal tissue of the male reproductive system but not in the seminal vesicle. Furthermore, the results indicated a weak expression of HLA-G in hyperplastic prostatic tissue. In summary, several of the findings reported in this study suggest an immunoregulatory role of HLA-G in the male reproductive system and in seminal plasma.

  17. Hantavirus antigen detection using human serum immunoglobulin M as the capturing antibody in an enzyme-linked immunosorbent assay.

    PubMed

    Alexeyev, O A; Elgh, F; Ahlm, C; Stigbrand, T; Settergren, B; Wadell, G; Juto, P

    1996-04-01

    An enzyme-linked immunosorbent assay (ELISA) was developed to detect different hantavirus antigens in cell culture; i.e. Puumala (PUU), Hantaan (HTN), and Dobrava (DOB) viruses. The assay was based on binding human serum immunoglobulin M (IgM) antibodies to the solid phase by use of goat anti-IgM antibodies. The captured IgM antibodies were present in the acute phase serum from two patients: one infected in Sweden and the other in Bosnia. Antigens being bound to the solid phase by the human anti-PUU and anti-DOB/HTN IgM antibodies were detected by a broadly reacting polyclonal rabbit anti PUU-recombinant nucleocapsid protein antiserum. The IgM isotype was proven to be at least five times more efficient than IgG when used as the capturing antibody. The sensitivity of the PUU antigen ELISA was approximately 0.5 ng/ml, as measured by titration with a PUU recombinant nucleoprotein antigen. Cell-associated PUU antigen in tissue culture was seen after 48 hr by the PUU-ELISA and after 96 hr by immunofluorescent assay. When tested for capacity to discriminate between PUU, DOB, and HTN viruses, significant differences were found: the Swedish serum detected PUU antigen at high titers, whereas no reactivity was found against DOB and HTN; the Bosnian serum detected both DOB and HTN at high titers but had a low reactivity to PUU. The method was also tested for its usefulness in detecting PUU antigen in bank vole (clethrionomys glareolus) lungs. Of 59 animals captured from the surroundings of patients with nephropathia epidemica, three became positive with a high activity in the PUU-ELISA, but with low reactivity in the DOB/HTN-ELISA. It is concluded that a sensitive ELISA has been developed to detect different hantaviruses in cell culture and lungs of bank voles.

  18. Human Leukocyte Antigen (HLA) Peptides Derived from Tumor Antigens Induced by Inhibition of DNA Methylation for Development of Drug-facilitated Immunotherapy.

    PubMed

    Shraibman, Bracha; Kadosh, Dganit Melamed; Barnea, Eilon; Admon, Arie

    2016-09-01

    Treatment of cancer cells with anticancer drugs often fails to achieve complete remission. Yet, such drug treatments may induce alteration in the tumor's gene expression patterns, including those of Cancer/Testis Antigens (CTA). The degradation products of such antigens can be presented as HLA peptides on the surface of the tumor cells and be developed into anticancer immunotherapeutics. For example, the DNA methyl transferase inhibitor, 5-aza-2'-deoxycytidine (Decitabine) has limited antitumor efficacy, yet it induces the expression of many genes, including CTAs that are normally silenced in the healthy adult tissues. In this study, the presentation of many new HLA peptides derived from CTAs and induced by Decitabine was demonstrated in three human Glioblastoma cell lines. Such presentation of CTA-derived HLA peptides can be exploited for development of new treatment modalities, combining drug treatment with anti-CTA targeted immunotherapy. The Decitabine-induced HLA peptidomes include many CTAs that are not normally detected in healthy tissues or in cancer cells, unless treated with the drug. In addition, the study included large-scale analyses of the simultaneous effects of Decitabine on the transcriptomes, proteomes and HLA peptidomes of the human Glioblastoma cells. It demonstrates the poor correlations between these three levels of gene expression, both in their total levels and in their response to the drug. The proteomics and HLA peptidomics data are available via ProteomeXchange with identifier PXD003790 and the transcriptomics data are available via GEO with identifier GSE80137.

  19. Cloning of the gene coding for a shared human melanoma antigen recognized by autologous T cells infiltrating into tumor.

    PubMed Central

    Kawakami, Y; Eliyahu, S; Delgado, C H; Robbins, P F; Rivoltini, L; Topalian, S L; Miki, T; Rosenberg, S A

    1994-01-01

    By cDNA expression cloning we have isolated a gene encoding a shared human melanoma antigen recognized by HLA-A2 restricted autologous and allogenic tumor-infiltrating lymphocytes (TILs) from patients with metastatic melanoma. By using both transient and stable expression systems, transfection of this gene into non-antigen-expressing HLA-A2+ cell lines resulted in recognition by the antigen-specific TILs. The sequence of this cDNA revealed a previously undescribed putative transmembrane protein whose expression was restricted to melanoma and melanocyte cell lines and human retina but no other fresh or cultured normal tissues tested or other tumor histologies. Thus, we have identified a gene encoding a melanocyte lineage-specific protein (MART-1; melanoma antigen recognized by T cells 1) that is a widely shared melanoma antigen recognized by the T lymphocytes of patients with established malignancy. Identification of this gene opens possibilities for the development of immunotherapies for patients with melanoma. PMID:8170938

  20. Guanine polynucleotides are self-antigens for human natural autoantibodies and are significantly reduced in the human genome.

    PubMed

    Fattal, Ittai; Shental, Noam; Ben-Dor, Shifra; Molad, Yair; Gabrielli, Armando; Pokroy-Shapira, Elisheva; Oren, Shirly; Livneh, Avi; Langevitz, Pnina; Zandman-Goddard, Gisele; Sarig, Ofer; Margalit, Raanan; Gafter, Uzi; Domany, Eytan; Cohen, Irun R

    2015-11-01

    In the course of investigating anti-DNA autoantibodies, we examined IgM and IgG antibodies to poly-G and other oligonucleotides in the sera of healthy persons and those diagnosed with systemic lupus erythematosus (SLE), scleroderma (SSc), or pemphigus vulgaris (PV); we used an antigen microarray and informatic analysis. We now report that all of the 135 humans studied, irrespective of health or autoimmune disease, manifested relatively high amounts of IgG antibodies binding to the 20-mer G oligonucleotide (G20); no participants entirely lacked this reactivity. IgG antibodies to homo-nucleotides A20, C20 or T20 were present only in the sera of SLE patients who were positive for antibodies to dsDNA. The prevalence of anti-G20 antibodies led us to survey human, mouse and Drosophila melanogaster (fruit fly) genomes for runs of T20 and G20 or more: runs of T20 appear > 170,000 times compared with only 93 runs of G20 or more in the human genome; of these runs, 40 were close to brain-associated genes. Mouse and fruit fly genomes showed significantly lower T20/G20 ratios than did human genomes. Moreover, sera from both healthy and SLE mice contained relatively little or no anti-G20 antibodies; so natural anti-G20 antibodies appear to be characteristic of humans. These unexpected observations invite investigation of the immune functions of anti-G20 antibodies in human health and disease and of runs of G20 in the human genome.

  1. Bovine leucocyte adhesion deficiency: a review of a modern disease and its implications.

    PubMed

    Gerardi, A S

    1996-11-01

    Bovine leucocyte adhesion deficiency (BLAD) is a genetic disease of cattle which affects the hematopoietic system. In the last decade BLAD has become a disease of economic importance in the dairy industry. This review describes the chronological developments and thinking that led to the elucidation of BLAD as a disease distinct from previous models in canine and human populations. All species affected show signs of chronic and recurrent infections. Necrotic and/or gangrenous infections of soft tissues are prevalent, in addition to secondary infections with bacteria or fungi. Low birthweight and unthriftiness are key signs in all species affected by leucocyte adhesion deficiency (LAD). Dermatomycoses and impaired pus formation are also common findings. The physiological basis for BLAD is a deficiency in the chemotactic and phagocytic properties of leucocytes and particularly neutrophils. The inhibition of diapedesis in the inflammatory response prevents normal immune reactions to invading pathogens. Chronic infections are a consequence of the faulty immune mechanisms. The biochemical aetiology of BLAD involves cell surface glycoprotein molecules known as integrins. These are responsible for the cell-cell interactions necessary for neutrophils to adhere to vascular endothelium in a normal individual. Experiments with monoclonal antibodies to block LFA-1, Mac-1, and p150,95 (three integrins vital for cell-cell interactions) mimic BLAD symptomatology and have led to the discovery of the reciprocal intercellular adhesion molecule (ICAM). Through pedigree analysis and biochemical detection with restrictive endonucleases, BLAD has been isolated genetically to a single gene locus. The economic significance and prophylaxis of the disease are briefly discussed. In addition, the beneficial aspects of the study of BLAD are considered. There are advantages in producing a BLAD-like state for preventing transplant rejection, ischaemia-reperfusion injury and other problems arising

  2. Cholesterol masks membrane glycosphingolipid tumor-associated antigens to reduce their immunodetection in human cancer biopsies.

    PubMed

    Novak, Anton; Binnington, Beth; Ngan, Bo; Chadwick, Karen; Fleshner, Neil; Lingwood, Clifford A

    2013-11-01

    Glycosphingolipids (GSLs) are neoplastic and normal/cancer stem cell markers and GSL/cholesterol-containing membrane rafts are increased in cancer cell plasma membranes. We define a novel means by which cancer cells can restrict tumor-associated GSL immunoreactivity. The GSL-cholesterol complex reorients GSL carbohydrate to a membrane parallel, rather than perpendicular conformation, largely unavailable for antibody recognition. Methyl-β-cyclodextrin cholesterol extraction of all primary human tumor frozen sections tested (ovarian, testicular, neuroblastoma, prostate, breast, colon, pheochromocytoma and ganglioneuroma), unmasked previously "invisible" membrane GSLs for immunodetection. In ovarian carcinoma, globotriaosyl ceramide (Gb3), the GSL receptor for the antineoplastic Escherichia coli-derived verotoxin, was increased throughout the tumor. In colon carcinoma, Gb3 detection was vastly increased within the neovasculature and perivascular stroma. In tumors considered Gb3 negative (neuroblastoma, Leydig testicular tumor and pheochromocytoma), neovascular Gb3 was unmasked. Tumor-associated GSL stage-specific embryonic antigen (SSEA)-1, SSEA-3, SSEA-4 and globoH were unmasked according to tumor: SSEA-1 in prostate/colon; SSEA-3 in prostate; SSEA-4 in pheochromocytoma/some colon tumors; globoH in prostate/some colon tumors. In colon, anti-SSEA-1 was tumor cell specific. Within the GSL-cholesterol complex, filipin-cholesterol binding was also reduced. These results may relate to the ill-defined benefit of statins on cancer prognosis, for example, prostate carcinoma. We found novel anti-tumor GSL antibodies circulating in 3/5 statin-treated, but not untreated, prostate cancer patients. Lowering tumor membrane cholesterol may permit immune recognition of otherwise unavailable tumor-associated GSL carbohydrate, for more effective immunosurveillance and active/passive immunotherapy. Our results show standard immunodetection of tumor GSLs significantly under assesses

  3. Association of human leukocyte antigen DQB1 and DRB1 alleles with chronic hepatitis B

    PubMed Central

    Doganay, Levent; Fejzullahu, Arta; Katrinli, Seyma; Yilmaz Enc, Feruze; Ozturk, Oguzhan; Colak, Yasar; Ulasoglu, Celal; Tuncer, Ilyas; Dinler Doganay, Gizem

    2014-01-01

    AIM: To investigate the effect of human leukocyte antigen (HLA) DRB1 and DQB1 alleles on the inactive and advanced stages of chronic hepatitis B. METHODS: Patient records at a single institution’s hepatology clinic were reviewed. Demographic data, laboratory results, endoscopy results, virological parameters, biopsy scores and treatment statuses were recorded. In total, 355 patients were eligible for the study, of whom 226 (63.7%) were male. Overall, 82 (23.1%) were hepatitis B early antigen (HBeAg) positive, 87 (24.5%) had cirrhosis, and 66 (18.6%) had inactive disease. The presence of DQB1 and DRB1 alleles was determined by polymerase chain reaction with sequence-specific primers. The distribution of the genotyped alleles among patients with cirrhosis and patients with chronic active hepatitis was analyzed. RESULTS: The most frequent HLA DQB1 allele was DQB1*03:01 (48.2%), and the most frequent HLA DRB1 allele was DRB1*13/14 (51.8%). DQB1*05:01 was more frequent in patients with active disease than in inactive patients (27% vs 9.1%; P = 0.002, Pc = 0.026). DRB1*07 was rare in patients with cirrhosis compared with non-cirrhotics (3.4% vs 16%; P = 0.002, Pc = 0.022). Older age (P < 0.001) and male gender (P = 0.008) were the other factors that affected the presence of cirrhosis. In a multivariate logistic regression analysis, DRB1*07 remained a significant negative predictor of cirrhosis (P = 0.015). A bioinformatics analysis revealed that a polymorphic amino acid sequence in DRB1*07 may alter interaction with the T-cell recognition site. CONCLUSION: This study demonstrates that HLA alleles may influence cirrhosis development and disease activity in Turkish chronic hepatitis B patients. PMID:25009391

  4. Small-angle neutron scattering study of recombinant yeast-derived human hepatitis B virus surface antigen vaccine particle

    NASA Astrophysics Data System (ADS)

    Sato, M.; Ito, Y.; Kameyama, K.; Imai, M.; Ishikawa, N.; Takagi, T.

    1995-02-01

    The overall and internal structure of recombinant yeast-derived human hepatitis B virus surface antigen vaccine particles was investigated by small-angle neutron scattering using the contrast variation method. The vaccine is a nearly spherical particle, and its contrast-matching point was determined to be at about 24% D 2O content, indicating that a large part of the vaccine particle is occupied by lipids and carbohydrates from the yeast. The Stuhrmann plot suggests that the surface antigens exist predominantly in the peripheral region of the particle, which is favorable to the induction of anti-virus antibodies.

  5. Human Leukocyte Antigen-G Within the Male Reproductive System: Implications for Reproduction.

    PubMed

    Hviid, Thomas Vauvert F

    2015-01-01

    In sexual reproduction in humans, a man has a clear interest in ensuring that the immune system of his female partner accepts the semi-allogenic fetus. Increasing attention has been given to soluble immunomodulatory molecules in the seminal fluid as one mechanism of ensuring this, possibly by "priming" the woman's immune system before conception and at conception. Recent studies have demonstrated the presence of the immunoregulatory and tolerance-inducible human leukocyte antigen (HLA)-G in the male reproductive organs. The expression of HLA-G in the blastocyst and by extravillous trophoblast cells in the placenta during pregnancy has been well described. Highly variable amounts of soluble HLA-G (sHLA-G) in seminal plasma from different men have been reported, and the concentration of sHLA-G is associated with HLA-G genotype. A first pilot study indicates that the level of sHLA-G in seminal plasma may even be associated with the chance of pregnancy in couples, where the male partner has reduced semen quality. More studies are needed to verify these preliminary findings.

  6. Inflammatory environment and oxidized LDL convert circulating human proangiogenic cells into functional antigen-presenting cells.

    PubMed

    Vinci, Maria Cristina; Piacentini, Luca; Chiesa, Mattia; Saporiti, Federica; Colombo, Gualtiero I; Pesce, Maurizio

    2015-09-01

    The function of human circulating PACs has been described extensively. However, little focus has been placed on understanding how these cells differ in their functions in the presence of microenvironments mimicking vascular inflammation. We hypothesized that exposure to proinflammatory cytokines or the oxLDL, an autoantigen abundant in advanced atherosclerotic plaques, converts PACs into immune-modulating/proinflammatory cells. Hence, we examined the effect of oxLDL and inflammatory stimuli on their phenotype by use of a functional genomics model based on secretome and whole genome transcriptome profiling. PACs obtained from culturing a PBMC fraction in angiogenic medium were primed with DC differentiation cytokines and then exposed to proinflammatory cytokines or oxLDL. Under these conditions, PACs converted into APCs, expressed maturation markers CD80 and CD83, and showed an increased up-regulation of CD86. APCcy and APCox induced a robust T cell BrdU incorporation. Despite a similar ability to induce lymphocyte proliferation, APCcy and APCox differed for the secretory pathway and mRNA expression. Analysis of the differentially expressed genes identified 4 gene "clusters," showing reciprocal modulation in APCcy vs. APCox, justifying, according to functional genomics analyses, a different putative function of the cells in antigen processing. Together, these data show that treatment with inflammatory cytokines or oxLDL converts human PAC phenotypes and functions into that of APCs with similar lymphocyte-activating ability but distinct maturation degree and paracrine functions.

  7. Tissue distribution and dependence of responsiveness of human antigen-specific memory B cells.

    PubMed

    Giesecke, Claudia; Frölich, Daniela; Reiter, Karin; Mei, Henrik E; Wirries, Ina; Kuhly, Rainer; Killig, Monica; Glatzer, Timor; Stölzel, Katharina; Perka, Carsten; Lipsky, Peter E; Dörner, Thomas

    2014-04-01

    Memory B cells (mBCs) are a key to immunologic memory, yet their distribution within lymphoid organs and the individual role of these for mBC functionality remain largely unknown. This study characterized the distribution and phenotype of human (Ag-specific) mBCs in peripheral blood (PB), spleen, tonsil, and bone marrow. We found that the spleen harbors most mBCs, followed by tonsils, BM, and PB, and we detected no major differences in expression of markers associated with higher maturity. Testing the distribution of tetanus toxoid-specific (TT(+)) mBCs revealed their presence in PB during steady state, yet absolute numbers suggested their largest reservoir in the spleen, followed by tonsils. To explore the role of both tissues in the maintenance of reactive B cell memory, we revaccinated controls and splenectomized and tonsillectomized individuals with TT. All donor groups exhibited comparable emergence of anti-TT IgG, TT(+) plasma cells, and TT(+) mBCs in the PB, together with similar molecular characteristics of TT(+) plasma cells. In summary, human mBCs recirculate through PB and reside in different lymphoid organs that do not reflect different mBC maturity stages. The spleen and tonsil, although harboring the largest number of overall and TT(+) mBCs, appear to be dispensable to preserve adequate responsiveness to secondary antigenic challenge.

  8. Selective Susceptibility of Human Skin Antigen Presenting Cells to Productive Dengue Virus Infection

    PubMed Central

    Cerny, Daniela; Haniffa, Muzlifah; Shin, Amanda; Bigliardi, Paul; Tan, Bien Keem; Lee, Bernett; Poidinger, Michael; Tan, Ern Yu; Ginhoux, Florent; Fink, Katja

    2014-01-01

    Dengue is a growing global concern with 390 million people infected each year. Dengue virus (DENV) is transmitted by mosquitoes, thus host cells in the skin are the first point of contact with the virus. Human skin contains several populations of antigen-presenting cells which could drive the immune response to DENV in vivo: epidermal Langerhans cells (LCs), three populations of dermal dendritic cells (DCs), and macrophages. Using samples of normal human skin we detected productive infection of CD14+ and CD1c+ DCs, LCs and dermal macrophages, which was independent of DC-SIGN expression. LCs produced the highest viral titers and were less sensitive to IFN-β. Nanostring gene expression data showed significant up-regulation of IFN-β, STAT-1 and CCL5 upon viral exposure in susceptible DC populations. In mice infected intra-dermally with DENV we detected parallel populations of infected DCs originating from the dermis and migrating to the skin-draining lymph nodes. Therefore dermal DCs may simultaneously facilitate systemic spread of DENV and initiate the adaptive anti-viral immune response. PMID:25474532

  9. Human papilloma virus (HPV) antigens and local immunologic reactivity in oral squamous cell tumors and hyperplasias.

    PubMed

    Syrjänen, K; Happonen, R P; Syrjänen, S; Calonius, B

    1984-08-01

    A series of 191 oral mucosal tumors including those with suspected HPV (Human Papilloma Virus) etiology; squamous cell papilloma (SQP), condyloma acuminatum (CA), focal epithelial hyperplasia (FEH), as well as those regarded as unrelated to HPV; fibrous hyperplasia (FH), papillary hyperplasia (PH), and true fibroma (TF), were analyzed for HPV structural proteins (by indirect immunoperoxidase IP-PAP technique), for the presence of epithelial dysplasia, and for the cellular composition (B and T lymphocytes, mononuclear phagocytes, (MPS cells] of their local inflammatory cell infiltrates using ANAE-(acid alpha-naphthyl acetate esterase) stain. HPV structural proteins were disclosed in 85% of FEH, in 75% of CA, and in 41% of SQP. These three lesions significantly differed from PH and FH with regard to the intensity and cellular composition of the local infiltrates, being most intense and B cell predominated in the latter two. Mild dysplasia was found in 20% of both CA and SQP lesions, the former also showing moderate dysplasia in 12% of cases. The HPV antigen positivity was negatively correlated with dysplasia in CA and SQP, the intensity of the infiltrate showing positive association with dysplasia. The results are discussed in terms of HPV etiology of CA, SQP and FEH, of the host immunologic reactivity against these lesions, as well as of the possible role of HPV in human squamous cell carcinogenesis.

  10. Nanogold-actuated biomimetic peroxidase for sensitized electrochemical immunoassay of carcinoembryonic antigen in human serum.

    PubMed

    Chen, Huafeng; Tang, Juan; Su, Biling; Chen, Guonan; Huang, Jianxin; Tang, Dianping

    2010-09-30

    A new electrochemical immunoassay protocol for the determination of carcinoembryonic antigen (CEA) in human serum was developed by means of immobilizing horseradish peroxidase-labeled anti-CEA antibodies (HRP-anti-CEA) on the nanogold/nano-Fe(3)O(4) particles-functionalized polyion complex (PIC) membrane. With a one-step immunoassay format, the formed immunocomplex inhibited partly the bioactive center of the immobilized HRP, and decreased the HRP toward the reduction of H(2)O(2). Nanogold-actuated nano-Fe(3)O(4) mimetic peroxidase was used for the sensitivity improvement of the electrochemical immunosensor with Prussian blue (PB) as mediators. Under optimal conditions, the electrochemical immunosensor displayed a wide dynamic range of 0.1-220 ng mL(-1) with a relative low detection limit (LOD) of 10 pg mL(-1) CEA (at 3σ). Intra- and inter-assay show good precisions with a coefficient of variation (CV) lower than 7.8% and 7.3%, respectively. No significant difference at the 95% confidence level was encountered in the analysis of 17 human serum specimens between the electrochemical immunosensor and automatic electrochemiluminescent (ECL) analyzer for the determination of CEA.

  11. Antigenic typing of Brazilian rabies virus samples isolated from animals and humans, 1989-2000.

    PubMed

    Favoretto, Silvana Regina; Carrieri, Maria Luiza; Cunha, Elenice Maria S; Aguiar, Elizabeth A C; Silva, Luzia Helena Q; Sodre, Miriam M; Souza, Maria Conceição A M; Kotait, Ivanete

    2002-01-01

    Animal and human rabies samples isolated between 1989 and 2000 were typified by means of a monoclonal antibody panel against the viral nucleoprotein. The panel had been previously established to study the molecular epidemiology of rabies virus in the Americas. Samples were isolated in the Diagnostic Laboratory of the Pasteur Institute and in other rabies diagnostic centers in Brazil. In addition to the fixed virus samples CVS-31/96-IP, preserved in mouse brain, and PV-BHK/97, preserved in cell culture, a total of 330 rabies virus samples were isolated from dogs, cats, cattle, horses, bats, sheep, goat, swine, foxes, marmosets, coati and humans. Six antigenic variants that were compatible with the pre-established monoclonal antibodies panel were defined: numbers 2 (dog), 3 (Desmodus rotundus), 4 (Tadarida brasiliensis), 5 (vampire bat from Venezuela), 6 (Lasiurus cinereus) and Lab (reacted to all used antibodies). Six unknown profiles, not compatible with the panel, were also found. Samples isolated from insectivore bats showed the greatest variability and the most commonly isolated variant was variant-3 (Desmodus rotundus). These findings may be related to the existence of multiple independent transmission cycles, involving different bat species. PMID:12048546

  12. Human peripheral blood leucocyte non-obese diabetic-severe combined immunodeficiency interleukin-2 receptor gamma chain gene mouse model of xenogeneic graft-versus-host-like disease and the role of host major histocompatibility complex

    PubMed Central

    King, M A; Covassin, L; Brehm, M A; Racki, W; Pearson, T; Leif, J; Laning, J; Fodor, W; Foreman, O; Burzenski, L; Chase, T H; Gott, B; Rossini, A A; Bortell, R; Shultz, L D; Greiner, D L

    2009-01-01

    Immunodeficient non-obese diabetic (NOD)-severe combined immune-deficient (scid) mice bearing a targeted mutation in the gene encoding the interleukin (IL)-2 receptor gamma chain gene (IL2rγnull) engraft readily with human peripheral blood mononuclear cells (PBMC). Here, we report a robust model of xenogeneic graft-versus-host-like disease (GVHD) based on intravenous injection of human PBMC into 2 Gy conditioned NOD-scid IL2rγnull mice. These mice develop xenogeneic GVHD consistently (100%) following injection of as few as 5 × 106 PBMC, regardless of the PBMC donor used. As in human disease, the development of xenogeneic GVHD is highly dependent on expression of host major histocompatibility complex class I and class II molecules and is associated with severely depressed haematopoiesis. Interrupting the tumour necrosis factor-α signalling cascade with etanercept, a therapeutic drug in clinical trials for the treatment of human GVHD, delays the onset and progression of disease. This model now provides the opportunity to investigate in vivo mechanisms of xenogeneic GVHD as well as to assess the efficacy of therapeutic agents rapidly. PMID:19659776

  13. IMMORTALIZATION OF HUMAN AND RHESUS MACAQUE PRIMARY ANTIGEN-SPECIFIC T CELLS BY RETROVIRALLY TRANSDUCED TELOMERASE REVERSE TRANSCRIPTASE

    PubMed Central

    Barsov, Eugene V.

    2011-01-01

    Human and rhesus macaque primary antigen-specific T cells derived from infected or immunized individuals or animals are a valuable material with which to study cellular immune responses against pathogens and tumors. Antigen-specific T cells can be expanded in vitro but have a finite proliferative life span. After a limited period in culture, primary T cells undergo replicative senescence and stop dividing. This restricts their applicability to short term experiments and complicates their use in adoptive immunotherapy. The proliferative life span of primary human and rhesus macaque T cells can be considerably extended by ectopically expressed human telomerase reverse transcriptase (TERT). Antigen-specific T cells transduced with TERT-expressing retroviral vectors can proliferate and expand in culture for long periods of time while maintaining their primary T cell characteristics including antigen-specific responses. Thus, TERT-immortalized T cells are an important and valuable resource for studying T cell immune responses and, potentially, for adoptive immunotherapy. PMID:22048804

  14. Evaluation of a di-O-methylated glycan as a potential antigenic target for the serodiagnosis of human toxocariasis.

    PubMed

    Elefant, G R; Roldán, W H; Seeböck, A; Kosma, P

    2016-04-01

    Serodiagnosis of human toxocariasis is based on the detection of specific IgG antibodies by the enzyme-linked immunosorbent assay (ELISA) using Toxocara larvae excretory-secretory (TES) antigens, but its production is a laborious and time-consuming process being also limited by the availability of adult females of T. canis as source for ova to obtain larvae. Chemical synthesis of the di-O-methylated (DiM) glycan structure found in the TES antigens has provided material for studying the antibody reactivity in a range of mammalian hosts, showing reactivity with human IgM and IgG. In this study, we have evaluated the performance of the DiM glycan against a panel of sera including patients with toxocariasis (n = 60), patients with other helminth infections (n = 75) and healthy individuals (n = 94), showing that DiM is able to detect IgG antibodies with a sensitivity and specificity of 91·7% and 94·7%, respectively, with a very good agreement with the TES antigens (kappa = 0·825). However, cross-reactivity was observed in some sera from patients with ascariasis, hymenolepiasis and fascioliasis. These results show that the DiM glycan could be a promising antigenic tool for the serodiagnosis of human toxocariasis.

  15. Evaluation of a di-O-methylated glycan as a potential antigenic target for the serodiagnosis of human toxocariasis.

    PubMed

    Elefant, G R; Roldán, W H; Seeböck, A; Kosma, P

    2016-04-01

    Serodiagnosis of human toxocariasis is based on the detection of specific IgG antibodies by the enzyme-linked immunosorbent assay (ELISA) using Toxocara larvae excretory-secretory (TES) antigens, but its production is a laborious and time-consuming process being also limited by the availability of adult females of T. canis as source for ova to obtain larvae. Chemical synthesis of the di-O-methylated (DiM) glycan structure found in the TES antigens has provided material for studying the antibody reactivity in a range of mammalian hosts, showing reactivity with human IgM and IgG. In this study, we have evaluated the performance of the DiM glycan against a panel of sera including patients with toxocariasis (n = 60), patients with other helminth infections (n = 75) and healthy individuals (n = 94), showing that DiM is able to detect IgG antibodies with a sensitivity and specificity of 91·7% and 94·7%, respectively, with a very good agreement with the TES antigens (kappa = 0·825). However, cross-reactivity was observed in some sera from patients with ascariasis, hymenolepiasis and fascioliasis. These results show that the DiM glycan could be a promising antigenic tool for the serodiagnosis of human toxocariasis. PMID:26896376

  16. Advances in alloimmune thrombocytopenia: perspectives on current concepts of human platelet antigens, antibody detection strategies, and genotyping

    PubMed Central

    Hayashi, Tomoya; Hirayama, Fumiya

    2015-01-01

    Alloimmunisation to platelets leads to the production of antibodies against platelet antigens and consequently to thrombocytopenia. Numerous molecules located on the platelet surface are antigenic and induce immune-mediated platelet destruction with symptoms that can be serious. Human platelet antigens (HPA) cause thrombocytopenias, such as neonatal alloimmune thrombocytopenia, post-transfusion purpura, and platelet transfusion refractoriness. Thirty-four HPA are classified into 28 systems. Assays to identify HPA and anti-HPA antibodies are critically important for preventing and treating thrombocytopenia caused by anti-HPA antibodies. Significant progress in furthering our understanding of HPA has been made in the last decade: new HPA have been discovered, antibody-detection methods have improved, and new genotyping methods have been developed. We review these advances and discuss issues that remain to be resolved as well as future prospects for preventing and treating immune thrombocytopenia. PMID:26057488

  17. "Danger" conditions increase sulfamethoxazole-protein adduct formation in human antigen-presenting cells.

    PubMed

    Lavergne, S N; Wang, H; Callan, H E; Park, B K; Naisbitt, D J

    2009-11-01

    Antigen-presenting cells (APC) are thought to play an important role in the pathogenesis of drug-induced immune reactions. Various pathological factors can activate APC and therefore influence the immune equilibrium. It is interesting that several diseases have been associated with an increased rate of drug allergy. The aim of this project was to evaluate the impact of such "danger signals" on sulfamethoxazole (SMX) metabolism in human APC (peripheral blood mononuclear cells, Epstein-Barr virus-modified B lymphocytes, monocyte-derived dendritic cells, and two cell lines). APC were incubated with SMX (100 microM-2 mM; 5 min-24 h), in the presence of pathological factors: bacterial endotoxins (lipopolysaccharide and staphylococcal enterotoxin B), flu viral proteins, cytokines [interleukin (IL)-1beta, IL-6, IL-10; tumor necrosis factor-alpha; interferon-gamma; and transforming growth factor-beta], inflammatory molecules (prostaglandin E2, human serum complement, and activated protein C), oxidants (buthionine sulfoximine and H(2)O(2)), and hyperthermia (37.5-39.5 degrees C). Adduct formation was evaluated by enzyme-linked immunosorbent assay and confocal microscopy. SMX-protein adduct formation was time- and concentration-dependent for each cell type tested, in both physiological and danger conditions. A danger environment significantly increased the formation of SMX-protein adducts and significantly shortened the delay for their detection. An additive effect was observed with a combination of danger signals. Dimedone (chemical selectively binding cysteine sulfenic acid) and antioxidants decreased both baseline and danger-enhanced SMX-adduct formation. Various enzyme inhibitors were associated with a significant decrease in SMX-adduct levels, with a pattern varying depending on the cell type and the culture conditions. These results illustrate that danger signals enhance the formation of intracellular SMX-protein adducts in human APC. These findings might be relevant

  18. Candida albicans Is Phagocytosed, Killed, and Processed for Antigen Presentation by Human Dendritic Cells

    PubMed Central

    Newman, Simon L.; Holly, Angela

    2001-01-01

    Candida albicans is a component of the normal flora of the alimentary tract and also is found on the mucocutaneous membranes of the healthy host. Candida is the leading cause of invasive fungal disease in premature infants, diabetics, and surgical patients, and of oropharyngeal disease in AIDS patients. As the induction of cell-mediated immunity to Candida is of critical importance in host defense, we sought to determine whether human dendritic cells (DC) could phagocytose and degrade Candida and subsequently present Candida antigens to T cells. Immature DC obtained by culture of human monocytes in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4 phagocytosed unopsonized Candida in a time-dependent manner, and phagocytosis was not enhanced by opsonization of Candida in serum. Like macrophages (Mφ), DC recognized Candida by the mannose-fucose receptor. Upon ingestion, DC killed Candida as efficiently as human Mφ, and fungicidal activity was not enhanced by the presence of fresh serum. Although phagocytosis of Candida by DC stimulated the production of superoxide anion, inhibitors of the respiratory burst (or NO production) did not inhibit killing of Candida, even when phagocytosis was blocked by preincubation of DC with cytochalasin D. Further, although apparently only modest phagolysosomal fusion occurred upon DC phagocytosis of Candida, killing of Candida under anaerobic conditions was almost equivalent to killing under aerobic conditions. Finally, DC stimulated Candida-specific lymphocyte proliferation in a concentration-dependent manner after phagocytosis of both viable and heat-killed Candida cells. These data suggest that, in vivo, such interactions between DC and C. albicans may facilitate the induction of cell-mediated immunity. PMID:11598054

  19. Anti-Human Leukocyte Antigen Antibody Induced Autoimmunity: Role In Chronic Rejection

    PubMed Central

    Nath, DS; Basha, H Ilias; Mohanakumar, T

    2009-01-01

    Purpose of review We provide evidence for the role of de novo development of immune responses to self-antigens in the post-transplant period and its possible induction by alloimmunity in the pathogenesis of chronic rejection following lung, heart and kidney transplantation. This review details recent findings for the two distinct yet inter-dependent immune processes in the immune-pathogenesis of chronic rejection. Recent findings The contribution of both humoral and cell mediated allo-immune responses against mismatched donor histocompatibility antigens (HLA) in the pathogenesis of chronic rejection is well established. Recent studies have focused on development of immune responses to self-antigens during the post-transplant period and its correlation with chronic rejection. These self-antigens include myosin and vimentin in cardiac, K-alpha-1-tubulin and collagen-V in lung and angiotensin II type 1 receptor, collagen-IV and VI in kidney transplants. During the post-transplant period, the development of immune responses to self-antigens is facilitated by induction of a distinct subset of auto-reactive T-helper cells referred to as Th17 cells. Summary Following organ transplantation, tissue injury and remodeling inflicted by Abs to HLA antigens is conducive to develop autoimmunity. Antibodies (Abs) to HLA and self-antigens are detectable in the serum of transplant recipients who develop chronic rejection. Anti-HLA Abs are often present transiently but precede the development of Abs to self-antigens. PMID:19898237

  20. A reliable method for avoiding false negative results with Luminex single antigen beads; evidence of the prozone effect.

    PubMed

    Carey, B Sean; Boswijk, Kim; Mabrok, Mazen; Rowe, Peter A; Connor, Andrew; Saif, Imran; Poles, Anthony

    2016-07-01

    Luminex single antigen bead (SAB) assays have become an essential tool in monitoring the status of antibody to the Human Leucocyte Antigen (HLA) of patients both before and after transplantation. In addition SAB data is used to aid risk stratification to assess immunological risk of humoral rejection in solid organ transplantation (CTAG/BTAG guidelines) [1]. Increasingly laboratories are reporting false negative results at high antibody titre due to a prozone effect. Here we report a case study where the prozone effect led to a false negative antibody result that could have resulted in adverse outcome. We describe a method to reliably remove the prozone effect through heat inactivation and the addition of Ethylenediaminetetraacetic acid (EDTA) to the Luminex wash buffer. PMID:27109036

  1. A new human tumor-associated antigen (TLP) is naturally expressed in rat DHD-K12 colorectal tumor cells.

    PubMed

    Rasi, G; Sinibaldi-Vallebona, P; Serafino, A; Bernard, P; Pierimarchi, P; Guarino, E; Faticanti-Scucchi, L; Graziano, P; Guadagni, F; Garci, E

    2000-02-15

    Renewed interest in cancer immunotherapy has been raised by the availability of a variety of tumor-associated antigens and animal models. We have recently described the presence of a new antigen, TLP, in sera and cancer tissue from lung and colorectal cancer patients. In order to develop an experimental model suitable for preclinical studies on cancer vaccines, we investigated the presence of TLP antigen in vitro, in the DHD-K12 cell line and in vivo, in metastases induced in syngeneic BDIX rats by DHD-K12 cell injection. TLP was not detected in any tissue of healthy rats nor in normal tissues of tumor-bearing rats. This is in agreement with our previous studies, in which we had demonstrated that TLP is expressed in human colorectal cancer and adenomas but not in normal colonic mucosa. Our results indicate TLP as a possible human tumor-specific antigen naturally expressed in DHD-K12 tumor syngeneic to immunocompetent BDIX rats.

  2. A computational method for identification of vaccine targets from protein regions of conserved human leukocyte antigen binding

    PubMed Central

    2015-01-01

    Background Computational methods for T cell-based vaccine target discovery focus on selection of highly conserved peptides identified across pathogen variants, followed by prediction of their binding of human leukocyte antigen molecules. However, experimental studies have shown that T cells often target diverse regions in highly variable viral pathogens and this diversity may need to be addressed through redefinition of suitable peptide targets. Methods We have developed a method for antigen assessment and target selection for polyvalent vaccines, with which we identified immune epitopes from variable regions, where all variants bind HLA. These regions, although variable, can thus be considered stable in terms of HLA binding and represent valuable vaccine targets. Results We applied this method to predict CD8+ T-cell targets in influenza A H7N9 hemagglutinin and significantly increased the number of potential vaccine targets compared to the number of targets discovered using the traditional approach where low-frequency peptides are excluded. Conclusions We developed a webserver with an intuitive visualization scheme for summarizing the T cell-based antigenic potential of any given protein or proteome using human leukocyte antigen binding predictions and made a web-accessible software implementation freely available at http://met-hilab.cbs.dtu.dk/blockcons/. PMID:26679766

  3. Meta-Analysis of the Association between Vitiligo and Human Leukocyte Antigen-A

    PubMed Central

    Ren, Jianwen; Niu, Xinwu; Xu, Qingqiang; Wang, Xiaopeng; Liu, Yale

    2016-01-01

    Objective. The objective of this study was to systematically evaluate the association between vitiligo and human leukocyte antigen- (HLA-) A. Methods. PubMed, Embase, Web of Science, Chinese National Knowledge Infrastructure, and reference lists were searched for relevant original articles. Results. Nineteen case-control studies comprising 3042 patients and 5614 controls were included, in which 33 HLA-A alleles were reported. Overall, three alleles (HLA-A⁎02, A⁎33, and Aw⁎31) were significantly associated with increased risk of vitiligo, two (HLA-A⁎09 and Aw⁎19) were associated with decreased risk, and the remaining 28 were unassociated. Twelve alleles, seven alleles, and 19 alleles were common to three ethnicities, both types of vitiligo, and both typing methods, respectively. In the subgroup analysis by ethnicity and typing methods, the association of six alleles and five alleles was inconsistent in three populations and both typing methods, respectively. In the subgroup analysis by clinical type, the association of all seven alleles was consistent in both types of vitiligo. Conclusion. The meta-analysis suggests that HLA-A⁎02, A⁎33, and Aw⁎31 are associated with increased risk of vitiligo, while HLA-A⁎09 and Aw⁎19 are associated with decreased risk of vitiligo. The association of some alleles varies in terms of ethnicity and typing methods.

  4. Profiling TRA-1-81 antigen distribution on a human embryonic stem cell

    SciTech Connect

    Qiu Dengli; Xiang Jialing; Li Zhaoxia; Krishnamoorthy, Aparna; Chen Liaohai; Wang Rong

    2008-05-02

    Human embryonic stem (hES) cells hold great promise in regenerative medicine. Although hES cells have unlimited self-renewal potential, they tend to differentiate spontaneously in culture. TRA-1-81 is a biomarker of undifferentiated hES cells. Quantitative characterization of TRA-1-81 expression level in a single cell helps capture the 'turn-on' signal and understand the mechanism of early differentiation. Here, we report on our examination of TRA-1-81 distribution and association on a hES cell membrane using an atomic force microscope (AFM). Our results suggest that aggregated distribution of TRA-1-81 antigen is characteristic for undifferentiated hES cells. We also evaluated the TRA-1-81 expression level at {approx}17,800 epitopes and {approx}700 epitopes per cell on an undifferentiated cell and a spontaneously differentiated cell, respectively. The method in this study can be adapted in examining other surface proteins on various cell types, thus providing a general tool for investigating protein distribution and association at the single cell level.

  5. Origins and relatedness of human leukocyte antigen class I allele supertypes.

    PubMed

    Naugler, Christopher

    2010-09-01

    Class I human leukocyte antigen (HLA) alleles can be classified into supertypes based on the epitope specificity of their peptide binding grooves. The evolutionary origin of these supertypes has been the topic of prior research and remains an important question because of the increasing interest in HLA supertypes in the contexts of infection and cancer epidemiology and vaccine development. Here I re-examine the origins of HLA class I supertypes using the nucleotide sequences of 88 HLA-A alleles and 117 HLA-B alleles. Phylogenetic trees with ancestral character state reconstruction show that the HLA-A02, A03, and A24 supertypes largely form clades with a single ancestral origin while HLA-A01 shows multiple independent origins all from HLA-A03 ancestors. HLA-B supertypes show multiple origins for the B07, B08, and B27 supertypes, while the B44, B58, and B62 supertypes largely form clades with a single ancestor. Supertypes arising multiple times show different amino acid substitutions in each clade. These findings suggest that convergent evolution has occurred in only a few HLA allele supertypes and may indicate different evolutionary pressures shaping certain supertypes.

  6. Inhibition of host immune response in colorectal cancer: Human leukocyte antigen-G and beyond

    PubMed Central

    Garziera, Marica; Toffoli, Giuseppe

    2014-01-01

    Colorectal cancer (CRC) is one of the most diffuse cancers worldwide and is still a clinical burden. Increasing evidences associate CRC clinical outcome to immune contexture represented by adaptive immune cells. Their type, density and location are summarized in the Immune Score that has been shown to improve prognostic prediction of CRC patients. The non-classical MHC class I human leukocyte antigen-G (HLA-G), is a crucial tumor-driven immune escape molecule involved in immune tolerance. HLA-G and soluble counterparts are able to exert inhibitory functions by direct interactions with inhibitory receptors present on both innate cells such as natural killer cells, and adaptive immune cells as cytotoxic T and B lymphocytes. HLA-G may play a prominent role in CRC strategies to avoid host immunosurveillance. This review highlights the current knowledge on HLA-G contribution in CRC, in related inflammatory diseases and in other type of cancers and disorders. HLA-G genetic setting (specific haplotypes, genotypes and alleles frequencies) and association with circulating/soluble profiles was highlighted. HLA G prognostic and predictive value in CRC was investigated in order to define a novel prognostic immune biomarker in CRC. PMID:24744572

  7. Meta-Analysis of the Association between Vitiligo and Human Leukocyte Antigen-A

    PubMed Central

    Ren, Jianwen; Niu, Xinwu; Xu, Qingqiang; Wang, Xiaopeng; Liu, Yale

    2016-01-01

    Objective. The objective of this study was to systematically evaluate the association between vitiligo and human leukocyte antigen- (HLA-) A. Methods. PubMed, Embase, Web of Science, Chinese National Knowledge Infrastructure, and reference lists were searched for relevant original articles. Results. Nineteen case-control studies comprising 3042 patients and 5614 controls were included, in which 33 HLA-A alleles were reported. Overall, three alleles (HLA-A⁎02, A⁎33, and Aw⁎31) were significantly associated with increased risk of vitiligo, two (HLA-A⁎09 and Aw⁎19) were associated with decreased risk, and the remaining 28 were unassociated. Twelve alleles, seven alleles, and 19 alleles were common to three ethnicities, both types of vitiligo, and both typing methods, respectively. In the subgroup analysis by ethnicity and typing methods, the association of six alleles and five alleles was inconsistent in three populations and both typing methods, respectively. In the subgroup analysis by clinical type, the association of all seven alleles was consistent in both types of vitiligo. Conclusion. The meta-analysis suggests that HLA-A⁎02, A⁎33, and Aw⁎31 are associated with increased risk of vitiligo, while HLA-A⁎09 and Aw⁎19 are associated with decreased risk of vitiligo. The association of some alleles varies in terms of ethnicity and typing methods. PMID:27689083

  8. Differential expression of a new tumor-associated antigen, TLP, during human colorectal cancer tumorigenesis.

    PubMed

    Guadagni, F; Graziano, P; Roselli, M; Mariotti, S; Bernard, P; Sinibaldi-Vallebona, P; Rasi, G; Garaci, E

    1999-04-01

    Tumour liberated particles (TLP) have been proposed as a potential new serum tumor marker. In particular, a high percentage of patients with early stages of lung cancer scored positive for serum TLP, suggesting its possible role as a marker for early diagnosis of disease. The aim of the present study was to analyze the expression of TLP in the colorectal adenoma-carcinoma sequence in order to determine whether its expression correlates with the various stages of cancer transformation. TLP distribution was assessed by immunohistochemistry in normal, premalignant, and malignant colorectal lesions. Normal colonic mucosa and hyperplastic polyps showed no positive staining, whereas adenomas and adenocarcinomas reacted to anti-TLP serum. The percentage of positive tumor cells increased from adenomas with mild dysplasia to adenomas with severe dysplasia. Moreover, a supranuclear staining pattern was observed mainly in adenomas with mild dysplasia, whereas adenomas with severe dysplasia as well as adenocarcinomas showed a characteristic diffuse staining pattern and a strong staining intensity. Only a few cases of adenocarcinoma were found to be TLP-negative and all were poorly differentiated. Our results suggest that TLP antigen expression may be considered as a marker of epithelial atypia in the colorectal tract and as a potential target for new diagnostic and/or therapeutic approaches to human colorectal cancer.

  9. Genetic screening for human leukocyte antigen alleles prior to carbamazepine treatment.

    PubMed

    Tan, Jeremy C K; Murrell, Dedee F; Hersch, Mark I

    2015-12-01

    We describe a 28-year-old Malaysian Australian man of Han Chinese descent with toxic epidermal necrolysis (TEN), occurring 2 weeks after commencing carbamazepine. He was subsequently found to be positive for human leukocyte antigen (HLA)-B*1502. Carbamazepine-induced Stevens-Johnson syndrome/TEN is strongly associated with the HLA-B*1502 allele, which is highly prevalent in Han Chinese, Malay, Thai and Indian populations. Prospective screening for the allele may prevent this cutaneous adverse drug reaction from occurring, but many neurologists and other medical practitioners are still unaware of the medico-legal risks of prescribing carbamazepine in susceptible populations and the availability of HLA-B*1502 testing. Performing HLA-B*1502 genotyping and avoiding carbamazepine in at-risk individuals has been proven to decrease incidences of drug-induced TEN. This test is widely available at most large pathology services in Australia, with results available within 2 weeks. The recommendation by regulatory bodies should be strengthened to ensure that the broad medical community is made more aware of this pertinent issue.

  10. Impact of killer immunoglobulin-like receptor-human leukocyte antigens ligand incompatibility among renal transplantation

    PubMed Central

    Alam, S.; Rangaswamy, D.; Prakash, S.; Sharma, R. K.; Khan, M. I.; Sonawane, A.; Agrawal, S.

    2015-01-01

    Killer immunoglobulin-like receptor (KIR) gene shows a high degree of polymorphism. Natural killer cell receptor gets activated once they bind to self-human leukocyte antigens (HLAs) with specific ligand. KIR gene and HLA ligand incompatibility due to the presence/absence of KIR in the recipient and the corresponding HLA ligand in the allograft may impact graft survival in solid organ transplantation. This study evaluates the effect of matches between KIR genes and known HLA ligands. KIR genotypes were determined using sequence specific primer polymerase chain reaction. Presence of certain KIR in a recipient, where the donor lacked the corresponding HLA ligand was considered a mismatch. The allograft was considered matched when both KIR receptor and HLA alloantigen reveald compatibility among recipient and donor. The data revealed better survival among individuals with matched inhibitory KIR receptors and their corresponding HLA ligands (KIR2DL2/DL3-HLAC2, KIR3DL1-HLABw4). On the contrary, no adverse effect was seen for matched activating KIR receptors and their corresponding HLA ligands. One of the activating gene KIR2DS4 showed risk (P = 0.0413, odds ratio = 1.91, 95% confidence interval = 1.02-3.57) association with renal allograft rejection. We conclude that the presence of inhibitory KIR gene leads to better survival; whereas activating motifs show no significant role in renal allograft survival. PMID:25684869

  11. Impact of killer immunoglobulin-like receptor-human leukocyte antigens ligand incompatibility among renal transplantation.

    PubMed

    Alam, S; Rangaswamy, D; Prakash, S; Sharma, R K; Khan, M I; Sonawane, A; Agrawal, S

    2015-01-01

    Killer immunoglobulin-like receptor (KIR) gene shows a high degree of polymorphism. Natural killer cell receptor gets activated once they bind to self-human leukocyte antigens (HLAs) with specific ligand. KIR gene and HLA ligand incompatibility due to the presence/absence of KIR in the recipient and the corresponding HLA ligand in the allograft may impact graft survival in solid organ transplantation. This study evaluates the effect of matches between KIR genes and known HLA ligands. KIR genotypes were determined using sequence specific primer polymerase chain reaction. Presence of certain KIR in a recipient, where the donor lacked the corresponding HLA ligand was considered a mismatch. The allograft was considered matched when both KIR receptor and HLA alloantigen reveald compatibility among recipient and donor. The data revealed better survival among individuals with matched inhibitory KIR receptors and their corresponding HLA ligands (KIR2DL2/DL3-HLAC2, KIR3DL1-HLABw4). On the contrary, no adverse effect was seen for matched activating KIR receptors and their corresponding HLA ligands. One of the activating gene KIR2DS4 showed risk (P = 0.0413, odds ratio = 1.91, 95% confidence interval = 1.02-3.57) association with renal allograft rejection. We conclude that the presence of inhibitory KIR gene leads to better survival; whereas activating motifs show no significant role in renal allograft survival.

  12. Loss of heterozygosity at the human leukocyte antigen locus in thymic epithelial tumors

    PubMed Central

    Chen, Yuan; Wang, Guojin; Zhang, Peng; Liu, Yimei; Yao, Yuanyuan; Wang, Hai; Wang, Yuanguo

    2015-01-01

    Background To study the relationship between loss of heterozygosity (LOH) at the human leukocyte antigen (HLA) locus and the pathogenicity and clinicopathological features of thymic epithelial tumors (TET). Methods Tumor and adjacent normal tissues were isolated from 36 TET patients. Five microsatellite loci (D6S1666, D6S265, D6S273, DS6276, and D6S291) within the HLA locus were amplified by polymerase chain reaction. DNA sequencing was used to measure the frequency of microsatellite LOH. Results LOH was identified in at least one locus in 83.6% of TET patients. LOH frequency at D6S1666, D6S265, D6S273, D6S276, and D6S291 was 44.4%, 16.7%, 30.5%, 38.9%, and 36.1% respectively. There was no significant association between LOH frequency in TET with tumor severity, or in the presence or absence of myasthenia gravis. Conclusions D6S1666, D6S265, D6S273, DS6S276, and D6S29 are sensitive loci for studying microsatellite LOH in TET. LOH within the HLA complex is implicated in the occurrence and development of TET, with the HLA-DQA1 gene likely involved. However, an understanding of the relationship between LOH and the clinicopathological features of TET requires a larger sample size than that of the present study. PMID:26557913

  13. Engineered Human Ferritin Nanoparticles for Direct Delivery of Tumor Antigens to Lymph Node and Cancer Immunotherapy

    PubMed Central

    Lee, Bo-Ram; Ko, Ho Kyung; Ryu, Ju Hee; Ahn, Keum Young; Lee, Young-Ho; Oh, Se Jin; Na, Jin Hee; Kim, Tae Woo; Byun, Youngro; Kwon, Ick Chan; Kim, Kwangmeyung; Lee, Jeewon

    2016-01-01

    Efficient delivery of tumor-specific antigens (TSAs) to lymph nodes (LNs) is essential to eliciting robust immune response for cancer immunotherapy but still remains unsolved. Herein, we evaluated the direct LN-targeting performance of four different protein nanoparticles with different size, shape, and origin [Escherichia coli DNA binding protein (DPS), Thermoplasma acidophilum proteasome (PTS), hepatitis B virus capsid (HBVC), and human ferritin heavy chain (hFTN)] in live mice, using an optical fluorescence imaging system. Based on the imaging results, hFTN that shows rapid LN targeting and prolonged retention in LNs was chosen as a carrier of the model TSA [red fluorescence protein (RFP)], and the flexible surface architecture of hFTN was engineered to densely present RFPs on the hFTN surface through genetic modification of subunit protein of hFTN. The RFP-modified hFTN rapidly targeted LNs, sufficiently exposed RFPs to LN immune cells during prolonged period of retention in LNs, induced strong RFP-specific cytotoxic CD8+ T cell response, and notably inhibited RFP-expressing melanoma tumor growth in live mice. This suggests that the strategy using protein nanoparticles as both TSA-carrying scaffold and anti-cancer vaccine holds promise for clinically effective immunotherapy of cancer. PMID:27725782

  14. Clinical implication of human leukocyte antigen (HLA)-F expression in breast cancer.

    PubMed

    Harada, Aya; Ishigami, Sumiya; Kijima, Yuko; Nakajo, Akihiro; Arigami, Takaaki; Kurahara, Hiroshi; Kita, Yoshiaki; Yoshinaka, Heiji; Natsugoe, Shoji

    2015-11-01

    Human leukocyte antigen (HLA)-F is one of the non-classical HLA class I molecules that protects the fetus in pregnancy. HLA-F expression was immunohistochemically examined and the association between clinical parameters and HLA-F expression was analyzed. Cancerous HLA-F and stromal HLA-F-positive infiltrating cells were detected in 91 (40.0%) and 186 (81.6%) cases, respectively. HLA-F positivity in cancer cells was significantly associated with tumor size (P < 0.05). There was a weak correlation between HLA-F positivity of cancer cells and HLA-F positive infiltrative cells (P < 0.01, r = 0.11). HLA-F positivity did not affect patients' survival in 209 breast cancer. However, confined to stage II breast cancer, the HLA-F positive group showed significantly poorer outcomes than the HLA-F negative group (P < 0.05). The present study provides the first evidence that HLA-F positivity in breast carcinoma affects clinicopathological factors and could be selected as a prognostic marker for limited clinical stage.

  15. Monoclonal antibodies to human growth hormone induce an allosteric conformational change in the antigen.

    PubMed Central

    Mazza, M M; Retegui, L A

    1989-01-01

    We re-investigated the properties of a monoclonal antibody (mAb), 4D11, to human growth hormone (hGH) that showed a very weak affinity, recognizing hGH only when the hormone was solubilized on a solid surface. MAb4D11 did not significantly bind 125I-hGH. It was found that three mAb directed to different hGH epitopes (mAb 3C11, 10C1 and NA71) were able to induce the binding of the soluble antigen to mAb 4D11. The co-operative effect could be demonstrated by the formation of binary complexes (Ag:Ab, 1:2) detected by high-performance liquid chromatography (HPLC) and by the increase of radioactivity found when the synergistic mAb were added to 125I-hGH incubated with mAb 4D11 immobilized on polyvinyl microplates. Other possible explanations, such as the formation of cyclic complexes or the generation of a new epitope in the Fc fragment of the first antibody (Ab), were dismissed because the Fab fragment of one of the enhancing mAb (3C11) gave the same effect as the intact Ab. The data suggest that the hGH molecule undergoes a localized conformational change after binding to mAb 3C11, NA71 or 10C1 and that mAb 4D11 binds with high affinity to the modified region of the hormone. The formation or not of ternary complexes (Ag:Ab, 1:3) was used to localize the 4D11 epitope on the surface of the Ag. It is suggested that mAb 4D11 recognizes a conformational change produced in the region defined by the AE5/AC8 epitopes, which is close to the hGH antigenic domain only expressed when the protein is immobilized on plastic surfaces. PMID:2473953

  16. Epigenetic Mechanisms Regulate MHC and Antigen Processing Molecules in Human Embryonic and Induced Pluripotent Stem Cells

    PubMed Central

    Suárez-Álvarez, Beatriz; Rodriguez, Ramón M.; Calvanese, Vincenzo; Blanco-Gelaz, Miguel A.; Suhr, Steve T.; Ortega, Francisco; Otero, Jesus; Cibelli, Jose B.; Moore, Harry; Fraga, Mario F.; López-Larrea, Carlos

    2010-01-01

    Background Human embryonic stem cells (hESCs) are an attractive resource for new therapeutic approaches that involve tissue regeneration. hESCs have exhibited low immunogenicity due to low levels of Mayor Histocompatibility Complex (MHC) class-I and absence of MHC class-II expression. Nevertheless, the mechanisms regulating MHC expression in hESCs had not been explored. Methodology/Principal Findings We analyzed the expression levels of classical and non-classical MHC class-I, MHC class-II molecules, antigen-processing machinery (APM) components and NKG2D ligands (NKG2D-L) in hESCs, induced pluripotent stem cells (iPSCs) and NTera2 (NT2) teratocarcinoma cell line. Epigenetic mechanisms involved in the regulation of these genes were investigated by bisulfite sequencing and chromatin immunoprecipitation (ChIP) assays. We showed that low levels of MHC class-I molecules were associated with absent or reduced expression of the transporter associated with antigen processing 1 (TAP-1) and tapasin (TPN) components in hESCs and iPSCs, which are involved in the transport and load of peptides. Furthermore, lack of β2-microglobulin (β2m) light chain in these cells limited the expression of MHC class I trimeric molecule on the cell surface. NKG2D ligands (MICA, MICB) were observed in all pluripotent stem cells lines. Epigenetic analysis showed that H3K9me3 repressed the TPN gene in undifferentiated cells whilst HLA-B and β2m acquired the H3K4me3 modification during the differentiation to embryoid bodies (EBs). Absence of HLA-DR and HLA-G expression was regulated by DNA methylation. Conclusions/Significance Our data provide fundamental evidence for the epigenetic control of MHC in hESCs and iPSCs. Reduced MHC class I and class II expression in hESCs and iPSCs can limit their recognition by the immune response against these cells. The knowledge of these mechanisms will further allow the development of strategies to induce tolerance and improve stem cell allograft acceptance

  17. Variable domain-linked oligosaccharides of a human monoclonal IgG: structure and influence on antigen binding.

    PubMed Central

    Leibiger, H; Wüstner, D; Stigler, R D; Marx, U

    1999-01-01

    The variable-domain-attached oligosaccharide side chains of a human IgG produced by a human-human-mouse heterohybridoma were analysed. In addition to the conserved N-glycosylation site at Asn-297, an N-glycosylation consensus sequence (Asn-Asn-Ser) is located at position 75 in the variable region of its heavy chain. The antibody was cleaved into its antigen-binding (Fab) and crystallizing fragments. The oligosaccharides of the Fab fragment were released by digestion with various endo- and exoglycosidases and analysed by anion-exchange chromatography and fluorophore-assisted carbohydrate electrophoresis. The predominant components were disialyl- bi-antennary and tetra-sialyl tetra-antennary complex carbohydrates. Of note is the presence in this human IgG of oligosaccharides containing N-glycolylneuraminic acid and N-acetylneuraminic acid in the ratio of 94:6. Furthermore, we determined N-acetylgalactosamine in the Fab fragment of this antibody, suggesting the presence of O-linked carbohydrates. A three-dimensional structure of the glycosylated variable (Fv) fragment was suggested using computer-assisted modelling. In addition, the influence of the Fv-associated oligosaccharides of the CBGA1 antibody on antigen binding was tested in several ELISA systems. Deglycosylation resulted in a decreased antigen-binding activity. PMID:10024532

  18. Sensitive immunoradiometric assay for the detection of Paracoccidioides brasiliensis antigens in human sera.

    PubMed Central

    Ferreira-da-Cruz, M F; Galvão-Castro, B; Daniel-Ribeiro, C T

    1991-01-01

    In the present study we report the standardization of an immunoradiometric assay (IRMA) for detection of Paracoccidioides brasiliensis circulating antigens that could be useful in the diagnosis and prognosis of paracoccidioidomycosis. For this purpose we studied the reactivities of P. brasiliensis and other mycotic antigens with rabbit polyclonal anti-P. brasiliensis antibodies (immunoglobulin G) in order to evaluate the sensitivity and specificity of an IRMA for detecting P. brasiliensis antigens. The results were compared with those obtained by the double immunodiffusion test, the standard technique for the serodiagnosis of paracoccidioidomycosis. By using the immunoglobulin G fraction of rabbit antisera (900 ng per well), it was possible to detect up to 3.6 ng (0.12 micrograms/ml) of cellular antigen and 360 ng (12 micrograms/ml) of metabolic antigen in contrast to the double immunodiffusion test that could detect only 12 micrograms (1.2 mg/ml) of both antigens. IRMA was shown to be feasible and very sensitive and may therefore help, together with clinical data, in establishing early diagnosis and assessing disease activity. It could also allow the study of relationships between P. brasiliensis circulating antigens and host defense mechanisms during the disease. PMID:1907608

  19. The challenge of producing skin test antigens with minimal resources suitable for human application against a neglected tropical disease; leprosy.

    PubMed

    Rivoire, Becky L; TerLouw, Stephen; Groathouse, Nathan A; Brennan, Patrick J

    2014-01-01

    True incidence of leprosy and its impact on transmission will not be understood until a tool is available to measure pre-symptomatic infection. Diagnosis of leprosy disease is currently based on clinical symptoms, which on average take 3-10 years to manifest. The fact that incidence, as defined by new case detection, equates with prevalence, i.e., registered cases, suggests that the cycle of transmission has not been fully intercepted by implementation of multiple drug therapy. This is supported by a high incidence of childhood leprosy. Epidemiological screening for pre-symptomatic leprosy in large endemic populations is required to facilitate targeted chemoprophylactic interventions. Such a test must be sensitive, specific, simple to administer, cost-effective, and easy to interpret. The intradermal skin test method that measures cell-mediated immunity was explored as the best option. Prior knowledge on skin testing of healthy subjects and leprosy patients with whole or partially fractionated Mycobacterium leprae bacilli, such as Lepromin or the Rees' or Convit' antigens, has established an acceptable safety and potency profile of these antigens. These data, along with immunoreactivity data, laid the foundation for two new leprosy skin test antigens, MLSA-LAM (M. leprae soluble antigen devoid of mycobacterial lipoglycans, primarily lipoarabinomannan) and MLCwA (M. leprae cell wall antigens). In the absence of commercial interest, the challenge was to develop these antigens under current good manufacturing practices in an acceptable local pilot facility and submit an Investigational New Drug to the Food and Drug Administration to allow a first-in-human phase I clinical trial. PMID:24874086

  20. Antigen capture and major histocompatibility class II compartments of freshly isolated and cultured human blood dendritic cells

    PubMed Central

    1995-01-01

    Dendritic cells (DC) represent potent antigen-presenting cells for the induction of T cell-dependent immune responses. Previous work on antigen uptake and presentation by human DC is based largely on studies of blood DC that have been cultured for various periods of time before analysis. These cultured cells may therefore have undergone a maturation process from precursors that have different capacities for antigen capture and presentation. We have now used immunoelectron microscopy and antigen presentation assays to compare freshly isolated DC (f-DC) and cultured DC (c-DC). f-DC display a round appearance, whereas c-DC display characteristic long processes. c-DC express much more cell surface major histocompatibility complex (MHC) class II than f-DC. The uptake of colloidal gold-labeled bovine serum albumin (BSA), however, is greater in f-DC, as is the presentation of 65-kD heat shock protein to T cell clones. The most striking discovery is that the majority of MHC class II molecules in both f-DC and c-DC occur in intracellular vacuoles with a complex shape (multivesicular and multilaminar). These MHC class II enriched compartments (MIIC) represent the site to which BSA is transported within 30 min. Although MIIC appear as more dense structures with less MHC class II molecules in f-DC than c-DC, the marker characteristics are very similar. The MIIC in both types of DC are acidic, contain invariant chain, and express the recently described HLA-DM molecule that can contribute to antigen presentation. CD19+ peripheral blood B cells have fewer MIIC and surface MHC class II expression than DCs, while monocytes had low levels of MIIC and surface MHC class II. These results demonstrate in dendritic cells the elaborate development of MIIC expressing several of the components that are required for efficient antigen presentation. PMID:7790816

  1. The Challenge of Producing Skin Test Antigens with Minimal Resources Suitable for Human Application against a Neglected Tropical Disease; Leprosy

    PubMed Central

    Rivoire, Becky L.; TerLouw, Stephen; Groathouse, Nathan A.; Brennan, Patrick J.

    2014-01-01

    True incidence of leprosy and its impact on transmission will not be understood until a tool is available to measure pre-symptomatic infection. Diagnosis of leprosy disease is currently based on clinical symptoms, which on average take 3–10 years to manifest. The fact that incidence, as defined by new case detection, equates with prevalence, i.e., registered cases, suggests that the cycle of transmission has not been fully intercepted by implementation of multiple drug therapy. This is supported by a high incidence of childhood leprosy. Epidemiological screening for pre-symptomatic leprosy in large endemic populations is required to facilitate targeted chemoprophylactic interventions. Such a test must be sensitive, specific, simple to administer, cost-effective, and easy to interpret. The intradermal skin test method that measures cell-mediated immunity was explored as the best option. Prior knowledge on skin testing of healthy subjects and leprosy patients with whole or partially fractionated Mycobacterium leprae bacilli, such as Lepromin or the Rees' or Convit' antigens, has established an acceptable safety and potency profile of these antigens. These data, along with immunoreactivity data, laid the foundation for two new leprosy skin test antigens, MLSA-LAM (M. leprae soluble antigen devoid of mycobacterial lipoglycans, primarily lipoarabinomannan) and MLCwA (M. leprae cell wall antigens). In the absence of commercial interest, the challenge was to develop these antigens under current good manufacturing practices in an acceptable local pilot facility and submit an Investigational New Drug to the Food and Drug Administration to allow a first-in-human phase I clinical trial. PMID:24874086

  2. Identification of a Novel 74-Kilodalton Immunodominant Antigen of Pythium insidiosum Recognized by Sera from Human Patients with Pythiosis

    PubMed Central

    Krajaejun, Theerapong; Kunakorn, Mongkol; Pracharktam, Rungnapa; Chongtrakool, Piriyaporn; Sathapatayavongs, Boonmee; Chaiprasert, Angkana; Vanittanakom, Nongnuch; Chindamporn, Ariya; Mootsikapun, Piroon

    2006-01-01

    The oomycetous, fungus-like, aquatic organism Pythium insidiosum is the etiologic agent of pythiosis, a life-threatening infectious disease of humans and animals that has been increasingly reported from tropical, subtropical, and temperate countries. Human pythiosis is endemic in Thailand, and most patients present with arteritis, leading to limb amputation and/or death, or cornea ulcer, leading to enucleation. Diagnosis of pythiosis is time-consuming and difficult. Radical surgery is the main treatment for pythiosis because conventional antifungal drugs are ineffective. The aims of this study were to evaluate the use of Western blotting for diagnosis of human pythiosis, to identify specific immunodominant antigens of P. insidiosum, and to increase understanding of humoral immune responses against the pathogen. We performed Western blot analysis on 16 P. insidiosum isolates using 12 pythiosis serum samples. These specimens were derived from human patients with pythiosis who had different forms of infection and lived in different geographic areas throughout Thailand. We have identified a 74-kDa immunodominant antigen in all P. insidiosum isolates tested. The 74-kDa antigen was also recognized by sera from all patients with pythiosis but not by control sera from healthy individuals, patients with thalassemia, and patients with various infectious diseases, indicating that Western blot analysis could facilitate diagnosis of pythiosis. Therefore, the 74-kDa antigen is a potential target for developing rapid serodiagnostic tests as well as a therapeutic vaccine for pythiosis. These advances could lead to early diagnosis and effective treatment, crucial factors for better prognosis for patients with pythiosis. PMID:16672392

  3. Transcription analysis of class II human leukocyte antigen genes from normal and immunodeficient B lymphocytes, using polymerase chain reaction.

    PubMed Central

    Bull, M; van Hoef, A; Gorski, J

    1990-01-01

    The RNA transcript levels of all human leukocyte antigen class II loci were determined from class II congenital immunodeficient B cells by polymerase chain reaction amplification of cDNA. No mRNA was observed under conditions in which 0.01% normal levels could be visualized. Pre-mRNA could be amplified from normal B cells but not from immunodeficient B cells, indicating a transcription defect. Images PMID:2113177

  4. [Cancer Immunotherapy Using Human Induced Pluripotent Stem Cell-Derived Dendritic Cells(iPSDCs)Expressing Carcinoembryonic Antigen].

    PubMed

    Kitadani, Junya; Ojima, Toshiyasu; Iwamoto, Hiromitsu; Tabata, Hiroaki; Nakamori, Mikihito; Nakamura, Masaki; Katsuda, Masahiro; Miyazawa, Motoki; Hayata, Keiji; Yamaue, Hiroki

    2016-09-01

    The difficulty in obtaining a sufficient number of functional dendritic cells(DCs)is a well-known serious problem in DCbased immunotherapy. Therefore, we used induced pluripotent stem cell-derived DCs(iPSDCs). We have reported that mouse iPSDCs are equivalent to BMDCs, in terms of maturation and antigen presentation. In this study, the antitumor immune response of human iPSDCs expressing the carcinoembryonic antigen was examined, to determine its clinical application in gastrointestinal cancer. Human iPS cells were established from healthy human fibroblasts using a Sendai virus vector, and human iPSDCs were differentiated under a feeder-free culture. Additionally, the surface marker expression, cytokine production, and migratory capacity of human iPSDCs were equivalent to those of monocyte-derived DCs(MoDCs). After 3 cycles of stimulation of autologous PBMCs by genetically modified DCs, the 51Cr-release assay was performed. The lymphocytes stimulated by iPSDCs-CEA showed cytotoxic activity against LCL-CEA and CEA652-pulsed LCL, but showed no cytotoxicity against LCL-LacZ. In addition, they showed cytotoxic activity against CEA-positive human cancer cell lines, MKN45 and HT29, but showed no cytotoxicity against CEA-negative human cancer cell line MKN1. In conclusion, CEA-specific CTLs responses could be induced by iPSDCs-CEA. This vaccination strategy may be useful in future clinical applications of cancer vaccines. PMID:27628546

  5. Allometric Scaling of Therapeutic Monoclonal Antibodies Using Antigen Concentration as a Correction Factor: Application to the Human Clearance Prediction.

    PubMed

    Wang, Lei; Qiang, Wei; Cheng, Zeneng

    2016-03-01

    Allometric scaling has been widely used for predictions of human pharmacokinetic (PK) parameters in the development of monoclonal antibody (mAb) drugs, and some correction factors have been proposed to improve the estimations. However, classic correction factors fail to offer a complete explanation of the additional differences among species besides the body weight and, thus, lack enough power to further improve the predictions. In this study, the antigen concentration was initially set as a new correction factor to predict the human clearance (CL) of mAbs. Bevacizumab was intravenously injected into 2 animal species and humans to obtain PK data to predict human CL from the animal data. Additionally, a new approach was also validated with data from 3 other mAbs which were collected through a literature review of published work. Accordingly, allometric scaling with a correction factor of the antigen concentration generated accurate estimations of the human CL of 4 mAbs, which were superior to the results obtained by other classic scaling methods. More importantly, the proposed method also achieved good predictions of individual human CL of bevacizumab. In conclusion, the potential of this method as a powerful tool for human PK estimation of mAbs in species translation has been demonstrated. PMID:26886347

  6. Human CD8+ herpes simplex virus-specific cytotoxic T-lymphocyte clones recognize diverse virion protein antigens.

    PubMed Central

    Tigges, M A; Koelle, D; Hartog, K; Sekulovich, R E; Corey, L; Burke, R L

    1992-01-01

    The role of the HLA class I-restricted, CD8+, herpes simplex virus (HSV)-specific cytotoxic T lymphocytes (CTL) in the control of human HSV infections is controversial because previous reports suggest that a substantial portion of the antigen-specific lytic response is mediated by CD4+ cells. To address this question directly, we isolated HSV-specific CD8+ CTL clones from a patient with recurrent genital herpes. These CTL were cloned by coculturing responder peripheral blood mononuclear cells (PBMC) with phytohemagglutinin-stimulated PBMC that had been infected with live HSV-2 and then irradiated prior to the addition of responder cells. After 1 week, CTL were cloned by limiting dilution using phytohemagglutinin stimulation and allogeneic feeder PBMC. Seven clones were isolated; all seven clones were CD8+ CD4- CD3+ DRbright, six lysed only HSV-2-infected targets, and one lysed both HSV-1- and HSV-2-infected targets. Antigen presentation was restricted by two to three different HLA class I loci. To determine the antigens recognized by these HSV-specific CTL, target cells were infected with HSV in the presence of acyclovir, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, or cycloheximide in a series of drug block/release protocols to limit the repertoire of viral gene expression to select transcriptional classes. Five of the clones exhibited a different pattern of cytotoxicity, suggesting that each recognized a distinct HSV antigen. One of the clones appears to be directed against an immediate-early antigen; six of the clones recognize virion proteins. Five of these clones recognized internal virion proteins that could be introduced into target cells by HSV infection in the absence of virus gene expression. Antigen specificity was further tested by using vaccinia virus vectors that express glycoproteins gD2 and gB2 or the tegument protein VP16. One clone lysed vaccinia virus/gD2-infected target cells; the remaining clones did not recognize any of these gene

  7. Immunodiagnosis of human cysticercosis (Taenia solium) with antigens purified by monoclonal antibodies.

    PubMed Central

    Nascimento, E; Tavares, C A; Lopes, J D

    1987-01-01

    Monoclonal antibodies were generated from mice immunized with scolex protein antigen of Cysticercus cellulosae. Three monoclonal antibodies specific for cysticercal antigens, which did not show any cross-reactivity with Taenia solium or Taenia saginata antigens, were selected. Each monoclonal antibody coupled to Sepharose could purify one antigen, which appeared as a single band on polyacrylamide gel electrophoresis. When antigens purified by monoclonal antibodies were used to detect antibody in serum samples taken from patients with cysticercosis, taeniasis, and other parasitic infections in an enzyme-linked immunosorbent assay, cross-reactivity was observed until a serum dilution of 1:128 was reached. Since serum samples from unexposed subjects showed positive reactions until a dilution of 1:64 was reached, we chose a discriminative dilution (1:128) above which no cross-reaction was observed. The percent positive serum samples from cysticercosis patients was 100% by the enzyme-linked immunosorbent assay with any of the antigens purified by monoclonal antibodies. Images PMID:3611310

  8. Antigen-specific expansion of human regulatory T cells as a major tolerance mechanism against mucosal fungi.

    PubMed

    Bacher, P; Kniemeyer, O; Schönbrunn, A; Sawitzki, B; Assenmacher, M; Rietschel, E; Steinbach, A; Cornely, O A; Brakhage, A A; Thiel, A; Scheffold, A

    2014-07-01

    Foxp3(+) regulatory T cells (Treg) have a central role for keeping the balance between pro- and anti-inflammatory immune responses against chronically encountered antigens at mucosal sites. However, their antigen specificity especially in humans is largely unknown. Here we used a sensitive enrichment technology for antigen-reactive T cells to directly compare the conventional vs. regulatory CD4(+) T-cell response directed against two ubiquitous mucosal fungi, Aspergillus fumigatus and Candida albicans. In healthy humans, fungus-specific CD4(+)CD25(+)CD127(-)Foxp3(+) Treg are strongly expanded in peripheral blood and possess phenotypic, epigenetic and functional features of thymus-derived Treg. Intriguingly, for A. fumigatus, the strong Treg response contrasts with minimal conventional T-cell memory, indicating selective Treg expansion as an effective mechanism to prevent inappropriate immune activation in healthy individuals. By contrast, in subjects with A. fumigatus allergies, specific Th2 cells were strongly expanded despite the presence of specific Treg. Taken together, we demonstrate a largely expanded Treg population specific for mucosal fungi as part of the physiological human T-cell repertoire and identify a unique capacity of A. fumigatus to selectively generate Treg responses as a potentially important mechanism for the prevention of allergic reactions.

  9. Characterization of a specific anti-human urokinase antibody: Development of a sensitive competition radioimmunoassay for urokinase antigen

    SciTech Connect

    Huber, K.; Kirchheimer, J.; Binder, B.R.

    1984-05-01

    Specific inhibiting IgG antibodies were raised in a rabbit using purified human high molecular weight urokinase as antigen. The antibodies reacted with both high molecular weight and low molecular weight human urokinase using an Ouchterlony double-immunodiffusion technique in such a way that one line of complete identity was obtained. Neither precipitation nor functional inhibition was observed for the tissue-type plasminogen activator. Kinetic studies with plasminogen as a natural high molecular weight substrate or the synthetic low molecular weight p-nitroanilide substrate pyro-Glu-Gly-Arg-pNA revealed, for both substrates, mainly a competitive type of inhibition for the Fab fragments of the specific anti-urokinase antibodies. This characterized anti-urokinase IgG was employed to develop a competitive radioimmunoassay, for human urokinase with /sup 125/I-labeled urokinase as tracer. In this radioimmunoassay, the lower detection limit for urokinase antigen was 10 pg/ml sample; the intrassay variation was 2.8%, and the interassay variation was 5.3%. Applying this radioimmunoassay to plasma samples obtained from healthy young volunteers, urokinase antigen could be measured in a concentration of 7.82 +/- 3.97 ng/ml for mean and 6.66 +/- 2.39 ng/ml for women (mean +/- SD).

  10. Defining antigen-specific plasmablast and memory B cell subsets in human blood after viral infection or vaccination.

    PubMed

    Ellebedy, Ali H; Jackson, Katherine J L; Kissick, Haydn T; Nakaya, Helder I; Davis, Carl W; Roskin, Krishna M; McElroy, Anita K; Oshansky, Christine M; Elbein, Rivka; Thomas, Shine; Lyon, George M; Spiropoulou, Christina F; Mehta, Aneesh K; Thomas, Paul G; Boyd, Scott D; Ahmed, Rafi

    2016-10-01

    Antigen-specific B cells bifurcate into antibody-secreting cells (ASCs) and memory B cells (MBCs) after infection or vaccination. ASCs (plasmablasts) have been extensively studied in humans, but less is known about B cells that become activated but do not differentiate into plasmablasts. Here we have defined the phenotype and transcriptional program of a subset of antigen-specific B cells, which we have called 'activated B cells' (ABCs), that were distinct from ASCs and were committed to the MBC lineage. We detected ABCs in humans after infection with Ebola virus or influenza virus and also after vaccination. By simultaneously analyzing antigen-specific ASCs and ABCs in human blood after vaccination against influenza virus, we investigated the clonal overlap and extent of somatic hypermutation (SHM) in the ASC (effector) and ABC (memory) lineages. Longitudinal tracking of vaccination-induced hemagglutinin (HA)-specific clones revealed no overall increase in SHM over time, which suggested that repeated annual immunization might have limitations in enhancing the quality of influenza-virus-specific antibody.

  11. Defining antigen-specific plasmablast and memory B cell subsets in human blood after viral infection or vaccination.

    PubMed

    Ellebedy, Ali H; Jackson, Katherine J L; Kissick, Haydn T; Nakaya, Helder I; Davis, Carl W; Roskin, Krishna M; McElroy, Anita K; Oshansky, Christine M; Elbein, Rivka; Thomas, Shine; Lyon, George M; Spiropoulou, Christina F; Mehta, Aneesh K; Thomas, Paul G; Boyd, Scott D; Ahmed, Rafi

    2016-10-01

    Antigen-specific B cells bifurcate into antibody-secreting cells (ASCs) and memory B cells (MBCs) after infection or vaccination. ASCs (plasmablasts) have been extensively studied in humans, but less is known about B cells that become activated but do not differentiate into plasmablasts. Here we have defined the phenotype and transcriptional program of a subset of antigen-specific B cells, which we have called 'activated B cells' (ABCs), that were distinct from ASCs and were committed to the MBC lineage. We detected ABCs in humans after infection with Ebola virus or influenza virus and also after vaccination. By simultaneously analyzing antigen-specific ASCs and ABCs in human blood after vaccination against influenza virus, we investigated the clonal overlap and extent of somatic hypermutation (SHM) in the ASC (effector) and ABC (memory) lineages. Longitudinal tracking of vaccination-induced hemagglutinin (HA)-specific clones revealed no overall increase in SHM over time, which suggested that repeated annual immunization might have limitations in enhancing the quality of influenza-virus-specific antibody. PMID:27525369

  12. Antigenic Protein In Microgravity-Grown Human Mixed Mullerian Tumor (LN1) Cells Preserved In RNA Stabilizing Agent

    NASA Technical Reports Server (NTRS)

    Hammond, Dianne K.; Becker, Jeanne; Holubec, K.; Baker, T. L.; Love, J. E.

    2004-01-01

    Cells treated with RNAlater(TradeMark) have previously been shown to contain antigenic proteins that can be visualized using Western blot analysis. These proteins seem to be stable for several months when stored in RNA stabilizer at 4 C. Antigenic protein can be recovered from cells that have been processed using an Ambion RNAqueous(Registered TradeMark) kit to remove RNA. In this set of experiments, human mixed Mullerian tumor (LN1) cells grown on the International Space Station during Expedition 3 were examined for antigenic stability after removal of RNA. The cells were stored for three months in RNAlater(TradeMark) and RNA was extracted. The RNA filtrate Containing the protein was precipitated, washed, and suspended in buffer containing sodium dodecyl sulfate (SDS). Samples containing equal concentrations of protein were loaded onto SDS-polyacrylamide gels. Proteins were separated by electrophoresis and transferred by Western blot to polyvinylidene fluoride (PVDF) membrane. The Western blots were stained with an enhanced chemiluminescent ECL(Registered TradeMark)Plus detection kit (Amersham) and scanned using a Storm 840 gel image analyzer (Amersham, Molecular Dynamics). ImageQuant(Registered TradeMark)a software was used to quantify the densities of the protein bands. The ground control and flight LN1 cell samples showed a similar staining pattern over time with antibodies to vimentin, glyceraldehyde-3-phosphate dehydrogenase, and epithelial membrane antigens.

  13. Antigenic Protein In Microgravity-Grown Human Mixed Mullerian Tumor (LN1) Cells Preserved In RNA Stabilizing Agent

    NASA Technical Reports Server (NTRS)

    Hammond, Dianne K.; Becker, Jeanne; Elliott, T. F.; Holubec, K.; Baker, T. L.; Love, J. E.

    2004-01-01

    Cells treated with RNAlater(TradeMark) have previously been shown to contain antigenic proteins that can be visualized using Western blot analysis. These proteins seem to be stable for several months when stored in RNA stabilizer at 4 C. Antigenic protein can be recovered from cells that have been processed using an Ambion RNAqueous(Registered TradeMark) kit to remove RNA. In this set of experiments, human mixed Mullerian tumor (LNI) cells grown on the International Space Station during Expedition 3 were examined for antigenic stability after removal of RNA. The cells were stored for three months in RNAlater(TradeMark) and RNA was extracted. The RNA filtrate containing the protein was precipitated, washed, and suspended in buffer containing sodium dodecyl sulfate (SDS). Samples containing equal concentrations of protein were loaded onto SDS-polyacrylamide gels. Proteins were separated by electrophoresis and transferred by Western blot to polyvinylidene fluoride (PVDF) membrane. The Western blots were stained with an enhanced chemiluminescent ECL(Registered Trademark) Plus detection kit (Amersham) and scanned using a Storm 840 gel image analyzer (Amersham, Molecular Dynamics). ImageQuant(Registered TradeMark) software was used to quantify the densities of the protein bands. The ground control and flight LN1 cell samples showed a similar staining pattern over time with antibodies to vimentin, glyceraldehyde-3-phosphate dehydrogenase, and epithelial membrane antigens.

  14. Recognition of Human Erythrocyte Receptors by the Tryptophan-Rich Antigens of Monkey Malaria Parasite Plasmodium knowlesi

    PubMed Central

    Tyagi, Kriti; Gupta, Deepali; Saini, Ekta; Choudhary, Shilpa; Jamwal, Abhishek; Alam, Mohd. Shoeb; Zeeshan, Mohammad; Tyagi, Rupesh K.; Sharma, Yagya D.

    2015-01-01

    Background The monkey malaria parasite Plasmodium knowlesi also infect humans. There is a lack of information on the molecular mechanisms that take place between this simian parasite and its heterologous human host erythrocytes leading to this zoonotic disease. Therefore, we investigated here the binding ability of P. knowlesi tryptophan-rich antigens (PkTRAgs) to the human erythrocytes and sharing of the erythrocyte receptors between them as well as with other commonly occurring human malaria parasites. Methods Six PkTRAgs were cloned and expressed in E.coli as well as in mammalian CHO-K1 cell to determine their human erythrocyte binding activity by cell-ELISA, and in-vitro rosetting assay, respectively. Results Three of six PkTRAgs (PkTRAg38.3, PkTRAg40.1, and PkTRAg67.1) showed binding to human erythrocytes. Two of them (PkTRAg40.1 and PkTRAg38.3) showed cross-competition with each other as well as with the previously described P.vivax tryptophan-rich antigens (PvTRAgs) for human erythrocyte receptors. However, the third protein (PkTRAg67.1) utilized the additional but different human erythrocyte receptor(s) as it did not cross-compete for erythrocyte binding with either of these two PkTRAgs as well as with any of the PvTRAgs. These three PkTRAgs also inhibited the P.falciparum parasite growth in in-vitro culture, further indicating the sharing of human erythrocyte receptors by these parasite species and the biological significance of this receptor-ligand interaction between heterologous host and simian parasite. Conclusions Recognition and sharing of human erythrocyte receptor(s) by PkTRAgs with human parasite ligands could be part of the strategy adopted by the monkey malaria parasite to establish inside the heterologous human host. PMID:26393350

  15. Genetic epistasis between killer immunoglobulin-like receptors and human leukocyte antigens in Kawasaki disease susceptibility.

    PubMed

    Bossi, G; Mannarino, S; Pietrogrande, M C; Salice, P; Dellepiane, R M; Cremaschi, A L; Corana, G; Tozzo, A; Capittini, C; De Silvestri, A; Tinelli, C; Pasi, A; Martinetti, M

    2015-10-01

    Kawasaki disease (KD) is a pediatric acute multisystemic vasculitis complicated by development of coronary artery lesions. The breakthrough theory on KD etiopathogenesis points to pathogens/environmental factors triggered by northeastern wind coming from China. Natural Killer cells and T lymphocytes express the inhibitory/activating Killer Immunoglobulin-like Receptors (KIR) to elicit an immune response against pathogens by binding to human leukocyte antigens (HLA) class I epitopes. We first report on the role of KIR/HLA genetic epistasis in a sample of 100 Italian KD children. We genotyped KIR, HLA-A, HLA-B and HLA-C polymorphisms, and compared KD data with those from 270 Italian healthy donors. The HLA-A*11 ligand for KIR2DS2/2DS4/3DL2 was a KD susceptibility marker by itself (odds ratio (OR)=3.85, confidence interval (CI)=1.55-9.53, P=0.004). Although no epistasis between HLA-A*11 and KIR2DS2/S4 emerged, HLA-A*11 also engages KIR3DL2, a framework gene encoding for a pathogen sensor of CpG-oligodeoxynucleotides (CpG-ODN), and KD blood mononuclear cells are actually prone to pathogen CpG-ODN activation in the acute phase. Moreover, carriers of KIR2DS2/HLA-C1 and KIR2DL2/HLA-C1 were more frequent among KD, in keeping with data demonstrating the involvement of these HLA/KIR couples in autoimmune endothelial damage. The highest KD risk factor was observed among carriers of KIR2DL2 and two or more HLA ligands (OR=10.24, CI=1.87-56.28; P=0.007). PMID:26335810

  16. A novel inhibition ELISA for the detection and monitoring of Penicillium marneffei antigen in human serum.

    PubMed

    Prakit, K; Nosanchuk, J D; Pruksaphon, K; Vanittanakom, N; Youngchim, S

    2016-04-01

    The thermally dimorphic fungus Penicillium marneffei is a causative agent of penicilliosis marneffei, a disease considered to be an acquired immune deficiency syndrome (AIDS)-defining illness in Southeast Asia and southern China. We have developed an inhibition enzyme-linked immunosorbent assay (inh-ELISA) incorporating the yeast phase specific mannoprotein-binding monoclonal antibody 4D1 for the detection of P. marneffei infection. In our sample set, the test detected antigenemia in all 45 (100 %) patients with P. marneffei, with a mean antigen concentration of 4.32 μg/ml. No cross-reactivity in this assay was found using serum from 44 additional patients with other fungal infections, such as Aspergillus fumigatus, Cryptococcus neoformans, and Candida albicans, as well as 44 patients with bacterial infections, such as Mycobacterium tuberculosis and Streptococcus suis. Additionally, no reactivity occurred using serum from 31 human immunodeficiency virus (HIV)-infected patients without a history of fungal infections and 113 healthy controls residing in endemic areas. To investigate the potential of the inh-ELISA for disease monitoring, we followed the reduction in antigenemia in six patients who clinically responded to itraconazole and P. marneffei was no longer isolated from their blood or tissues. In contrast, we correlated increased concentrations of antigenemia in patients with relapsed P. marneffei infection with the progression of their clinical symptoms and the isolation of P. marneffei from their clinical specimens. In summary, the P. marneffei inh-ELISA is a promising new assay for the rapid diagnosis of P. marneffei, as well as a tool for evaluating clinical response and clearance of the fungus during treatment. PMID:26838686

  17. Human leukocyte antigen-B27 alleles in Xinjiang Uygur patients with ankylosing spondylitis.

    PubMed

    Zou, H-Y; Yu, W-Z; Wang, Z; He, J; Jiao, M

    2015-05-25

    We investigated the distribution of human leukocyte antigen (HLA)-B27 subtypes in Uygur ankylosing spondylitis patients in Xinjiang. B27-positive patients with ankylosing spondylitis were subtyped by using polymerase chain reaction-sequence-based typing. The HLA-B27 subtype frequencies of Uygur patients were compared with those in Han patients in Xinjiang and the other areas of China. B*2705 was the predominant subtype in Uygur patients with a frequency of 58.95%, which was much higher than that in Han patients in Xinjiang (31.58%, P < 0.05) and the other areas of China (excluding the Shandong region, which was 63.89%). The frequency of B*2704 (27.37%) in Uygur patients was the lowest and significantly lower than that in Han patients (61.18%, P < 0.05) and in 8 other areas of China. B*2710 has not been previously reported in Uygur ankylosing spondylitis patients; B*2704 was the main (61.18%) subtype in Han patients in Xinjiang, followed by B*2705 (31.58%) and was similar to the characteristics of Han patients in the other areas of China. B*2724 in Han ankylosing spondylitis patients has not been previously reported. Additionally, the B*2702/B*2705 homozygote was identified in Uygur patients. B*2702/B*2704, B*2704/B*2705, and B*2705/B*2705 homozygotes were identified in 3 Han patients. The distribution of HLAB27 subtypes in Uygur ankylosing spondylitis patients in Xinjiang significantly differed from that in Han patients. Understanding the distribution of HLAB27 subtypes in ethnic minority populations of Xinjiang is important for anthropological genetic studies and for analyzing the impact of genetic background on ankylosing spondylitis susceptibility.

  18. Prevalence of human leukocyte antigen (HLA) DRB1 alleles in Kuwaiti children with juvenile rheumatoid arthritis.

    PubMed

    Alsaeid, Khaled; Haider, M Z; Kamal, H; Srivastva, B S; Ayoub, E M

    2002-02-01

    The prevalence of human leukocyte antigen (HLA) DR alleles has been determined in 69 Kuwaiti Arab children with juvenile rheumatoid arthritis (JRA) and compared to that in 212 ethnically matched normal healthy controls using a PCR-sequence specific primers (PCR-SSP) method. A very high incidence of DR3 was detected in JRA patients compared to the controls (P < 0.0001, RR = 2.235). The high incidence of HLA-DR3 in JRA patients was accounted for mainly by an excess of DRB1*0307 (P < 0.05, RR = 3.072) and DRB1*0308 (P < 0.009, RR = 2.663) compared to the controls. Moreover, DR3 was more prevalent when patients with ANA-positive JRA were analysed separately; 73% compared to 58% for the whole JRA patient group. The frequency of DR1 was also higher in the JRA group compared to controls (P = 0.019, RR = 3.585). Although the incidence of some alleles was higher in the control group (DR13 and DR7), none reached a statistically significant level. All the patients with iridocyclitis had either a DR1 or DR3 allele, except for one child. The frequency of DRB1*03 was found to be much higher in the polyarticular subtype of Kuwaiti JRA cases compared to the oligoarticular subgroup and the controls. Also, a non-significant increase in the frequency of the DRB1*04, *11 and *15 alleles was detected in the polyarticular subtype of the Kuwaiti JRA cases compared to the controls.

  19. Associations of human leukocyte antigens with autoimmune diseases: challenges in identifying the mechanism.

    PubMed

    Miyadera, Hiroko; Tokunaga, Katsushi

    2015-11-01

    The mechanism of genetic associations between human leukocyte antigen (HLA) and susceptibility to autoimmune disorders has remained elusive for most of the diseases, including rheumatoid arthritis (RA) and type 1 diabetes (T1D), for which both the genetic associations and pathogenic mechanisms have been extensively analyzed. In this review, we summarize what are currently known about the mechanisms of HLA associations with RA and T1D, and elucidate the potential mechanistic basis of the HLA-autoimmunity associations. In RA, the established association between the shared epitope (SE) and RA risk has been explained, at least in part, by the involvement of SE in the presentation of citrullinated peptides, as confirmed by the structural analysis of DR4-citrullinated peptide complex. Self-peptide(s) that might explain the predispositions of variants at 11β and 13β in DRB1 to RA risk have not currently been identified. Regarding the mechanism of T1D, pancreatic self-peptides that are presented weakly on the susceptible HLA allele products are recognized by self-reactive T cells. Other studies have revealed that DQ proteins encoded by the T1D susceptible DQ haplotypes are intrinsically unstable. These findings indicate that the T1D susceptible DQ haplotypes might confer risk for T1D by facilitating the formation of unstable HLA-self-peptide complex. The studies of RA and T1D reveal the two distinct mechanistic basis that might operate in the HLA-autoimmunity associations. Combination of these mechanisms, together with other functional variations among the DR and DQ alleles, may generate the complex patterns of DR-DQ haplotype associations with autoimmunity. PMID:26290149

  20. Transcription analysis, physical mapping, and molecular characterization of a nonclassical human leukocyte antigen class I gene.

    PubMed Central

    Chorney, M J; Sawada, I; Gillespie, G A; Srivastava, R; Pan, J; Weissman, S M

    1990-01-01

    The human major histocompatibility complex contains approximately 20 class I genes, pseudogenes, and gene fragments. These include the genes for the three major transplantation antigens, HLA-A, HLA-B, and HLA-C, as well as a number of other genes or pseudogenes of unknown biological significance. Most of the latter have C + G-rich sequences in their 5' ends that are unmethylated in the B-lymphoblastoid cell line 3.1.0. We investigated one of these genes, HLA-H, in more detail. The gene is, overall, strongly homologous in sequence to HLA-A but differs in several potentially significant ways, including changes in conserved promoter sequences, a single-base deletion producing a translation termination codon in exon 4, and a region of sequence divergence downstream of the transcribed portion of the gene. Nevertheless, mouse L cells transfected with the gene accumulated small amounts of apparently full-length polyadenylated RNA. A portion of this RNA begins at the transcription site predicted by analogy to certain class I cDNA clones, while another portion appears to begin shortly upstream. L cells transfected with a hybrid gene containing the first three exons of HLA-H and the last five exons of HLA-B27 accumulated full-length HLA transcripts at the same level as cells transfected with an HLA-B27 gene; both levels are at least 15- to 20-fold higher than that directed by HLA-H alone. In addition, we isolated a cDNA clone for HLA-H that contains a portion of intron 3 attached to a normally spliced sequence comprising exons 4 through 8. These results suggest that low levels of translatable mRNA for the truncated class I heavy chain encoded by HLA-H are produced under physiologic circumstances and that sequences 3' of intron 3 decrease the levels of stable transcripts. Images PMID:2294403

  1. The Human Minor Histocompatibility Antigen1 Is a RhoGAP

    PubMed Central

    de Kreuk, Bart-Jan; Schaefer, Antje; Anthony, Eloise C.; Tol, Simon; Fernandez-Borja, Mar; Geerts, Dirk; Pool, Jos; Hambach, Lothar; Goulmy, Els; Hordijk, Peter L.

    2013-01-01

    The human minor Histocompatibility Antigen HMHA-1 is a major target of immune responses after allogeneic stem cell transplantation applied for the treatment of leukemia and solid tumors. The restriction of its expression to hematopoietic cells and many solid tumors raised questions regarding its cellular functions. Sequence analysis of the HMHA-1 encoding HMHA1 protein revealed the presence of a possible C-terminal RhoGTPase Activating Protein (GAP) domain and an N-terminal BAR domain. Rho-family GTPases, including Rac1, Cdc42, and RhoA are key regulators of the actin cytoskeleton and control cell spreading and migration. RhoGTPase activity is under tight control as aberrant signaling can lead to pathology, including inflammation and cancer. Whereas Guanine nucleotide Exchange Factors (GEFs) mediate the exchange of GDP for GTP resulting in RhoGTPase activation, GAPs catalyze the low intrinsic GTPase activity of active RhoGTPases, resulting in inactivation. Here we identify the HMHA1 protein as a novel RhoGAP. We show that HMHA1 constructs, lacking the N-terminal region, negatively regulate the actin cytoskeleton as well as cell spreading. Furthermore, we show that HMHA1 regulates RhoGTPase activity in vitro and in vivo. Finally, we demonstrate that the HMHA1 N-terminal BAR domain is auto-inhibitory as HMHA1 mutants lacking this region, but not full-length HMHA1, showed GAP activity towards RhoGTPases. In conclusion, this study shows that HMHA1 acts as a RhoGAP to regulate GTPase activity, cytoskeletal remodeling and cell spreading, which are crucial functions in normal hematopoietic and cancer cells. PMID:24086303

  2. Human neutrophil antigen profiles in Banjar, Bugis, Champa, Jawa and Kelantan Malays in Peninsular Malaysia

    PubMed Central

    Manaf, Siti M.; NurWaliyuddin, Hanis Z.A.; Panneerchelvam, Sundararajulu; Zafarina, Zainuddin; Norazmi, Mohd N.; Chambers, Geoffrey K.; Edinur, Hisham A.

    2015-01-01

    Background Human neutrophil antigens (HNA) are polymorphic and immunogenic proteins involved in the pathogenesis of neonatal alloimmune neutropenia, transfusion-related acute lung injury (TRALI) and transfusion-related alloimmune neutropenia. The characterisation of HNA at a population level is important for predicting the risk of alloimmunisation associated with blood transfusion and gestation and for anthropological studies. Materials and methods Blood samples from 192 healthy, unrelated Malays were collected and genotyped using polymerase chain reaction-sequence specific primers (HNA-1, -3, -4) and polymerase chain reaction-restriction fragment length polymorphisms (HNA-5). The group comprised 30 Banjar, 37 Bugis, 51 Champa, 39 Jawa and 35 Kelantan Malays. Results The most common HNA alleles in the Malays studied were HNA-1a (0.641–0.765), -3a (0.676–0.867), -4a (0.943–1.000) and -5a (0.529–0.910). According to principal coordinate plots constructed using HNA allele frequencies, the Malay sub-ethnic groups are closely related and grouped together with other Asian populations. The risks of TRALI or neonatal neutropenia were not increased for subjects with HNA-1, -3 and -4 loci even for donor and recipient or pairs from different Malay sub-ethnic groups. Nonetheless, our estimates showed significantly higher risks of HNA alloimmunisation during pregnancy and transfusion between Malays and other genetically differentiated populations such as Africans and Europeans. Discussion This study reports HNA allele and genotype frequencies for the five Malay sub-ethnic groups living in Peninsular Malaysia for the first time. These Malay sub-ethnic groups show closer genetic relationships with other Asian populations than with Europeans and Africans. The distributions of HNA alleles in other lineages of people living in Malaysia (e.g. Chinese, Indian and Orang Asli) would be an interesting subject for future study. PMID:26057487

  3. Profiling leucocyte subsets in tuberculosis-diabetes co-morbidity.

    PubMed

    Kumar, Nathella Pavan; Moideen, Kadar; Dhakshinraj, Sharmila D; Banurekha, Vaithilingam V; Nair, Dina; Dolla, Chandrakumar; Kumaran, Paul; Babu, Subash

    2015-10-01

    The immune system plays an important role in the pathogenesis of pulmonary tuberculosis-type 2 diabetes mellitus (PTB-DM) co-morbidity. However, the phenotypic profile of leucocyte subsets at homeostasis in individuals with active or latent tuberculosis (LTB) with coincident diabetes is not known. To characterize the influence of diabetes on leucocyte phenotypes in PTB or LTB, we examined the frequency (Fo ) of leucocyte subsets in individuals with TB with (PTB-DM) or without (PTB) diabetes; individuals with latent TB with (LTB-DM) or without (LTB) diabetes and non-TB-infected individuals with (NTB-DM) or without (NTB) diabetes. Coincident DM is characterized by significantly lower Fo of effector memory CD4(+) T cells in LTB individuals. In contrast, DM is characterized by significantly lower Fo of effector memory CD8(+) T cells and significantly higher Fo of central memory CD8(+) T cells in PTB individuals. Coincident DM resulted in significantly higher Fo of classical memory B cells in PTB and significantly higher Fo of activated memory and atypical B cells in LTB individuals. Coincident DM resulted in significantly lower Fo of classical and intermediate monocytes in PTB, LTB and NTB individuals. Finally, DM resulted in significantly lower Fo of myeloid and plasmacytoid dendritic cells in PTB, LTB and NTB individuals. Our data reveal that coincident diabetes alters the cellular subset distribution of T cells, B cells, dendritic cells and monocytes in both individuals with active TB and those with latent TB, thus potentially impacting the pathogenesis of this co-morbid condition.

  4. Profiling leucocyte subsets in tuberculosis–diabetes co-morbidity

    PubMed Central

    Kumar, Nathella Pavan; Moideen, Kadar; Dhakshinraj, Sharmila D; Banurekha, Vaithilingam V; Nair, Dina; Dolla, Chandrakumar; Kumaran, Paul; Babu, Subash

    2015-01-01

    The immune system plays an important role in the pathogenesis of pulmonary tuberculosis–type 2 diabetes mellitus (PTB-DM) co-morbidity. However, the phenotypic profile of leucocyte subsets at homeostasis in individuals with active or latent tuberculosis (LTB) with coincident diabetes is not known. To characterize the influence of diabetes on leucocyte phenotypes in PTB or LTB, we examined the frequency (Fo) of leucocyte subsets in individuals with TB with (PTB-DM) or without (PTB) diabetes; individuals with latent TB with (LTB-DM) or without (LTB) diabetes and non-TB-infected individuals with (NTB-DM) or without (NTB) diabetes. Coincident DM is characterized by significantly lower Fo of effector memory CD4+ T cells in LTB individuals. In contrast, DM is characterized by significantly lower Fo of effector memory CD8+ T cells and significantly higher Fo of central memory CD8+ T cells in PTB individuals. Coincident DM resulted in significantly higher Fo of classical memory B cells in PTB and significantly higher Fo of activated memory and atypical B cells in LTB individuals. Coincident DM resulted in significantly lower Fo of classical and intermediate monocytes in PTB, LTB and NTB individuals. Finally, DM resulted in significantly lower Fo of myeloid and plasmacytoid dendritic cells in PTB, LTB and NTB individuals. Our data reveal that coincident diabetes alters the cellular subset distribution of T cells, B cells, dendritic cells and monocytes in both individuals with active TB and those with latent TB, thus potentially impacting the pathogenesis of this co-morbid condition. PMID:26095067

  5. Defective production of leucocytic endogenous mediator (interleukin 1) by peripheral blood leucocytes of patients with systemic sclerosis, systemic lupus erythematosus, rheumatoid arthritis and mixed connective tissue disease.

    PubMed Central

    Whicher, J T; Gilbert, A M; Westacott, C; Hutton, C; Dieppe, P A

    1986-01-01

    Leucocytic endogenous mediator is one of the activities ascribed to the cytokine or family of cytokines known as interleukin 1. In this study we have examined the ability of circulating blood leucocytes from patients with rheumatic diseases to produce this mediator in vitro. Leucocytic endogenous mediator production was found to be significantly decreased below normal values (mean 107 units, s.e.m. +/- 25) in systemic sclerosis (-6 units +/- 18), systemic lupus erythematosus (-25 units +/- 13), rheumatoid arthritis (-3 units +/- 13) and mixed connective tissue disease (-4 units +/- 40). A control group of ill patients with cancer produced 57 units +/- 8. PMID:3491701

  6. Polymorphonuclear leucocyte motility in men with ankylosing spondylitis.

    PubMed Central

    Pease, C T; Fennell, M; Brewerton, D A

    1989-01-01

    The polymorphonuclear leucocyte (PMN) response to a chemotactic or chemokinetic stimulus is enhanced in men with ankylosing spondylitis (AS). This effect does not parallel the severity of disease activity or the size of the acute phase response, and it is independent of non-steroidal anti-inflammatory drug treatment. Polymorph function is normal in HLA-B27 positive brothers of probands with AS and in other HLA-B27 positive individuals in the absence of disease. Polymorph motility is also normal in patients with psoriasis vulgaris or Crohn's disease, indicating that enhanced PMN motility is not a non-specific consequence of all inflammatory disorders. PMID:2784306

  7. Leucocyte arylsulphatase A activity and subtypes of chronic schizophrenia.

    PubMed

    Heavey, A M; Philpot, M P; Fensom, A H; Jackson, M; Crammer, J L

    1990-07-01

    Previous studies have suggested that arylsulphatase A (ASA - the biochemical marker of metachromatic leucodystrophy) deficiency may be present in a sizeable proportion of patients with chronic psychosis. This study surveyed leucocyte ASA activity in a group of chronic psychotic patients and compared ASA activity in 3 subgroups fulfilling Research Diagnostic Criteria for schizophrenia (undifferentiated), paranoid schizophrenia and schizoaffective psychosis. Three of 45 patients had significantly reduced ASA activity but none had metachromatic leucodystrophy. Although ASA levels did not differ significantly between the groups, schizophrenics without a family history of schizophrenia had significantly lower ASA levels than those with. The implications of these findings are discussed.

  8. Purification of antibodies to O antigen of Salmonella Typhimurium from human serum by affinity chromatography.

    PubMed

    O'Shaughnessy, Colette M; Micoli, Francesca; Gavini, Massimiliano; Goodall, Margaret; Cobbold, Mark; Saul, Allan; Maclennan, Calman A

    2013-01-31

    Nontyphoidal Salmonellae (NTS) are a common cause of bacteraemia in children and HIV-infected adults in Sub-Saharan Africa. We have previously shown that antibodies play a key role in both bactericidal and cellular mechanisms of immunity to NTS, but found that high concentrations of antibody to Salmonella Typhimurium O antigen (OAg) in the serum of some HIV-infected African adults is associated with impaired killing of NTS. To further investigate the function of antibodies to the OAg of NTS, we developed a method to purify these antibodies from human serum by affinity chromatography. Purified Salmonella Typhimurium OAg was activated with adipic acid dihydrazide (ADH) via two different chemistries before linking to N-hydroxysuccinamide-Sepharose resin: one ADH molecule was introduced per OAg chain on its terminal 3-deoxy-D-manno-octulosonic acid sugar (OAg-ADH), or multiple ADH molecules were attached along the OAg chain after oxidation with sodium periodate (OAgoxADH). Both resulting columns worked well when tested with commercial polyclonal anti-O:4,5 antibodies from rabbit serum. Over 90% of the applied antibodies bound to the resin and 89% of these antibodies were then eluted as detected by ELISA. OAg-ADH was preferred as the method for OAg derivatisation as it does not modify the saccharide chain and can be applied to OAg from different bacteria. Both columns were able to bind OAg-specific antibodies in human serum, but antibody recovery was initially low. Different elution buffers were tested and different amounts of OAg-ADH were linked to the resin to improve the yield. Optimal recovery (51%) was obtained by loading 1mg of activated OAg per ml of resin and eluting with 0.1M glycine, 0.1M NaCl pH2.4. The column matrix could be regenerated following elution with no detectable loss in performance for over ten uses. This method offers the potential to purify antibodies to Salmonella OAg from polyclonal serum following vaccination or natural exposure to Salmonella

  9. Association of Human Leukocyte Antigen Alleles and Nevirapine Hypersensitivity in a Malawian HIV-Infected Population

    PubMed Central

    Carr, Daniel F.; Chaponda, Mas; Jorgensen, Andrea L.; Castro, Elena Cornejo; van Oosterhout, Joep J.; Khoo, Saye H.; Lalloo, David G.; Heyderman, Robert S.; Alfirevic, Ana; Pirmohamed, Munir

    2013-01-01

    Background. The nonnucleoside reverse transcriptase inhibitor nevirapine is the cornerstone of treatment for human immunodeficiency virus (HIV) in many sub-Saharan African countries. However, nevirapine is associated with a 6%–10% risk of developing a hypersensitivity reaction, with different phenotypes, including the blistering conditions Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). Our aim was to identify predictive human leukocyte antigen (HLA) markers that are associated with nevirapine hypersensitivity. Methods. We identified 117 HIV-infected Malawian adults with nevirapine hypersensitivity (15 drug-induced liver injury [DILI], 33 SJS/TEN, 20 hypersensitivity syndrome, and 46 nevirapine-induced rash plus 3 with both DILI and SJS phenotype) and 155 age-, sex- and ethnicity-matched nevirapine-exposed controls. HLA typing for 5 loci (A, B, C, DRB1, and DQB1) was undertaken using a sequence-based high-resolution protocol. Logistic regression analysis included CD4+ cell count as a covariate. Results. HLA-C*04:01 was found to markedly increase the risk for SJS (odds ratio [OR] = 17.52; 95% confidence interval, 3.31–92.80) and all hypersensitivity phenotypes (OR = 2.64; 95% CI, 1.13–6.18) when compared to the baseline rare allele group in a binary logistic regression model. The OR for absolute risk of SJS/TEN associated with carriage of HLA-C*04:01 was 5.17 (95% CI, 2.39–11.18). Positive predictive value was 2.6% and negative predictive value was 99.2%. In addition, a number of alleles within the HLA-DQB1 loci protected against nevirapine-induced hypersensitivity phenotypes. Conclusions. Our study has identified HLA-C*04:01 carriage as a risk factor for nevirapine-induced SJS/TEN in a Malawian HIV cohort. Validation of these findings in a larger cohort of patients and mechanistic investigation of the pathogenesis are required. PMID:23362284

  10. Human leukocyte antigen-G overexpression predicts poor clinical outcomes in low-grade gliomas.

    PubMed

    Fan, Xing; Wang, Yinyan; Zhang, Chuanbao; Liu, Xing; Qian, Zenghui; Jiang, Tao

    2016-05-15

    Overexpression of human leukocyte antigen-G (HLA-G), a non-classical major histocompatibility complex class-I molecule associated with immunosuppression, has been reported in various human malignancies. In the present study, we examined the role of HLA-G in gliomas. Clinical characteristics, mRNA expression microarrays and follow-up data pertaining to 293 patients with histologically confirmed gliomas were analyzed. The expression levels of HLA-G were compared between different grades of gliomas and correlated with progression-free survival (PFS) and overall survival (OS) to evaluate its prognostic value. We found that HLA-G was overexpressed in gliomas as compared to that in normal brain tissue samples (-1.288±0.265). The highest expression levels were in glioblastomas (GBMs), anaplastic gliomas (AGs) and low-grade gliomas (LGGs), in that order (0.328±0.778, 0.176±0.881, -0.388±0.686, respectively). Significant inter-group differences were observed between low-grade and high-grade glioma tissues (p<0.001 and p<0.001, t-test, AGs and GBMs, respectively). More astrocytoma patients exhibited increased HLA-G expression as compared to other LGG patients (p=0.004, Chi-square test). Significant differences were observed with respect to PFS and OS (p=0.009 and 0.032, log-rank test, for PFS and OS, respectively) between the high- and low-expression subgroups in patients with LGGs. On Cox regression analysis, overexpression of HLA-G appeared to be an independent predictor of clinical outcomes (p=0.007 and 0.026, for PFS and OS, respectively). Our results suggest that HLA-G expression may serve as a potential biomarker for predicting aggressive tumor grades of gliomas and for histological subtype of LGGs. Elevated HLA-G expression could serve as an independent predictor of poor clinical outcomes in patients with low-grade gliomas.

  11. Polycyclic aromatic hydrocarbons-induced ROS accumulation enhances mutagenic potential of T-antigen from human polyomavirus JC.

    PubMed

    Wilk, Anna; Waligórski, Piotr; Lassak, Adam; Vashistha, Himanshu; Lirette, David; Tate, David; Zea, Arnold H; Koochekpour, Shahriar; Rodriguez, Paulo; Meggs, Leonard G; Estrada, John J; Ochoa, Augusto; Reiss, Krzysztof

    2013-11-01

    Polycyclic aromatic hydrocarbons (PAHs) are the products of incomplete combustion of organic materials, which are present in cigarette smoke, deep-fried food, and in natural crude oil. Since PAH-metabolites form DNA adducts and cause oxidative DNA damage, we asked if these environmental carcinogens could affect transforming potential of the human Polyomavirus JC oncoprotein, T-antigen (JCV T-antigen). We extracted DMSO soluble PAHs from Deepwater Horizon oil spill in the Gulf of Mexico (oil-PAHs), and detected several carcinogenic PAHs. The oil-PAHs were tested in exponentially growing cultures of normal mouse fibroblasts (R508), and in R508 stably expressing JCV T-antigen (R508/T). The oil-PAHs were cytotoxic only at relatively high doses (1:50-1:100 dilution), and at 1:500 dilution the growth and cell survival rates were practically unaffected. This non-toxic dose triggered however, a significant accumulation of reactive oxygen species (ROS), caused oxidative DNA damage and the formation of DNA double strand breaks (DSBs). Although oil-PAHs induced similar levels of DNA damage in R508 and R508/T cells, only T-antigen expressing cells demonstrated inhibition of high fidelity DNA repair by homologous recombination (HRR). In contrast, low-fidelity repair by non-homologous end joining (NHEJ) was unaffected. This potential mutagenic shift between DNA repair mechanisms was accompanied by a significant increase in clonal growth of R508/T cells chronically exposed to low doses of the oil-PAHs. Our results indicate for the first time carcinogenic synergy in which oil-PAHs trigger oxidative DNA damage and JCV T-antigen compromises DNA repair fidelity. PMID:23558788

  12. Memory-Like Antigen-Specific Human NK Cells from TB Pleural Fluids Produced IL-22 in Response to IL-15 or Mycobacterium tuberculosis Antigens

    PubMed Central

    Fu, Xiaoying; Yu, Sifei; Yang, Binyan; Lao, Suihua; Li, Baiqing; Wu, Changyou

    2016-01-01

    Our previous result indicated that memory-like human natural killer (NK) cells from TB pleural fluid cells (PFCs) produced large amounts of IFN-γ in response to Bacille Calmette Guerin (BCG). Furthermore, recent studies have shown that human lymphoid tissues harbored a unique NK cell subset that specialized in production of interleukin (IL)-22, a proinflammatory cytokine that mediates host defense against pathogens. Yet little information was available with regard to the properties of IL-22 production by memory-like human NK cells. In the present study, we found that cytokines IL-15 induced and IL-12 enhanced the levels of IL-22 by NK cells from TB PFCs. In addition, IL-22 but not IL-17 was produced by NK cells from PFCs in response to BCG and M.tb-related Ags. More importantly, the subset of specific IL-22-producing NK cells were distinct from IFN-γ-producing NK cells in PFCs. CD45RO+ or CD45RO- NK cells were sorted, co-cultured with autologous monocytes and stimulated with BCG for the production of IL-22. The result demonstrated that CD45RO+ but not CD45RO- NK cells produced significantly higher level of IL-22. Anti-IL-12Rβ1 mAbs (2B10) partially inhibit the expression of IL-22 by NK cells under the culture with BCG. Consistently, BCG specific IL-22-producing NK cells from PFCs expressed CD45ROhighNKG2Dhighgranzyme Bhigh. In conclusion, our data demonstrated that memory-like antigen-specific CD45RO+ NK cells might participate in the recall immune response for M. tb infection via producing IL-22, which display a critical role to fight against M. tb. PMID:27031950

  13. Antigenic variation of pilin regulates adhesion of Neisseria meningitidis to human epithelial cells.

    PubMed

    Nassif, X; Lowy, J; Stenberg, P; O'Gaora, P; Ganji, A; So, M

    1993-05-01

    Pili have been shown to play an essential role in the adhesion of Neisseria meningitidis to epithelial cells. However, among piliated strains, both inter- and intrastrain variability exist with respect to their degree of adhesion to epithelial cells in vitro (Virji et al., 1992). This suggests that factors other than the presence of pili per se are involved in this process. The N. meningitidis pilin subunit undergoes extensive antigenic variation. Piliated low- and high-adhesive derivatives of the same N. meningitidis strain were selected and the nucleotide sequence of the pilin gene expressed in each was determined. The highly adhesive derivatives had the same pilin sequence. The alleles encoding the pilin subunit of the low-adhesive derivatives were completely different from the one found in the high-adhesive isolates. Using polyclonal antibodies raised against one hyperadhesive variant, it was confirmed that the low-adhesive piliated derivatives expressed pilin variants antigenically different from the highly adhesive strains. The role of antigenic variation in the adhesive process of N. meningitidis was confirmed by performing allelic exchanges of the pilE locus between low- and high-adhesive isolates. Antigenic variation has been considered a means by which virulent bacteria evade the host immune system. This work provides genetic proof that a bacterial pathogen, N. meningitidis, can use antigenic variation to modulate their degree of virulence.

  14. Simian virus 40 large tumor antigen-immortalized normal human liver epithelial cells express hepatocyte characteristics and metabolize chemical carcinogens.

    PubMed Central

    Pfeifer, A M; Cole, K E; Smoot, D T; Weston, A; Groopman, J D; Shields, P G; Vignaud, J M; Juillerat, M; Lipsky, M M; Trump, B F

    1993-01-01

    Normal human liver tissue and cultured human hepatocytes are valuable models to study xenobiotic metabolism and toxicity, but they only have a limited in vitro life-span and are not readily available. This report describes the establishment of replicative cultures of human adult liver epithelial cells in serum-free medium. The longevity of three of these cultures, derived from different donors, was extended by introduction of the simian virus 40 large T antigen gene. Two cell lines, THLE-2 and -3, established with a recombinant simian virus 40 large T antigen virus have undergone > 100 population doublings, are nontumorigenic when injected into athymic nude mice, have near-diploid karyotypes, and do not express alpha-fetoprotein. The cells express cytokeratin 18 and albumin in early passage, whereas higher-passage cells in logarithmic-phase growth also express cytokeratin 19. THLE-2 and -3 cells metabolize benzo[a]pyrene, N-nitrosodimethylamine, and aflatoxin B1 to their ultimate carcinogenic metabolites that adduct DNA, which indicates functional cytochrome P450 pathways. Other enzymes involved in metabolism of chemical carcinogens, such as epoxide hydrolase, NADPH cytochrome P450 reductase, superoxide dismutase, catalase, glutathione S-transferases, and glutathione peroxidase are also retained by THLE cells. Thus, these immortalized human liver cells constitute an in vitro model for pharmacotoxicological studies and for the investigation of etiology and pathogenesis of human hepatocellular carcinoma. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7685115

  15. Role of the MHC restriction during maturation of antigen-specific human T cells in the thymus.

    PubMed

    Hesnard, Leslie; Legoux, François; Gautreau, Laetitia; Moyon, Melinda; Baron, Olivier; Devilder, Marie-Claire; Bonneville, Marc; Saulquin, Xavier

    2016-03-01

    In the thymus, a T-cell repertoire able to confer protection against infectious and noninfectious agents in a peptide-dependent, self-MHC-restricted manner is selected. Direct detection of Ag-specific thymocytes, and analysis of the impact of the expression of the MHC-restricting allele on their frequency or function has never been studied in humans because of the extremely low precursor frequency. Here, we used a tetramer-based enrichment protocol to analyze the ex vivo frequency and activation-phenotype of human thymocytes specific for self, viral and tumor-antigens presented by HLA-A*0201 (A2) in individuals expressing or not this allele. Ag-specific thymocytes were quantified within both CD4CD8 double or single-positive compartments in every donor. Our data indicate that the maturation efficiency of Ag-specific thymocytes is poorly affected by HLA-A2 expression, in terms of frequencies. Nevertheless, A2-restricted T-cell lines from A2(+) donors reacted to A2(+) cell lines in a highly peptide-specific fashion, whereas their alloreactive counterparts showed off-target activity. This first ex vivo analysis of human antigen-specific thymocytes at different stages of human T-cell development should open new perspectives in the understanding of the human thymic selection process. PMID:26635029

  16. Human Monoclonal Islet Cell Antibodies From a Patient with Insulin- Dependent Diabetes Mellitus Reveal Glutamate Decarboxylase as the Target Antigen

    NASA Astrophysics Data System (ADS)

    Richter, Wiltrud; Endl, Josef; Eiermann, Thomas H.; Brandt, Michael; Kientsch-Engel, Rosemarie; Thivolet, Charles; Jungfer, Herbert; Scherbaum, Werner A.

    1992-09-01

    The autoimmune phenomena associated with destruction of the β cell in pancreatic islets and development of type 1 (insulin-dependent) diabetes mellitus (IDDM) include circulating islet cell antibodies. We have immortalized peripheral blood lymphocytes from prediabetic individuals and patients with newly diagnosed IDDM by Epstein-Barr virus transformation. IgG-positive cells were selected by anti-human IgG-coupled magnetic beads and expanded in cell culture. Supernatants were screened for cytoplasmic islet cell antibodies using the conventional indirect immunofluorescence test on cryostat sections of human pancreas. Six islet cell-specific B-cell lines, originating from a patient with newly diagnosed IDDM, could be stabilized on a monoclonal level. All six monoclonal islet cell antibodies (MICA 1-6) were of the IgG class. None of the MICA reacted with human thyroid, adrenal gland, anterior pituitary, liver, lung, stomach, and intestine tissues but all six reacted with pancreatic islets of different mammalian species and, in addition, with neurons of rat cerebellar cortex. MICA 1-6 were shown to recognize four distinct antigenic epitopes in islets. Islet cell antibody-positive diabetic sera but not normal human sera blocked the binding of the monoclonal antibodies to their target epitopes. Immunoprecipitation of 35S-labeled human islet cell extracts revealed that a protein of identical size to the enzyme glutamate decarboxylase (EC 4.1.1.15) was a target of all MICA. Furthermore, antigen immunotrapped by the MICA from brain homogenates showed glutamate decarboxylase enzyme activity. MICA 1-6 therefore reveal glutamate decarboxylase as the predominant target antigen of cytoplasmic islet cell autoantibodies in a patient with newly diagnosed IDDM.

  17. Human and guinea pig immune responses to Legionella pneumophila protein antigens OmpS and Hsp60.

    PubMed Central

    Weeratna, R; Stamler, D A; Edelstein, P H; Ripley, M; Marrie, T; Hoskin, D; Hoffman, P S

    1994-01-01

    We studied the immune responses of guinea pigs and humans to two Legionella pneumophila antigens. Guinea pigs surviving a lethal intraperitoneal challenge dose of virulent L. pneumophila exhibited strong cutaneous delayed-type hypersensitivity (DTH) reactions to purified OmpS (28-kDa major outer membrane protein) and Hsp60 (heat shock protein or common antigen), while weak DTH reactions were noted for extracellular protease (major secretory protein [MSP] [ProA]) and no reaction was observed with an ovalbumin (OA) control. Lymphocyte proliferation responses (LPRs) were measured for peripheral blood and spleen lymphocytes from guinea pigs surviving sublethal and lethal challenge doses of L. pneumophila. Lymphocytes from uninfected animals showed no proliferation to Hsp60 or OmpS, while lymphocytes from sublethally and lethally challenged animals exhibited strong proliferative responses to Hsp60 and OmpS. Guinea pigs vaccinated with purified OmpS exhibited low antibody titers and strong DTH and LPRs to OmpS, whereas lymphocytes from animals vaccinated with Hsp60 exhibited weak DTH responses and high antibody titers to Hsp60. All guinea pigs immunized with OmpS survived experimental challenge with L. pneumophila (two of two in a pilot study and seven of seven in trial 2) versus zero of seven OA-immunized controls (P = 0.006 by Fisher's exact test). In three vaccine trials in which animals were vaccinated with Hsp60, only 1 guinea pig of 15 survived lethal challenge. Peripheral blood lymphocytes (PBLs) from humans with legionellosis showed stronger LPRs to OmpS than PBLs from humans with no history of legionellosis (P = 0.0002 by Mann-Whitney test). PBLs of humans surviving legionellosis exhibited a lower but highly significant proliferative response to Hsp60 (P < 0.0001 compared with controls by Mann-Whitney test). These studies indicate that OmpS and Hsp60 are important antigens associated with the development of protective cellular immunity. However, as determined in

  18. [Detection of common determinants of HLA antigens A2 and B17 by absorption using human HLA serum].

    PubMed

    Májský, A; Korínková, P

    1983-01-01

    Attempts of cross absorption where sera of anti-HLA A2 + B17, anti-HLA A2 and anti-HLA B17 with thrombocytes were absorbed from donors of HLA A2 positive, B17 negative and HLA A2 negative, B17 positive, revealed that anti-HLA A2 and anti-HLA B17 could be eliminated from the sera of both HLA types on the platelets. Thus, the findings allow the existence of a common determinant of HLA A2 and B17-antigens to be assumed. This is the first case where the evidence of a cross reaction between antigens of two different HLA loci with human sera could be established.

  19. Discriminating antigen and non-antigen using proteome dissimilarity: bacterial antigens

    PubMed Central

    Ramakrishnan, Kamna; Flower, Darren R

    2010-01-01

    It has been postulated that immunogenicity results from the overall dissimilarity of pathogenic proteins versus the host proteome. We have sought to use this concept to discriminate between antigens and non-antigens of bacterial origin. Sets of 100 known antigenic and nonantigenic peptide sequences from bacteria were compared to human and mouse proteomes. Both antigenic and non-antigenic sequences lacked human or mouse homologues. Observed distributions were compared using the non-parametric Mann-Whitney test. The statistical null hypothesis was accepted, indicating that antigen and non-antigens did not differ significantly. Likewise, we were unable to determine a threshold able to separate meaningfully antigen from non-antigen. Thus, antigens cannot be predicted from pathogen genomes based solely on their dissimilarity to the human genome. PMID:20975907

  20. Serological survey in the Finnish human population implies human-to-human transmission of Ljungan virus or antigenically related viruses.

    PubMed

    Jääskeläinen, A J; Voutilainen, L; Lehmusto, R; Henttonen, H; Lappalainen, M; Kallio-Kokko, H; Vaheri, A; Vapalahti, O

    2016-04-01

    Ljungan virus (LV) is a picornavirus related to human parechoviruses (HPeV). The virus has been found in bank voles (Myodes glareolus) and several other rodent species, and suggested to have zoonotic potential. Thus far, seroepidemiological data on LV infections in humans are scarce. In this study, we aimed to characterize the demographic and geographical distribution of LV-reactive antibodies in Finland, and to investigate its occurrence in patients suspected of having a rodent-borne disease, nephropathia epidemica (NE) caused by Puumala hantavirus (PUUV). Using an immunofluorescence assay (LV strain 145SLG), we screened human sera (n = 1378) and found LV-reactive antibodies in 36% of samples. The probability of possessing LV-reactive antibodies peaked at age of 14 years, suggesting that most infections occur in childhood. The prevalence of LV-reactive antibodies was significantly higher in the urbanized area surrounding Helsinki than in more rural Central Finland. These findings are uncharacteristic of a rodent-borne pathogen, and therefore we consider human-to-human transmission of one or several Ljungan-like viruses as a likely cause for most of the observed antibody responses. PMID:26489898

  1. Human La antigen is required for the hepatitis C virus internal ribosome entry site-mediated translation.

    PubMed

    Ali, N; Pruijn, G J; Kenan, D J; Keene, J D; Siddiqui, A

    2000-09-01

    The 5'-noncoding region (5'-NCR) of the hepatitis C virus (HCV) RNA genome serves as an internal ribosome entry site (IRES) and mediates translation initiation in a cap-independent manner. Previously, we reported the interaction between La antigen and the HCV IRES, which appeared to occur in the context of initiator AUG. It was further shown that HCV IRES-mediated translation was stimulated in the presence of human La antigen. In this study, we have defined the cis- and trans-acting elements responsible for La-5'-NCR interactions and established the dependence of the HCV IRES efficiency on cellular La antigen. During the La-IRES interaction, initiator AUG but not the neighboring codons was found to be the direct target of La binding. The C terminus effector domain-dependent modulation of La binding to the HCV IRES is demonstrated by deletion and substitution mutagenesis of the protein. An RNA systematic evolution of ligands by exponential enrichment (SELEX), generated against La protein that selectively binds La in HeLa lysates and competes for the protein binding to the 5'-NCR, was used to demonstrate the requirement of La for the HCV IRES function in the context of mono- and dicistronic mRNAs. Sequestration of La antigen by the RNA SELEX in HeLa translation lysates blocked the HCV and poliovirus IRES-mediated translation in vitro. The functional requirement of La protein for the HCV IRES activity was further established in a liver-derived cell line and in an add-back experiment in which the inhibited IRES was rescued by recombinant human La. These results strongly argue for the novel role of La protein during selection of the initiator AUG and its participation during internal initiation of translation of the HCV RNA genome. PMID:10856291

  2. Immunohistochemical Analysis of Leucocyte Subsets in the Sinonasal Mucosa of Cats with Upper Respiratory Tract Aspergillosis.

    PubMed

    Whitney, J L; Krockenberger, M B; Day, M J; Beatty, J A; Dhand, N K; Barrs, V R

    2016-01-01

    Leucocyte populations in the sinonasal mucosa of cats with and without upper respiratory tract aspergillosis were compared using immunohistochemistry and computer-aided morphometry. Inflammation was identified in the nasal mucosa of all affected cats, comprising predominantly of lymphoplasmacytic infiltration of the lamina propria associated with epithelial proliferation and degeneration. There was intense and diffuse expression of class II antigens of the major histocompatibility complex, associated with sites of hyphal invasion with hyperplasia and ulceration of the epithelium adjacent to fungal elements. Significantly more CD79b(+) cells, total lymphocytes, immunoglobulin (Ig)-expressing cells and MAC387(+) cells infiltrated the epithelium and more IgG(+) cells and total Ig-expressing cells infiltrated the lamina propria in affected cats compared with controls. Importantly, the inflammatory profile in affected cats was not consistent with the T helper (Th)1 and Th17 cell-mediated response that confers protective acquired immunity against invasive aspergillosis in dogs and people and in murine models of the infection. This finding may help to explain the development of invasive aspergillosis in systemically immunocompetent cats. PMID:27576043

  3. Leucocyte adhesion deficiency type 1 with developmental delay secondary to CMV infection and filiation questions.

    PubMed

    Strickler, Alexis; Gallo, Silvanna; King, Alejandra; Rosenzweig, Sergio D

    2015-04-09

    Leucocyte adhesion deficiency (LAD) is a group of rare autosomal recessive (<1:1 000 000 births) inherited disorders characterised by immune deficiency and peripheral neutrophilia. Three types of LAD syndrome have been distinguished. LAD type 1 (LAD-I) is the most common. It results from a mutation in the integrin β 2 (ITGB2) gene that codes the ITGB subunit (CD18 antigen). Since 1970, it has been reported in more than 300 children worldwide. It is characterised by delayed separation of the umbilical cord, recurrent bacterial and fungal infections, defective wound healing, blood neutrophilia and a high mortality rate at an early age. We report the second fatal case of an infant with LAD-I diagnosed in Chile, with developmental delay associated with a congenital cytomegalovirus infection. CD18/CD11 expression was normal. Genetic analysis of CD18 revealed a homozygous mutation in ITGB2, viz.c.1835G>T; p.C612F, and led us to suspect a biological parent other than the legal father and, therefore, an unwanted social situation.

  4. Leucocyte adhesion deficiency type 1 with developmental delay secondary to CMV infection and filiation questions.

    PubMed

    Strickler, Alexis; Gallo, Silvanna; King, Alejandra; Rosenzweig, Sergio D

    2015-01-01

    Leucocyte adhesion deficiency (LAD) is a group of rare autosomal recessive (<1:1 000 000 births) inherited disorders characterised by immune deficiency and peripheral neutrophilia. Three types of LAD syndrome have been distinguished. LAD type 1 (LAD-I) is the most common. It results from a mutation in the integrin β 2 (ITGB2) gene that codes the ITGB subunit (CD18 antigen). Since 1970, it has been reported in more than 300 children worldwide. It is characterised by delayed separation of the umbilical cord, recurrent bacterial and fungal infections, defective wound healing, blood neutrophilia and a high mortality rate at an early age. We report the second fatal case of an infant with LAD-I diagnosed in Chile, with developmental delay associated with a congenital cytomegalovirus infection. CD18/CD11 expression was normal. Genetic analysis of CD18 revealed a homozygous mutation in ITGB2, viz.c.1835G>T; p.C612F, and led us to suspect a biological parent other than the legal father and, therefore, an unwanted social situation. PMID:25858935

  5. Human prostate-specific antigen: structural and functional similarity with serine proteases.

    PubMed

    Watt, K W; Lee, P J; M'Timkulu, T; Chan, W P; Loor, R

    1986-05-01

    The complete amino acid sequence of the prostate-specific antigen (PA) from human seminal plasma has been determined from analyses of the peptides generated by cyanogen bromide, hydroxylamine, endoproteinases Arg-C and Lys-C. The single polypeptide chain of PA contains 240-amino acid residues and has a calculated Mr of 26,496. An N-linked carbohydrate side chain is predicted at asparagine-45, and O-linked carbohydrate side chains are possibly attached to serine-69, threonine-70, and serine-71. The primary structure of PA shows a high degree of sequence homology with other serine proteases of the kallikrein family. The active site residues of histidine, aspartic acid, and serine comprising the charge-relay system of typical serine proteases were found in similar positions in PA (histidine-41, aspartic acid-96, and serine-192). At pH 7.8, PA hydrolyzed insulin A and B chains, recombinant interleukin 2, and--to a lesser extent--gelatin, myoglobin, ovalbumin, and fibrinogen. The cleavage sites of these proteins by PA were chemically analyzed as the alpha-carboxyl side of some hydrophobic residues, tyrosine, leucine, valine, and phenylalanine, and of basic residues histidine, lysine, and arginine. The chymotrypsin-like activity of PA exhibited with the chromogenic substrate N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine p-nitroanilide yielded a specific activity of 9.21 microM per min per mg of PA and Km and kcat values of 15.3 mM and 0.075s-1, respectively. "Trypsin-like" activity of PA was also detected with N alpha-benzoyl-DL-arginine p-nitroanilide and gave a specific activity of 1.98 microM per min per mg of PA. Protease inhibitors such as phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate, L-1-tosylamido-2-phenylethyl chloromethyl ketone, aprotinin, leupeptin, soybean trypsin inhibitor as well as Zn2+ and spermidine were effective inhibitors of PA enzymatic activity.

  6. Human Leukocyte Antigen Class I and II Alleles and Cervical Adenocarcinoma

    PubMed Central

    Safaeian, Mahboobeh; Johnson, Lisa G.; Yu, Kai; Wang, Sophia S.; Gravitt, Patti E.; Hansen, John A.; Carrington, Mary; Schwartz, Stephen M.; Gao, Xiaojiang; Hildesheim, Allan; Madeleine, Margaret M.

    2014-01-01

    Background: Associations between human leukocyte antigens (HLA) alleles and cervical cancer are largely representative of squamous cell carcinoma (SCC), the major histologic subtype. We evaluated the association between HLA class I (A, B, and C) and class II (DRB1 and DQB1) loci and risk of cervical adenocarcinoma (ADC), a less common but aggressive histologic subtype. Methods: We pooled data from the Eastern and Western US Cervical Cancer studies, and evaluated the association between individual alleles and allele combinations and ADC (n = 630 ADC; n = 775 controls). Risk estimates were calculated for 11 a priori (based on known associations with cervical cancer regardless of histologic type) and 38 non a priori common alleles, as odds ratios (OR) and 95% confidence intervals (CI), adjusted for age and study. In exploratory analysis, we compared the risk associations between subgroups with HPV16 or HPV18 DNA in ADC tumor tissues in the Western US study cases and controls. Results: Three of the a priori alleles were significantly associated with decreased risk of ADC [DRB1*13:01 (OR = 0.61; 95% CI: 0.41–0.93), DRB1*13:02 (OR = 0.49; 95% CI: 0.31–0.77), and DQB1*06:03 (OR = 0.64; 95% CI: 0.42–0.95)]; one was associated with increased risk [B*07:02 (OR = 1.39; 95% CI: 1.07–1.79)]. Among alleles not previously reported, DQB1*06:04 (OR = 0.46; 95% CI: 0.27–0.78) was associated with decreased risk of ADC and remained significant after correction for multiple comparisons, and C*07:02 (OR = 1.41; 95% CI: 1.09–1.81) was associated with increased risk. We did not observe a difference by histologic subtype. ADC was most strongly associated with increased risk with B*07:02/C*07:02 alleles (OR = 1.33; 95% CI: 1.01–1.76) and decreased risk with DRB1*13:02/DQB1*06:04 (OR = 0.41; 95% CI: 0.21–0.80). Conclusion: Results suggest that HLA allele associations with cervical ADC are similar to those for cervical SCC. An intriguing

  7. Isolation and sequencing of a cDNA coding for the human DF3 breast carcinoma-associated antigen

    SciTech Connect

    Siddiqui, J.; Abe, M.; Hayes, D.; Shani, E.; Yunis, E.; Kufe, D. )

    1988-04-01

    The murine monoclonal antibody (mAb) DF3 reacts with a high molecular weight glycoprotein detectable in human breast carcinomas. DF3 antigen expression correlates with human breast tumor differentiation, and the detection of a cross-reactive species in human milk has suggested that this antigen might be useful as a marker of differentiated mammary epithelium. To further characterize DF3 antigen expression, the authors have isolated a cDNA clone from a {lambda}gt11 library by screening with mAb DF3. The results demonstrate that this 309-base-pair cDNA, designated pDF9.3, codes for the DF3 epitope. Southern blot analyses of EcoRI-digested DNAs from six human tumor cell lines with {sup 32}P-labeled pDF9.3 have revealed a restriction fragment length polymorphism. Variations in size of the alleles detected by pDF9.3 were also identified in Pst I, but not in HindIII, DNA digests. Furthermore, hybridization of {sup 32}P-labeled pDF9.3 with total cellular RNA from each of these cell lines demonstrated either one or two transcripts that varied from 4.1 to 7.1 kilobases in size. The presence of differently sized transcripts detected by pDF9.3 was also found to correspond with the polymorphic expression of DF3 glycoproteins. Nucleotide sequence analysis of pDF9.3 has revealed a highly conserved (G + C)-rich 60-base-pair tandem repeat. These findings suggest that the variation in size of alleles coding for the polymorphic DF3 glycoprotein may represent different numbers of repeats.

  8. Use of Heteropolymeric Monoclonal Antibodies to Attach Antigens to the C3b Receptor of Human Erythrocytes: A Potential Therapeutic Treatment

    NASA Astrophysics Data System (ADS)

    Taylor, Ronald P.; Sutherland, William M.; Reist, Craig J.; Webb, Donna J.; Wright, Eleanor L.; Labuguen, Ronald H.

    1991-04-01

    We have prepared bispecific, cross-linked monoclonal antibodies (heteropolymers) with specificity for both targeted antigens and the human erythrocyte (RBC) complement receptor. These heteropolymers facilitate binding of target antigens (human IgG and dinitrophenylated bovine γ globulin) to human RBCs under conditions that either allow or preclude complement activation. Quantitative analyses of this binding agree well with the number of complement receptors per RBC. In vitro "whole-blood" model experiments indicate heteropolymer-facilitated binding of antigens to RBCs is rapid and stable at 37^circC. It may be possible to extend these prototype experiments to the in vivo situation and use heteropolymer-attached RBCs for the safe and rapid binding, neutralization, and removal from the circulation of pathogenic antigens associated with infectious disease.

  9. Use of heteropolymeric monoclonal antibodies to attach antigens to the C3b receptor of human erythrocytes: A potential therapeutic treatment

    SciTech Connect

    Taylor, R.P.; Sutherland, W.M.; Reist, C.J.; Webb, D.J.; Wright, E.L.; Labuguen, R.H. )

    1991-04-15

    The authors prepared bispecific, cross-linked monoclonal antibodies (heteropolymers) with specificity for both targeted antigens and the human erythrocyte (RBC) complement receptor. These heteropolymers facilitate binding of target antigens (human IgG and dinitrophenylated bovine {gamma} globulin) to human RBCs under conditions that either allow or preclude complement activation. Radioimmuno-assay analyses of this binding agree well with the number of complement receptors per RBC. In vitro whole-blood model experiments indicate heteropolymer-facilitated binding of antigens to RBCs is rapid and stable at 37C. It may be possible to extend these prototype experiments to the in vivo situation and use heteropolymer-attached RBCs for the safe and rapid binding, neutralization, and removal from the circulation of pathogenic antigens associated with infectious disease.

  10. Thalidomide inhibits granulocyte responses in healthy humans after ex vivo stimulation with bacterial antigens.

    PubMed

    Juffermans, N P; Verbon, A; Schultz, M J; Hack, C E; van Deventer, S J; Speelman, P; van der Poll, T

    2001-05-01

    Ingestion of thalidomide was associated with a reduction in the upregulation of the granulocyte activation marker CD11b and a reduced capacity to release elastase and lactoferrin after stimulation with lipopolysaccharide or lipoteichoic acid. A single oral dose of thalidomide attenuates neutrophil activation upon ex vivo stimulation with bacterial antigens.

  11. Human cell-mediated immune responses to antigenic fractions of Salmonella typhi.

    PubMed Central

    Pérez, C; Calderón, G M; Ximénez, C; Melendro, E I

    1996-01-01

    The proliferative responses of peripheral blood mononuclear cells of 10 subjects that had typhoid fever, and healthy volunteers without history of typhoid fever or immunization against disease, were analysed with antigen fractions from two protein extracts of Salmonella typhi. Fractions from each extract were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis, transferred to nitrocellulose filters by electroblotting and processed to obtain antigen-bearing nitrocellulose particles for use in lymphocyte cultures. Although the individual proliferative responses were heterogeneous we identified two main immunogenic regions of 29-32 10(3) MW and 45-56 x 10(3) MW for both extracts. Even though there was no one particular antigenic fraction capable of stimulating lymphocytes from all individuals with a previous history of typhoid fever, the combination of three fractions 29-32, 41-45, 63-71 x 10(3) MW could be stimulatory for cells of 90% of these individuals. Also, four subjects that did not respond to unfractionated antigens gave proliferative responses to several fractions of the same extract. We have identified the main immunogenic fractions of S. typhi that might play a role during typhoid infection and postinfection immunity, and merit further purification and characterization. Images Figure 1 Figure 2 Figure 3 Figure 5 PMID:8943724

  12. Antigen-presenting cells in human cutaneous leishmaniasis due to Leishmania major.

    PubMed Central

    ElHassan, A M; Gaafar, A; Theander, T G

    1995-01-01

    In this study biopsies from skin lesions and draining lymph nodes of patients suffering from cutaneous leishmaniasis caused by Leishmania major were examined by immunohistochemistry, and by light and electron microscopy to identify the types of antigen-presenting cells (APC) and their location. APC, identified morphologically and by their expression of specific cell markers, included Langerhans cells, macrophages, follicular dendritic cells, and interdigitating reticulum cells of the paracortex of lymph nodes. These cells expressed MHC class II antigens and contained Leishmania antigen. Since some keratinocytes and endothelial cells also showed these characteristics, they may also act as APC. By examining tissue samples from skin lesions and draining lymph nodes it was possible to follow the probable route of trafficking of various inflammatory cells between the skin lesion and lymph nodes. Leishmania antigen containing Langerhans cells were found in the epidermis, dermis and the regional lymph nodes. We believe these cells translocate from the epidermis to the dermis, where they take up antigen and migrate to the paracortex of the regional lymph nodes. There they are intimately associated with cells of the paracortex, and could be involved in the generation of Leishmania-specific T memory cells. LFA-1-positive T cells of the CD45RO phenotype were found in the skin lesion. Venular endothelium in the skin lesions expressed intercellular adhesion molecule-1 (ICAM-1), which is the ligand for LFA-1. The migration of lymphocytes from the vascular lumen to the site of inflammation is possibly a result of the interaction of these two adhesion molecules. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7882568

  13. Evidence that verotoxins (Shiga-like toxins) from Escherichia coli bind to P blood group antigens of human erythrocytes in vitro.

    PubMed Central

    Bitzan, M; Richardson, S; Huang, C; Boyd, B; Petric, M; Karmali, M A

    1994-01-01

    The interaction of verotoxins (VTs) with human erythrocytes (RBCs) in vitro was investigated, with particular reference to the role of P blood group glycolipids that are structurally related to the known VT receptors. RBC binding of purified VT1, VT2, VT2c, and VT2e was detected by direct and indirect immunofluorescence. Glycolipids were extracted from defined RBCs, separated by thin-layer chromatography, and assessed for VT binding in an overlay assay by adding toxin and specific antibodies. All VTs bound to P1 phenotype (Pk, P, and P1 antigens) and P2 phenotype (Pk and P antigens) RBCs but not to p phenotype (lacking the Pk, P, and P1 antigens) RBCs. Binding of VT1 and VT2 was approximately 10-fold greater to P1 and the rare Pk2 (Pk antigen but no P1 or P antigen) phenotype cells than to P2 phenotype RBCs, whereas VT2e bound equally well to P1 and P2 phenotype cells. The VT1 and VT2 immunofluorescence results correlated with the detection of P1 and/or increased amounts of Pk (globotriaosylceramide) antigen; VT2e immunofluorescence correlated with the detection of P (globotetraosylceramide) antigen. The Pk band pattern and VT binding observed in the thin-layer chromatogram of human P1 and P phenotype RBC extracts varied from that of human kidney and Pk1 phenotype (Pk and P1 antigens) RBCs. We conclude that each VT binds to human RBCs in vitro by utilizing specific P blood group glycolipids as receptors. On P1 and P phenotype RBCs, the accessibility of the Pk antigen for VTs appeared to be restricted. The occurrence of VT-RBC binding in natural VT-producing Escherichia coli disease and its relevance for the pathophysiology of hemolytic uremic syndrome remain to be established. Images PMID:8039905

  14. Invariant chain as a vehicle to load antigenic peptides on human MHC class I for cytotoxic T-cell activation.

    PubMed

    Wälchli, Sébastien; Kumari, Shraddha; Fallang, Lars-Egil; Sand, Kine M K; Yang, Weiwen; Landsverk, Ole J B; Bakke, Oddmund; Olweus, Johanna; Gregers, Tone F

    2014-03-01

    Protective T-cell responses depend on efficient presentation of antigen (Ag) in the context of major histocompatibility complex class I (MHCI) and class II (MHCII) molecules. Invariant chain (Ii) serves as a chaperone for MHCII molecules and mediates trafficking to the endosomal pathway. The genetic exchange of the class II-associated Ii peptide (CLIP) with antigenic peptides has proven efficient for loading of MHCII and activation of specific CD4(+) T cells. Here, we investigated if Ii could similarly activate human CD8(+) T cells when used as a vehicle for cytotoxic T-cell (CTL) epitopes. The results show that wild type Ii, and Ii in which CLIP was replaced by known CTL epitopes from the cancer targets MART-1 or CD20, coprecipitated with HLA-A*02:01 and mediated colocalization in the endosomal pathway. Furthermore, HLA-A*02:01-positive cells expressing CLIP-replaced Ii efficiently activated Ag-specific CD8(+) T cells in a TAP- and proteasome-independent manner. Finally, dendritic cells transfected with mRNA encoding IiMART-1 or IiCD20 primed naïve CD8(+) T cells. The results show that Ii carrying antigenic peptides in the CLIP region can promote efficient presentation of the epitopes to CTLs independently of the classical MHCI peptide loading machinery, facilitating novel vaccination strategies against cancer.

  15. Expression of senescent antigen on erythrocytes infected with a knobby variant of the human malaria parasite Plasmodium falciparum

    SciTech Connect

    Winograd, E.; Greenan, J.R.T.; Sherman, I.W.

    1987-04-01

    Erythrocytes infected with a knobby variant of Plasmodium falciparum selectively bind IgG autoantibodies in normal human serum. Quantification of membrane-bound IgG, by use of /sup 125/I-labeled protein A, revealed that erythrocytes infected with the knobby variant bound 30 times more protein A than did noninfected erythrocytes; infection with a knobless variant resulted in less than a 2-fold difference compared with noninfected erythrocytes. IgG binding to knobby erythrocytes appeared to be related to parasite development, since binding of /sup 125/I-labeled protein A to cells bearing young trophozoites (less than 20 hr after parasite invasion) was similar to binding to uninfected erythrocytes. By immunoelectron microscopy, the membrane-bound IgG on erythrocytes infected with the knobby variant was found to be preferentially associated with the protuberances (knobs) of the plasma membrane. The removal of aged or senescent erythrocytes from the peripheral circulation is reported to involve the binding of specific antibodies to an antigen (senescent antigen) related to the major erythrocyte membrane protein band 3. Since affinity-purified autoantibodies against band 3 specifically bound to the plasma membrane of erythrocytes infected with the knobby variant of P. falciparum, it is clear that the malaria parasite induces expression of senescent antigen.

  16. Functional Recombinant Extra Membrane Loop of Human CD20, an Alternative of the Full Length CD20 Antigen

    PubMed Central

    Anbouhi, Mahdi Habibi; Baraz, Aida Feiz; Bouzari, Saeid; Abolhassani, Mohsen; Khanahmad, Hossein; Golkar, Majid; Aghasadeghi, Mohammad Reza; Behdani, Mahdi; Najafabadi, Ali Jahanian; Shokrgozar, Mohammad Ali

    2012-01-01

    Background: Targeting of CD20 antigen with monoclonal antibodies has become the mainstay in the treatment of non-Hodgkin's lymphomas and immunotherapeutic depletion of malignant B cells. Accessibility of antigen is one of the crucial factors in development of monoclonal antibodies against this antigen. One major problem in expression of full length CD20 is aggregation and misfolding. Therefore, production of an alternative polypeptide is easer and favorable comparing to that of a full length transmembrane protein CD20. Methods: In this study, we expressed the extra membrane loop of hCD20 (exCD20) consisting of a non-glycosylated 47-amino acids region. The exCD20 coding sequence was amplified by PCR and cloned in pET32a(+) expression vector. The desired protein was expressed in fusion with thioredoxin and 6× His tag in E. coli Origami strain. ELISA and Western-blotting data were performed to indicate the functionality of this protein. Results: We have obtained the exCD20 recombinant protein which can be detected in ELISA and Western-blot experiments. This recombinant fusion protein was soluble and stable without aggregation and misfolding problems. Conclusion: The recombinant extra membrane loop of human CD20 protein in fusion with thioredoxin (exCD20) can be used in function assays and some applications such as ELISA, immuneblotting, affinity purification, immunization, screening, and development of anti-CD20 antibodies. PMID:23023212

  17. T suppressor cells are required for the maintenance of the antigen-induced B-cell unresponsive state in humans

    SciTech Connect

    Benveniste, E.; Stevens, R.H.

    1983-04-01

    Tetanus toxoid immunization of humans generates circulating B cells which secrete IgG anti-tetanus toxoid antibodies (IgG-Tet) when stimulated in vitro with T cells and pokeweed mitogen (PWM). A unique property of these cells is the inhibition of maturation into antibody-secreting plasma cells following a 1-hr in vitro pulse with tetanus toxoid. Studies were undertaken to determine if different T-cell subsets could modulate the in vitro generated B-cell unresponsive state. The addition of OKT4+/OKT8- cells to antigen-treated B cells resulted in a partial reversal of the antigen-induced inhibition of IgG-Tet synthesis. The addition of OKT4-/OKT8+ cells to the treated B cells caused a suppression of IgG-Tet synthesis comparable to that seen in cultures containing unfractionated T cells. These results indicate that (1) the B-cell unresponsive state generated by antigen treatment is not absolute, (2) the degree of B-cell unresponsiveness results from a balance of suppressor and helper signals, and (3) T-suppressor cells need to be present to induce and maintain the B-cell unresponsive state.

  18. Fever-range whole-body heat treatment stimulates antigen-specific T-cell responses in humans.

    PubMed

    Kobayashi, Yasunobu; Ito, Yusuke; Ostapenko, Valentina V; Sakai, Mayuko; Matsushita, Norimasa; Imai, Kenichiro; Shimizu, Koichi; Aruga, Atsushi; Tanigawa, Keishi

    2014-11-01

    Increase in body temperature has been thought to play an important role in the regulation of immune responses, although its precise mechanisms are still under investigation. Here, we examined the effects of physiologically relevant thermal stress on the cytokine production from human peripheral T cells. Volunteers were heated using a whole-body hyperthermia device, the rectal temperature was maintained above 38.5 °C for more than 60 min, and peripheral blood mononuclear cells (PBMCs) were obtained before and after the treatment. When T cells were stimulated with anti-CD3/CD28 antibodies, marked increases in the production of interferon-γ (IFN-γ) and interleukin-2 were observed in PBMCs prepared immediately after and 24h after the treatment. Similarly, enhanced production of IFN-γ in response to the tuberculin purified protein derivative or antigenic viral peptides was also observed immediately after and 24h after the treatment. Fluorescence photo-bleaching analyses showed heat-induced increase of membrane fluidity in T cells, which probably enables them to induce rapid and efficient cluster formation of molecules involved in antigen recognition and signal transduction for T-cell stimulation. We concluded that physiologically relevant thermal stress could efficiently modify T-cell responsiveness to various stimuli, including enhanced responses to specific antigens.

  19. Pheophorbide a-Mediated Photodynamic Therapy Triggers HLA Class I-Restricted Antigen Presentation in Human Hepatocellular Carcinoma1

    PubMed Central

    Tang, Patrick Ming-Kuen; Bui-Xuan, Ngoc-Ha; Wong, Chun-Kwok; Fong, Wing-Ping; Fung, Kwok-Pui

    2010-01-01

    The immunomodulatory effects of photodynamic therapy (PDT) have been reported in several photosensitizers. Pheophorbide a (Pa), a chlorophyll derivative, shows antitumor effects on a number of human cancers in a PDT approach (Pa-PDT); however, the potential effect of Pa-PDT on the anticancer immunity has never been studied. In the present work, the underlying action mechanism of Pa-PDT was systemically investigated with a human hepatoma cell line HepG2. We found that Pa-PDT significantly inhibited the growth of HepG2 cells with a half maximal inhibitory concentration/endoplasmic reticulum of 0.35 µM at 24 hours by the induction of apoptosis, as shown by externalization of phosphatidylserine, release of mitochondrial cytochrome c, and activation of the caspases cascade in the treated cells. Interestingly, using two-dimensional polyacrylamide gel electrophoresis analysis, a 57-kDa disulfide-isomerase-like ER resident protein (ERp57) that belongs to the HLA class I-restricted antigen-processing machinery was found to be mediated during the Pa-PDT treatment. This activation of antigen presentation was confirmed by Western blot analysis and immunostaining. Furthermore, a cross-presentation of antigen with HLA class I proteins and 70-kDa heat shock protein was found in Pa-PDT-treated cells, as shown by the confocal microscopic observation and immunoprecipitation assay. Nevertheless, the immunogenicity of HepG2 cells was increased by Pa-PDT treatment that triggered phagocytic capture by human macrophages. Our findings provide the first evidence that Pa-PDT can trigger both apoptosis and cancer immunity in the tumor host. PMID:20360936

  20. Estimation of Group B Streptococcus Type III Polysaccharide-Specific Antibody Concentrations in Human Sera Is Antigen Dependent

    PubMed Central

    Bhushan, Reva; Anthony, Bascom F.; Frasch, Carl E.

    1998-01-01

    The presence of immunoglobulin G (IgG) antibodies against group B streptococcus (GBS) type III polysaccharide (PS) has been correlated with protection against GBS disease. The GBS type III PS is structurally similar to the pneumococcal type 14 PS, differing only in the presence of sialic acid residues. Four different preparations of GBS type III PS were evaluated for their specificity in enzyme-linked immunosorbent assay (ELISA): free PS, free PS mixed with methylated human serum albumin (mHSA), PS conjugated to biotin and PS conjugated to human serum albumin. Three groups of human sera were used to evaluate these PS preparations: sera from recipients of a GBS PS vaccine, sera from women receiving a GBS type III PS-tetanus toxoid conjugate vaccine, and sera from nonimmunized healthy women of childbearing age. Estimated antibody concentrations were different depending on the PS preparation used. Using any of the four preparations, we were able to measure ≤0.05 μg of IgG antibody to the GBS type III PS per ml. The specificity of the assay was determined by competitive inhibition with homologous and heterologous PS. The pneumococcal type 14 PS did not inhibit binding of antibody to the native GBS type III PS in sera from adults receiving the GBS PS vaccine or in sera from nonimmunized adults (except serum G9). The pneumococcal type 14 PS inhibited 50% in sera from recipients of GBS type III conjugate vaccine and in serum G9 when GBS type III PS conjugated to biotin or to HSA was used as antigen in ELISA. These data show that free GBS type III PS or PS mixed with mHSA is a sensitive and specific antigen for ELISA and that conjugation can alter the antigenic specificity of a PS. PMID:9826364

  1. Analysis of gene selection in reassortant formation between canine rotavirus K9 and human rotaviruses with different antigenic specificities.

    PubMed

    Kobayashi, N; Taniguchi, K; Urasawa, T; Urasawa, S

    1993-01-01

    A number of antigenic mosaic reassortants which have neutralization proteins VP4 and VP7 derived from different parental strains were analysed in order to study gene selection in reassortant formation between animal and human rotaviruses (HRV). These reassortants were isolated from mixed infection of MA-104 cells with canine rotavirus strain K9 (subgroup I and G serotype 3) and HRV strains (with subgroup I or II antigen and G serotype 1-4, 9 or 12 antigen), through repeated selections with anti-VP4 and anti-VP7 neutralizing monoclonal antibodies directed specifically at HRV and K9, respectively. By serological and genomic analyses, all the isolated clones were found to be antigenic mosaic reassortants possessing VP4 of K9 and VP7 of HRV. In the reassortants between strain K9 and one of the six strains of subgroup II HRV, a single or a few genotypes with particular constellations of RNA segments were predominant, with only a few RNA segments including gene 4 (encoding VP4) being derived from K9. In contrast, in the reassortants between strain K9 and any one of the subgroup I HRV, more than nine different genotypes were identified and various RNA segments, except for segments 8 and 10, were derived from K9. These findings indicated that the RNA segments of K9 might be reassorted more readily with those of subgroup I HRV than with those of subgroup II HRV, suggesting the possible existence of functional mechanisms which determine the extent of diversity of genome selection depending on the pairs of parent strains in the reassortant formation. PMID:7506839

  2. Francisella tularensis Schu S4 O-antigen and capsule biosynthesis gene mutants induce early cell death in human macrophages.

    PubMed

    Lindemann, Stephen R; Peng, Kaitian; Long, Matthew E; Hunt, Jason R; Apicella, Michael A; Monack, Denise M; Allen, Lee-Ann H; Jones, Bradley D

    2011-02-01

    Francisella tularensis is capable of rampant intracellular growth and causes a potentially fatal disease in humans. Whereas many mutational studies have been performed with avirulent strains of Francisella, relatively little has been done with strains that cause human disease. We generated a near-saturating transposon library in the virulent strain Schu S4, which was subjected to high-throughput screening by transposon site hybridization through primary human macrophages, negatively selecting 202 genes. Of special note were genes in a locus of the Francisella chromosome, FTT1236, FTT1237, and FTT1238. Mutants with mutations in these genes demonstrated significant sensitivity to complement-mediated lysis compared with wild-type Schu S4 and exhibited marked defects in O-antigen and capsular polysaccharide biosynthesis. In the absence of complement, these mutants were phagocytosed more efficiently by macrophages than wild-type Schu S4 and were capable of phagosomal escape but exhibited reduced intracellular growth. Microscopic and quantitative analyses of macrophages infected with mutant bacteria revealed that these macrophages exhibited signs of cell death much earlier than those infected with Schu S4. These data suggest that FTT1236, FTT1237, and FTT1238 are important for polysaccharide biosynthesis and that the Francisella O antigen, capsule, or both are important for avoiding the early induction of macrophage death and the destruction of the replicative niche.

  3. 'Attached cell' antigen 28.3.7 mapping to human chromosome 15 characterises TPA-induced differentiation of the promyelocytic HL-60 cell line to give macrophage/monocyte populations.

    PubMed Central

    Blaineau, C; Avner, P; Tunnacliffe, A; Goodfellow, P

    1983-01-01

    Human cells growing in vitro attached to the substratum express a cell antigen called 28.3.7 identified by a species-specific monoclonal antibody. This antigen is not expressed on human cells growing in suspension. The antigen has a mol. wt. in reduced SDS-polyacrylamide gel electrophoresis gels of 95 000 and in human-mouse somatic cell hybrids, expression of the antigen is controlled by a gene, MIC7, mapping to human chromosome 15. The antigen functions as a marker for macrophage differentiation. In vitro differentiation of the 28.3.7 antigen-negative human promyelocytic leukaemia line HL-60 induced by phorbol ester, results in the formation of a macrophage/monocyte population and the concomitant expression of the 28.3.7 antigen on this adherent cell population. Images Fig. 1. PMID:6641710

  4. Oral delivery of human biopharmaceuticals, autoantigens and vaccine antigens bioencapsulated in plant cells

    PubMed Central

    Kwon, Kwang-Chul; Verma, Dheeraj; Singh, Nameirakpam D.; Herzog, Roland; Daniell, Henry

    2012-01-01

    Among 12 billion injections administered annually, unsafe delivery leads to >20 million infections and >100 million reactions. In an emerging new concept, freeze-dried plant cells (lettuce) expressing vaccine antigens/biopharmaceuticals are protected in the stomach from acids/enzymes but are released to the immune or blood circulatory system when plant cell walls are digested by microbes that colonize the gut. Vaccine antigens bioencapsulated in plant cells upon oral delivery after priming, conferred both mucosal and systemic immunity and protection against bacterial, viral or protozoan pathogens or toxin challenge. Oral delivery of autoantigens was effective against complications of type 1diabetes and hemophilia, by developing tolerance. Oral delivery of proinsulin or exendin-4 expressed in plant cells regulated blood glucose levels similar to injections. Therefore, this new platform offers a low cost alternative to deliver different therapeutic proteins to combat infectious or inherited diseases by eliminating inactivated pathogens, expensive purification, cold storage/transportation and sterile injections. PMID:23099275

  5. Diagnostic Accuracy of Antigen 5-Based ELISAs for Human Cystic Echinococcosis

    PubMed Central

    Pagnozzi, Daniela; Addis, Maria Filippa; Biosa, Grazia; Roggio, Anna Maria; Tedde, Vittorio; Mariconti, Mara; Tamarozzi, Francesca; Meroni, Valeria; Masu, Gabriella; Masala, Giovanna; Brunetti, Enrico; Uzzau, Sergio

    2016-01-01

    Background Clinical diagnosis and follow up of cystic echinococcosis (CE) are based on imaging complemented by serology. Several immunodiagnostic tests are commercially available, but the development of new tools is still needed to overcome the lack of standardization of the target antigen, generally consisting of a crude extract of Echinococcus granulosus hydatid cyst fluid. In a previous work, we described a chromatographic method for the preparation of a highly enriched Antigen 5 fraction from hydatid cyst fluid. The high reactivity of patient sera against this preparation prompted us to evaluate further this antigen for the serodiagnosis of CE on a larger cohort of samples. Methodology/Principal Findings A total of 327 sera from CE patients with heterogeneous conditions for cyst stage, cyst number, organ localization, drug therapy, and surgical intervention, together with 253 sera from healthy controls, were first analyzed by an ELISA based on the Ag5 preparation in two different experimental setups and, in parallel, by a commercial ELISA routinely used in clinical laboratories for CE serodiagnosis. The Ag5 ELISAs revealed different sensitivity (88.3% vs 95.3%) without significant differences in specificity (94.1% vs 92.5%), for the two setups, respectively. Moreover, possible relationships between the Ag5 ELISA absorbance results and clinical variables were investigated. Chi squared test, bivariate logistic regression and multiple regression analyses highlighted differences in the serology reactivity according to pharmacological treatment, cyst activity, and cyst number. Conclusions/Significance The two Ag5 ELISAs revealed different performances depending on the setup. The good diagnostic sensitivity and the high reliability of the Ag5 preparation method make this antigen a promising candidate for the serodiagnosis of CE. Further studies will be needed to evaluate the ability of our test to provide useful information on specific CE clinical traits. PMID

  6. Differential expression of HLA-DR antigens in subsets of human CFU-GM.

    PubMed

    Griffin, J D; Sabbath, K D; Herrmann, F; Larcom, P; Nichols, K; Kornacki, M; Levine, H; Cannistra, S A

    1985-10-01

    Expression of HLA-DR surface antigens by granulocyte/monocyte colony-forming cells (CFU-GM) may be important in the regulation of proliferation of these cells. Using immunological techniques to enrich for progenitor cells, we investigated the expression of HLA-DR in subsets of CFU-GM. "Early" (day 14) CFU-GM express higher levels of HLA-DR than do "late" (day 7) CFU-GM. Among late CFU-GM, cells destined to form monocyte (alpha-naphthyl acetate esterase-positive) colonies express higher levels of HLA-DR than do CFU-GM destined to form granulocyte (chloroacetate esterase-positive) colonies. Because high-level expression of DR antigen was a marker for monocyte differentiation, we examined several lymphokines for their effects on both DR expression and in vitro commitment to monocyte differentiation by myeloid precursor cells. DR antigen density could be increased by more than twofold over 48 hours upon exposure to gamma-interferon (gamma-IFN), whereas colony-stimulating factors had no effect. This was associated with a dose-dependent inhibition of total CFU-GM number, and a relative, but not absolute, increase in the ratio of monocyte colonies to granulocyte colonies. Similarly, in day 7 suspension cultures of purified myeloid precursor cells, gamma-IFN inhibited cell proliferation and increased the ratio of monocytes to granulocytes. Thus, despite the induction of high levels of HLA-DR antigen on precursor cells (a marker of monocyte commitment), the dominant in vitro effect of gamma-IFN was inhibition of granulocyte differentiation.

  7. Deletion of antigens of the Lewis a/b blood group family in human prostatic carcinoma.

    PubMed Central

    Young, W. W.; Mills, S. E.; Lippert, M. C.; Ahmed, P.; Lau, S. K.

    1988-01-01

    The expression of antigens of the blood group Lewis a/b family were studied in a series of 42 prostatectomy specimens from patients with adenocarcinoma clinically confined to the prostate; 19 of these were later reclassified as pathologic Stage C. Staining of normal or hyperplastic versus neoplastic epithelium was assessed in routinely processed, paraffin-embedded tissue using murine monoclonal antibodies and an avidin-biotin immunoperoxidase technique. Antigens screened and the antibodies used to recognize them were Lewis a (CF4C4), Lewis b and Type 1 H (NS10), monosialosyl Lewis a I (19.9), and disialosyl Lewis a and monosialosyl Lewis a II (FH7). FH7 strongly stained the benign epithelium of all 39 Lewis positive cases, suggesting that the sialyltransferase responsible for synthesis of FH7-reactive determinants is highly active in benign prostatic tissue. When compared to the reactivity of benign epithelium in Lewis positive cases, the staining of the carcinomas was markedly reduced in 18 cases (46%) and absent in 16 cases (41%). This reduction or loss of staining of the malignant epithelium was observed for all antibodies that stained the corresponding benign epithelium of each case. In only five of the cases (13%) was the intensity of staining in the carcinoma equal to that of the surrounding benign epithelium. No cases in this latter group had recurrence of disease, whereas in the other staining groups 25-33% of the cases had recurrences; median follow-up for the entire group was 78 months. No correlation was apparent between Gleason score and the staining pattern with these antigens. In summary, antigens of the Lewis a/b family are deleted in a high percentage of cases of prostatic adenocarcinoma. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:2454582

  8. Comparison of transformation and T antigen induction in human cell lines.

    PubMed

    Potter, C W; Potter, A M; Oxford, J S

    1970-03-01

    Skin fibroblast cultures were established from eight individuals. These cell cultures, together with WI-38 cells, were examined for susceptibility to transformation by SV40 virus. Four transformation-susceptible cell lines (TS), established from patients with Down's syndrome, were found to be three to four times more susceptible to transformation than transformation-resistant cell lines (TR) from normal individuals. TR and TS cell lines were compared for their susceptibility to induction of SV40 T antigen. For dividing cells T antigen was detected in a higher percentage of TS cells than TR cells. For nondividing cells, the reverse was found; T antigen was detected in 10-fold more cells of the TR lines than in cells of the TS lines. Similar results were obtained after infection of cells with CELO virus. Titration of vaccinia virus and influenza virus A2/Scotland/49/57 indicated that TR and TS cells were equally sensitive to the former virus, but TR cells were three to five times more sensitive to influenza virus A2/Scotland/49/57 than were TS cells.

  9. An improved DNA test for bovine leucocyte adhesion deficiency.

    PubMed

    Tammen, I; Klippert, H; Kuczka, A; Treviranus, A; Pohlenz, J; Stöber, M; Simon, D; Harlizius, B

    1996-05-01

    A modified DNA test, based on the polymerase chain reaction, was developed for the monogenic recessive disease bovine leucocyte adhesion deficiency (BLAD). The test was improved by the selection of new primers which facilitated the interpretation of the results. An easily scorable banding pattern makes the test useful in cattle breeding schemes and for clinical diagnosis. A total of 2381 samples was analysed over a period of three years. The carrier rate among young bulls at artificial insemination (AI) stations decreased from 11.6 per cent in 1993 to 9.9 per cent in the first five months of 1995. Continuous screening of young bulls before entering AI is still recommended unless both parents are proven to be genetically free of BLAD. The carrier rate among clinically suspect animals was not increased, and carriers are therefore not expected to be immunodeficient. Despite all efforts to eradicate the disease, calves with BLAD were still observed in 1995. PMID:8735510

  10. The promoter of the human cystic fibrosis transmembrane conductance regulator gene directing SV40 T antigen expression induces malignant proliferation of ependymal cells in transgenic mice.

    PubMed

    Perraud, F; Yoshimura, K; Louis, B; Dalemans, W; Ali-Hadji, D; Schultz, H; Claudepierre, M C; Chartier, C; Danel, C; Bellocq, J P

    1992-05-01

    Transgenic mice bearing a human cystic fibrosis transmembrane conductance regulator (CFTR) promoter-SV40 T antigen fusion transgene were generated in order to localize in vivo the potential oncogenesis linked to the tissue-specific activity of the promoter for the CFTR gene. Surprisingly, the only site of tumors resulting from expression of the reporter onc gene was ependymal cells lining the brain ventricles. SV40 T antigen expression in these cells led to a consistent pathology in the first weeks of age: ependymoma and consequent hydrocephaly. Tumor-derived cell lines were established, characterized and shown to originate from SV40 T antigen-induced ependymoma. No pathological alterations were found in other organs, such as lungs and pancreas, in which cystic fibrosis is pathologically manifest in humans. Such transgenic mice and derived cell lines may represent valid models for analysing (1) the role of SV40 T antigen in ependymoma formation and (2) CFTR function in ependymal cells. PMID:1373882

  11. The Role of XCR1 and its Ligand XCL1 in Antigen Cross-Presentation by Murine and Human Dendritic Cells

    PubMed Central

    Kroczek, Richard A.; Henn, Volker

    2012-01-01

    Recently, the chemokine receptor XCR1 has been found to be exclusively expressed on a subset of dendritic cell (DC) known to be involved in antigen cross-presentation. This review aims to summarize the known biology of the XCR1 receptor and its chemokine ligand XCL1, both in the mouse and the human. Further, any involvement of this receptor–ligand pair in antigen uptake, cross-presentation, and induction of innate and adaptive cytotoxic immunity is explored. The concept of antigen delivery to DC via the XCR1 receptor is discussed as a vaccination strategy for selective induction of cytotoxic immunity against certain pathogens or tumors. PMID:22566900

  12. Leucocyte function in Crohn's disease. Studies on mobilisation using a quantitative skin window technique and on the function of circulating polymorphonuclear leucocytes in vitro.

    PubMed Central

    Wandall, J H; Binder, V

    1982-01-01

    Leucocyte function was evaluated by mobilisation to skin windows with chambers and by the chemotactic, phagocytic, and nitro blue tetrazolium (NBT) reducing activity of circulating leucocytes in vitro in 20 patients with Crohn's disease, 21 healthy volunteers, and nine patients with sarcoidosis or tuberculosis. Leucocytes had been mobilised in significantly reduced numbers at 12, 24, 36, and 48 hours in Crohn's disease compared with healthy volunteers (P less than 0.01) and patients with sarcoidosis/tuberculosis (P less than 0.01). The leucocyte migration rate showed that mobilisation in Crohn's disease begins after a prolonged lag phase and is reduced compared with healthy volunteers (P less than 0.01) and patients with sarcoidosis/tuberculosis (P less than 0.02). The reduced mobilisation was not correlated with disease activity. In vitro random migration by leucocytes was slightly lower in Crohn's disease (P less than 0.05) than in healthy volunteers, but there was no difference after removal of the autologous plasma. Chemotactic response to casein did not differ between the groups studied. Serum independent and dependent phagocytosis did not differ from control groups. Serum independent phagocytosis was positively and significantly correlated to the disease activity (rho 0.4812, P less than 0.05). Resting leucocyte NBT reduction was increased in Crohn's disease and sarcoidosis/tuberculosis (P less than 0.01), but during phagocytosis a lower NBT reduction was found in Crohn's disease than in healthy volunteers (P less than 0.02). The inflammatory response in Crohn's disease, with reduced leucocyte accumulation, differs from patients with other granulomatous reactions and is independent of the disease activity. Our data suggest that the defect is not cellular. They support the hypothesis that a pathogenic factor in Crohn's disease may be foreign material that is normally eliminated remaining in the tissue and eliciting a chronic inflammatory response. PMID:7040174

  13. Generation of multi-functional antigen-specific human T-cells by lentiviral TCR gene transfer.

    PubMed

    Perro, M; Tsang, J; Xue, S-A; Escors, D; Cesco-Gaspere, M; Pospori, C; Gao, L; Hart, D; Collins, M; Stauss, H; Morris, E C

    2010-06-01

    T-cell receptor (TCR) gene transfer is an attractive strategy to generate antigen-specific T-cells for adoptive immunotherapy of cancer and chronic viral infection. However, current TCR gene transfer protocols trigger T-cell differentiation into terminally differentiated effector cells, which likely have reduced ability to mediate disease protection in vivo. We have developed a lentiviral gene transfer strategy to generate TCR-transduced human T-cells without promoting T-cell differentiation. We found that a combination of interleukin-15 (IL15) and IL21 facilitated lentiviral TCR gene transfer into non-proliferating T-cells. The transduced T-cells showed redirection of antigen specificity and produced IL2, IFNgamma and TNFalpha in a peptide-dependent manner. A significantly higher proportion of the IL15/IL21-stimulated T-cells were multi-functional and able to simultaneously produce all three cytokines (P<0.01), compared with TCR-transduced T-cells generated by conventional anti-CD3 plus IL2 stimulation, which primarily secreted only one cytokine. Similarly, IL15/IL21 maintained high levels of CD62L and CD28 expression in transduced T-cells, whereas anti-CD3 plus IL2 accelerated the loss of CD62L/CD28 expression. The data demonstrate that the combination of lentiviral TCR gene transfer together with IL15/IL21 stimulation can efficiently redirect the antigen specificity of resting primary human T-cells and generate multi-functional T-cells.

  14. Identification of human leukemia antigen A*0201-restricted epitopes derived from epidermal growth factor pathway substrate number 8

    PubMed Central

    TANG, BAISHAN; ZHOU, WEIJUN; DU, JINGWEN; HE, YANJIE; LI, YUHUA

    2015-01-01

    T-cell-mediated immunotherapy of hematological malignancies requires selection of targeted tumor-associated antigens and T-cell epitopes contained in these tumor proteins. Epidermal growth factor receptor pathway substrate 8 (EPS8), whose function is pivotal for tumor proliferation, progression and metastasis, has been found to be overexpressed in most human tumor types, while its expression in normal tissue is low. The aim of the present study was to identify human leukemia antigen (HLA)-A*0201-restricted epitopes of EPS8 by using a reverse immunology approach. To achieve this, computer algorithms were used to predict HLA-A*0201 molecular binding, proteasome cleavage patterns as well as translocation of transporters associated with antigen processing. Candidate peptides were experimentally validated by T2 binding affinity assay and brefeldin-A decay assay. The functional avidity of peptide-specific cytotoxic T lymphocytes (CTLs) induced from peripheral blood mononuclear cells of healthy volunteers were evaluated by using an enzyme-linked immunosorbent spot assay and a cytotoxicity assay. Four peptides, designated as P455, P92, P276 and P360, had high affinity and stability of binding towards the HLA-A*0201 molecule, and specific CTLs induced by them significantly responded to the corresponding peptides and secreted IFN-γ. At the same time, the CTLs were able to specifically lyse EPS8-expressing cell lines in an HLA-A*0201-restricted manner. The present study demon-strated that P455, P92, P276 and P360 were CTL epitopes of EPS8, and were able to be used for epitope-defined adoptive T-cell transfer and multi-epitope-based vaccine design. PMID:25936538

  15. Generation of multi-functional antigen-specific human T-cells by lentiviral TCR gene transfer.

    PubMed

    Perro, M; Tsang, J; Xue, S-A; Escors, D; Cesco-Gaspere, M; Pospori, C; Gao, L; Hart, D; Collins, M; Stauss, H; Morris, E C

    2010-06-01

    T-cell receptor (TCR) gene transfer is an attractive strategy to generate antigen-specific T-cells for adoptive immunotherapy of cancer and chronic viral infection. However, current TCR gene transfer protocols trigger T-cell differentiation into terminally differentiated effector cells, which likely have reduced ability to mediate disease protection in vivo. We have developed a lentiviral gene transfer strategy to generate TCR-transduced human T-cells without promoting T-cell differentiation. We found that a combination of interleukin-15 (IL15) and IL21 facilitated lentiviral TCR gene transfer into non-proliferating T-cells. The transduced T-cells showed redirection of antigen specificity and produced IL2, IFNgamma and TNFalpha in a peptide-dependent manner. A significantly higher proportion of the IL15/IL21-stimulated T-cells were multi-functional and able to simultaneously produce all three cytokines (P<0.01), compared with TCR-transduced T-cells generated by conventional anti-CD3 plus IL2 stimulation, which primarily secreted only one cytokine. Similarly, IL15/IL21 maintained high levels of CD62L and CD28 expression in transduced T-cells, whereas anti-CD3 plus IL2 accelerated the loss of CD62L/CD28 expression. The data demonstrate that the combination of lentiviral TCR gene transfer together with IL15/IL21 stimulation can efficiently redirect the antigen specificity of resting primary human T-cells and generate multi-functional T-cells. PMID:20164855

  16. A compact phage display human scFv library for selection of antibodies to a wide variety of antigens

    PubMed Central

    Pansri, Potjamas; Jaruseranee, Nanthnit; Rangnoi, Kuntalee; Kristensen, Peter; Yamabhai, Montarop

    2009-01-01

    Background Phage display technology is a powerful new tool for making antibodies outside the immune system, thus avoiding the use of experimental animals. In the early days, it was postulated that this technique would eventually replace hybridoma technology and animal immunisations. However, since this technology emerged more than 20 years ago, there have only been a handful reports on the construction and application of phage display antibody libraries world-wide. Results Here we report the simplest and highly efficient method for the construction of a highly useful human single chain variable fragment (scFv) library. The least number of oligonucleotide primers, electroporations and ligation reactions were used to generate a library of 1.5 × 108 individual clones, without generation of sub-libraries. All possible combinations of heavy and light chains, among all immunoglobulin isotypes, were included by using a mixture of primers and overlapping extension PCR. The key difference from other similar libraries was the highest diversity of variable gene repertoires, which was derived from 140 non-immunized human donors. A wide variety of antigens were successfully used to affinity select specific binders. These included pure recombinant proteins, a hapten and complex antigens such as viral coat proteins, crude snake venom and cancer cell surface antigens. In particular, we were able to use standard bio-panning method to isolate antibody that can bind to soluble Aflatoxin B1, when using BSA-conjugated toxin as a target, as demonstrated by inhibition ELISA. Conclusion These results suggested that by using an optimized protocol and very high repertoire diversity, a compact and efficient phage antibody library can be generated. This advanced method could be adopted by any molecular biology laboratory to generate both naïve or immunized libraries for particular targets as well as for high-throughput applications. PMID:19175944

  17. Analysis of a cDNA clone expressing a human autoimmune antigen: full-length sequence of the U2 small nuclear RNA-associated B antigen

    SciTech Connect

    Habets, W.J.; Sillekens, P.T.G.; Hoet, M.H.; Schalken, J.A.; Roebroek, A.J.M.; Leunissen, J.A.M.; Van de Ven, W.J.M.; Van Venrooij, W.J.

    1987-04-01

    A U2 small nuclear RNA-associated protein, designated B'', was recently identified as the target antigen for autoimmune sera from certain patients with systemic lupus erythematosus and other rheumatic diseases. Such antibodies enabled them to isolate cDNA clone lambdaHB''-1 from a phage lambdagt11 expression library. This clone appeared to code for the B'' protein as established by in vitro translation of hybrid-selected mRNA. The identity of clone lambdaHB''-1 was further confirmed by partial peptide mapping and analysis of the reactivity of the recombinant antigen with monospecific and monoclonal antibodies. Analysis of the nucleotide sequence of the 1015-base-pair cDNA insert of clone lambdaHB''-1 revealed a large open reading frame of 800 nucleotides containing the coding sequence for a polypeptide of 25,457 daltons. In vitro transcription of the lambdaHB''-1 cDNA insert and subsequent translation resulted in a protein product with the molecular size of the B'' protein. These data demonstrate that clone lambdaHB''-1 contains the complete coding sequence of this antigen. The deduced polypeptide sequence contains three very hydrophilic regions that might constitute RNA binding sites and/or antigenic determinants. These findings might have implications both for the understanding of the pathogenesis of rheumatic diseases as well as for the elucidation of the biological function of autoimmune antigens.

  18. Phosphorylation in vitro of Escherichia coli-produced 235R and 266R tumor antigens encoded by human adenovirus type 12 early transformation region 1A.

    PubMed Central

    Lucher, L A; Loewenstein, P M; Green, M

    1985-01-01

    The tumor (T) antigens encoded by the human adenovirus early transforming region 1A (E1A) are gene regulatory proteins whose functions can immortalize cells. We have recently described the synthesis in Escherichia coli and the purification of the complete T antigens encoded by the adenovirus type 12 (Ad12) E1A 12S mRNA (235-residue [235R] T antigen) and 13S mRNA (266R T antigen). In this study, we show that the Ad12 E1A T antigens are extensively phosphorylated in Ad12-infected mammalian cells but are not phosphorylated in E. coli. Inasmuch as posttranslational phosphorylation at specific amino acid sites may be important for biological activity, we have studied the phosphorylation of the E. coli-produced T antigens in vitro by using a kinase activity isolated from cultured human KB cells. The kinase was purified about 300-fold and appears to be a cyclic AMP-independent, Ca2+-independent protein kinase requiring only ATP and Mg2+ for activity. To determine which amino acids are phosphorylated and whether phosphorylation in vitro occurs at the same amino acid sites that are phosphorylated in vivo, the Ad12 E1A T-antigen species synthesized by infected cells were metabolically labeled with 32Pi and compared with the E. coli-produced E1A T antigens labeled in vitro with [gamma-32P]ATP by using the partially purified kinase. Partial V8 proteolysis analysis gave similar patterns for in vivo- and in vitro-phosphorylated T antigen. Two-dimensional maps of tryptic phosphopeptides and of chymotryptic phosphopeptides suggested that mainly the same amino acid sites are phosphorylated in vitro and in vivo and that phosphorylation occurred at multiple sites distributed throughout the T-antigen molecule. Serine was the only amino acid that was phosphorylated both in vivo and in vitro, and, surprisingly, most serines appeared to be phosphorylated. The feasibility of faithfully phosphorylating T antigens in vitro suggests that the E. coli-produced Ad12 E1A 235R and 266R T antigens

  19. Proinsulin multi-peptide immunotherapy induces antigen-specific regulatory T cells and limits autoimmunity in a humanized model.

    PubMed

    Gibson, V B; Nikolic, T; Pearce, V Q; Demengeot, J; Roep, B O; Peakman, M

    2015-12-01

    Peptide immunotherapy (PIT) is a targeted therapeutic approach, involving administration of disease-associated peptides, with the aim of restoring antigen-specific immunological tolerance without generalized immunosuppression. In type 1 diabetes, proinsulin is a primary antigen targeted by the autoimmune response, and is therefore a strong candidate for exploitation via PIT in this setting. To elucidate the optimal conditions for proinsulin-based PIT and explore mechanisms of action, we developed a preclinical model of proinsulin autoimmunity in a humanized HLA-DRB1*0401 transgenic HLA-DR4 Tg mouse. Once proinsulin-specific tolerance is broken, HLA-DR4 Tg mice develop autoinflammatory responses, including proinsulin-specific T cell proliferation, interferon (IFN)-γ and autoantibody production. These are preventable and quenchable by pre- and post-induction treatment, respectively, using intradermal proinsulin-PIT injections. Intradermal proinsulin-PIT enhances proliferation of regulatory [forkhead box protein 3 (FoxP3(+))CD25(high) ] CD4 T cells, including those capable of proinsulin-specific regulation, suggesting this as its main mode of action. In contrast, peptide delivered intradermally on the surface of vitamin D3-modulated (tolerogenic) dendritic cells, controls autoimmunity in association with proinsulin-specific IL-10 production, but no change in regulatory CD4 T cells. These studies define a humanized, translational model for in vivo optimization of PIT to control autoimmunity in type 1 diabetes and indicate that dominant mechanisms of action differ according to mode of peptide delivery. PMID:26206289

  20. Microbe-specific unconventional T-cells induce human neutrophil differentiation into antigen cross-presenting cells

    PubMed Central

    Liuzzi, Anna Rita; Tyler, Christopher J.; Khan, Mohd Wajid A.; Szakmany, Tamas; Hall, Judith E.; Moser, Bernhard; Eberl, Matthias

    2014-01-01

    The early immune response to microbes is dominated by the recruitment of neutrophils whose primary function is to clear invading pathogens. However, there is emerging evidence that neutrophils play additional effector and regulatory roles. The present study demonstrates that human neutrophils assume antigen cross-presenting functions, and suggests a plausible scenario for the local generation of APC-like neutrophils through the mobilization of unconventional T-cells in response to microbial metabolites. Vγ9/Vδ2 T-cells and MAIT cells are abundant in blood, inflamed tissues and mucosal barriers. Here, both human cell types responded rapidly to neutrophils after phagocytosis of Gram-positive and Gram-negative bacteria producing the corresponding ligands, and in turn mediated the differentiation of neutrophils into APCs for both CD4+ and CD8+ T-cells through secretion of GM-CSF, IFN-γ and TNF-α. In patients with acute sepsis, circulating neutrophils displayed a similar APC-like phenotype and readily processed soluble proteins for cross-presentation of antigenic peptides to CD8+ T-cells, at a time when peripheral Vγ9/Vδ2 T-cells were highly activated. Our findings indicate that unconventional T-cells represent key controllers of neutrophil-driven innate and adaptive responses to a broad range of pathogens. PMID:25165152

  1. A sandwich-type immunosensor using Pd-Pt nanocrystals as labels for sensitive detection of human tissue polypeptide antigen

    NASA Astrophysics Data System (ADS)

    Wang, Yaoguang; Wei, Qin; Zhang, Yong; Wu, Dan; Ma, Hongmin; Guo, Aiping; Du, Bin

    2014-02-01

    A sandwich-type immunosensor was developed for the detection of human tissue polypeptide antigen (hTPA). In this work, a graphene sheet (GS) was synthesized to modify the surface of a glassy carbon electrode (GCE), and Pd-Pt bimetallic nanocrystals were used as secondary-antibody (Ab2) labels for the fabrication of the immunosensor. The amperometric response of the immunosensor for catalyzing hydrogen peroxide (H2O2) was recorded. And electrochemical impedance spectroscopy was used to characterize the fabrication process of the immunosensor. The anti-human tissue polypeptide antigen primary antibody (Ab1) was immobilized onto the GS modified GCE via cross-linking with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide (EDC/NHS). With Ab1 immobilized onto the GS modified GCE and Ab2 linked on Pd-Pt bimetallic nanocrystals, the immunosensor demonstrated a wide linear range (0.0050-15 ng ml-1), a low detection limit (1.2 pg ml-1), good reproducibility, good selectivity and acceptable stability. This design strategy may provide many potential applications in the detection of other cancer biomarkers.

  2. SDS-PAGE/immunoblot detection of Abeta multimers in human cortical tissue homogenates using antigen-epitope retrieval.

    PubMed

    Rosen, Rebecca F; Tomidokoro, Yasushi; Ghiso, Jorge A; Walker, Lary C

    2010-01-01

    The anomalous folding and polymerization of the beta-amyloid (Abeta) peptide is thought to initiate the neurodegenerative cascade in Alzheimer's disease pathogenesis(1). Abeta is predominantly a 40- or 42-amino acid peptide that is prone to self-aggregation into beta-sheet-rich amyloid fibrils that are found in the cores of cerebral senile plaques in Alzheimer's disease. Increasing evidence suggests that low molecular weight, soluble Abeta multimers are more toxic than fibrillar Abeta amyloid(2). The identification and quantification of low- and high-molecular weight multimeric Abeta species in brain tissue is an essential objective in Alzheimer's disease research, and the methods employed also can be applied to the identification and characterization of toxic multimers in other proteopathies(3). Naturally occurring Abeta multimers can be detected by SDS-polyacrylamide gel electrophoresis followed by immunoblotting with Abeta-specific antibodies. However, the separation and detection of multimeric Abeta requires the use of highly concentrated cortical homogenates and antigen retrieval in small pore-size nitrocellulose membranes. Here we describe a technique for the preparation of clarified human cortical homogenates, separation of proteins by SDS-PAGE, and antigen-epitope retrieval/Western blotting with antibody 6E10 to the N-terminal region of the Abeta peptide. Using this protocol, we consistently detect Abeta monomers, dimers, trimers, tetramers, and higher molecular weight multimers in cortical tissue from humans with Alzheimer's pathology. PMID:20418805

  3. Engineered antigen-specific human regulatory T cells: immunosuppression of FVIII-specific T- and B-cell responses

    PubMed Central

    Kim, Yong Chan; Zhang, Ai-Hong; Su, Yan; Rieder, Sadiye Amcaoglu; Rossi, Robert J.; Ettinger, Ruth A.; Pratt, Kathleen P.; Shevach, Ethan M.

    2015-01-01

    Expansion of human regulatory T cells (Tregs) for clinical applications offers great promise for the treatment of undesirable immune responses in autoimmunity, transplantation, allergy, and antidrug antibody responses, including inhibitor responses in hemophilia A patients. However, polyclonal Tregs are nonspecific and therefore could potentially cause global immunosuppression. To avoid this undesirable outcome, the generation of antigen-specific Tregs would be advantageous. Herein, we report the production and properties of engineered antigen-specific Tregs, created by transduction of a recombinant T-cell receptor obtained from a hemophilia A subject’s T-cell clone, into expanded human FoxP3+ Tregs. Such engineered factor VIII (FVIII)-specific Tregs efficiently suppressed the proliferation and cytokine production of FVIII-specific T-effector cells. Moreover, studies with an HLA-transgenic, FVIII-deficient mouse model demonstrated that antibody production from FVIII-primed spleen cells in vitro were profoundly inhibited in the presence of these FVIII-specific Tregs, suggesting potential utility to treat anti-FVIII inhibitory antibody formation in hemophilia A patients. PMID:25498909

  4. Binding to histo-blood group antigen-expressing bacteria protects human norovirus from acute heat stress

    PubMed Central

    Li, Dan; Breiman, Adrien; le Pendu, Jacques; Uyttendaele, Mieke

    2015-01-01

    This study aims to investigate if histo-blood group antigen (HBGA) expressing bacteria have any protective role on human norovirus (NoV) from acute heat stress. Eleven bacterial strains were included, belonging to Escherichia coli, Enterobacter cloacae, Enterobacter aerogenes, Clostridium difficile, Bifidobacterium adolescentis, and B. longum. HBGA expression of the bacteria as well as binding of human NoV virus-like particles (VLPs, GI.1, and GII.4 strains) to the bacteria were detected by flow cytometry. NoV VLPs pre-incubated with HBGA expressing or non-HBGA expressing bacteria were heated and detected by both direct ELISA and porcine gastric mucin-binding assay. The NoV-binding abilities of the bacteria correlated well with their HBGA expression profiles. Two HBGA expressing E. coli (LMG8223 and LFMFP861, both GI.1 and GII.4 binders) and one non-HBGA expressing E. coli (ATCC8739, neither GI.1 nor GII.4 binder) were selected for the heat treatment test with NoV VLPs. Compared with the same cell numbers of non-HBGA expressing E. coli, the presence of HBGA-expressing E. coli could always maintain higher antigen integrity, as well as mucin-binding ability of NoV VLPs of both GI.1 and GII.4 after heat-treatment at 90°C for 2 min. These results indicate that HBGA-expressing bacteria may protect NoVs during the food processing treatments, thereby facilitating their transmission. PMID:26191052

  5. STUDIES OF PHAGOCYTOSIS OF GROUP A STREPTOCOCCI BY POLYMORPHONUCLEAR LEUCOCYTES IN VITRO

    PubMed Central

    Hirsch, James G.; Church, Alice B.

    1960-01-01

    Studies have been made on phagocytosis and killing of Group A streptococci during mixing with suspensions of leucocytes in vitro. Under appropriate test conditions an anti-phagocytic effect can be demonstrated for the streptococcal hyaluronic acid capsule as well as for its M protein. The results obtained suggest an explanation for the suitability of human, but not rabbit, blood for opsonophagocytic tests designed to measure type-specific streptococcal antibodies. Human sera contain a factor which counteracts the anti-phagocytic effects of streptococcal hyaluronic acid capsules, and hence human blood serves well for detection of antibodies which combine with the only other phagocytosis-resisting component of this microorganism, namely M protein. In contrast, rabbit sera contain none of this factor, and addition of antibody to M protein to phagocytic test systems employing rabbit serum does not necessarily render the streptococci susceptible to engulfment by white cells, since the hyaluronic acid capsule may continue to interfere with phagocytosis. The nature of the human serum factor which opsonizes encapsulated streptococci is unknown. It does not appear to be an antibody or an enzyme capable of depolymerizing hyaluronic acid. PMID:13714578

  6. Human antibody responses to VlsE antigenic variation protein of Borrelia burgdorferi.

    PubMed

    Lawrenz, M B; Hardham, J M; Owens, R T; Nowakowski, J; Steere, A C; Wormser, G P; Norris, S J

    1999-12-01

    VlsE is a 35-kDa surface-exposed lipoprotein of Borrelia burgdorferi that was shown previously to undergo antigenic variation through segmental recombination of silent vls cassettes with vlsE during experimental mouse infections. Previous data had indicated that sera from North American Lyme disease patients and experimentally infected animals contained antibodies reactive with VlsE. In this study, sera from patients with Lyme disease, syphilis, and autoimmune conditions as well as from healthy controls were examined for reactivity with VlsE by Western blotting and enzyme-linked immunosorbent assay (ELISA). Strong Western blot reactivity to a recombinant VlsE cassette region protein was obtained consistently with Lyme disease sera. Although sera from Lyme disease patients also reacted with a band corresponding to VlsE in B. burgdorferi B31-5A3, interpretation was complicated by low levels of VlsE expression in in vitro-cultured B. burgdorferi and by the presence of comigrating bands. An ELISA using recombinant VlsE was compared with an ELISA using sonically disrupted B. burgdorferi as the antigen. For a total of 93 Lyme disease patient sera examined, the VlsE ELISA yielded sensitivities of 63% for culture-confirmed erythema migrans cases and 92% for later stages, as compared to 61 and 98%, respectively, for the "whole-cell" ELISA. The specificities of the two assays with healthy blood donor sera were comparable, but the VlsE ELISA was 90% specific with sera from syphilis patients, compared to 20% specificity for the whole-cell ELISA with this group. Neither assay showed reactivity with a panel of sera from 20 non-Lyme disease arthritis patients or 20 systemic lupus erythematosus patients. Our results indicate that VlsE may be useful in the immunodiagnosis of Lyme disease and may offer greater specificity than ELISAs using whole B. burgdorferi as the antigen.

  7. Human Antibody Responses to VlsE Antigenic Variation Protein of Borrelia burgdorferi

    PubMed Central

    Lawrenz, M. B.; Hardham, J. M.; Owens, R. T.; Nowakowski, J.; Steere, A. C.; Wormser, G. P.; Norris, S. J.

    1999-01-01

    VlsE is a 35-kDa surface-exposed lipoprotein of Borrelia burgdorferi that was shown previously to undergo antigenic variation through segmental recombination of silent vls cassettes with vlsE during experimental mouse infections. Previous data had indicated that sera from North American Lyme disease patients and experimentally infected animals contained antibodies reactive with VlsE. In this study, sera from patients with Lyme disease, syphilis, and autoimmune conditions as well as from healthy controls were examined for reactivity with VlsE by Western blotting and enzyme-linked immunosorbent assay (ELISA). Strong Western blot reactivity to a recombinant VlsE cassette region protein was obtained consistently with Lyme disease sera. Although sera from Lyme disease patients also reacted with a band corresponding to VlsE in B. burgdorferi B31-5A3, interpretation was complicated by low levels of VlsE expression in in vitro-cultured B. burgdorferi and by the presence of comigrating bands. An ELISA using recombinant VlsE was compared with an ELISA using sonically disrupted B. burgdorferi as the antigen. For a total of 93 Lyme disease patient sera examined, the VlsE ELISA yielded sensitivities of 63% for culture-confirmed erythema migrans cases and 92% for later stages, as compared to 61 and 98%, respectively, for the “whole-cell” ELISA. The specificities of the two assays with healthy blood donor sera were comparable, but the VlsE ELISA was 90% specific with sera from syphilis patients, compared to 20% specificity for the whole-cell ELISA with this group. Neither assay showed reactivity with a panel of sera from 20 non-Lyme disease arthritis patients or 20 systemic lupus erythematosus patients. Our results indicate that VlsE may be useful in the immunodiagnosis of Lyme disease and may offer greater specificity than ELISAs using whole B. burgdorferi as the antigen. PMID:10565921

  8. Immunological evaluation of a 26-kDa antigen from Taenia solium larvae for specific immunodiagnosis of human neurocysticercosis.

    PubMed

    Ev, L V; Maia, A A; Pianetti, G; Nascimento, E

    1999-02-01

    Human neurocysticercosis, due to infection of the central nervous system by cysticerci of Taenia solium, is a severe form of neurologic disease occurring in Central and South America. Specific proteins from scolex antigen from cysticerci were purified by polyacrylamide gel electrophoresis and electroelution and recognized in Western blots by antibodies present in sera from patients with neurocysticercosis. The proteins appeared as 13-, 17-, and 26-kDa bands on Coomassie blue-stained gels and proved to be specific to cysticerci of T. solium. No cross-reactivity with sera from patients with taeniasis or hydatidosis was observed. Enzyme-linked immunosorbent assay using the purified proteins of 13, 17, and 26 kDa demonstrated rates of 53%, 88%, and 100% specificity, respectively, at the cutoff serum dilution of 1:32 for the specific immunodiagnosis of human neurocysticercosis.

  9. Antigenic analysis of divergent genotypes human Enterovirus 71 viruses by a panel of neutralizing monoclonal antibodies: current genotyping of EV71 does not reflect their antigenicity.

    PubMed

    Chen, Yixin; Li, Chuan; He, Delei; Cheng, Tong; Ge, Shengxiang; Shih, James Wai-Kuo; Zhao, Qinjian; Chen, Pei-Jer; Zhang, Jun; Xia, Ningshao

    2013-01-01

    In recent year, Enterovirus 71 (EV71)-associated hand, foot and mouth disease (HFMD) has become an important public health issue in China. EV71 has been classified into genotypes A, B1-B5 and C1-C5. With such genetic diversity, whether the convalescent or recovery antibody responses can cross-protect infections from other genotypes remains a question. Understanding of the antigenicity of such diverse genetic EV71 isolates is crucial for the EV71 vaccine development. Here, a total of 186 clones anti-EV71 MAbs was generated and characterized with Western blot and cell-based neutralization assay. Forty neutralizing anti-EV71 MAbs were further used to analyze the antigenic properties of 18 recent EV71 isolates representing seven genotypes in neutralization assay. We found that most neutralizing anti-EV71 MAbs are specific to conformational epitopes. We also classified the 40 neutralizing anti-EV71 MAbs into two classes according to their reactivity patterns with 18 EV71 isolates. Class I MAb can neutralize all isolates, suggesting conserved epitopes are present among EV71. Class II MAb includes four subclasses (IIa-IId) and neutralizes only subgroups of EV71 strains. Conversely, 18 EV71 strains were grouped into antigenic types 1 and four antigenic subtypes (2.1-2.4). These results suggest that the current genotyping of EV71 does not reflect their antigenicity which may be important in the selection of EV71 vaccine strains. This panel of neutralizing anti-EV71 MAbs may be useful for the recognition of emerging antigenic variants of EV71 and vaccine development.

  10. Sporozoite and liver stage antigen Plasmodium falciparum peptides bind specifically to human hepatocytes.

    PubMed

    Puentes, Alvaro; García, Javier; Vera, Ricardo; López, Ramsés; Suarez, Jorge; Rodríguez, Luis; Curtidor, Hernando; Ocampo, Marisol; Tovar, Diana; Forero, Martha; Bermudez, Adriana; Cortes, Jimena; Urquiza, Mauricio; Patarroyo, Manuel E

    2004-03-12

    Sporozoite and Liver Stage Antigen (SALSA) sequence synthetic peptides were used in HepG2 cell binding assays to identify regions involved in parasite invasion. SALSA 20608 ( 21IWASEKKDEKEASEQGEESHY40) and 20611 ( 64KKDDGTDKVQEKVLEKSPKY83) peptides were determined as having high binding activity in HepG2 cell assays, some of them were located in immunogenic regions. Immune-fluorescence antibody test with 24276 (20608 peptide analogue, CGIWSSMKMDEKMAAMQGEESHCG) showed sporozoite and merozoite reactivity. This data suggests SALSA high activity binding peptides' (HABPs) possible role in hepatic cell invasion and merozoite invasion of erythrocytes.

  11. Genome Sequence of Enterobacter cloacae Strain SENG-6, a Bacterium Producing Histo-Blood Group Antigen-Like Substances That Can Bind with Human Noroviruses

    PubMed Central

    Amarasiri, Mohan; Hashiba, Satoshi; Yang, Peiyi; Okabe, Satoshi

    2016-01-01

    Enterobacter sp. strain SENG-6, isolated from healthy human feces, produces histo-blood group antigen (HBGA)-like substances that can bind with human noroviruses. Based on the genome sequence analysis, strain SENG-6 belongs to the species Enterobacter cloacae. The genome sequence of this strain should help identify genes associated with the production of HBGA-like substances. PMID:27563051

  12. Genome Sequence of Enterobacter cloacae Strain SENG-6, a Bacterium Producing Histo-Blood Group Antigen-Like Substances That Can Bind with Human Noroviruses.

    PubMed

    Ishii, Satoshi; Amarasiri, Mohan; Hashiba, Satoshi; Yang, Peiyi; Okabe, Satoshi; Sano, Daisuke

    2016-01-01

    Enterobacter sp. strain SENG-6, isolated from healthy human feces, produces histo-blood group antigen (HBGA)-like substances that can bind with human noroviruses. Based on the genome sequence analysis, strain SENG-6 belongs to the species Enterobacter cloacae The genome sequence of this strain should help identify genes associated with the production of HBGA-like substances. PMID:27563051

  13. Genome Sequence of Enterobacter cloacae Strain SENG-6, a Bacterium Producing Histo-Blood Group Antigen-Like Substances That Can Bind with Human Noroviruses.

    PubMed

    Ishii, Satoshi; Amarasiri, Mohan; Hashiba, Satoshi; Yang, Peiyi; Okabe, Satoshi; Sano, Daisuke

    2016-01-01

    Enterobacter sp. strain SENG-6, isolated from healthy human feces, produces histo-blood group antigen (HBGA)-like substances that can bind with human noroviruses. Based on the genome sequence analysis, strain SENG-6 belongs to the species Enterobacter cloacae The genome sequence of this strain should help identify genes associated with the production of HBGA-like substances.

  14. Differentiation induced by physiological and pharmacological stimuli leads to increased antigenicity of human neuroblastoma cells.

    PubMed

    Carlson, Lena-Maria; Påhlman, Sven; De Geer, Anna; Kogner, Per; Levitskaya, Jelena

    2008-03-01

    Sympathetic neuronal differentiation is associated with favorable prognosis of neuroblastoma (NB), the most common extra-cranial solid tumor of early childhood. Differentiation agents have proved useful in clinical protocols of NB treatment, but using them as a sole treatment is not sufficient to induce tumor elimination in patients. Therefore, complementary approaches, such as immunotherapy, are warranted. Here we demonstrate that differentiation of NB cell lines and ex vivo isolated tumor cells in response to physiological or pharmacological stimuli is associated with acquisition of increased antigenicity. This manifests as increased expression of surface major histocompatibility class I complexes and ICAM-1 molecules and translates into increased sensitivity of NB cells to lysis by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. The latter is paralleled by enhanced ability of differentiated cells to form immune conjugates and bind increased amounts of granzyme B to the cell surface. We demonstrate, for the first time, that, regardless of the stimulus applied, the differentiation state in NBs is associated with increased tumor antigenicity that enables more efficient elimination of tumor cells by cytotoxic lymphocytes and paves the way for combined application of differentiation-inducing agents and immunotherapy as an auxiliary approach in NB patients.

  15. Antibodies to porin antigens of Salmonella typhi induced during typhoid infection in humans.

    PubMed Central

    Calderón, I; Lobos, S R; Rojas, H A; Palomino, C; Rodríguez, L H; Mora, G C

    1986-01-01

    Immunoglobulin G (IgG)- and IgM-specific antibody titers against Salmonella typhi Ty2 porins have been measured in 30 paired typhoid sera by enzyme-linked immunosorbent assay. These studies have found that IgG serum titers of acute and convalescent sera were 625 and 5,000 times higher, respectively than the control serum titers. The same typhoid sera were titrated with S. typhi Ty2 flagellin and S. typhi lipopolysaccharide. The titers against these antigens were considerably lower than those against the porins. The highest IgM-specific titer has also been found against porins in convalescent-phase sera. However, the largest increase in IgM-specific titer compared with the control group titer was obtained against flagellin during the acute phase of typhoid. The lowest increases in antibody titer were obtained with the IgM-specific anti-lipopolysaccharide in both types of sera. This may be because many normal individuals in endemic areas already have IgM titers against lipopolysaccharide. This study has provided good evidence that porins are excellent antigens and that IgG-specific antiporin titers may be of diagnostic value in typhoid infections in endemic areas. PMID:3007360

  16. Receptor Binding Sites and Antigenic Epitopes on the Fiber Knob of Human Adenovirus Serotype 3

    PubMed Central

    Liebermann, Herbert; Mentel, Renate; Bauer, Ulrike; Pring-Åkerblom, Patricia; Dölling, Rudolf; Modrow, Susanne; Seidel, Werner

    1998-01-01

    The adenovirus fiber knob causes the first step in the interaction of adenovirus with cell membrane receptors. To obtain information on the receptor binding site(s), the interaction of labeled cell membrane proteins to synthetic peptides covering the adenovirus type 3 (Ad3) fiber knob was studied. Peptide P6 (amino acids [aa] 187 to 200), to a lesser extent P14 (aa 281 to 294), and probably P11 (aa 244 to 256) interacted specifically with cell membrane proteins, indicating that these peptides present cell receptor binding sites. Peptides P6, P11, and P14 span the D, G, and I β-strands of the R-sheet, respectively. The other reactive peptides, P2 (aa 142 to 156), P3 (aa 153 to 167), and P16 (aa 300 to 319), probably do not present real receptor binding sites. The binding to these six peptides was inhibited by Ad3 virion and was independent of divalent cations. We have also screened the antigenic epitopes on the knob with recombinant Ad3 fiber, recombinant Ad3 fiber knob, and Ad3 virion-specific antisera by enzyme-linked immunosorbent assay. The main antigenic epitopes were presented by P3, P6, P12 (aa 254 to 269), P14, and especially the C-terminal P16. Peptides P14 and P16 of the Ad3 fiber knob were able to inhibit Ad3 infection of cells. PMID:9765458

  17. Glycan analysis of Fonsecaea monophora from clinical and environmental origins reveals different structural profile and human antigenic response.

    PubMed

    Burjack, Juliana R; Santana-Filho, Arquimedes P; Ruthes, Andrea C; Riter, Daniel S; Vicente, Vania A; Alvarenga, Larissa M; Sassaki, Guilherme L

    2014-01-01

    Dematiaceous fungi constitute a large and heterogeneous group, characterized by having a dark pigment, the dihydroxynaftalen melanin-DHN, inside their cell walls. In nature they are found mainly as soil microbiota or decomposing organic matter, and are spread in tropical and subtropical regions. The fungus Fonsecaea monophora causes chromoblastomycosis in humans, and possesses essential mechanisms that may enhance pathogenicity, proliferation and dissemination inside the host. Glycoconjugates confer important properties to these pathogenic microorganisms. In this work, structural characterization of glycan structures present in two different strains of F. monophora MMHC82 and FE5p4, from clinical and environmental origins, respectively, was performed. Each one were grown on Minimal Medium (MM) and Czapeck-Dox (CD) medium, and the water soluble cell wall glycoconjugates and exopolysaccharides (EPS) were evaluated by NMR, methylation and principal component analysis (PCA). By combining the methylation and 2D NMR analyses, it was possible to visualize the glycosidic profiles of the complex carbohydrate mixtures. Significant differences were observed in β-D-Galf-(1→5) and (1→6) linkages, α- and β-D-Glcp-(1→3), (1→4), and (1→6) units, as well as in α-D-Manp. PCA from (1)H-NMR data showed that MMHC82 from CD medium showed a higher variation in the cell wall carbohydrates, mainly related to O-2 substituted β-D-Galf (δ 106.0/5.23 and δ 105.3/5.23) units. In order to investigate the antigenic response of the glycoconjugates, these were screened against serum from chromoblastomycosis patients. The antigen which contained the cell wall of MMHC82 grown in MM had β-D-Manp units that promoted higher antigenic response. The distribution of these fungal species in nature and the knowledge of how cell wall polysaccharides and glycoconjugates structure vary, may contribute to the better understanding and the elucidation of the pathology caused by this fungus.

  18. Soluble intercellular adhesion molecules in human schistosomiasis: correlations with disease severity and decreased responsiveness to egg antigens.

    PubMed Central

    Secor, W E; dos Reis, M G; Ramos, E A; Matos, E P; Reis, E A; do Carmo, T M; Harn, D A

    1994-01-01

    Granuloma formation, the principal pathologic consequence of infection with Schistosoma mansoni, is a complex process involving intricate cell-cell interactions in which intercellular adhesion molecules are likely to participate. To examine this possibility, sera of schistosomiasis patients in various clinical groups were assayed for the presence of soluble intercellular adhesion molecule 1 (sICAM-1) and soluble E-selectin (sE-selectin). Comparisons were made between groups with different infection intensities (as predicted by fecal egg count) as well as between groups with severe (hepatosplenic) or milder (intestinal) pathology. All groups had elevated levels of sICAM-1 compared with controls. Also, patients in the high egg-excreting and hepatosplenic groups had significantly higher levels of serum sICAM-1 than patients in the low-egg-excreting and intestinal groups, respectively. The levels of sE-selectin were significantly elevated in the sera of all patients except those in the hepatosplenic group compared with controls. Patients in the intestinal group had significantly higher levels of sE-selectin in their sera than did hepatosplenic group patients, but serum sE-selectin levels of high- and low-egg-excreting patients were comparable. A striking finding of this study was the inverse correlation observed between sICAM-1 levels and peripheral blood mononuclear cell responses to schistosome soluble egg antigens (SEA) but not with responses to other schistosome antigens, purified protein derivative, or mitogen. Because ICAM-1 can perform a costimulatory function in antigen-presenting cell-T cell interactions, it is possible that shedding of ICAM-1 in the granuloma microenvironment interrupts proper costimulation, leading to unresponsive SEA-specific T cells. In this way, sICAM-1 could be one factor contributing to the observed modulation of cellular responses to SEA in chronic human schistosomiasis. PMID:7516309

  19. The Dynamics of the Human Leukocyte Antigen Head Domain Modulates Its Recognition by the T-Cell Receptor

    PubMed Central

    García-Guerrero, Estefanía; Pérez-Simón, José Antonio; Sánchez-Abarca, Luis Ignacio; Díaz-Moreno, Irene; De la Rosa, Miguel A.; Díaz-Quintana, Antonio

    2016-01-01

    Generating the immune response requires the discrimination of peptides presented by the human leukocyte antigen complex (HLA) through the T-cell receptor (TCR). However, how a single amino acid substitution in the antigen bonded to HLA affects the response of T cells remains uncertain. Hence, we used molecular dynamics computations to analyze the molecular interactions between peptides, HLA and TCR. We compared immunologically reactive complexes with non-reactive and weakly reactive complexes. MD trajectories were produced to simulate the behavior of isolated components of the various p-HLA-TCR complexes. Analysis of the fluctuations showed that p-HLA binding barely restrains TCR motions, and mainly affects the CDR3 loops. Conversely, inactive p-HLA complexes displayed significant drop in their dynamics when compared with its free versus ternary forms (p-HLA-TCR). In agreement, the free non-reactive p-HLA complexes showed a lower amount of salt bridges than the responsive ones. This resulted in differences between the electrostatic potentials of reactive and inactive p-HLA species and larger vibrational entropies in non-elicitor complexes. Analysis of the ternary p-HLA-TCR complexes also revealed a larger number of salt bridges in the responsive complexes. To summarize, our computations indicate that the affinity of each p-HLA complex towards TCR is intimately linked to both, the dynamics of its free species and its ability to form specific intermolecular salt-bridges in the ternary complexes. Of outstanding interest is the emerging concept of antigen reactivity involving its interplay with the HLA head sidechain dynamics by rearranging its salt-bridges. PMID:27124285

  20. The Dynamics of the Human Leukocyte Antigen Head Domain Modulates Its Recognition by the T-Cell Receptor.

    PubMed

    García-Guerrero, Estefanía; Pérez-Simón, José Antonio; Sánchez-Abarca, Luis Ignacio; Díaz-Moreno, Irene; De la Rosa, Miguel A; Díaz-Quintana, Antonio

    2016-01-01

    Generating the immune response requires the discrimination of peptides presented by the human leukocyte antigen complex (HLA) through the T-cell receptor (TCR). However, how a single amino acid substitution in the antigen bonded to HLA affects the response of T cells remains uncertain. Hence, we used molecular dynamics computations to analyze the molecular interactions between peptides, HLA and TCR. We compared immunologically reactive complexes with non-reactive and weakly reactive complexes. MD trajectories were produced to simulate the behavior of isolated components of the various p-HLA-TCR complexes. Analysis of the fluctuations showed that p-HLA binding barely restrains TCR motions, and mainly affects the CDR3 loops. Conversely, inactive p-HLA complexes displayed significant drop in their dynamics when compared with its free versus ternary forms (p-HLA-TCR). In agreement, the free non-reactive p-HLA complexes showed a lower amount of salt bridges than the responsive ones. This resulted in differences between the electrostatic potentials of reactive and inactive p-HLA species and larger vibrational entropies in non-elicitor complexes. Analysis of the ternary p-HLA-TCR complexes also revealed a larger number of salt bridges in the responsive complexes. To summarize, our computations indicate that the affinity of each p-HLA complex towards TCR is intimately linked to both, the dynamics of its free species and its ability to form specific intermolecular salt-bridges in the ternary complexes. Of outstanding interest is the emerging concept of antigen reactivity involving its interplay with the HLA head sidechain dynamics by rearranging its salt-bridges.

  1. Presentation of human minor histocompatibility antigens by HLA-B35 and HLA-B38 molecules.

    PubMed Central

    Yamamoto, J; Kariyone, A; Akiyama, N; Kano, K; Takiguchi, M

    1990-01-01

    Cytotoxic T lymphocyte (CTL) clones specific for human minor histocompatibility antigens (hmHAs) were produced from a patient who had been grafted with the kidneys from his mother and two HLA-identical sisters. Of eight CTL clones generated, four recognized an hmHA (hmHA-1) expressed on cells from the mother and sister 3 (second donor); two recognized another antigen (hmHA-2) on cells from the father, sister 2 (third donor), and sister 3; and the remaining two clones recognized still another antigen (hmHA-3) on cells from the father and sister 3. Panel studies revealed that CTL recognition of hmHA-1 was restricted by HLA-B35 and that of hmHA-2 and hmHA-3 was restricted by HLA-B38. The HLA-B35 restriction of the hmHA-1-specific CTL clones was substantiated by the fact that they killed HLA-A null/HLA-B null Hmy2CIR targets transfected with HLA-B35 but not HLA-B51, -Bw52, or -Bw53 transfected Hmy2CIR targets. These data demonstrated that the five amino acids substitutions on the alpha 1 domain between HLA-B35 and -Bw53, which are associated with Bw4/Bw6 epitopes, play a critical role in the relationship of hmHA-1 to HLA-B35 molecules. The fact that the hmHA-1-specific CTLs failed to kill Hmy2CIR cells expressing HLA-B35/51 chimeric molecules composed of the alpha 1 domain of HLA-B35 and other domains of HLA-B51 indicated that eight residues on the alpha 2 domain also affect the interaction of hmHA-1 and the HLA-B35 molecules. PMID:2157206

  2. The Dynamics of the Human Leukocyte Antigen Head Domain Modulates Its Recognition by the T-Cell Receptor.

    PubMed

    García-Guerrero, Estefanía; Pérez-Simón, José Antonio; Sánchez-Abarca, Luis Ignacio; Díaz-Moreno, Irene; De la Rosa, Miguel A; Díaz-Quintana, Antonio

    2016-01-01