Association of class II HLA alleles with susceptibility to develop immune-mediated diseases in Paraguayan patients

Genetic and nongenetic factors are involved in the pathogenesis of immune-mediated inflammatory diseases (IMIDs). The best-known genetic factor for susceptibility to IMIDs is the human leukocyte antigen (HLA). The aim of the present study was to evaluate the association of HLA class II genes with the risk of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and systemic sclerosis (SSc) in the Paraguayan population. We included 254 patients with IMIDs (101 SLE, 103 RA, and 50 SSc) and 50 healthy controls. The haplotypes of five genes corresponding to HLA class II genes and their relationship to the IMIDs studied were determined. Note that 84.6% were women, with a mean age of 43.4 ± 14 years. Among the associated HLA alleles, we found the previously identified risk factors in other populations like HLA-DRB1*03:01 and HLA-DRB1*14:02 for RA, as well as new ones not previously identified, such as DPA1*02:01 for SLE and, DB1*02:01 for RA and SSc. In the genetic association analysis, already known associations have been replicated, and unpublished associations have been identified in Paraguayan patients with IMIDs. This is the first genetic association study in Paraguayan patients with IMIDs.     

Prevalence and specificity of immunoglobulin G and immunoglobulin A non-complement-binding anti-HLA alloantibodies in retransplant candidates

The role of complement-binding donor-directed anti-human leukocyte antigen (HLA) antibodies in graft rejection is well established, whereas the prevalence and relevance of non-complement-binding (NCB) anti-HLA antibodies are less well defined. The aim of our study was to establish a sensitive and reliable test system for the detection and the specification of these NCB anti-HLA antibodies. Sera from 60 patients awaiting retransplantation were analysed for the presence of anti-HLA class I alloantibodies with complement-dependent cytotoxicity (CDC) tests. Immunoglobulin (Ig)G(all) anti-HLA class I and class II alloantibodies were differentiated on generic level by plate-based solid phase enzyme-linked immunosorbent assay. Subsequently, a modified bead-based (Luminex) assay was applied, allowing the investigation of IgG(2/4) NCB isotypes as well as IgA(1/2). The anti-HLA specificities of the NCB alloantibodies were determined and compared with known mismatches from previous transplants. Seventeen of the 60 sera (28%) were positive in the CDC increasing to 26 of 60 (43%) in the class I and 33 of 60 (55%) in the class II plate-based assay. Using the modified bead-based system 24 of 60 sera (40%) contained NCB IgG(2/4), which were mostly donor specific. In addition, a high prevalence of NCB IgA antibodies was detected (26 of 60 sera), which occurred independently of IgG(2/4) NCB, and half of which were donor specific. NCB anti-HLA alloantibodies, including the IgA isotype, can reliably be detected using the modified bead-based test system. These NCB alloantibodies had a high prevalence in retransplant candidates and were mostly donor specific.     

A new enzyme-linked immunosorbent assay (ELISA) for measuring immunoconglutinins directed against the third component of human complement. Findings in systemic lupus erythematosus

A new enzyme-linked immunosorbent assay, using purified activation product of the third component of human complement (C3b), detects immunoglobulins of the IgG isotype which demonstrate affinity for C3b (C3b-immunoconglutinins; C3b-IK). This assay offers significantly improved specificity compared to previous immunoconglutinin (IK) assays in that it not only defines the isotype and antigenic specificity of the IK but also eliminates false positive results associated with immune complexes or aggregated human gamma globulin, or with natural antibodies directed at heterologous reagents. Using this assay, we observed elevated C3b-IK levels in serum of 34 systemic lupus erythematosus (SLE) patients when compared to serum of 13 healthy controls. Comparing sera from patients with clinically active and clinically inactive lupus showed greater immunoconglutinin levels in the active group. Immunoconglutinin levels did not correlate with the erythrocyte sedimentation rate, total hemolytic complement, or with circulating immune complex levels by the Raji cell and C1q-binding assays.     

Decreased levels of autoantibodies against apolipoprotein B‐100 antigens are associated with cardiovascular disease in systemic lupus erythematosus

Increased production of autoantibodies is a characteristic feature of systemic lupus erythematosus (SLE) and there is evidence that several of these autoantibodies may contribute to increased cardiovascular disease (CVD) in SLE. Autoantibodies against the apolipoprotein (apo) B‐100 peptides p45 and p210 have been associated with a lower CVD risk in non‐SLE cohorts. The aim of the present study was to investigate how SLE affects the occurrence of these potentially protective autoantibodies. The study cohort consisted of 434 SLE patients and 322 age‐ and sex‐matched population controls. Antibodies against native and malondialdehyde (MDA)‐modified p45 and p210 were measured by enzyme‐linked immunosorbent assay (ELISA). SLE patients had significantly lower levels of p210 immunoglobulin (Ig)G and p45 IgM (both the native and malondialdehyde (MDA)‐modified forms). SLE patients with manifest CVD (myocardial infarction, ischaemic cerebrovascular disease or peripheral vascular disease) had lower levels p210 IgG and p45 IgM than SLE patients without CVD. Decreased levels of these autoantibodies were also observed in SLE patients with permanent organ damage, as assessed by the Systemic Lupus International Collaborating Clinics/American College of Rheumatology (ACR) Damage Index (SDI). The present findings show that patients with SLE, a condition generally characterized by abundance of autoantibodies of multiple specificities, have reduced levels of antibodies against the apo B‐100 antigens p45 and p210 and that the levels of these antibodies are reduced further in SLE patients with CVD. These observations suggest the possibility that an impaired antibody‐mediated removal of damaged LDL particles may contribute to the development of vascular complications and organ damage in SLE.     

检测抗人疱疹病毒6型IgG的间接免疫荧光试验的建立及其应用

目的 :建立检测血清中人疱疹病毒 6型 (HHV 6 )IgG的间接免疫荧光试验 (IFA)。方法 :用HHV 6国内分离株感染人脐带血单个核细胞制备抗原片 ,建立检测HHV 6IgG的IFA方法 ,并用于育龄期妇女血清流行病学调查。结果 :建立的IFA具有特异性。对 116份育龄期妇女血清标本检测表明 ,HHV 6IgG的阳性率为 72 .4 % ,几何平均滴度 (GMT)为 1∶6 1;在孕妇和正常未孕妇女之间 ,以及不同孕期的孕妇之间 ,HHV 6IgG的阳性率和GMT均无差异 (P >0 .0 5 )。结论 :建立了具有特异性的IFA法 ,可用于对育龄妇女HHV 6感染率的流行病学调查     

Human immunoglobulin production in immunodeficient mice: enhancement by immunosuppression of host and in vitro activation of human mononuclear cells.

The affect of host and donor related factors on successful engraftment of human cells into mice was examined to minimize the variability that has been observed in successful development of human-mouse chimera for the study of human disease and immune physiology and regulation. Human immunoglobulin production in severe combined immunodeficiency (SCID) mice engrafted with human peripheral blood mononuclear cells (PBMC) was augmented by immunosuppressing recipient mice and activating donor PBMC. Immunosuppression of recipient mice with 3 Gy of gamma-irradiation induced a 10-fold increase in human IgG in the sera of engrafted SCID mice. Variation in production of human IgG in recipient mice correlated with preinjection phenotype and activation status of injected PBMC. Mice injected with PBMC with a low CD4/CD8 ratio (less than 0.5) produced no detectable circulating human immunoglobulin. When the CD4/CD8 ratio was greater than 1.5, human IgG was detected in sera of PBMC-recipient SCID mice. Serum IgG increased 10-fold following in vitro activation of donor PBMC with anti-CD3, IL-2 and Staphylococcus aureus. Successful engraftment and serum IgG production was evidenced by an increase in the recovery of activated human IgG+ cells in the spleens of mice with maximal IgG production. Optimization of functional engraftment required modification of both the host (SCID mice) and the donor cells.     

Pharmacodynamics of recombinant human DNase I in serum

Recombinant human deoxyribonuclease I (rhDNase) may be an effective therapeutic for the treatment of systemic lupus erythematosus (SLE). The pharmacodynamics of rhDNase in serum was investigated using two activity assays: one based on hydrolysis of a radiolabelled phage DNA and the other based on hydrolysis of human chromatin. The concentration of endogenous immunoreactive DNase in sera from 16 normal subjects was 3.2 ± 1.4 ng/ml (mean ± s.d.); however, low levels or no nuclease activity were detected in the same sera, suggesting the presence of DNase inhibitors. We assessed the ability of rhDNase to degrade DNA in undiluted serum, since the observed inhibition of endogenous DNase was reversed upon dilution. Addition of rhDNase to undiluted serum at a concentration of 50–100 ng/ml was necessary for degradation of radiolabelled phage DNA. The activity of rhDNase added to serum from normal subjects and SLE patients was similar. rhDNase degraded human chromatin and chromatin/anti-DNA immune complexes in serum with similar potency (EC₅₀ ≈ 100–200 ng/ml). A 500-fold variation in the chromatin/anti-DNA stoichiometry did not significantly affect the digestion of these immune complexes by rhDNase in buffer. These results indicate that a minimum rhDNase concentration of 50–100 ng/ml in serum was required to achieve detectable catalytic activity and that the presence of antibodies to DNA did not inhibit the degradation of DNA/anti-DNA immune complexes.     

Analysis of human C4A and C4B binding to an immune complex in serum.

Previous studies using isolated complement proteins have shown that more C4A than C4B binds to certain types of immune complexes. However, the in vivo binding of the C4 isoforms to an immune complex has not been investigated in detail and may differ from events when measured with the isolated proteins. We report here the binding of C4A and C4B to an immune complex of bovine serum albumin (BSA) anti-BSA as it occurs in serum. We found that when using the isolated C4 proteins more C4A than C4B bound to the complex, but in serum similar amounts of C4A and C4B were found to bind. Furthermore, these results were not explainable by a difference in activity between isoforms. In an attempt to explain these results a number of unexpected observations were noted. First C4A, but not C4B, bound specifically to a yet unidentified 38-kD serum protein. Second, when both covalent and non-covalent binding was assessed, we found that as serum concentration increased there followed a concomitant decrease in covalent binding and C4B was more affected than C4A. The potential biological significance of these findings is discussed.     

Detection of human and murine common idiotypes of DNA antibodies in tissues and sera of patients with autoimmune diseases.

The expression in tissue and serum of a panel of murine and human common DNA antibody idiotypes (Ids) (BEG 2, PR 4, F-423, I-402, II-28, IV-228, V-88) has been investigated. The murine V-88 Id was detected in eight out of 10 and the human BEG 2 Id in five out of 10 labial biopsies from patients with Sjögren''s syndrome. The murine F-423, I-402 and IV-228 Ids were identified in one out of 10 biopsies. In each case the pattern of staining was similar with staining of the acinar basement membrane and a cell population. Using double-labelling immunohistochemistry this cell population were identified as plasma cells. No staining was seen in four normal labial biopsies. The V-88 Id was detected on the epithelial aspect of the thickened basement membrane in three out of nine renal biopsies from patients with systemic lupus erythematosus (SLE). None of the other Ids (BEG 2, PR4, IV-228, F-423 or I-402) could be detected in renal tissue. None of the Ids were found in skin biopsies from SLE patients. Id V-88 may, like the 16/6 Id to which it is phenotypically related, play a role in the pathogenesis of renal lesions in SLE. The BEG 2 Id could be detected in the serum of patients with rheumatoid arthritis (RA) and active untreated tuberculosis. Ids II-28, V-88 and I-402 were elevated in serum from patients with Sjögren''s syndrome and II-28 Id in serum from patients with myositis and RA. None of the Ids were elevated in serum from patients with SLE. Apart from the BEG 2 Id, none of the Ids were elevated in serum from patients with tuberculosis or Gram-negative infections. The presence of murine Ids in human tissue and serum suggests that they are cross-species idiotypes and have been conserved through evolution.     

HLA class I serotypes and cytotoxic T-lymphocyte responses among human immunodeficiency virus-1-uninfected Thai volunteers immunized with ALVAC-HIV in combination with monomeric gp120 or oligomeric gp160 protein boosting

Antigen-induced cellular immunogenicity may vary between populations due to differences in human leukocyte antigen (HLA) diversity and, hence, may play a critical role in the protection afforded by vaccines. In the setting of two, phase I/II human immunodeficiency virus-1 vaccine trials of a recombinant canarypox prime, and boosting with either recombinant monomeric gp120 or oligomeric gp160, we assessed the association between specific human leukocyte antigen (HLA) class I serotypes and the presence of cytotoxic T-lymphocyte response measured by 51Cr-release assay. HLA class I serotypes A11, A24, A33, B46, and B75 were the most common, present in 10% or more of 245 individuals studied. Forty of 187 (21.4%) Thai adults who received either ALVAC-HIV with gp120 or oligomeric gp160 or ALVAC alone had a precursor cytolytic CD8 T-cell response (pCTL). HLA-B44 was positively and significantly associated with a pCTL response (odds ratio 7.6, 95% CI: 2.7-21.2), whereas B46 was negatively associated but not robust when adjusted for multiple comparisons. Responses to Env proteins accounted for the majority (nine of 11) of pCTL activity among those persons with B44. This HLA class I serotype occurred in 9.4% of participants overall (including the placebo group), less commonly than what is reported from populations of European ancestry. These results strengthen the importance of assessing HLA class I distributions in conjunction with studies of vaccines designed to elicit cellular immunity in different populations.     

广州地区人腺病毒5型血清中和抗体阳性率初步调查

目的评估广州地区人群中人腺病毒5型（HAdV-5）中和抗体阳性率情况。方法采用以β-半乳糖苷酶（LacZ）为报告基因,结合CMV启动子的人重组腺病毒5型载体,运用化学发光法,检测209份免疫功能正常的成人血清样本中HAdV-5的中和抗体。结果 HAdV-5中和抗体的阳性率为82.3%（172/209）。HAdV-5中和抗体在〈1：20（低滴度）、1：40-1：160（中滴度）、1：320-1：1280（高滴度）、〉1：1280（超高滴度）时均能检测到,而且随着滴度的增加,阳性率逐渐升高。在20～40岁时HAdV-5中和抗体阳性率最高,在〈20岁时HAdV-5阳性率最低。结论广州地区人群中针对HAdV-5的先存中和抗体阳性率高,应用基于该种DNA病毒作为载体进行基因疫苗及基因治疗的研究时,具有一定的局限性。     

Production of subgroup-specific monoclonal antibodies against human rotaviruses and their application to an enzyme-linked immunosorbent assay for subgroup determination

Nonneutralizing monoclonal antibodies were prepared against two strains, S2 and YO, of human rotaviruses isolated in cell culture. S2-37 and YO-5 antibodies had subgroup I and subgroup II specificities, respectively. The remaining antibodies (S2-65, YO-71, YO-89, and YO-156) reacted commonly with all the rotaviruses examined. All of the monoclonal antibodies agglutinated exclusively single-shelled particles and immunoprecipitated 42,000-dalton protein, a major component of inner capsid. Using the three monoclonal antibodies (S2-37, YO-5, and YO-156), an enzyme-linked immunosorbent assay was developed for detecting and subgrouping human rotavirus isolates.     

HLA class I and class II frequencies in patients with sarcoidosis from Croatia: role of HLA-B8, -DRB1*0301, and -DQB1*0201 haplotype in clinical variations of the disease

Sarcoidosis is an immune-mediated, multiorgan, granulomatous disease triggered by a combination of environmental and genetic factors. Numerous studies have reported about an association of human leukocyte antigen (HLA) alleles with sarcoidosis, with variation of alleles in different ethnic groups. Therefore, we investigated 142 Croatian sarcoidosis patients treated at the University Hospital for Lung Diseases Jordanovac, Zagreb, Croatia. Diagnosis was based on the presence of typical clinical features, chest X-ray findings and biopsy evidence of granuloma. Patients and control subjects (n = 190) were typed for HLA class I antigens by serology, while for HLA class II, they were tested by the polymerase chain reaction-sequence specific primers (PCR-SSP) method. Results indicated that HLA-B8, -DRB1*0301, and -DQB1*0201 positive patients have a significantly higher risk of acute onset of the disease (AOD), radiological stage I erythema nodosum (EN), L?fgren's syndrome, no-medicament therapy, and pulmonary sarcoidosis. On the other hand, the group of non-treated patients (corticosteroids and/or immunosuppressive) showed a significantly lower presence of HLA-B15 antigen in comparison to controls and treated patients (P = 0.0490 and P = 0.0379, respectively) and for DRB1*04 specificity (P = 0.0078 and P = 0.0065, respectively). In the group of patients with AOD, those who were positive for DRB1*16 specificity have a statistically significant chance to develop EN, as opposed to those who are positive for DRB1*15 specificity.     

Loss of heterozygosity at 6p21 underling HLA class I downregulation in Chinese primary esophageal squamous cell carcinomas

Loss or downregulation of human leukocyte antigen (HLA) class I molecules is a widespread mechanism used by tumor cells to avoid tumor recognition by cytotoxic T lymphocytes favoring tumor immune escape. Multiple molecular mechanisms are responsible for these altered HLA class I tumor phenotypes, such as the loss of heterozygosity (LOH) at chromosome region 6p21.3 . In this study, we used immunohistological techniques with a highly selective panel of anti-HLA monoclonal antibodies to analyze the expression of HLA class I molecules in 84 formalin-fixed, paraffin-embedded section and 49 frozen-fresh tissues of primary esophageal squamous cell carcinomas (pESCC) from Chinese patients. To elucidate the underlying mechanism of HLA class I loss or downregulation, we also analyzed LOH of previously selected microsatellite markers located in chromosomes 6 and 15 by polymerase chain reaction. DNA was obtained from frozen-fresh tumor tissues and surrounding stroma to define the LOH associated with chromosomes 6p21 and 15q21 . Our results showed that HLA-A, HLA-B/C, HLA class I heavy chain, β2-microglobuline, and HLA class I complex were lost or downregulated in pESCC ( P  < 0.0001), and were moderately associated with the microsatellite alterations in HLA class I gene regions, correlated with patients' age, tumor's location, and stage, and indicated that LOH at 6p21.3 is a frequent mechanism that leads to HLA class I abnormalities in pESCC.     

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

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 florescence 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.     

IgA class antibodies to 2-oxo-acid dehydrogenase complex are not predictive markers of histopathological progression in primary biliary cirrhosis

Although antimitochondrial antibody (AMA) is the characteristic serological feature of primary biliary cirrhosis (PBC), its pathogenic role remains unclear. In our previous study, we reported a positive correlation between immunoglobulin (Ig) A class anti-2-oxo-acid dehydrogenase complex (2-OADC) and histopathological stage. To determine whether the appearance of IgA class anti-2-OADC by immunoblotting represents an early marker of more aggressive disease or whether it is late finding during the disease course of PBC, we tested not only the entire IgA class but also IgA1, IgA2 and secretory IgA class anti-2-OADC in serial serum samples from 15 patients with PBC. During the median observation period of 51 months, four cases showed histopathological progression (from stage 1 to 2, stage 1 to 3, stage 1 to 4 and stage 2 to 4). There was no statistically significant correlation between the above IgA class anti-2-OADCs and histopathological progression. There was no significant correlation between histopathological stages and IgA2 class anti-2-OADC or secretory IgA class anti-2-OADC by immunoblotting. IgA class anti-2-OADC was more frequent in stages 3–4 than in stages 1–2 (p = 0.0049), but IgA1 class anti-2-OADC was more frequent in stages 1–2 than in stages 3–4 (p = 0.0232). Our present study demonstrated that serum IgA class 2-OADC was not a predictive marker of histopathological progression but was associated with the histopathological stage of PBC. Although the IgA class AMA may have a specific pathogenic role for PBC, the discrepant results between IgA and IgA1 class anti-2-OADC should be further assessed to investigate different functional activities depending on their molecular form.     

Evaluation of an enzyme-linked immunosorbent assay that measures rhinovirus-specific antibodies in human sera and nasal secretions

Rhinovirus-specific antibodies have traditionally been detected by their ability to neutralise the homologous rhinovirus serotype in tissue culture. Recently, however, we have described an enzyme-linked immunosorbent assay that detects rhinovirus-specific antibodies in sera and nasal secretions [Barclay and Al-Nakib, 1987]. Here we describe an evaluation of the ELISA in a study involving 71 adult volunteers inoculated intranasally with human rhinovirus type 2 (HRV-2). Pre- and post-inoculation serum samples and pre-inoculation nasal washings were tested for the presence of HRV-2-specific antibodies by ELISA. Such antibodies were associated with protection against infection when present locally in nasal secretions, but when also present in the serum they were associated with protection against both infection and the development of illness. The antibody concentrations showed strong correlation with each other and with that of antibodies detected by the neutralisation test. Following HRV-2 infection, rises in HRV-2-specific IgA in sera detected by ELISA occurred more frequently than rises in neutralising antibody. These results suggest that the ELISA is a sensitive and reliable indicator of recent infection, as well as a predictor of homologous immune status.