One-way matching of HLA antigens between recipient and donor as a risk factor of transfusion-associated GVHD

Adsorption of basic fibroblast growth factor (bFGF) onto a plain fabric Dacron vascular prosthesis (Micron, InterVascular Co., Ltd., Clearwater, FL, U.S.A.) and its release properties from the graft were examined using labeled bFGF, and its biological efficacy was evaluated in an animal study. In an in vitro study, 6 pieces of a Dacron graft were soaked in [(125)I]-bFGF solution for 30 min. Then these pieces were soaked in 500 ml saline solution for 15 days, and the radioactivity of each piece was counted at various times. The initial amount of adsorbed bFGF was 2.48 ng/cm2. At 24 h, 41% of the adsorbed bFGF was released and 59% remained. On the third day 55% remained, on the seventh day 52%, and on the 15th day 50% remained on the Dacron surface. Using the atmospheric glow discharge (APG) plasma treatment to render hydrophilic properties, no significant difference between plasma-treated grafts and non-plasma-treated grafts regarding the adsorption of bFGF was observed. In an in vivo study, 6 Dacron pieces adsorbed with bFGF were implanted in the subcutaneous layer of 2 dogs and removed 5 days after implantation. Six Dacron pieces without bFGF were also implanted as a control into the 2 dogs. Fibroblast migration with capillary ingrowth was observed in the specimens with bFGF compared to the controls. These results indicate that the simple adsorption of bFGF onto Dacron fabric is a useful method for the acceleration of host cell migration and capillary ingrowth into Dacron fabric vascular prostheses.     

Ligation of HLA class I molecules on smooth muscle cells with anti-HLA antibodies induces tyrosine phosphorylation, fibroblast growth factor receptor expression and cell proliferation

The development of transplant atherosclerosis, a manifestation of chronic rejection, is the major obstacle to long-term survival of cardiac and renal allografts. The incidence of transplant atherosclerosis is increased in transplant recipients producing antidonor HLA antibodies following transplantation, suggesting that anti-HLA antibodies play a role in the pathogenesis of the disease. We have postulated that anti-HLA antibodies mediate the development of transplant atherosclerosis by binding to class I molecules on the endothelium and smooth muscle of the graft and transducing signals which stimulate cell proliferation. In this report we demonstrate that anti-HLA class I antibodies transduce signals in smooth muscle cells stimulating increased tyrosine phosphorylation of intracellular proteins and up-regulation of fibroblast growth factor (FGF) receptors. Antibody binding to class I molecules on smooth muscle cells is also accompanied by increased responsiveness to basic FGF and augmented cell proliferation. These findings may explain the increased occurrence of transplant atherosclerosis in recipients producing anti-donor HLA antibodies.     

The lack of long-term detrimental effects on liver allografts caused by donor-specific anti-HLA antibodies

Recent reports indicate a higher incidence of both acute and chronic liver allograft rejection when, at the time of transplantation, the recipients serum contains donor-specific anti-HLA antibodies. From 9/89 to 5/91, 133 liver allografts were performed at our institution. Thirteen liver recipients had donor-specific IgG anti-HLA antibodies (complement-fixing) at the time of transplantation. In eleven patients, antibodies reacted to donor class I antigens while in 1 patient the donor-specific antibody had class II reactivity. Twelve patients have been followed for a minimum of 12 months (median 18 months, range 28-12 months). No hyperacute rejection was seen in any of the cases and four patients had acute rejections. Thus far only one of the twelve patients has biopsy evidence suggestive of chronic liver injury. The remaining have normal liver enzymes and bilirubin. Three of these twelve patients died (one from a myocardial infarction and the others from sepsis) accounting for a one-year graft survival of 75%. There was no significant statistical difference in the one-year graft survival in those recipients without donor-specific antibodies (i.e., 80.5%). In eight of the twelve patients, pretransplant preformed antibody level (PRA) was > 50%. In six of the thirteen patients donor-specific antibody was present at dilutions greater than 1:64. As previously reported, the donor-specific antibody disappeared from the serum posttransplant within hours and did not reappear. In vitro studies demonstrated no factor in portal or hepatic artery blood that could inhibit rabbit complement mediated lysis of anti-HLA antibodies. We conclude that it is not a contraindication to do liver transplants in the presence of donor-specific anti-HLA antibodies.     

Selection and identification of single domain antibody fragments from camel heavy-chain antibodies

OBJECTIVE: Our purpose was to determine whether women who were themselves small for gestational age at birth are at risk of giving birth to a small-for-gestational-age child and whether women who were themselves preterm at birth are at risk for preterm delivery. STUDY DESIGN: Women born in Copenhagen as subjects in the Danish Perinatal Study (1959 to 1961) were traced through the Danish Population Register. Information was obtained on their pregnancies during 1974 through 1989. RESULTS: A total of 25% of the children of small-for-gestational-age women were small for gestational age compared with 11% of the children of non-small-for-gestational-age women. Eleven percent of the children of preterm women were preterm compared with 7% of the children of women born at term. The adjusted odds ratios were 2.0 (95% confidence interval 1.4 to 3.0) for women who were small for gestational age to have small-for-gestational-age children and 1.5 (95% confidence interval 0.9 to 2.5) for women who were born preterm to have preterm children. Small-for-gestational-age women were not at significantly increased risk of preterm delivery (odds ratio 1.2), and preterm women were not at significantly increased risk of having small-for-gestational-age children (odds ratio 1.3). CONCLUSIONS: Reduced intrauterine growth of the mother is a risk factor for reduced intrauterine growth of her children. However, preterm birth of the mother is not strongly associated with preterm birth of her children.     

An immunohistochemical method of detecting Mycoplasma species antigens by use of monoclonal antibodies on paraffin sections of pneumonic bovine and caprine lungs

Lung samples from pneumonic lesions in cattle and goats, naturally or experimentally infected with strains of the Mycoplasma mycoides cluster, were fixed in formalin and embedded in paraffin. An immunohistochemical technique using monoclonal or polyclonal antibodies was performed on tissue sections in order to detect Mycoplasma antigens. Four monoclonal antibodies (MAbs), one (2A3) raised against M. mycoides ssp. mycoides small colony (SC) and large colony (LC), two (1D3 and 5E5) against M. mycoides ssp. capri, and one (5A10) against M. bovis, were used. A range of polyclonal antibodies, raised to the individual subspecies of the M. mycoides cluster, and one to Pasteurella haemolytica, was also used. The MAb 2A3 showed positive immunostaining in lung sections from cattle and goats naturally and experimentally infected with M. mycoides ssp. mycoides SC and LC, but not with pneumonic lesions of cattle and goats due to other members of the M. mycoides cluster, M. bovis or Pasteurella spp. The MAb 1D3 showed immunostaining in lung sections from goats naturally and experimentally infected with M. mycoides ssp. capri, but again not with pneumonic lesions caused by other members of the M. mycoides cluster, M. bovis or Pasteurella spp. The MAb 5E5 immunoreacted in sections from pneumonic lesions from all animals infected with one of the three M. mycoides cluster subspecies used in the study, but not with M. bovis or Pasteurella infected tissue. Immunoreaction was mainly found in the cell debris around necrotic areas, as well as in macrophages, neutrophils and epithelial cells. The localization of antigens of the M. mycoides cluster using polyclonal antisera followed basically the same pattern as that obtained with the monoclonals. However, a wide cross reactivity was found between different antisera and relatively high background immunostaining was also seen, especially in necrotic areas. The results suggest that immunohistochemical methods using monoclonal antibodies are useful tools for the diagnosis and study of the pathogenesis of pneumonia caused by the Mycoplasmas of the M. mycoides cluster.     

Immunochemical methods for identification of Babesia bovis antigens expressed on the erythrocyte surface

The aim of this study was to compare and improve standard methods to determine nitrite (NO2-), nitrate (NO3-) and S-nitrosothiol (RSNO) levels in cell culture supernatants, sera, and urine. We modified the conventional Griess reaction by replacing sulfanilamide with dapsone (4,4'-diamino-diphenylsulfone) and compared the NO2- levels in our study samples with a commercially available NO2- assay kit. Our modification, along with ultrafiltration of the samples, resulted in an enhanced sensitivity to measure NO2- down to 0.2 microM. The detection limit was further improved to 0.02 microM when NO2- was identified by the fluorochrome 2,3-diaminonaphthalene (DAN). To measure the stable end product NO3- by the Griess reaction or the DAN method, this anion must be reduced to NO2-. We compared the capacity of bacterial nitrate reductase with the reducing metal cadmium to convert NO3- to NO2-. After reduction, NO2- levels were determined either by the DAN method or by our modified Griess reaction. We found that there was a high correlation (r2 = 0.998) in total NO2- concentrations in the study samples using both methods for reducing NO3- to NO2-. The simultaneous determination of NO2- and NO3- was achieved by using anion-exchange chromatography (HPLC; Polyspher IC AN-1 column). The detection limit of this assay for each anion is 0.5 microM, and it can be applied equally well to sera, urine, and culture media. We also adapted the DAN method to determine RSNO levels in our study samples. Using this approach, we were able to measure RSNO levels down to 0.15 microM. As result we discovered that RSNO levels were markedly increased in urine from septic patients and in supernatants from cytokine-stimulated human tumor cell lines. L-Citrulline, a coproduct of NO biosynthesis, was measured using a colorimetric assay with a sensitivity limit of 3.0 microM. Increased L-citrulline levels in media from cultured cells, but not in sera or urine, correlated with increased NO production. Although all methods studied were suitable for quantifying end products of NO in biological fluids and media, the use of bacterial reductase and the modified Griess reaction proved successful to provide the greatest sensitivity and linear range for routine measurements of NO2- and NO3-.     

The importance of the HLA complex antigens in systemic rheumatoid diseases

The submitted paper reviews contemporary knowledge on the clinical impact of assessment of HLA-complex antigens in systemic rheumatic diseases. The authors explain the term "relative risk" and its practical importance. As to antigens of the HLA-complex class I, attention was paid in particular to the importance of HLA B 27 in ankylosing spondylitis, other spondylarthropathies and reactive arthritis; in this part the review is supplemented by results of the authors' own research. As to antigens of the HLA-complex class II, attention was paid to the sub-area DR in rheumatoid arthritis and other diffuse affections of connective tissue. The advance in knowledge of the clinical impact of investigations of selected HLA-complex antigens in systemic rheumatic diseases is that in many instances it reveals an association not only at the level of the nosological unit but frequently also sub-unit with prognostically important clinical and biochemical manifestations, a typical autoantibody profile and sometimes also with the risk of a greater organ toxicity of drugs.     

Analysis of antibodies that recognize the B-epitopes formed on protein antigens by glutaraldehyde treatment: an efficient method of their neutralization

Immunochemical analysis of antigenic determinants (AD) formed on protein macromolecules by glutaraldehyde (GA) treatment with subsequent block of free aldehyde groups with glycine was performed. The structure of the epitopes was analyzed using BSA and ovalbumin (Ova) treated with GA with subsequent block of the active groups with glycine and other amino acids. The products of reaction of GA with Gly, Lys, Pro, and His were used as hapten-like antigens that mimic GA antigenic determinants (GA-AD). Indirect and competitive enzyme-linked immunoassay and immunoaffinity chromatography revealed two regions of epitope density in the structure of the GA determinant. It is shown that the product of GA reaction with Gly efficiently blocks the antibodies raised against GA-treated proteins. Based on this finding, a method of assay of the peptide-specific antibodies of the immune complexes with the peptide--protein conjugate in the presence of antibodies to the linking agents is suggested.     

The role of HLA antigens in predicting the active course of multiple myeloma

AIM: Detection of associations between carrying some HLA-antigens class I in patients with multiple myeloma (MM) and activity of the malignant process. MATERIALS AND METHODS: 76 MM patients received polychemotherapy. Its efficacy was assessed after one, three, six and twelve courses by reduced blood and/or urine levels of monoclonal protein, signs of bone healing, reestablishment of normal number of plasma cells in the bone marrow. Identification of HLA-antigens was made in two-stage lymphocytotoxic complement-mediated test using the standard panel of the anti-HLA sera. Data on HLA-typing of 865 blood donors served control. The findings were statistically processed. RESULTS: All the patients were divided into 3 groups: with indolent (n = 18), active (n = 25) and aggressive (n = 37) MM course. In patients with aggressive MM course high chi-square values were estimated for three HLA specificities: HLA-B13, HLA-B40, HLA-B5. Only HLA-B13 proved significant. No significant differences in carrying HLA-antigens were revealed for patients with active MM course. CONCLUSION: The survival of MM patients depends on the degree of the malignant process activity. Patients with aggressive MM course significantly more frequently carry HLA-B13, therefore it can be considered a genetic marker of MM. Its detection can serve a criterion for determination of adequate polychemotherapy.     

Workshop on a detailed characterization of Trichinella spiralis antigens: a platform for future studies on antigens and antibodies to this parasite

In order to characterize immunodominant components of T. spiralis a workshop was organized. In this the reactivity of monoclonal and polyclonal antibodies, provided by different research groups, towards total extracts from adult, new born larvae and muscle larvae as well as to excretory/secretory components of muscle larvae were tested by ELISA, Western blot and immunoprecipitation assays. As a result of this workshop T. spiralis ML antigens have been classified into eight groups (TSL-1-TSL-8) according to their recognition by monoclonal and polyclonal antibodies. Among them, TSL-1 antigens have been the most extensively characterized both biochemically and immunologically. These antigens are stage specific, originate in the muscle stichosome and are abundant in both E/S and on the larval cuticular surface. The TSL-1 antigens share an immunodominant carbohydrate epitope (tyvelose), which is unique for Trichinella and is not associated with phosphorylcholine. The data collected in this workshop has allowed both the unification of the nomenclature for T. spiralis antigens and their biochemical characterization. It also has provided a platform for further studies on the characterization of other T. spiralis antigens and indeed for other Trichinella species.     

Schizophrenic psychoses and HLA antigens. Personal data and review of the literature

Genetic factors play probably an important part in the development of schizophrenic psychoses. As a consequence the use of a genetic marker as the HLA system appears to be interesting in determining the disease susceptibility gene of these psychoses. Methods and results of an investigation about the frequencies of 33 HLA alleles observed in 51 patients considered as paranoid schizophrenics are presented. The frequency of HLA-A 29 was diminished while the one of HLA-B 15 was increased but the differences were no longer significant when p was corrected. However when all the results published from 1974 to 1980 were pooled in a combined statistical analysis, some associations became significant. It seems that schizophrenia as a whole, once paranoid and hebephrenic sub-types have to be distinguished. It may be concluded from these data that correlations between schizophrenia and HLA antigens which remain doubtful could be explained with a biological genetic heterogeneity of schizophrenic disorders. Review of literature concerning the identification of HLA haplotypes in schizophrenics pedigrees and about the HLA system as a genetic marker for the clinical response to neuroleptics in schizophrenic patients or in vitro, is also discussed.     

An improved method for the production of antibodies to lipophilic carboxylic hapten using small amount of hapten-carrier conjugate

Normal human diploid cells have a limited proliferative lifespan in in vitro cultures. Changes in gene expression have been examined for understanding control mechanisms of limited proliferative lifespan. and enhanced expression of growth suppressing genes such as p21 was reported in late-passaged cells. We screened genes which were expressed preferentially in mid-passaged cells by the differential plaque screening of the subtracted cDNA libraries prepared from young, life-extended, and immortalized SV40-transformed human fibroblasts. Among isolated clones, ASF/SF2, which was known to affect alternative splicing, was expressed in normal fibroblasts with a peak at mid-passage. Relative expression levels of SC35 and hnRNPA1, which are also known to affect alternative splicing, was also highest at mid-passage. Changes in alternative splicing at mid-passage, if it occurred, may play a crucial role in the process of cellular senescence.     

基于GPR反射波信号多维分析的隧道病害智能辨识

随着我国隧道工程建设的快速发展,由隧道病害引发的隧道质量和安全问题越发常见.通过地质雷达探测隧道病害对于减少隧道质量和安全问题具有十分重要的意义,为了提高病害探测的效率及可靠性,基于雷达反射波信号多维度分析,提出一种隧道病害智能辨识的新方法.根据反射波信号时域、频域及时频域分析结果提取病害信号辨识的6个典型特征,利用支持向量机算法对典型特征的训练构建病害信号的二分类模型,实现了病害水平分布范围的自动辨识;再依据病害信号的第一本征模态函数分量振幅包络计算病害深度分布范围,最终实现隧道病害的智能辨识.结合某隧道回填层雷达实测数据对智能辨识算法的性能进行评价,与人工辨识结果的对比表明,该智能算法对于病害的辨识能力较强,病害的识别率高达100%,但辨识结果中同时存在少量误判,准确率达78.6%,满足工程应用的需求.该算法可用于隧道工程各类地质雷达探测数据中病害的智能辨识,而对于其他领域的地质雷达探测数据,本文研究成果亦可为不同类型探测目标智能辨识算法的设计提供可行思路.     

Analysis of mutational changes at the HLA locus in single human sperm

Using a simple and efficient single sperm PCR and direct sequencing method, we screened for HLA-DPB1 gene mutations that may give rise to new alleles at this highly polymorphic locus. More than 800 single sperm were studied from a heterozygous individual whose two alleles carried 16 nucleotide sequence differences clustered in six polymorphic regions. A potential microgene conversion event was detected. Unrepaired heteroduplex DNA similar to that which gives rise to postmeiotic segregation events in yeast was observed in three cases. Control experiments also revealed unusual sperm from DPB1 homozygous individuals. The data may help explain allelic diversity in the MHC and suggest that a possible source of human mosaicism may be incomplete DNA mismatch repair during gametogenesis.     

Development of cancer immunotherapies based on identification of the genes encoding cancer regression antigens

Tumor-infiltrating lymphocytes (TILs) have been grown from patients with metastatic melanoma and administered to the autologous patients to identify those TIL populations capable of mediating tumor regression. These TILs have been used to clone the genes that encode melanoma antigens. With the use of this strategy, we have identified six different genes encoding antigens restricted by multiple HLA alleles that appear to be related to tumor regression in patients. These antigens are now being used to develop immunization approaches for the treatment of patients with metastatic melanoma. The availability of genes encoding unique cancer antigens is opening new possibilities for the development of immunotherapies for the treatment of patients with cancer.     

Use of immunoplot analysis for the identification of immunodominant non-variant antigens of Trypanosoma brucei rhodesiense

BACKGROUND: Common elements in many different types of amyloid may have important roles in amyloidogenesis. The proteinaceous tissue deposits have a common appearance in polarized light and other similar features. The present investigation describes for the first time the relation between beta 2-microglobulin (beta 2-M)-type amyloidosis and colocalized materials, as demonstrated using specific antibodies and hyaluronan-binding protein. EXPERIMENTAL DESIGN: Amyloid-rich carpal tunnel synovium was obtained surgically from 28 patients who were being treated by maintenance hemodialysis. Serial sections were examined using a hyaluronan (hyaluronic acid)-binding protein and antibodies against heparan sulfate-glycosaminoglycan, chondroitin sulfate-proteoglycan, dermatan sulfate-proteoglycan, alpha 1-antichymotrypsin, alpha 1-antitrypsin, inter-alpha-trypsin inhibitor, haptoglobin, and ubiquitin. RESULTS: Accumulation of hyaluronan was of three types, namely, localization around beta 2-M deposits, colocalization with deposition of beta 2-M itself and localization at a small distance from beta 2-M deposits. Immunostaining for heparan sulfate glycosaminoglycan was demonstrated at the sites of beta 2-M plaques. Chondroitin sulfate-proteoglycan did not show specific patterns of immunostaining, resembling hyaluronan rather than heparan sulfate. The other materials tested, alpha 1-antichymotrypsin, alpha 1-antitrypsin, inter-alpha-trypsin, haptoglobin and ubiquitin, were not immunostained at sites of beta 2-M plaques. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting revealed that the molecular weight of heparan sulfate-glycosaminoglycan was 16,000. CONCLUSIONS: These results suggest that HS has an important role in hemodialysis-associated amyloidosis as it does in other types of amyloidosis. Moreover, accumulation of hyaluronan may be an indication of inflammation of the carpal synovium.     

The effect of tolerance to noninherited maternal HLA antigens on the survival of renal transplants from sibling donors

BACKGROUND: During pregnancy and nursing, a baby's developing immune system is intimately exposed to the mother's antigens. To determine whether this exposure is of clinical benefit to patients who later receive an allograft as an adult, we analyzed the outcome of primary renal transplantations from sibling donors. METHODS: We retrospectively studied graft survival and rejection episodes in 205 patients who had received renal transplants at nine centers between 1966 and 1996 from sibling donors bearing maternal or paternal HLA antigens not inherited by the recipient. The sibling donors were categorized by analysis of family HLA-typing data. RESULTS: In the multicenter analysis, graft survival was higher at 5 years and at 10 years after transplantation in recipients of kidneys from siblings expressing maternal HLA antigens not inherited by the recipient than in recipients of kidneys from siblings expressing paternal HLA antigens not inherited by the recipient (86 percent vs. 67 percent at 5 years and 77 percent vs. 49 percent at 10 years, P=0.006 for both). Paradoxically, there was a higher incidence of early rejection in the former group, suggesting that fetal and neonatal exposure to maternal antigens results in immunologic priming. Pretransplantation transfusions of donor blood reduced the incidence of acute rejection while preserving the beneficial effect of tolerance to noninherited maternal antigens on graft survival. Since 1986, new immunosuppressive drugs have lessened the short-term, but not the long-term, survival advantage of grafts expressing maternal HLA antigens not inherited by the recipient. CONCLUSIONS: In the transplantation of a kidney from a sibling donor who is mismatched with the recipient for one HLA haplotype, graft survival is higher when the donor has maternal HLA antigens not inherited by the recipient than when the donor has paternal HLA antigens not inherited by the recipient.