Immunoblot analysis of humoral immune response to Helicobacter pylori in children with and without duodenal ulcer

Several studies have demonstrated that enzyme-linked immunosorbent assay is not a sensitive and specific method to diagnose Helicobacter pylori infection in children, especially in the younger ones. Since serum immune response can also be determined by immunoblotting and it permits the detection of antibodies to virulence factors such as CagA and VacA, we evaluated the accuracy of a commercial immunoblotting test to diagnose H. pylori infection and to assess the humoral immune response to different H. pylori antigens in 122 children who underwent upper gastrointestinal endoscopy. The presence of H. pylori was determined in antral biopsy specimens by culture, preformed urease test, and histological analysis. H. pylori was identified by microbiological and histopathological methods in 66 children (including all of the 21 who had duodenal ulcer). Antibodies to H. pylori were detected in 63 infected children and in 8 noninfected ones. The sensitivity, specificity, and positive and negative predictive values of the immunoblotting test were 95.5, 85.7, 88.7, and 94.1%, respectively. The number of immunoreactive bands increased with age (P = 0.003), and the bands of 35 kDa (P = 0.013); 89 kDa, the VacA antigen (P = 0.001); and 116 kDa, the CagA antigen (P = 0.00004) were more frequently observed in older children. The frequency of the bands of 89 kDa (P = 0.001) and 116 kDa (P = 0.03) was higher in children with duodenal ulcer than in H. pylori-positive children without the disease. In conclusion, the immunoblotting test appears to be useful for the diagnosis of H. pylori infection in children, even in the younger ones.     

Putative consequences of exposure to Helicobacter pylori infection in patients with coronary heart disease in terms of humoral immune response and inflammation

Introduction

Pathogens, including Helicobacter pylori (Hp), have been suggested to contribute to the development of coronary heart disease (CHD), although the evidence still remains insufficient. The study was focused on the exposure of CHD patients to Hp and resulting anti-Hp heat shock protein B HspB antibody production in relation to the level of serum lipopolysaccharide binding protein (LBP) as a marker of inflammation.

Material and methods

One hundred seventy CHD patients and 58 non-CHD individuals participated in this study. Coronary angiography confirmed the atheromatic background of CHD. The panel of classical risk factors included: arterial hypertension, diabetes, total cholesterol, low-density lipoprotein (LDL)/high-density lipoprotein (HDL) cholesterol, triglycerides, obesity and nicotinism. The Hp status was estimated by ¹³C urea breath test and serology. Immunoblot and ELISA were used for screening the sera samples for anti-Hp HspB immunoglobulins (Igs) and LBP.

Results

Coronary heart disease patients were exposed to Hp more frequently than non-CHD individuals. This was associated with increased levels of specific anti-Hp IgG2 and IgA as well as total IgA. Hp infected CHD and non-CHD donors produced anti-Hp HspB IgG cross-reacting with human Hsp 60. In CHD patients the LBP level was significantly higher in comparison to non-CHD donors. This was related to the severity of the disease. Type I Hp strains stimulated higher LBP levels than less pathogenic type II isolates.

Conclusions

Lipopolysaccharide binding protein secreted in excess together with anti-Hp HspB, cross-reacting with human Hsp60, may increase the risk of vascular pathologies in Hp-exposed CHD patients.     

幽门螺杆菌相关抗原及宿主免疫应答的研究进展

幽门螺杆菌与多种胃肠道疾病诸如慢性胃炎,胃肠溃疡,胃腺瘤及胃癌等密切相关的结论已得到证实,预防和治疗幽门螺杆菌感染是控制其广泛传播的有效途径,此项工作虽早已开始,但至今未找到可行的方案.近来对幽门螺杆菌相关毒素的研究越来越多,这可作为研究幽门螺杆菌菌苗的重要依据;幽门螺杆菌诱发的宿主免疫应答以TH1反应为主,幽门螺杆菌感染者可出现系统和局部的抗体反应,这些抗体反应对机体不具保护作用且自身抗体对宿主上皮细胞还可能带来不良影响,阐明宿主免疫应答的机制对预防和治疗幽门螺杆菌感染具特殊意义.因此,本文主要介绍了脲素酶、空泡细胞毒素、cag相关基因蛋白,中性粒细胞蛋白等几种毒素及宿主免疫应答的研究现状,并对幽门螺杆菌的研究前景进行综述.     

Impact of Helicobacter pylori infection on the humoral immune response to MUC1 peptide in patients with chronic gastric diseases and gastric cancer

Many investigators have demonstrated alteration of gastric mucins in H. pylori infected individuals. The inflammatory environment induced by H. pylori leading to aberrant glycosylation of MUC1 and demasking of core peptide MUC1 epitope could enhance immune responses to MUC1. IgG and IgM immune response to MUC1 in patients with gastric cancer (n = 214) chronic gastroduodenal diseases (n = 160) and healthy blood donors (n = 91) was studied with ELISA using bovine serum albumin-MUC1 60-mer peptide as antigen. H. pylori serologic status was evaluated with ELISA and CagA status by immunoblotting. Gastric mucosa histology was scored according to the Sydney system. Compared to H. pylori seronegative individuals, higher levels of IgG antibody to MUC1 were found in H. pylori seropositive patients with benign gastric diseases (p < 0.01) and blood donors (p < 0.03). Higher MUC1 IgG antibody levels were associated with a higher degree of gastric corpus mucosa inflammation in patients with chronic gastroduodenal diseases (p < 0.0025). There was a positive correlation between the levels of anti-H. pylori IgG and MUC1 IgG antibody levels in blood donors (p = 0.03), and in patients with benign diseases (p < 0.0001). In patients with gastric cancer (n = 214) a significantly higher level of anti-MUC1 IgG than in blood donors was observed (p < 0.001) irrespective of H. pylori status or stage of cancer. MUC1 IgM antibody levels were not related to the H. pylori serology. IgG immune response to tumor-associated MUC1 is up regulated in H. pylori infected individuals. This increase is associated with a higher IgG immune response to H. pylori and with a higher degree of gastric mucosa inflammation. High levels of MUC1 IgG antibody irrespective of H. pylori serologic status characterized patients with gastric cancer. The findings suggest that, in some individuals, the H. pylori infection may stimulate immune response to tumor-associated MUC1 peptide antigen thus modulating tumor immunity.     

Cell-mediated immune response to bacterial products in human tonsils and peripheral blood lymphocytes.

Lymphoproliferative responses of tonsillar tissue lymphocytes and peripheral blood lymphocytes to phytohemagglutinin and specific bacterial product antigens were studied in children undergoing tonsillectomy and adenoidectomy. Tonsillar tissue lymphocytes responded to optimal concentrations of phytohemagglutinin. Varidase, and streptolysin-O in a manner similar to peripheral blood lymphocytes. Higher base-line mitogenic activity in tonsillar lymphocytes was frequently associated with the presence of Staphylococcus aureus in the tonsils. Tonsillar tissue lymphocytes from 23% of the subjects with the highest base-line mitogenic activity manifested a decreased response to in vitro stimulation with mitogens or antigens. In subjects with such preactivated tonsillar lymphocytes, the proliferative responsiveness of blood lymphocytes to mitogen and antigens was markedly increased after tonsillectomy and adenoidectomy. These observations suggest the existence of in vitro correlates of cellular immunity to bacterial products in the mucosal surfaces. In addition, it is proposed that tonsils may possess immunosuppressive activity for peripheral blood lymphocytes, which may be related to local tonsillar infections.     

Influence of age and duration of infection on bacterial load and immune responses to Helicobacter pylori infection in a murine model

Using a murine model, we previously showed that Helicobacter pylori infects and colonizes offspring via maternal transmission during the nursing period. The aim of this study was to investigate the influence of age and duration of infection on inflammatory and immune responses to H. pylori in infant and adult mice. During the breast-feeding period, the number of bacteria was significantly suppressed in 1-week-old mice infected with H. pylori at an early stage of nursing, compared with adult mice, suggesting that breast-milk induces such low colonization. In addition, these mice had weaker gastric inflammation, especially Th1 cytokine and humoral responses than in mice infected with H. pylori after weaning in spite of elevated levels of Th1 cytokines. Although infant mice showed low inflammatory responses against H. pylori, they produced H. pylori-specific antibodies following vaccination with oral or parenteral adjuvant. Our results suggest the importance of age at the time of primary infection on bacterial load, gastric inflammation and humoral responses in a murine model of H. pylori infection.     

B-cell and T-cell immune responses to experimental Helicobacter pylori infection in humans

The acute antibody and T-cell immune response to Helicobacter pylori infection in humans has not been studied systematically. Serum from H. pylori-naive volunteers challenged with H. pylori and cured after 4 or 12 weeks was tested by enzyme-linked immunosorbent assays for anti-H. pylori-specific immunoglobulin M (IgM) and IgA established using bacterial lysates from homologous (the infecting strain) and heterologous H. pylori. Proteins recognized by IgM antibody were identified by mass spectrometry of immunoreactive bands separated by two-dimensional gel electrophoresis. Mucosal T-cell subsets (CD4, CD8, CD3, and CD30 cells) were assessed by immunohistochemistry. All 18 infected volunteers developed H. pylori-specific IgM responses to both homologous or heterologous H. pylori antigens. H. pylori antigens reacted with IgM antibody at 4 weeks postinfection. IgM Western blotting showed immunoreactivity of postinfection serum samples to multiple H. pylori proteins with molecular weights ranging between 9,000 (9K) to 150K with homologous strains but only a 70K band using heterologous antigens. Two-dimensional electrophoresis demonstrated that production of H. pylori-specific IgM antibodies was elicited by H. pylori flagellins A and B, urease B, ABC transporter binding protein, heat shock protein 70 (DnaK), and alkyl hydroperoxide reductase. Mucosal CD3, CD4, and CD8 T-cell numbers increased following infection. IgM antibody responses were detected to a range of homologous H. pylori antigens 2 to 4 weeks postchallenge. The majority of H. pylori proteins were those involved in motility and colonization and may represent targets for vaccine development.     

Serum immunoglobulin G immune response to Helicobacter pylori antigens in Mongolian gerbils

The Mongolian gerbil model for Helicobacter pylori infection is an animal model that mimics human disease. We examined the serum immune response to H. pylori infection in gerbils by enzyme-linked immunosorbent assay (ELISA) and Western blotting, both with whole-cell (H. pylori) extracts. A total of 66 7-week-old specific-pathogen-free male gerbils were inoculated orogastrically with H. pylori strain ATCC 43504. Sera were collected 1, 2, 4, 8, 12, 26, 38, and 52 weeks after H. pylori inoculation. Sixty-nine noninfected gerbils and their sera were used as controls. The specificity of the ELISA was 95.7%. The frequency of seropositivity increased over time: 2 of 10 (20%), 7 of 10 (70%), and 7 of 7 (100%) samples of sera from inoculated gerbils were positive for H. pylori at 2, 4, and 8 weeks postinoculation, respectively. Western blot assays showed that the primary immunoglobulin G (IgG) response against low-molecular-mass (25-, 30-, and 20-kDa) proteins appeared after a lag period of 2 to 8 weeks after inoculation. Antibodies against 160-, 150-, 110-, 120-, 80-, 66-, and 63-kDa proteins were observed 12 weeks after inoculation. The early reactive 30-kDa protein was identified as a urease alpha subunit by N-terminal amino acid sequencing. After 26 weeks, two groups of animals could be distinguished: one group developed ulcers (n = 5), and the other developed hyperplastic polyps without ulcers (n = 19). Gerbils in the gastric ulcer group showed significantly higher serum anti-H. pylori IgG levels than did gerbils in the hyperplastic group (P = 0.001) as measured by ELISA. Furthermore, a higher proportion of animals developed antibodies to H. pylori proteins of 26, 25, and 20 kDa in the ulcer group than those animals with hyperplastic polyps (75 to 100% versus 17 to 50%) in Western blot assays. These results highlight the importance of the immune response of the host in the development of H. pylori-related gastric lesions.     

利用人外周血白细胞端粒长度推断年龄

目的：探讨人外周咀白细胞端粒长度与供体年龄的关系,为法医学通过微量软组织推断年龄提供依据。方法：选取105例0～81岁健康人外周血样本,采用Southern印迹法检测其端粒限制性片段(terminal restriction fragment,TRF)平均长度,并作相关及回归分析。结果：外周血白细胞TRF长度随年龄增长逐渐缩短,并呈不均衡趋势;端粒DNA平均每年缩短51bp;得到推断年龄回归方程：Y=-16．539X 236．287±9．832。结论：人外周血白细胞TRF长度与年龄呈负相关,此规律为通过端粒DNA长度推断个体年龄提供了可能。     

根除儿童口腔幽门螺杆菌预防胃内幽门螺杆菌感染的多中心研究

目的:探讨根除儿童口腔幽门螺杆菌（Hp）预防胃内Hp感染的可能性。方法:采用多中心前瞻随机研究,选取口腔Hp阳性但胃内Hp阴性的幼儿园儿童共计427例,随机分为使用“无幽梅”牙膏组与普通牙膏组,分别接受“无幽梅”牙膏和普通牙膏。疗程结束后,再次检测口腔Hp,将口腔Hp阳性及阴性患者各分为一组,1年后行C13呼气试验检查,分析两组患者胃内Hp感染情况。口腔Hp检测方法采用特异度及敏感度双高的套式PCR方法。结果:随访1年,口腔Hp阴性组胃内Hp感染率为0.51%,口腔Hp阳性组胃内Hp感染率为6.51%,两组统计差异具有显著性（P<0.01）。结论:儿童根除口腔Hp可以降低胃内Hp感染的发生。     

Helicobacter pylori induces lymphocyte activation in peripheral blood cultures.

Immunohistological assessment of Kupffer cells was made using the antibody MAC387 and an antibody to lysozyme. Autopsy liver samples from 13 fetuses aged from 17 weeks gestation to term, and from 10 neonates and children aged 1 day to 18 months, were studied. For comparison, 10 normal adult autopsy liver specimens were included. The number of positively staining cells per unit area was counted for periportal sinusoids (zone 1) and centrilobular sinusoids (zone 3). No difference was found between zone 1 and zone 3 macrophage numbers with either antibody at any stage of development. Hepatic sinusoidal macrophage numbers were low during early gestation but increased during intra-uterine life to reach approximately normal adult values in the neonatal period. The numbers of cells staining with MAC387 or lysozyme were similar in each case except for hepatic sinusoidal macrophages in fetuses of less than 30 weeks gestation. Here anti-lysozyme stained significantly fewer cells, suggesting that lysozyme production may be low in immature fetuses. No difference was found between infants of similar maturity who had died immediately or had lived for more than 48 h and hence been exposed to gut antigens.     

Cross-talk between gammadelta T lymphocytes and immune cells in humoral response.

The role of gamma delta T cells in immunoregulation is largely unknown. In the current study we noted that gamma delta T cells play a positive role in the humoral response. These positively acting gamma delta T cells are required for the successful adoptive cell transfer of the humoral response, as well as for in vitro generation of plaque-forming cells (PFC). The presented results show that gammadelta T cells cause an increase in interleukin-10 (IL-10) production, which partly elucidates the mechanism of action of these cells. However, experiments with cell culture inserts strongly suggest that direct cell-cell contact between immune and gamma delta H-2-compatible regulatory T cells is critical to the exertion of the positive immunoregulatory function of gamma delta cells. The mechanism of cross-talk between these two cell populations is still not clear but we regard as most likely that the positively acting gamma delta T cells may interact with a complex of heat-shock protein-non-polymorphic MHC (IB) on the surface of T helper type 2 and/or B cells. This could provide, by direct cell-cell contact, the cognate recognition between gamma delta T-cell receptors and heat-shock protein-MHC that leads to positive internal signalling in the immune cells.     

Effect of antioxidants on the immune response of Helicobacter pylori

Antioxidants are substances capable of inhibiting oxidation. In chronic diseases, inflammatory response cells produce oxygen free radicals. Oxygen free radicals cause DNA damage, and this may lead to gene modifications that might be carcinogenic. Chronic Helicobacter pylori infection causes the production of DNA-damaging free radicals. In recent years, various groups have studied the effects of antioxidants, especially on H. pylori -associated gastric cancer. In most of the studies, it has been shown that H. pylori infection does affect the level of antioxidants measured in the gastric juice, but there are also controversial results. Recent experimental studies, both in vivo and in vitro, have shown that vitamin C and astaxanthin, a carotenoid, are not only free radical scavengers but also show antimicrobial activity against H. pylori. It has been shown that astaxanthin changes the immune response to H. pylori by shifting the Th1 response towards a Th2 T-cell response. Very few experimental studies support the epidemiologic studies, and further studies are needed to describe the effect and the mechanism of antioxidants in the H. pylori immune response.     

The humoral immune response of mouse bone marrow lymphocytes in vitro.

Mouse bone marrow (BM) small lymphocytes are shown to contain competent precursors for a primary haemolytic plaque forming cell (PFC) response to heterologous red blood cells and TNP in an in vitro culture system. Their response is dependent on T co-operative factors, which can be provided by irradiated spleen cells activated by concanavalin A or the supernatant of an allogeneic culture, added at the beginning or after 24 h of culture. The frequency of PFC precursors for the response to SRBC is found to be equal or higher in BM than spleen cultures. However, BM lymphocyte cultures stimulated by E. coli lipopolysaccharide show an increase of DNA synthesis but contain only few polyclonal PFC, in contrast to spleen.     

Reaction of peripheral blood lymphocytes to mitogens from the age aspect

The reaction of peripheral blood lymphocytes from healthy blood donors aged from 19 to 49 years to the mitogens phytohemagglutinin, concanavalin A (con A), and rabbit serum against human thymocytes (ATS) was investigated. A significant decrease in the proliferative response of the lymphocytes to con A was found in subjects over 30 years old. Significant negative correlation was found between the indices of the proliferative response and the age of the donors during stimulation by con A for the whole group of subjects, but for stimulation by ATS only for subjects aged 30–49 years. Analysis of the intensity of incorporation of [³H]-thymidine shows a decrease with age in the proportion of cells with intensively labeled nuclei and an increase in the number of cells with weak labeling of the nucleus both with a decrease in the indices of the proliferative response with age and also when no significant differences were found in these indices.Laboratory of General Pathophysiology, Institute of Psychiatry, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR A. M. Chernukh.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 84, No. 11, pp. 600–602, November, 1977.     

Immunoglobulin G antibody response to infection with coccoid forms of Helicobacter pylori

An increasing number of studies support a potential role for coccoid forms in Helicobacter pylori infection. Evidence for this was obtained through scanning microscopy, genetic analysis for virulence traits, examination of the presence and activity of key enzymes, and other methods. We studied the serum immunoglobulin G responses to coccoid H. pylori forms by enzyme-linked immunosorbent assay (ELISA) and immunoblotting and compared them with those of bacillary cells. Sera from a total of 295 infected individuals were studied; these included sera from 100 patients with duodenal ulcers, 98 patients with nonulcer dyspepsia, 11 patients with gastroduodenal cancer, and 86 asymptomatic individuals. Initially, we characterized and selected coccoid and bacillary antigenic preparations by one-dimensional (1-D) and 2-D gel electrophoresis and immunoblotting. Data showed that coccoid and bacillary preparations with comparable protein contents have similar patterns in 1-D and 2-D electrophoresis gels and antigenic recognition at blotting. These results revealed that coccoid and spiral antigens in ELISA can equally recognize specific antibodies to H. pylori in sera from infected individuals. The analysis of the spiral and coccoid preparations by Western blotting showed no major differences in antigen recognition. No specific bands or profiles associated with a single gastric condition were identified.     

Inhibition of the pokeweed mitogen-induced response of normal peripheral blood lymphocytes by humoral components of colostrum.

Colostral whey at dilutions up to 1 : 100 inhibited both the uptake of tritium-labelled thymidine and the differentiation of pokeweed mitogen-stimulated peripheral blood lymphocytes from normal adults into immunoglobulin-containing plasma cells. Upon gel filtration (Sephadex G-200), inhibitory activity was associated with high molecular weight fractions. Secretory component, but not monomeric, polymeric or colostral IgA, IgM, IgG, lactoferrin, casein or alpha-lactalbumin, inhibited the response to mitogen. Supernatants from cultures of colostral cells did not induce the differentiation of adult peripheral or cord blood lymphocytes into IgA-containing cells and did not stimulate the uptake of tritium-labelled thymidine.     

Profiling the humoral immune response to Borrelia burgdorferi infection with protein microarrays

To determine the cell envelope proteins of Borrelia burgdorferi recognized by immune sera of patients with late Lyme disease, we developed a Borrelia microarray containing proteins encoded by 90 cell envelope genes and their homologs described in the annotated genomic sequence of B. burgdorferi, strain B31. The protein microarray was used to profile the humoral immune response using sera from 13 patients with late Lyme disease and four normal controls. Although there was considerable heterogeneity in the individual sera responses, 25 of the cell envelope proteins were recognized by seven or more samples. Sera from non-infected individuals lacked reactivity against any of the proteins on the array. Among the most antigenic envelope proteins, BLAST search revealed little sequence homology to known microbial proteins from other species. The proteins that were highly seropositive included several members of the Erp gene families, BBA24 (decorin binding protein A (DbpA)) and members of the Borrelia gene family Pfam113 that code for the Mlp lipoprotein gene family. Several novel, uncharacterized B. burgdorferi antigens identified in this study were BBA14, BBG23, BB0108, BB0442 and BBQ03. The accurate diagnosis of Lyme disease depends on correlating objective clinical abnormalities with serological evidence of exposure to B. burgdorferi. A protein array of the envelope proteins of Borrelia burgdorferi may be very useful in specifically identifying patients with Lyme disease. This approach could contribute to a more rapid discovery of antigens not expressed in vitro that may be useful for the development of vaccine and diagnostics.