Riitta Karttunen

Detection of Chlamydia pneumoniae–Reactive T Lymphocytes in Human Atherosclerotic Plaques of Carotid Artery

Linkage between Chlamydia pneumoniae infection and atherosclerosis has been confirmed in several studies, but the precise role of this organism in the disease process is not known. We investigated the relation and reactivity of T lymphocytes of human carotid plaques to C pneumoniae antigens. Tissue specimens were obtained from 17 patients who underwent carotid endarterectomy. Immunohistological staining and/or in situ hybridization revealed the presence of C pneumoniae in 11 (64%) of the 17 of the cases. Inflammatory infiltration seen in the vessel walls consisted primarily of CD45RO+ T-memory lymphocytes (median 80%, range 50% to 90%), whereas CD20+ B cells and monocytes were in minor proportion. In vivo activated T lymphocytes were propagated from the specimens with interleukin-2, and the antigen specificity of the established T-cell lines (TLLs) was analyzed against C pneumoniae elementary body antigen. TLLs were established from all 17 carotid tissues but none from the control specimens of ascending aorta. C pneumoniae was recognized as a specific T-cell-stimulating antigen in 7 (41%) of 17 cases. Further analyses of the C pneumoniae-reactive TLLs showed that chlamydial 60-kDa heat-shock protein induced specific proliferation in 5 (71%) of 7 cases and revealed 2 haplotype (DRB1*1502 and DQB1*06) binding motifs in human 60-kDa heat-shock protein. C pneumoniae was identified as a specific microbial antigen recognized by 41% of TLLs propagated from in vivo activated plaque T cells. Our results suggests that cell-mediated immunity to C pneumoniae plays a role in the atherosclerotic process and that this response may involve autoimmunity.

Genetic variations in IL6 associate with intervertebral disc disease characterized by sciatica

&NA; Intervertebral disc disease (IDD) characterized by sciatica is a common disorder affecting about 5% of individuals. Environmental factors can predispose to this disease, but IDD has a strong genetic background. Recent evidence suggests that inflammation is one of the key factors in the etiology of IDD. Here, a possible role of the inflammatory mediator genes was studied in 155 patients with IDD‐related sciatica and 179 controls. Forty‐eight patients were analyzed for mutations in the IL1A, IL1B, IL6 and TNFA genes, and 16 polymorphisms in 10 candidate cytokine genes (IL1A, IL1B, IL1RN, TNFA, IL2, IL4, IL4R, IL6, IL10, IFNG) were genotyped from all subjects. No disease‐causing mutations were identified in IL1A, IL1B, IL6 or TNFA. Allele frequencies were, however, significantly different between the two groups for IL6 SNP, T15A in exon 5 (P=0.007). Furthermore, the genotypes AA and AT of the exon 5 SNP were more common in the patients (P=0.011; OR=4.4, 95% CI=1.2–15.7; AR=7.5%, 1.6–13.1%). Haplotypes were then generated for four IL6 SNPs, G‐597A, G‐572C, G‐174C, and T15A in exon 5. Haplotype GGGA was more common in the patients (P=0.011; OR=4.8, 95% CI=1.6–14.5). To evaluate attributable risk, haplotype pairs were assigned for the individuals. The presence of GGGA/GGGA or GGGA/other genotypes had an OR of 5.4 (95% CI=1.5–19.2). Association of GGGA with disease was highly significant (P=0.0033), and the associated AR was 6.8% (1.9–11.5%). These findings support the role of IL‐6 genetic variations in discogenic pain.

Helicobacter pylori induces lymphocyte activation in peripheral blood cultures

Helicobacter pylori‐induced, in vitro stimulation of mononuclear cells was characterized by measuring DNA synthesis response, interferon‐gamma (IFN‐γ) secretion and the number of immunoglobulin‐secreting cells. The strength of these responses was measured in 51 subjects comprising 36 dyspeptic patients from the Gastroenterological Unit and 15 members of the laboratory staff. Nineteen subjects had antibodies to H. pylori and 32 did not. The responses were compared with respect to H. pylori antibody status. Positive stimulation of DNA synthesis (stimulation index > 2) was obtained in 96% of the subjects, and the bacterium induced IFN‐γ secretion in all of them. The induction of immunoglobulin‐secreting cells revealed B cell in vitro stimulation. The antibody‐positive subjects had a tendency to synthesize less DNA (P not significant) and secrete less IFN‐γ (P < 0.05) in response to H. pylori than did the antibody‐negative ones, but the differences were not marked. The results show that the whole H. pylori bacterium stimulates mononuclear cells. The nature of the stimulation is not yet characterized (non‐specific versus specific).

Blood lymphocyte proliferation, cytokine secretion and appearance of T cells with activation surface markers in cultures with Helicobacter pylori. Comparison of the responses of subjects with and without antibodies to H. pylori

A whole inactivated H. pylori bacterium preparation was found to stimulate blood mononuclear cells from both antibody‐positive and antibody‐negative subjects, but the antibody‐positive subjects tended to have lower proliferation responses. The present study was designed to characterize T cell activation further by measuring several components of the response. Eighty‐seven subjects (80 dyspeptic patients and seven healthy persons from the laboratory staff) with or without antibodies to H. pylori were studied by measuring the DNA synthesis induced by several H. pylori concentrations 1–23 μg/ml) and the control stimulants PPD, tetanus toxoid and pokcweed mitogen (PWM). H. pylori‐induced secretion of interleukin‐2 (IL‐2), tumour necrosis factor‐alpha (TNF‐α), interleukin‐4 (IL‐4), soluble CD8 and IL‐2 receptor (IL‐2R) molecules and H. pylori‐ and PPD‐induced appearances of IL‐2R+ and HLA‐DR+ T cells were measured in a smaller number of subjects. H. pylori‐induced DNA synthesis was again lower in the antibody/bacterium‐positive subjects, while no differences between the two groups were found in cultures stimulated by unrelated antigens or PWM. Soluble IL‐2R and TNF‐α were detectable in cultures with H. pylori from all subjects, while the amount of IL‐2 did not differ from that in the background culture. No differences were found in the amounts of IL‐2 or soluble IL‐2R between the antibody‐positive and negative subjects; while the former tended to secrete more soluble CD8 molecules, a difference which was significant with the smaller H. pylori concentration used (P<0.01). The numbers of HLA‐DR+ and IL‐2R+ T cells increased in cultures with H. pylori or PPD from all the subjects, the majority of both cells having the CD4 phenotype. Numbers of DR+ and IL‐2R+ T cells were similar in the cultures of the antibody‐positive and negative subjects, but the respective CD8 subsets were increased in the former. The confirmed decrease in proliferation in the antibody‐positive subjects does not seem to be connected with lower IL‐2/IL‐2R responses but may involve CD8 cell activation.

Effect of CD14 promoter polymorphism and H. pylori infection and its clinical outcomes on circulating CD14

CD14 is a pattern recognition receptor on the membranes of monocytes and macrophages for several microbial products, of which lipopolysaccharide (LPS) is the best known. A shed form of CD14 is present in serum. As the CD14 gene promoter polymorphism –159C/T and some bacterial infections may affect the sCD14 levels, we compared the impact of both the CD14 promoter polymorphism and Helicobacter pylori infection on serum sCD14 levels in 201 dyspeptic patients (group 1) who had undergone gastroscopy, and 127 staff members (group 2) with no endoscopy. sCD14 was measured from the sera by a commercial enzyme immunoassay (EIA), and CD14 genotyping was carried out with PCR. Helicobacter pylori infection was detected by serology and/or culture or PCR. sCD14 levels were elevated in the subjects carrying the T allele (CT or TT genotype) in both groups when compared with subjects with the CC genotype. Overall, H. pylori‐positive subjects tended to have higher sCD14 levels compared with H. pylori‐negative subjects. In group 1 consisting of dyspeptic patients, those with gastric ulcer, gastric erosion or duodenal ulcer had significantly elevated levels of sCD14 compared with the patients with normal endoscopic findings or macroscopic gastritis. The recent use of NSAIDs was also associated with enhanced sCD14. Thus, we were able to show several factors, one genetic and the other environmental (H. pylori infection and mucosal lesion), to have an impact on sCD14.

Gastrointestinal complaints and diagnosis in children: a population-based study.

Aim: To find out the extent to which children at 10–11 y of age suffer from various gastrointestinal complaints and how often a food‐induced or other diagnostic disorder might be assessed behind them, we carried out a population‐based survey of 404 children in a rural Finnish town. Methods: A questionnaire filled in retrospectively by their parents was used to describe the frequency of various abdominal symptoms during the previous 2 y and to select the symptomatic subjects for closer clinical examination. In the clinical investigation of the children, an elimination challenge with milk protein and lactose intolerance tests, as well as endoscopic examinations in selected cases and blood tests, were performed. Results: In all, 110 (27%) subjects reported some gastrointestinal (GI) complaints during the last 2 y; 64 (16%) meeting the Apley criteria for recurrent abdominal pain. A specific organic or functional disorder was found in 26 subjects (6%), two having no GI symptoms. Milk protein intolerance was the most common specific disorder diagnosed in nine subjects (2.2%), followed by lactose intolerance in eight (2%), coeliac disease in five (1.2%) and Helicobacter pylori infection in three (0.7%). An endoscopic examination performed on 17 subjects (4.2%) and a colonoscopy on three revealed significant findings in 11; lymphonodular changes being most common, occurring in five subjects. Subjects with milk protein‐induced disorders showed significantly lower IgA‐class antibodies to milk and its fractions than the non‐symptomatic controls. Chronic diseases, short breastfeeding, GI problems and food intolerance during the first year of life were observed as significant risk factors in determining whether a subject belonged to the group experiencing any GI complaints.

Chlamydial heat shock protein 60-specific T cells in inflamed salpingeal tissue☆

OBJECTIVE To evaluate the role of chlamydial heat shock protein 60 (CHSP60)-specific T-lymphocytes in tubal factor infertility. DESIGN Case series of patients with tubal factor infertility. SETTING Infertility Clinic, Department of Obstetrics and Gynecology, Helsinki University Central Hospital and Laboratory of Cell-Mediated Immunity, National Public Health Institute, Oulu, Finland. PATIENT(S) Five patients with tubal factor infertility who underwent elective salpingectomy because of hydrosalpinges. INTERVENTION Collection of salpingeal tissue specimens for in vitro culture of T-lymphocytes. MAIN OUTCOME MEASURE(S) Cloning of Chlamydia trachomatis and CHSP60-specific T-lymphocyte lines derived from inflamed salpingeal tissue. Cytokine production analysis of the established T-lymphocyte clones. RESULT(S) Seventy-seven (34%) of the 229 T-lymphocyte clones recognized C. trachomatis and C. pneumoniae elementary bodies as target antigens. One-third of these Chlamydia genus-specific T-lymphocyte clones further recognized CHSP60 as the target antigen. Most of the CHSP60-specific T-lymphocyte clones produced predominantly IL-10. CONCLUSION(S) CHSP60 may be an important T-lymphocyte antigen involved in the immunopathogenesis of tubal damage associated with chronic C. trachomatis infection.

Serum levels of interleukin-10 and tumour necrosis factor-α in chronic periodontitis

AIMS To investigate, using a cross-sectional study design, whether the extent of periodontal inflammation associates with the serum levels of cytokine interleukin (IL)-10 and tumour necrosis factor (TNF)-α and their ratio. MATERIAL AND METHODS The study group consisted of 61 subjects with chronic periodontitis and 30 control subjects with minimally inflamed periodontal tissues. Probing pocket depth (PD), bleeding on probing (BOP) and periodontal attachment level (AL) were measured. The serum IL-10 (pg/ml) and TNF-α (U/l) levels were analysed using enzyme-linked immunosorbent assays. After categorization of the subjects, associations between serum IL-10 and TNF-α levels and the extent of periodontal inflammation were studied using linear regression models adjusted for age, gender, body mass index and smoking. RESULTS A negative, partly dose-dependent association existed between the extent of BOP, PD ≥ 4 mm and AL ≥ 4 mm and serum IL-10 level. The subjects in the periodontitis group presented significantly higher serum TNF-α levels and their TNF-α/IL-10 ratio was approximately threefold when compared with the ratio in the control group. CONCLUSIONS The significantly higher serum TNF-α/IL-10 ratio in the subjects with chronic periodontitis when compared with the ratio in the controls is indicative of a stronger systemic pro-inflammatory state in chronic periodontitis.

Human leucocyte antigen and TNFα polymorphism association in microscopic colitis

Objectives Coeliac disease (CD) is common in patients with microscopic colitis (MC). The human leucocyte antigen (HLA)-DR3-DQ2 haplotype is strongly associated with CD, and there is evidence for an association with MC. We analysed the genetic background of MC by assessing the haplotypes of HLA-DR3-DQ2 and HLA-DR4-DQ8. In addition, TNF&agr; gene polymorphism (−308) associated with susceptibility to several autoimmune diseases was studied. Methods Eighty patients with MC including 29 with collagenous colitis (CC) and 51 with lymphocytic colitis (LC) were typed for HLA-DR3-DQ2, and HLA-DR4-DQ8 molecule encoding genes using either an allele-specific PCR, or hybridization with sequence-specific oligonucleotides. Duodenal biopsies (N=78) confirmed the diagnosis of CD in 15 (18.8%) patients. TNF&agr;(308) alleles were analyzed in 78 patients with MC (27 with CC and 51 with LC). A control group of 3627 patients was used in the HLA study and 178 patients in the TNF&agr; study. Results HLA-DR3-DQ2 haplotype was more frequent in patients with MC (43.8%) including both subgroups (LC, 44.8%; CC, 43.1%; P<0.001), and MC with CD (86.7%; P<0.001) and without CD (33.3%; P=0.003), compared with the controls (18.1%). Similarly, the TNF2 carrier rate was higher in MC (46.2%; P<0.001) including both CC (44.4%; P=0.031) and LC (47.1%; P=0.001), and both MC patients with CD (66.7%; P=0.001) and without CD (39.3%; P=0.019), compared with the controls (23%). Conclusion Both CC and LC are associated with the HLA-DR3-DQ2 haplotype and with TNF2 allele carriage. These associations are present also in MC patients without CD. The shared predisposing HLA-DR3-DQ2 haplotype and the high prevalence of CD in patients with MC suggest an epidemiological overlap, and probably some similarities in the pathogenesis of CD and MC.

Gastric inflammation is enhanced in children with CagA-positive Helicobacter pylori infection

BACKGROUND Helicobacter pylori infection is likely to be acquired at an early age. The factors leading to active inflammation in childhood, however, are largely unknown. SUBJECTS AND METHODS We determined the CagA status, the best characterized virulence factor of H. pylori, and serum antibodies of IgG and IgA classes to H. pylori in 39 infected children. RESULTS Mononuclear cell infiltration in the antrum but not in the gastric body was more intense in CagA-positive children than in CagA-negative children. The degree of polymorphonuclear cell infiltration on the other hand was independent of the CagA status. The antibody titers of IgG and IgA classes to H. pylori were higher in CagA-positive than in CagA-negative infections (P<0.001 and P<0.01, respectively). IgG antibody titers to H. pylori correlated directly with the density of mononuclear and polymorphonuclear cell infiltration in the gastric antrum but not in the gastric body. CONCLUSION H. pylori-infected children with CagA antibodies seem to have a more severe inflammation in the gastric antrum than CagA-negative children as shown by an increase in the density of antral mononuclear cells. A finding of higher serum antibody titers to H. pylori in CagA-positive children may be related to this enhancement of inflammation.