Peripheral blood mononuclear cell proliferative responses to soluble and particulate heat shock protein 65 in health and inflammatory bowel disease

Objectives: The aims of this study were to determine, in peripheral blood mononuclear cells (PBMC), whether particulate antigen triggers (i) an amplified cell proliferative response compared to soluble antigen and (ii) a dysfunctional response in cells derived from patients with chronic inflammation and specifically in those with inflammatory bowel disease (IBD). Subjects: Healthy volunteers (n = 17), inflammatory controls (n = 8) and patients with IBD (n = 17) were recruited from St Thomas’ and Guys’ Hospital, London, UK. Methods: Following optimisation of experimental conditions (0.1–10.0 μg/ml antigen), PBMC were stimulated with (i) 10.0 μg/ml recombinant soluble heat shock protein 65 (hsp 65) and (ii) 1.0 and 10.0 μg/ml hsp 65 conjugated to microparticles (0.5 μm diameter). PBMC proliferative responses were measured by ³H-Thymidine incorporation at day 5 and results compared between groups using unpaired t-test. Results: Conjugation to microparticles of low dose hsp 65 significantly increased overall proliferative responses by 2–11 fold compared to soluble antigen alone (p < 0.05). However, no specific PBMC proliferative dysregulation was noted in cells from subjects with IBD. Conclusions: Low dose antigen, in microparticulate form, leads to amplified cell proliferation in primary human cells, as showed previously in cell lines and animal studies. However there is no abnormal proliferative response in cells from subjects with IBD. Received 8 February 2006; returned for revision 7 March 2006; accepted by G. Wallace 25 October 2006     

Inflammation activates self hsp60-specific T cells

Injection of incomplete Freund's adjuvant (IFA) into the footpads of BALB/c mice induced an acute inflammation. Draining popliteal lymph nodes showed major histocompatibility complex (MHC) class II-restricted proliferation when challenged in vitro with recombinant Mycobacterium bovis 65-kDa heat shock protein (hsp65). αβ Tcell receptor-positive, CD4⁺, hsp65-specific T cell lines and clones were generated from these lymph nodes, and 87% of clones responded to a P galactosidase fusion protein containing residues 238–573 of human hsp60. Seventy percent of these hsp60-responsive clones also responded to a synthetic peptide corresponding to residues 412–423 of the mouse hsp60. This peptide also induced significant responses in IFA-primed lymph node cells but not in lymphoid cells from unimmunized mice. These results demonstrate that T cells specific for epitopes in self hsp60 are activated during inflammatory responses induced in the absence of exogenous bacterial hsp65. The findings of this study may provide a basis for understanding the often reported isolation of mycobacterial hsp65-responsive T cells from inflammatory sites of arthritis patients, and the protective effects of preimmunization with hsp65 in experimental models of arthritis.     

Frequency of anti-hsp60, -65 and -70 antibodies in sera of patients with juvenile idiopathic arthritis

Cross-reactivity between microbial and human heat shock proteins (hsps) led to the concept that hsp might be involved in the etiopathogenesis of autoimmune diseases. We investigated antibodies to recombinant human hsp60, recombinant Mycobacterium bovis hsp65 and to stress-inducible recombinant human hsp70 using enzyme-linked immunosorbent assay (ELISA) in sera of 209 juvenile idiopathic arthritis (JIA) patients and 50 healthy controls. Anti-hsp60 antibodies did not exceed the control level in any JIA patient. The numbers of JIA patients (16/209, 7.6%) who raised anti-hsp65 antibodies was equal to healthy controls (4/50, 8%). Elevated levels of antibodies against hsp70 were found in a cohort of patients with JIA (36.8%) when compared with age-matched healthy individuals (2%). These antibodies were predominantly of IgG isotype in systemic disease and IgM isotype in oligoarthritis. In polyarthritis both IgG and IgM antibodies frequently occurred. Significantly higher anti-hsp70 antibody levels were found in RF-positive JIA patients. The levels of anti-hsp70 antibodies correlated with the severity of disease evaluated on the basis of Steinbrocker's functional classification and rtg staging system. No association between anti-hsp70 antibody levels and ANA, HLA B27 and disease duration (less than 2 years x more than 2 years) was observed except IgM anti-hsp70 antibody where significantly higher levels were also detected in HLA B27-positive patients. The prevalence of anti-hsp70 antibodies is much higher in JIA patients when compared with healthy controls, suggesting their possible role in pathological mechanism of the disease.     

Identification of mycobacteria of the MAIS complex and M. tuberculosis by restriction fragment length polymorphism analysis of hsp65 gene

Restriction fragment length polymorphism analysis of hsp65 gene was performed on museum strains of mycobacteria using Hin6I restrictase. Study of restriction profiles allowed us to distinguish mycobacterial species of the MAIS complex and several strains of nontuberculous mycobacteria. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 142, No. 8, pp. 188–191, August, 2006     

<Emphasis Type="Italic">Eimeria bovis</Emphasis> infection enhances adhesion of peripheral blood mononuclear cells to and their transmigration through an infected bovine endothelial cell monolayer in vitro

The first schizogony of Eimeria bovis takes place in lymphatic endothelial cells of the ileum, resulting in the formation of macroschizonts within 2–3 weeks. In this study, we analyse early cellular immune responses to infected host cells on the basis of peripheral blood mononuclear cell (PBMC) adhesion on and transmigration through infected bovine umbilical vein endothelial cell (BUVEC) monolayers. Adhesion of PBMC was upregulated by an E. bovis infection. Most marked effects were observed 1 day p.i.; thereafter, PBMC adhesion declined reaching control levels from day 8 p.i. onward. CD8⁺ T cells adhered more frequently to infected BUVEC (42%) than CD4⁺ T cells (25%). About one third of attached PBMC were represented by γδ-TCR⁺ T cells. Adhesion of T cells was not restricted to parasitised host cells, but occurred almost equally on non-infected BUVEC within the same monolayer. Furthermore, we found moderately enhanced levels of PBMC transmigration through infected BUVEC monolayers, in particular on day 2 p.i. The data presented here suggest that E. bovis infection of BUVEC induces endothelial cell-derived proinflammatory reactions, which appear suitable for the initiation of both adaptive and innate immune responses.     

Accumulation of human heat shock protein 60-reactive T cells in the gingival tissues of periodontitis patients

Heat shock protein 60s (hsp60) are remarkably immunogenic, and both T-cell and antibody responses to hsp60 have been reported in various inflammatory conditions. To clarify the role of hsp60 in T-cell responses in periodontitis, we examined the proliferative response of peripheral blood mononuclear cells (PBMC), as well as the cytokine profile and T-cell clonality, for periodontitis patients and controls following stimulation with recombinant human hsp60 and Porphyromonas gingivalis GroEL. To confirm the infiltration of hsp60-reactive T-cell clones into periodontitis lesions, nucleotide sequences within complementarity-determining region 3 of the T-cell receptor (TCR) beta-chain were compared between hsp60-reactive peripheral blood T cells and periodontitis lesion-infiltrating T cells. Periodontitis patients demonstrated significantly higher proliferative responses of PBMC to human hsp60, but not to P. gingivalis GroEL, than control subjects. The response was inhibited by anti-major histocompatibility complex class II antibodies. Analysis of the nucleotide sequences of the TCR demonstrated that human hsp60-reactive T-cell clones and periodontitis lesion-infiltrating T cells have the same receptors, suggesting that hsp60-reactive T cells accumulate in periodontitis lesions. Analysis of the cytokine profile demonstrated that hsp60-reactive PBMC produced significant levels of gamma interferon (IFN-gamma) in periodontitis patients, whereas P. gingivalis GroEL did not induce any skewing toward a type1 or type2 cytokine profile. In control subjects no significant expression of IFN-gamma or interleukin 4 was induced. These results suggest that periodontitis patients have human hsp60-reactive T cells with a type 1 cytokine profile in their peripheral blood T-cell pools.     

Differential rat T cell recognition of cathepsin D-released fragments of mycobacterial 65 kDa heat-shock protein after immunization with either the recombinant protein or whole mycobacteria

T cells specific for the mycobacterial 65 kDa heat-shock protein(hsp65) play a pivotal role in the development of adjuvant arthritis(AA) in Lewis rats. Upon adoptive transfer, CD4⁺ T cells recognizinga particular hsp65 epitope trigger the onset of disease. Activationof hsp65-reactlve T cells can be achieved by immunization withheat-killed mycobacteria in mineral oil—complete Freund'sadjuvant (CFA)—or with purified recombinant hsp65. Arthritis,however, will only develop after immunization with CFA. In fact,prelmmunlzatlon with hsp65 protects against any subsequent attemptto induce AA. In this study, we examined polyclonal lymph nodecell responses in Lewis rats, Immunized with either CFA or purifiedrecombinant hsp65 in incomplete Freund's adjuvant, to a setof hsp65 fragments generated by a mild digestion with cathepsinD. Prollferatlve responses to several hsp65 fragments variedwith the type of antigen used for immunization. A cathepsinD-released fragment, Identified as residues 376–408, preferentiallytriggered proliferation of rat T cells after hsp65 Immunization.Prelmmunlzatlon of Lewis rats with this peptlde delayed theonset and reduced the severity of AA. Prelmmunlzatlon with anotherfragment which was preferentially recognized after CFA immunization,representing residues 40–60, did not have such a protectiveeffect. Our findings suggest the presence of mycobacterial hsp65determinants that selectively trigger AA-regulatlng T cellsand illustrate that cathepsin D may be used as an experimentaltool to generate such determinants.     

T cell reactivity to an epitope of the mycobacterial 65-kDa heat-shock protein (hsp 65) corresponds with arthritis susceptibility in rats and is regulated by hsp 65-specific cellular responses.

Adjuvant arthritis (AA) can be induced in genetically susceptible rats by immunization with heat-killed mycobacteria suspended in mineral oil. From our analysis of arthritogenic T cell clone A2b, obtained from an arthritic Lewis rat and specific for the 180-188 epitope of mycobacterial 65-kDa heat-shock protein (hsp 65), the possible origin of AA was explained by the existence of a molecular mimicry of the 180-188 epitope with a cartilage-associated self antigen. We now have shown that Lewis rats respond to the 180-188 epitope after Mycobacterium tuberculosis immunization and that arthritis-resistant Fisher and (Lewis x Fisher)F1 rats, although major histocompatibility complex class II identical with Lewis, do not respond to this epitope. However, in rare cases of arthritis in Fisher rats, responses to the epitope were seen. We obtained no evidence for a defect at the level of antigen processing and presentation or for suppression in Fisher rats. Thus, non-responsiveness in Fisher rats was likely due to a difference at the level of the T cell repertoire. Previously, we have reported that pretreatment with hsp 65 in experimental arthritis, and not only in AA, caused resistance to arthritis induction. We now present evidence that immunization with hsp 65 or in vitro stimulation with hsp 65 may lead to inhibition of responses specific for epitope 180-188. Thus the hsp 65-induced resistance to arthritis is probably caused by the induction of regulatory control specifically targeted at the 180-188 epitope. Especially in rats that tend to focus their responses on the critical 180-188 sequence, such as Lewis, regulation seems to develop following immunization with hsp 65. Since recent evidence suggests that hsp 65 and also the 180-188 epitope have a role in human arthritic conditions, the present findings are expected to contribute to further experimentation directed at exploiting hsp 65 or its epitopes for the development of new therapeutical approaches in humans.     

Antibody Response of Restricted Isotype to Heat Shock Proteins in Juvenile Chronic Arthritis

Synovial fluid and peripheral blood mononuclear cells from juvenile chronic arthritis (JCA) patients have previously been shown to exhibit substantial proliferative responses to both human and mycobacterial heat shock protein (hsp) 65. We investigated the nature of the antibody response to mycobacterial and E. coli hsp 65 and human and E. coli hsp 70 in 56 JCA patients using an ELISA. Elevated levels of antibodies to both human and E. coli hsp 70 were demonstrated. With hsp 65, raised levels of antibodies to the mycobacterial but not the E. coli protein were detected. Overall, 48% of patient serum samples contained antibodies of at least one isotype to mycobacterial hsp 65. These antibodies were predominantly of IgG and IgM isotype, a finding in contrast to adult rheumatoid arthritis, where IgA and IgG isotypes are most often detected.     

Cell-Mediated Immunity to Human and Escherichia coli 60-kDa Heat Shock Protein in Women: Association with A History of Spontaneous Abortion and Endometriosis

PROBLEM: Heat shock proteins are expressed during early pregnancy and in peritoneal fluids from women with endometriosis. The relationship between a cell-mediated immune response to human 60-kDa heat shock protein (hsp60), spontaneous abortion, and endometriosis was examined. METHOD OF STUDY: Peripheral blood mononuclear cells (PBMCs) from 110 female partners of infertile couples undergoing in vitro fertilization and 41 fertile control subjects were incubated with human hsp60 or Escherichia coli hsp60. PBMC proliferation was measured by [³H]thymidine incorporation, and a stimulation index was calculated. RESULTS: Lymphocytes from 21.8% of the infertile women, as opposed to 7.3% of the fertile women, proliferated in response to human hsp60 (P = 0.05). In contrast, proliferation in response to the E. coli hsp60 was equivalent in both groups. Within the infertile group, the response to human hsp60 was 40.7% among women with a history of spontaneous abortion and only 12% in those with no history of spontaneous abortion (P = 0.003). There was no association between immunity to E. coli hsp60 and spontaneous abortion or between immunity to human hsp60 and therapeutic abortion or the cause of infertility. Immunity to the E. coli hsp60 was associated with endometriosis. CONCLUSIONS: A cell-mediated autoimmune response to human hsp60 is associated with a history of spontaneous abortion, whereas immunity to E. coli hsp60 was most prevalent in women with endometriosis.     

Modulation of Mycobacterium bovis-Specific Responses of Bovine Peripheral Blood Mononuclear Cells by 1,25-Dihydroxyvitamin D3

Historically, administration of vitamin D has been considered beneficial in the treatment of tuberculosis. The interaction of this vitamin {i.e., 1,25-dihdroxyvitamin D₃ [1,25(OH)₂D₃]} with the antitubercular immune response, however, is not clear. In the present study, in vitro recall responses of peripheral blood mononuclear cells (PBMC) from cattle infected with Mycobacterium bovis were used to study the immune-modulatory effects of 1,25(OH)₂D₃ on M. bovis-specific responses in vitro. Addition of 1 or 10 nM 1,25(OH)₂D₃ inhibited M. bovis-specific proliferative responses of PBMC from M. bovis-infected cattle, affecting predominately the CD4⁺ cell subset. In addition, 1,25(OH)₂D₃ inhibited M. bovis-specific gamma interferon (IFN-γ) production yet enhanced M. bovis-specific nitric oxide (NO) production. Lymphocyte apoptosis, measured by flow cytometry using annexin-V staining, was diminished by addition of 1,25(OH)₂D₃ to PBMC cultures. These findings support the current hypothesis that 1,25(OH)₂D₃ enhances mycobacterial killing by increasing NO production, a potent antimicrobial mechanism of activated macrophages, and suggest that 1,25(OH)₂D₃ limits host damage by decreasing M. bovis-induced IFN-γ production.     

TRH receptor on immune cells:In vitro andin Vivo stimulation of human lymphocyte and rat splenocyte DNA synthesis by TRH

This work examined whether (1) immune cells express thyrotrophin releasing hormone (TRH) receptor mRNA and (2) TRH modulates lymphocyte activation. By Northern blot of RNA extracted from human peripheral blood mononuclear cells (PBMC) and rat splenocytes, a single TRH receptor mRNA band of about 3.8 kb (identical to that obtained from pituitary cells) was obtained, under both basal and stimulated conditions. A significant increase in DNA synthesis was observed in phytohemagglutinin-stimulated PBMC and concanavalin A (Con A) stimulated splenocytes when TRH (10^–6 M-10^–12 M) was added. After 5, 30, 60, 180 min and 24 h of TRH administrationin vivo, a significant increase in the rat splenocyte proliferative response to Con A was observed.In vivo administration of anti-rat TSH antibody (1/1000) blocked the increase observed after 30 min of TRH administration on the Con A stimulated splenocyte response. TRH possess immunostimulatory functions directly via its receptor and indirectly via release of other immunostimulatory factors such as thyrotrophin.     

Preferential Binding withEscherichia colihsp60 of Antibodies Prevalent in Sera from Patients with Rheumatoid Arthritis

One hundred thirty-two patients with various connective tissue disorders, including 60 with rheumatoid arthritis (RA), had antibodies against human as well asEscherichia colihsp60 in titers significantly higher than those of normal controls. There was a correlation between titers of antibody to human hsp60 and those toE. colihsp60. Levels of antibodies against human andE. colihsp60 were lower in joint fluids than in sera, indicating little production of antibodies in the joint. Antibodies affinity-purified withE. colihsp60 bound strongly with the homologous hsp60, but weakly with human hsp60. However, antibodies affinity-purified with human hsp60 bound comparably with bothE. colihsp60 and human hsp60. Antibodies affinity-purified withMycobacterium tuberculosishsp65 bound to human hsp60 with a reactivity similar to the reactivity of those affinity-purified with human hsp60. The reactivity to the three hsp60 species was lost when sera were absorbed withE. colihsp60, while the reactivity toE. colihsp60 remained after extensive absorption withM. tuberculosishsp65 or human hsp60. These results indicate that anti-hsp60 antibodies in patients with RA and other connective tissue disorders are raised by infection with intestinal microorganisms such asE. coli.They may represent another example of autoimmune responses triggered by antigenic mimicry of host proteins to microbes and suggest that the reactivity of antibodies from RA patients withM. tuberculosishsp65 might have been a cross-reaction with theE. colihomologue.     

<Emphasis Type="Italic">Ex vivo</Emphasis> production of interferon-γ, tumor necrosis factor-α, and interleukin-6 by mouse macrophages during infection with <Emphasis Type="Italic">M. bovis</Emphasis> and <Emphasis Type="Italic">M. tuberculosis</Emphasis> H37Rv

Ex vivo production of IFN-γ, TNF-α, and IL-6 by mouse peritoneal macrophages was studied during successive infection with the vaccine strain M. bovis BCG and virulent strain M. tuberculosis H37Rv. The increase in the concentrations of TNF-α and IL-6 did not depend on the sequence of macrophage infection with the vaccine or virulent strain, but was related to the presence of the vaccine strain M. bovis BCG in the medium. IFN-γ production depended on infection of macrophages with the virulent strain M. tuberculosis H37Rv. The concentration of IFN-γ was maximum during primary infection with the virulent strain and did not increase after successive infection with the virulent and vaccine strain. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 144, No. 11, pp. 552–555, November, 2007     

ANTIBODIES AGAINST THE HUMAN HEAT SHOCK PROTEIN hsp70 IN PATIENTS WITH SEVERE CORONARY ARTERY DISEASE

Heat shock proteins (hsps) play complex role in the function of the immune system, they can activate both humoral and cellular immune response, as well the complement system. Although autoimmunity to hsp70 was implicated in certain autoimmune diseases and other conditions, the exact role of anti-hsp70 antibodies is not known. It was demonstrated by our previous work and other's findings that antibodies against the 60 kDa hsps are strongly associated with coronary atherosclerosis and carotis disease. It is also known that there is increased hsp70 expression at different sites of atherosclerosis. Therefore our aim was to study whether level of anti-hsp70 antibodies correlate with the presence of severe coronary artery disease (CAD). We measured and compared anti-hsp70 IgG antibody levels in CAD patients (n=99) and healthy subjects (n=99) with ELISA. The frequency of these antibodies was high in both groups and there was no significant difference in the median level of anti-hsp70 antibodies between patients with severe CAD and controls (653 (400–1141) vs. 630 (326–1152) AU/mL, P=0.337). Adjustment for age, sex, BMI and lipid parameters did not change this result. Furthermore we did not find a correlation between anti-hsp70 antibody levels and certain risk factors of CAD (age, lipid parameters, body mass index, C-reactive protein, gender, smoking, diabetes and anti-hsp60 antibodies). By contrast, in accordance with our previous findings, anti-hsp60 and anti-hsp65 antibody levels were significantly higher in CAD patients, compared to this control group (p<0.0001 for both variables). We did not find any correlation between the levels of anti-hsp70 and anti-hsp60 or anti-hsp65 antibodies either in the patients or the controls. The exact role of hsp70 in atherosclerosis is controversial, but we suggest that humoral immunity against human hsp70 does not contribute to coronary atherosclerosis in contrast to antibodies against 60 kDa hsps.     

T-lymphocyte reactivity to the recombinant mycobacterial 65- and 70-kDa heat shock proteins in multiple sclerosis

Owing to their conservation and immunogenicity, heat shock proteins (hsps) represent a class of potential autoantigens. Moreover, they could be targets for gamma delta T lymphocytes, which are prominent in various immune disorders. We studied the T cell proliferative primary responses to recombinant M. bovis 65 kDa hsp (hsp65) and M. tuberculosis 70 kDa hsp (hsp70) in 31 patients with multiple sclerosis (MS), 19 patients with other neurological diseases (OND) and 19 healthy individuals. Positive responses to hsp70, but not to hsp65 were significantly more frequent in patients with MS than in patients with OND or in healthy individuals. In order to verify and refine these results and to characterize the hsp reactive T lymphocytes, we screened 147 PPD-specific long-term T cell lines (76 from 10 patients with MS and 71 from 12 healthy donors) for their proliferative response to hsp65 and hsp70. hsp70-reactive T lines were significantly more common in patients with MS than in healthy controls. The number of T lines responding to hsp65 increased in the MS group only slightly. In 19 T lymphocyte lines from patients with MS and healthy donors, a cytofluorometric analysis was performed with special attention paid to distinct T cell receptor gamma delta determinants. With one exception, in each line the population of gamma delta T cells remained a minority. We conclude that an increased T cell response to mycobacterial hsp70 may be present in patients with multiple sclerosis.     

Antibodies against the human heat shock protein hsp70 in patients with severe coronary artery disease

Heat shock proteins (hsps) play complex role in the function of the immune system, they can activate both humoral and cellular immune response, as well the complement system. Although autoimmunity to hsp70 was implicated in certain autoimmune diseases and other conditions, the exact role of anti-hsp70 antibodies is not known. It was demonstrated by our previous work and other's findings that antibodies against the 60 kDa hsps are strongly associated with coronary atherosclerosis and carotis disease. It is also known that there is increased hsp70 expression at different sites of atherosclerosis. Therefore our aim was to study whether level of anti-hsp70 antibodies correlate with the presence of severe coronary artery disease (CAD). We measured and compared anti-hsp70 IgG antibody levels in CAD patients (n = 99) and healthy subjects (n = 99) with ELISA. The frequency of these antibodies was high in both groups and there was no significant difference in the median level of anti-hsp70 antibodies between patients with severe CAD and controls (653 (400-1141) vs. 630 (326-1152) AU/mL, P = 0.337). Adjustment for age, sex, BMI and lipid parameters did not change this result. Furthermore we did not find a correlation between anti-hsp70 antibody levels and certain risk factors of CAD (age, lipid parameters, body mass index, C-reactive protein, gender, smoking, diabetes and anti-hsp60 antibodies). By contrast, in accordance with our previous findings, anti-hsp60 and anti-hsp65 antibody levels were significantly higher in CAD patients, compared to this control group (p < 0.0001 for both variables). We did not find any correlation between the levels of anti-hsp70 and anti-hsp60 or anti-hsp65 antibodies either in the patients or the controls. The exact role of hsp70 in atherosclerosis is controversial, but we suggest that humoral immunity against human hsp70 does not contribute to coronary atherosclerosis in contrast to antibodies against 60kDa hsps.     

Calcium-activated chloride conductance in a pancreatic adenocarcinoma cell line of ductal origin (HPAF) and in freshly isolated human pancreatic duct cells

 Using the whole-cell patch-clamp technique, a calcium-activated chloride conductance (CACC) could be elicited in HPAF cells by addition of 1 μM ionomycin to the bath solution (66 ± 22 pA/pF;V _m + 60 mV) or by addition of 1 μM calcium to the pipette solution (136 ± 17 pA/pF; V _m + 60 mV). Both conductances had similar biophysical characteristics, including time-dependent inactivation at hyperpolarising potentials and a linear/slightly outwardly rectifying current/voltage (I/V) curve with a reversal potential (E _rev) close to the calculated cloride equilibrium potential. The anion permeability sequence obtained from shifts in E _rev was I > Br ≥ Cl. 4,4′-Diisothiocyanatostilbene disulphonic acid (DIDS, 500 μM) caused a 13% inhibition of the current (V _m + 60 mV) while 100 μM glibenclamide, 30 nM TS-TM-calix[4]arene and 10 μM tamoxifen, all chloride channel blockers, had no marked effects (8%, –6% and –2% inhibition respectively). Niflumic acid (100 μM) caused a voltage-dependent inhibition of the current of 48% and 17% (V _m ± 60 mV, respectively). In freshly isolated human pancreatic duct cells (PDCs) a CACC was elicited with 1 μM calcium in the pipette solution (260 ± 62 pA/pF; V _m + 60 mV). The presence of this CACC in human PDCs could provide a possible therapeutic pathway for treatment of pancreatic insufficiency of the human pancreas in cystic fibrosis. Received: 2 October 1997 / Received after revision: 28 November 1997 / Accepted: 1 December 1997     

Low levels of antibodies against <Emphasis Type="Italic">E. coli</Emphasis> and mycobacterial 65kDa heat shock proteins in patients with inflammatory bowel disease

Objective and design: The aim of the present study was to support and extend our initial observation, where we found low levels of antibodies against mycobacterial 65kD heat shock proteins in patients with inflammatory bowel disease (IBD). For this purpose we tested a new group of 124 patients with IBD, and beside measuring antibodies to Mycobacterium bovis 65kD heat shock protein (Hsp65) and human 60kD heat shock protein (Hsp60) as described previously, we also determined IgG antibody levels to Hsp65 from E. coli, called GroEL.Patients and control subjects: seventy-four patients with Crohns disease (CD) (30 males, 44 females, 33 (27–45) years old, median (interquartile range)) and 50 patients with ulcerative colitis (UC) (22 males, 28 females, 38 (30–50) years old) were involved in the study. 110 healthy subjects (34 males, 76 females, 47 (37–53) years old) served as controls. Study subjects were consecutive patients referred to an IBD center for complex treatment of the disease.Methods and statistical analysis: The amounts of IgG-type antibodies reacting with proteins of the chaperonin 60 family were assessed by ELISA. Since the antibody levels to heat-shock proteins as variables were not normally distributed, non-parametric Mann-Whitney test and Dunn post hoc test were used for group comparisons.Results: Median levels of anti-GroEL (7,5 (3,5–18,3)) and anti-Hsp65 (4,8 (2,1–7,85)) were significantly (GroEL p = 0,008; and Hsp65 p < 0,001) lower in the IBD patients than in the healthy subjects (GroEL: 10,0 (5,4–31,0); Hsp65: 7,04 (4,66–12,77)). However this difference was found to be restricted to the CD patients (GroEL: 7,5 (3,7–14,2); p < 0,05; Hsp65: 4,35 (1,90–6,94); p < 0,001). We did not find difference in the concentration of anti-human Hsp60 IgG levels between patients (Hsp60: 45,5 (24,9–69,0)) and healthy controls (38,4 (21,6–69,4). Regarding the serum concentrations of each antibody tested there was no significant difference between the active and inactive stage of disease.Conclusion: Our present findings support conclusion of our previous work, antibody levels not only for Mycobacterium bovis hsp65 but for E. coli GroEl were found to be decreased as well. In contrast no changes in the concentrations of human anti-hsp60 antibodies were observed. These findings indicate that production of antibodies to 65 kDa bacterial heat shock proteins is selectively impaired in IBD.Received 9 March 2004; returned for revision 9 April 2004; accepted by A. Falus 18 May 2004     

<Emphasis Type="Italic">Mycoplasma pneumoniae</Emphasis> infection in primary care investigated by real-time PCR in England and Wales

Real-time PCR was employed to detect a conserved region of the P1 cytadhesin gene of Mycoplasma pneumoniae in combined nose and throat swabs collected from patients attending GP surgeries during 2005–2009 with symptoms of respiratory tract infection (RTI). Samples were collected as part of an annual winter epidemiological and virological linked study in England and Wales. A total of 3,987 samples were tested, 65 (1.7%, 95%CI 1.3–2.1) had detectable M. pneumoniae DNA. Positive patients were detected of both gender, aged from 9 months to 78 years, who had clinical signs of upper RTI, fever and/or myalgia, an influenza-like illness to lower RTI. Mixed infections were identified in four cases, two with influenza A H1, one with H3 and one with influenza B. Children aged 5–14 years were more likely to have detectable M. pneumoniae in samples than all other age groups (Fishers p = 0.03), attributed to the 2005–2006 season in which 6.0% (12/200, 95%CI 3.4–10.3) of 5–14 year olds had detectable M. pneumoniae in comparison to 2.2% in 2006–2007 (3/141 95%CI 0.5–6.4), 2.2% in 2007–2008 (2/89 95%CI 0.1–8.3) and 0% in 2008-2009 (0/151 95%CI 0–2.9).