bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanisms of villous atrophy in autoimmune enteropathy and coeliac disease
Authors: CICCOCIOPPO R , D'ALÒ S , SABATINO A D I , PARRONI R , ROSSI M , DOGLIONI C , CIFONE M G , CORAZZA G R
Published: March 2003

Since in coeliac disease mucosal flattening has been suggested to result from an increased enterocyte apoptosis triggered by Fas/Fas ligand system and perforin cytolytic granules, we looked for a similar mechanism in autoimmune enteropathy. Moreover, we tried to assess whether enterocyte autoantibodies, which are the hallmark of autoimmune enteropathy, may be involved in triggering enterocyte apoptosis in this condition. Immunohistochemical staining with anti-Fas, -FasL and -perforin MoAb, and TUNEL technique were applied on endoscopic duodenal biopsies of two autoimmune enteropathy patients, two untreated coeliac patients and two biopsied controls. Cytotoxicity assays were carried out by incubating peripheral blood mononuclear cells from a healthy subject (effectors) with enterocytes primed with patient or control sera (targets). In autoimmune enteropathy a large number of enterocytes were apoptotic, as in coeliac disease, whereas neither Fas/Fas ligand or perforin expressions were up-regulated. On the other hand, antibody-dependent cellular cytotoxicity assay revealed the ability of sera from patients with autoimmune enteropathy to mediate enterocyte death through apoptosis. These results point to enterocyte autoantibody-dependent cellular cytotoxicity as the prevalent mechanism of increased enterocyte apoptosis in autoimmune enteropathy but not in coeliac disease.
Combination of molecular mimicry and aberrant autoantigen expression is important for development of anti-Fas ligand autoantibodies in patients with systemic lupus erythematosus
Authors: MIHARA S , SUZUKI N , TAKEBA Y , SOEJIMA K , YAMAMOTO S
Published: March 2003

We have reported previously that circulating anti-Fas ligand (FasL) autoantibodies able to inhibit Fas/FasL-mediated apoptosis were present in patients with systemic lupus erythematosus (SLE). In the present study, we describe the epitopes recognized by these anti-FasL autoantibodies. Rabbit antihuman antibody, raised against a FasL fragment consisting of amino acids (aa) 103–179 (fragment 2·0), inhibited Fas/FasL-mediated apoptosis, whereas an antibody against a FasL aa 103–146 fragment (fragment 1·0) did not. This suggested that an epitope around aa 146–179 was important for Fas/FasL interaction. Epitope mapping of anti-FasL autoantibodies using deletion mutants indicated that the epitope was located around aa 163–179. Three-dimensional molecular modelling of the Fas/FasL complex revealed that the aa 162–169 region was located on the outermost side of FasL, which suggested that the anti-FasL autoantibody would easily have access to the epitope. FasL point mutants involving aa positions 162–169 resulted in complete loss of apoptosis-inducing capability, which suggested that the aa 162–169 region was important for Fas/FasL interaction. A synthetic FasL peptide consisting of aa 161–170 blocked the binding of anti-FasL autoantibodies to FasL fragment 2·0 (aa 103–179). The FasL aa 161–170 sequence was found to be highly homologous with aa sequences from several infectious agents. Synthetic peptides derived from some of these microorganisms cross-reacted with the epitope recognized by the autoantibodies, suggesting that several foreign infectious agent-derived proteins may share an epitope with human FasL. As lymphocytes from SLE patients aberrartly expressed FasL, it is possible that infection by one of several infectious agents may trigger cross-reactive antibody responses, after which aberrantly expressed endogenous FasL might induce the shift from a cross-reactive response to an authentic autoimmune response. Therefore, a combination of molecular mimicry and aberrant autoantigen expression may be important for the development of anti-FasL autoantibodies in SLE patients.
The effects of CD40- and interleukin (IL-4)-activated CD23+ cells on the production of IL-10 by mononuclear cells in Graves’ disease: the role of CD8+ cells
Authors: UCHIMURA K , ITOH M , YAMAMOTO K , IMAMURA S , MAKINO M , KATO T , FUJIWARA K , SAWAI Y
Published: March 2003

The possible roles of CD8+ cells in the abnormal T cell-dependent B-cell activation in Graves’ disease were investigated by analysing lymphocyte subsets in peripheral blood mononuclear cells (PBMC) and their production of soluble factors and cytokines such as IL-10 in patients with Graves’ disease, Hashimoto's thyroiditis and normal controls. The PBMC were separated into CD8+ and CD8-depleted cells by magnetic separation columns, and cultured for 7 days with or without anti-CD40 monoclonal antibodies and IL-4. The culture supernatant was assayed for sCD23 and IL-10 using EIA, and the remaining cells were analysed by flow cytometry. Stimulation with anti-CD40 antibody together with IL-4 increased sCD23 levels and the number of CD23+ cells. The latter was further augmented by depletion of CD8+ cells. This combination of B cell stimulants increased production of IL-10 by PBMC from patients with Graves’ disease. The CD40- and IL-4-activated production of IL-10 was decreased by CD8+ cell depletion. In contrast, constitutive production of IL-10 was increased after CD8+ cell depletion in a group of patients with low basal secretion levels (<35 ng/ml). It was, however, decreased in a group with higher basal production levels, but such a relationship was not found in the normal control group. Thus, T cell-dependent B-cell activation via a CD40 pathway activates CD23+ cells, leading to over-production of IL-10 and a shift of the Th1/Th2 balance to Th2 dominance, while CD8+ cells may suppress this activation to counteract the Th2 deviation in Graves’ disease.
Effects of human placental lactogen on the expression of CD163 and CD14 on human monocytes in culture
Authors: CRANNY A , CROWLEY P , WHELAN A
Published: March 2003

The effect of human placental lactogen (hPL), a member of the somatomammotrophin family, on the regulation of the scavenger receptor molecules CD14 and CD163 on human monocytes cultured for 48h was investigated. Cells were cultured in the presence or absence of the hormone and also in the presence or absence of IFN-γ and dexamethasone. Monocytes cultured in the presence of hPL showed a significant increase in the expression of CD14 in both males and females compared to background. When IFN-γ and dexamethasone were added to the cultures, CD14 expression was decreased and was not rescued by the presence of hPL. hPL alone had no effect on the expression of CD163 on cultured monocytes from either gender, although cells cultured in the presence of IFN-γ and dexamethasone showed a profound increase in their expression of CD163. This expression was augmented further by the presence of hPL in the cultures over a 48-h period. These results support the hypothesis of a potential role of this hormone in the regulation of the innate immune response.
The Th1/Th2 cytokine balance changes with the progress of the immunopathological lesion of Sjogren's syndrome
Authors: MITSIAS D I , TZIOUFAS A G , VEIOPOULOU C , ZINTZARAS E , TASSIOS I K , KOGOPOULOU O , MOUTSOPOULOS H M , THYPHRONITIS G
Published: March 2003

Expression of type-1 and type-2 cytokines at the mRNA level in labial salivary glands (LSG) of patients with Sjogren's syndrome (SS), as reported by several groups, have generated conflicting results. In the present study we have directly examined the production of IL-4, IL-13 and IFN-γ by lymphocytes infiltrating the LSG of 44 consecutive patients referred for SS evaluation. Cytokines production was evaluated following in vitro culture of LSG in the presence of IL-2. IFN-γ and IL-13 were detected in the majority of SN (24/44 and 26/44, respectively) while IL-4 was present in 5/44 SN. The presence of IFN-γ was significantly higher in SS patients, as opposed to patients who did not fulfil the criteria for SS (P < 0·01). In addition, almost all cultured lymphocytes expressed mRNA for IFN-γ (17/19 cultures) and IL-13 (18/19) while IL-4 mRNA was also expressed at high frequency (14/19 cultures). Interestingly, the IFN-γ mRNA copies in cultured lymphocytes correlated significantly with the intensity of lymphocytic infiltration as evaluated by Chisholm's score (P < 0·01). Furthermore, RT-PCR of RNA extracted from whole LSG from 14 SS patients also demonstrated the presence of all cytokines in the majority of the cases and the prevalence of IFN-γ in LSG with high-grade infiltration. Because IL-13 was produced by the majority of the cultured LSG, IgE production was also evaluated. Interestingly, IgE was detected in 21/44 LSG culture SN and mainly in those biopsies that had Chisholm's score less than 0·5 (P < 0·05). We conclude that lymphocytes infiltrating the LSG are capable of producing both Th1 and Th2 cytokines and that the balance between them shifts in favour of Th1 in LSG with high infiltration score and in patients with SS.
Molecular study of an IgG1κcryoglobulin yielding organized microtubular deposits and glomerulonephritis in the course of chronic lymphocytic leukaemia
Authors: GALEA H R , BRIDOUX F , ALDIGIER J-C , PARAF F , BORDESSOULE D , TOUCHARD G , COGNÉ M
Published: March 2003

Glomerulonephritis with organized microtubular monoclonal immunoglobulin deposits (GOMMID) and glomerulonephritis related to type I cryoglobulin are well-known but rare complications of B cell derived chronic lymphocytic leukaemia. In these disorders, monoclonal Ig have never been studied at the molecular level. We conducted a pathological and molecular analysis in a patient with chronic lymphocytic leukaemia, glomerulonephritis and a single circulating monoclonal Ig. Unusual IgG1κ kidney deposits were observed. The heavy and light chain variable region sequences of that cryoprecipitating monoclonal Ig were characterized. Light microscopy revealed glomerulonephritis typical of cryoglobulinaemia, with neutrophil and macrophage infiltration, endocapillary hyperplasia and few protein thrombi. Electron microscopic study clearly evidenced numerous subepithelial mixed granular and organized deposits with a unique microtubular organization, reminiscent of the GOMMID. The Ig molecule sequence revealed alterations of charge and hydrophobicity potentially promoting a crystal-like aggregation and the aggregation of microtubules. This description suggests that common mechanisms are involved in various forms of precipitation and/or deposition of complete Ig molecules, with a variable extent of organization and with a possible overlap between pathological patterns of either glomerulonephritis with microtubular deposits or type I cryoglobulinic glomerulonephritis.
Continuous nasal administration of antigen is critical to maintain tolerance in adoptively transferred autoimmune arthritis in SCID mice
Authors: BÁRDOS T , CZIPRI M , VERMES C , ZHANG J , MIKECZ K , GLANT T T
Published: March 2003

Mucosal tolerance is a natural mechanism that prevents immunological reactions to antigens by altering the activity of immune cells of pathogenic clones without modulating the entire immune system. This ‘natural immune suppression’ can be exploited when antigen(s) of the target organ in an autoimmune disease is used for mucosal treatment. Being inspired by the experimental results in animal models, clinical trials using type II collagen for mucosal treatment have been conducted in rheumatoid arthritis. High-density proteoglycan (aggrecan) is another major macromolecular component in articular cartilage, and may be a candidate autoantigen for provoking immune reactions in patients with rheumatoid arthritis. Indeed, like type II collagen, systemic immunization of genetically susceptible mice with proteoglycan (PG) aggrecan induces progressive autoimmune polyarthritis. Here, we investigated whether intranasally applied PG can be effective in suppressing PG-induced arthritis (PGIA) in BALB/c mice. We found that nasal administration of 100μg PG exerted a strong suppressive effect on both the incidence and severity of the disease, most probably by reducing responsiveness towards the immunizing PG antigen. When we transferred PGIA into genetically matched but immunodeficient SCID mice, we were able to establish a tolerized state, but only if the recipient SCID mice received lymphocytes from tolerized animals and intranasal treatment with PG was continued. Without nasally administered antigen, the transferred anergic cells recovered and arthritis rapidly developed in a severe form. Intranasal PG treatment of recipient SCID mice was ineffective when cells from non-tolerized arthritic donors were transferred, in which case the regular weekly ‘tolerizing’ dose of PG made the disease worse. Our results suggest that mucosal treatment in an already existing disease may result in paradoxical outcomes.
Allelic family-specific humoral responses to merozoite surface protein 2 (MSP2) in Gabonese residents withPlasmodium falciparuminfections
Authors: EKALA M-T , JOUIN H , LEKOULOU F , MERCEREAU-PUIJALON O , NTOUMI F
Published: March 2003

Merozoite surface protein 2 (MSP2) expressed by Plasmodium falciparum asexual blood stages has been identified as a promising vaccine candidate. In order to explore allelic family-specific humoral responses which may be responsible for parasite neutralization during natural infections, isolates from individuals with either asymptomatic infections or uncomplicated malaria and residing in a Central African area where Plasmodium transmission is high and perennial, were analysed using MSP2 as polymorphic marker. The family-specific antibody responses were assessed by ELISA using MSP2 synthetic peptides. We observed an age-dependence of P. falciparum infection complexity. The decrease of infection complexity around 15 years of age was observed simultaneously with an increase in the mean number of MSP2 variants recognized. No significant difference in the P. falciparum genetic diversity and infection complexity was found in isolates from asymptomatic subjects and patients with uncomplicated malaria. The longitudinal follow-up showed a rapid development of immune responses to various regions of MSP2 variants within one week. Comparing humoral responses obtained with the other major antigen on the merozoite surface, MSP1, our findings suggest that different pathways of responsiveness are involved in antibody production to merozoite surface antigens.
Down-regulation of specific antigen-driven cytokine production in a population with endemicSchistosoma japonicuminfection
Authors: SHEN L , ZHANG Z S , WU H W , WEIR R E , XIE Z W , HU L S , CHEN S Z , JI M J , SU C , ZHANG Y , BICKLE Q D , COUSENS S N , TAYLOR M G , WU G L
Published: March 2003

Schistosome antigen-driven cytokine responses and antischistosome antibody levels of residents of a Schistosoma japonicum endemic island in Poyang Lake, Jiangxi Province were studied before and 45 days after treatment with praziquantel. IL-4, IL-5, IL-10 and INF-γ were all detected in the supernatants of whole-blood cultures after stimulation with schistosome soluble egg antigen (SEA) and soluble worm antigen preparation (SWAP). The percentages of subjects producing detectable amounts of each cytokine assayed were higher in the group who were negative by stool examination at the start of the study than in those who were initially stool positive. After praziquantel treatment the percentages of subjects producing both type I and type II cytokines increased. This suggests that the production of both types of cytokine was down-regulated in the presence of live, egg-laying S. japonicum adult worms but that this was reversible by treatment. In contrast, the antibody studies showed higher levels of SWAP and SEA-specific antibodies (IgE, total IgG, IgG4, IgM) in subjects who were originally stool-positive than in those who were stool-negative. After treatment specific IgE responses were elevated, but total IgG and IgG4 anti-SEA and IgM anti-SWAP antibody levels all fell significantly.
Interference of PR3-ANCA with the enzymatic activity of PR3: differences in patients during active disease or remission of Wegener's granulomatosis
Authors: VAN DER GELD Y M , TOOL A T J , VIDELER J , DE HAAS M , COHEN TERVAERT J W , STEGEMAN C A , LIMBURG P C , KALLENBERG C G M , ROOS D
Published: March 2003

Anti-neutrophil cytoplasmic antibodies (ANCA) against proteinase 3 (PR3) are strongly associated with Wegener's granulomatosis (WG) and are thought to be involved in its pathogenesis. Levels of PR3-ANCA do not always correspond to clinical disease activity.

To investigate the relationship between functional effects of PR3-ANCA and disease activity, we tested the effect of IgG samples from sera of 43 WG patients, taken during active disease or remission, for their capacity to interfere with the proteolytic activity of PR3. Furthermore, longitudinal sera of seven WG patients were included. The enzymatic activity of PR3 was determined (1) with casein or with a small synthetic substrate and (2) by complexation of PR3 with α1-antitrypsin (α1-AT).

With a fixed concentration (100 μg/ml) of IgG, PR3-ANCA from patients during an active phase of WG had a higher inhibitory capacity towards the proteolytic activity of PR3 and complexation of PR3 with α1-AT than did PR3-ANCA from WG patients during remission. However, the number of PR3-ANCA units that gave 50% inhibition of the PR3 enzymatic activity and its complexation with α1-AT was lower for patients during remission than for patients during an active phase of WG, indicating a stronger inhibitory capacity at a molar base.

In conclusion, PR3-ANCA from patients during remission had a relatively higher inhibitory capacity towards the enzymatic activity of PR3 than PR3-ANCA from patients during an active phase. This may indicate that during active disease the ANCA titre is increased, but the number of active ANCA molecules that recognize the enzyme-inhibiting epitopes is not increased.
CD57+ T cells augment IFN-γproduction in a one-way mixed lymphocyte reaction and their expansion after stem cell transplantation in paediatric patients
Authors: KOIKE Y , SEKI S , OHKAWA T , KANEKO T , KOGAWA K , FUJITSUKA S , HIRAIDE H , SEKINE I
Published: March 2003

To clarify the immune response of CD57+ T cells (most of them are CD8+) in peripheral blood (PB) against alloantigens in order to elucidate the T helper 1 (Th 1) immune response, we assessed the role of CD57+ T cells in IFN-γ (one of the representative Th 1 cytokines) production in a one-way mixed lymphocyte reaction (MLR). In this study, we showed that CD57+ T cells in responder cells were essential for effective IFN-γ production in allogeneic MLR due partly to the augmentation of the alloresponse of regular T cells. Furthermore, IFN-γ production in MLR correlated with the proportions of CD57+ T cells in PB regardless of the responders’ age. We also showed that the extent of the expansion of CD57+ T cells in paediatric patients after haematopoietic stem cell transplantation (HSCT) was markedly lower than that in adult patients. In addition, CD57+ T cells purified and activated with a combination of cytokines showed a greater cytotoxicity than regular T cells against human umbilical vein endothelial cells. Because IFN-γ production in one-way MLR is a useful predictor of graft-versus-host disease (GVHD), especially in the acute phase that occurs after allogeneic HSCT, our findings suggested that CD57+ T cells play a role in the development of GVHD and thus may explain the reason as to why a higher donor age is associated with an increased risk of developing GVHD while, in addition, the incidence of severe GVHD in paediatric patients is lower than that in adult patients.
IL-10 induces mesangial cell proliferation via a PDGF-dependent mechanism
Authors: ROBERTSON T E , NIKOLIC-PATERSON D J , HURST L A , ATKINS R C , CHADBAN S J
Published: March 2003

Interleukin-10 (IL-10) is a mesangial cell growth factor in vivo and in vitro. However, the mechanism by which IL-10 exerts its mitogenic activity is not known. The aim of this study was to determine whether IL-10 induces mesangial cell proliferation in a PDGF-dependent or independent fashion. A well-­characterized rat mesangial cell line (1097) was used in a series of cell proliferation experiments in which cells were serum-starved and then incubated with recombinant IL-10 in the presence or absence of STI 571 (a specific inhibitor of signalling via the PDGF-α and β receptors) or a neutralizing anti-PDGF-AB antibody. IL-10 induced significant mesangial cell proliferation at 24 and 48 h after cytokine addition. This response was inhibited totally by the addition of STI-571, demonstrating that IL-10 mitogenic activity has an absolute requirement for signalling through the PDGF receptor. In further studies, it was found that STI-571 could be added 24 h after IL-10 stimulation and still exert a profound inhibition of IL-10 mitogenic activity. The ability of a neutralizing anti-PDGF-AB antibody to inhibit completely IL-10-induced mesangial cell proliferation confirmed that IL-10 acts via induction of an autocrine PDGF response rather than the possibility that IL-10 may transactivate the PDGF receptor in a PDGF-independent fashion. In conclusion, this study has demonstrated that IL-10 induces mesangial cell proliferation via an autocrine PDGF-mediated mechanism. Thus, therapies which antagonize PDGF signalling will also inhibit any contribution of IL-10 to mesangial proliferation.
Direct modulatory effect of C-reactive protein on primary human monocyte adhesion to human endothelial cells
Authors: WOOLLARD K J , PHILLIPS D C , GRIFFITHS H R
Published: March 2003

C-reactive protein (CRP) is the prototypic acute phase serum protein in humans. The effects of CRP on primary human monocyte adhesion molecule expression and interaction with the endothelium have not been studied. Herein, we describe an investigation into the phenotypic and functional consequences of CRP binding to peripheral blood monocytes ex vivo. Peripheral whole blood was collected from healthy, non-smoking males. Mononuclear cells (MNC) and monocytes were isolated by differential centrifugation using lymphoprep and Dynal negative isolation kit, respectively. Cells were exposed to CRP from 0 to 250 µg/ml for 0–60 min at 37°C and analysed for (a) CD11b, PECAM-1 (CD31) and CD32 expression by flow cytometry and (b) adhesion to LPS (1 µg/ml; 0–24 h) treated human umbilical vein endothelial cells (HUVEC). CD14+ monocyte expression of CD11b increased significantly up to twofold when exposed to CRP, compared to controls. There was no significant difference in CD32 expression, whereas CD31 expression decreased after exposure to CRP. CRP treatment of monocytes inhibited their adhesion to early LPS-activated HUVEC (0–5 h). However, the adhesion of CRP-treated monocytes to HUVEC was significantly greater to late activation antigens on HUVEC (24 h, LPS) compared to controls. We have shown that CRP can affect monocyte activation ex vivo and induce phenotypic changes that result in an altered recruitment to endothelial cells. This study provides the first evidence for a further role for C-reactive protein in both monocyte activation and adhesion, which may be of importance during an inflammatory event.
Adjuvant modulation of the immune responses and the outcome of infection withChlamydia pneumoniae
Authors: BANDHOLTZ L , KREUGER M R , SVANHOLM C , WIGZELL H , ROTTENGERG M E
Published: March 2003

Immunization with different adjuvants resulted in antithetic outcomes of infection with Chlamydia pneumoniae. Immunization with the outer major protein-2 from C. pneumoniae (OMP-2) emulsified in Freund's complete adjuvant (FCA) thus increased the susceptibility of mice to infection with the bacteria. The detrimental effect was not observed upon inoculation of irrelevant antigens or major outer membrane protein (MOMP) in FCA, but was also observed after immunization with FCA–chlamydial heat shock protein-60 (HSP-60). The harmful effect of FCA-OMP-2 depended on the presence of both CD4+ and CD8+ cells and was mediated by IL-10, as shown using gene-ablated mice. The increased susceptibility to infection caused by FCA-OMP-2 immunization was long-lasting and observed in mice infected 4 months after the last dose of immunogen. In contrast, partial protection against C. pneumoniae was observed when FCA was replaced with oligodeoxynucleotides containing immunostimulatory CpG motifs mixed with Freund's incomplete adjuvant (FIA-IS-CpG). These polar outcomes of infection related to the cytokine pattern: antigen-stimulated spleen cells from FCA-OMP-2-immunized mice showed higher IL-10/IFN-γ ratios than FIA-IS-CpG-OMP-2-immunized animals. In agreement, sera from FCA-OMP-2 showed higher anti-OMP-2 IgG1/IgG2a ratios than FIA-IS-CpG-OMP-2-immunized animals. Finally, OMP-2 also generated a protective response when delivered by a eukaryotic expression vector in tandem with CTLA4, a procedure that targeted OMP-2 to antigen-presenting cells.
Involvement of CD27/CD70 interactions in antigen-specific cytotoxic T-lymphocyte (CTL) activity by perforin-mediated cytotoxicity
Authors: YAMADA S , SHINOZAKI K , AGEMATSU K
Published: March 2003

CD27 molecules are shown to be essential in the regulation of the death, activation and differentiation of T and B cells. However, the influence of CD27 on cytotoxic T-cell function remains obscure. Autologous EBV transformed B-cell lines (LCL), which highly express CD27 ligand CD70, here stimulated T cells and induced the cytotoxic T-lymphocyte (CTL) activity via T-cell antigen receptors (TCR). The cytotoxicity against LCL was diminished when anti-CD70 blocking MoAb was added initially in the culture. Resting T cells killed more CD70-transfected P815 cells than wild type P815 cells in the presence of anti-CD3 MoAb as measured by a 4-h 51Cr release assay, and the cytotoxicity of both of the cell populations completely disappeared in the presence of concanamycin A (CMA). The expression of the perforin by the LCL-induced CTL in the presence of anti-CD70 blocking MoAb was diminished as compared with that without the blockage of CD27/CD70 interactions. The CTL induced by LCL did not kill Fas-transfected WR cells. CD27 signalling in the T cells did not affect Fas ligand (FasL) mRNA expression, LAK activity and IFN-γ synthesis in humans. Our data demonstrate that CD27/CD70 interactions enhance the cytotoxicity of CTL in the induction phase through enhancement of killing activity induced via the perforin-dependent mechanism, but not via the Fas/FasL system.
Reactive arthritis and serum levels of mannose binding lectin – lack of association
Authors: LOCHT H , CHRISTIANSEN M , LAURSEN I
Published: March 2003

The purpose was to evaluate the possible association of serum mannose binding lectin (s-MBL) levels on type of triggering microbe, duration of diarrhoea, incidence and course of reactive arthritis (ReA) caused by Salmonella, Yersinia and Campylobacter. Sixty patients with ReA of 1–228 months duration, 173 patients with ReA or uncomplicated enterocolitis caused by Campylobacter, 226 sera from patients with elevated antibody levels against Salmonella, Yersinia or Campylobacter, and 114 blood donors were tested for s-MBL using ELISA technique, both direct mannan binding assay and sandwich ELISA. s-MBL was compared with C-reactive protein (CRP) levels and with the ability of activating complement C4. Among the 114 donors 9% had s-MBL <50 µg/l, 16% had from 50–500 µg/l and 75% had >500 µg/l. The distribution of s-MBL levels in the three-patient groups did not differ significantly from the controls. There were no indications that low s-MBL was associated with prolonged duration of arthritis, diarrhoea or individual bacterial infections. The two MBL assays were comparable with respect to serum concentrations, indicating that the actual circulating MBL was also functionally active. s-MBL exhibited acute phase reactant behaviour and correlated to CRP level, but only in patients with s-MBL concentrations exceeding 1000 µg/l. MBL in 10 randomly selected ReA sera were tested for the ability to activate complement C4. The results did not differ from those of donor controls. This study demonstrates that the distributions of s-MBL levels in serum among patients with ReA are not different from donor controls. The course, outcome or triggering bacteria are not associated with a particular level of s-MBL.
Vaginal T lymphocyte population kinetics during experimental vaginal candidosis: evidence for a possible role of CD8+ T cells in protection against vaginal candidosis
Authors: GHALEB M , HAMAD M , ABU-ELTEEN K H
Published: March 2003

Vaginal candidosis represents a significant health problem to women of childbearing age worldwide. It has been postulated that localized T cells play a role in protection against vaginal candidosis. In an attempt to evaluate the role of vaginal T cells in protection against vaginal candidosis, T cell population kinetics was evaluated using an oestrogen-dependent vaginal candidosis murine model. Vaginal T lymphocytes were isolated at different time points post C. albicans inoculation, viable cells were enumerated, phenotypically analysed for the expression of CD3, CD4 and CD8 T cell markers and absolute numbers of T cell subsets were calculated. Oestrogen-induced persistence of vaginal candidosis resulted in a significant increase in the total number of vaginal lymphocytes within 24–48 h post infection; increased vaginal lymphocyte numbers persisted throughout the infection period. The number of CD3+ T cells dramatically increased following C. albicans administration and was maintained at high levels throughout the infection period. The majority of CD3+ T cells were of the CD8+ type; however, considerable numbers of both CD4+ T cells and CD4+CD8+ T cells were also observed throughout the infection period. The considerable and persistent increase in vaginal T cell numbers in general and that of CD8+ T cells in particular are evidence of the possible role played by localized T cells in protection against vaginal candidosis.
High circulating levels of biologically inactive IL-6/SIL-6 receptor complexes in systemic juvenile idiopathic arthritis: evidence for serum factors interfering with the binding to gp130
Authors: PIGNATTI P , CIAPPONI L , GALLE P , HANSEN M B , MASSA M , MEAZZA C , PAONESSA G , NOVICK D , CILIBERTO G , MARTINI A , DE BENEDETTI F
Published: March 2003

We previously demonstrated that high levels of IL-6/sIL-6R complexes are present in sera of patients with systemic juvenile idiopathic arthritis (s-JIA) and that the amount of IL-6 estimated in the IL-6/sIL-6R complexes is markedly higher than that measured by the B9 assay. Here, we show that two additional bioassays, employing human myeloma XG-1 cells and human hepatoma Hep3B cells, detected serum IL-6 levels similar to those measured by the B9 assay and approximately 10-fold lower than the IL-6 levels estimated to be present in the IL-6/sIL-6R complex. Using an assay for the measurement of the amount of circulating IL-6 complexed with the sIL-6R and available for binding to gp130 (gp130 binding activity), we show that the IL-6/gp130 binding activity is similar to that detected by the bioassays and again significantly lower than that estimated to be present in the IL-6/sIL-6R complex. Addition of recombinant human IL-6 (rhIL-6) to sera of patients or controls results in a markedly lower increase in the gp130 binding activity in patients than in controls. Moreover, sera from s-JIA patients inhibited in a dose dependent manner the gp130 binding activity assay. These results show that sera from patients with s-JIA contain a factor, or factors, that inhibit(s) the binding of the IL-6/sIL-6R complex to gp130. This inhibitory activity does not appear to be due to soluble gp130, C-reactive protein or autoantibodies to IL-6.
High positive frequency of antibodies to metallothionein and heat shock protein 70 in sera of patients with metal allergy
Authors: JIN G-B , NAKAYAMA H , SHMYHLO M , INOUE S , KONDO M , IKEZAWA Z , OUCHI Y , CYONG J-C
Published: March 2003

Two principal types of stress protein, heat shock proteins (hsps) and metallothionein (MT), are induced in cells responding to a variety of stresses. They play an important role in protecting cells from these stresses. However, many reports indicate that antibodies to hsps are present in human serum and are associated with several autoimmunity diseases. Metals, which are commonly allergenic to humans, induce both MT and hsp70 (one of the hsps family). Until now, there has been no report of any antibody to MT in human serum. In the present study, serum samples from healthy controls (Group I), and patients suffering from atopic dermatitis without (Group II) or with (Group III) metal allergy, were measured for antibodies to MT and hsp70, using an enzyme-linked immunosorbent assay (ELISA). Metal allergy was confirmed by patch testing. We first found that antibody to MT exists in human serum. We also found a high positive frequency of antibody to MT (51·3%) and to hsp70 (43·6%) in the sera of Group III, compared to those of Group I (3·8% and 5·1%) or Group II (6·4% and 5·1%). Furthermore, there was a strong positive correlation between antibody to MT and antibody to hsp70 in Group III (P = 0·0013), but not in Group I and Group II. Our results indicate that antibody to MT exists in human serum, as do antibodies to hsps, and suggest that elevated levels of MT and hsp70 antibodies are associated with metal allergy in atopic patients.
A family with hereditary extra‐adrenal paragangliomas without evidence for mutations in the von Hippel‐Lindau disease or ret genes
Authors: Sköldberg  , Grimelius  , Woodward  , Rorsman  , Van Schothorst  , Winqvist  , Karlsson  , Åkerström  , Kämpe  , Husebye
Published: March 2003

OBJECTIVES

To characterize a family with hereditary paraganglioma, and to search for germline mutations in the von Hippel‐Lindau disease (VHL) tumour suppressor gene and the ret proto‐oncogene.

DESIGN

Patient records and histopathological reports were reviewed. Available tumour samples were reinvestigated using immunohistochemical techniques. The VHL gene was investigated by single strand conformational polymorphism analysis of PCR products amplified from exons 1, 2 and 3 and the 3′ untranslated region. The ret gene was analysed by amplifying and sequencing exons 10, 11 and 16.

PATIENTS

A family with paragangliomas in three consecutive generations was investigated.

RESULTS

The affected individuals were found to have multiple extra‐adrenal paragangliomas. All three affected individuals had retroperitoneal tumours, and two also had paraganglioma in the neck. No mutations of the VHL or ret genes were detected.

CONCLUSIONS

The described family may represent a novel dominantly inherited neuroendocrine tumour syndrome.
Normal final height and apparent cure after pituitary irradiation for Cushing's disease in childhood: long‐term follow‐up of anterior pituitary function
Authors: Johnston L. B. , Grossmann A. B. , Plowman P. N. , Besser G. M. , Savage M. O.
Published: March 2003

Pituitary‐dependent Cushing's syndrome is rare in childhood. Two patients are reported who had unsuccessful transsphenoidal microadenomectomy and therefore underwent pituitary radiotherapy. In both patients there was progressive normalization of hypothalamo–pituitary–adrenal function and at 8 and 10 years after radiotherapy their Cushing's syndrome remained in remission clinically and biochemically. Most importantly, they have both achieved adult heights within their respective expected ranges with close clinical observation and replacement growth hormone therapy. Thyroid and gonadal function is normal and neither patient requires any other hormone replacement therapy. Pituitary radiotherapy is thus a safe, effective and well tolerated second‐line therapy for paediatric Cushing's disease. Collaboration between the paediatric and adult departments and an experienced radiotherapist contributed to the successful outcome of these two patients.
Genotype‐phenotype analysis in late onset 21‐hydroxylase deficiency in comparison to the classical forms
Authors: Rumsby  , Avey  , Conway  , Honour
Published: March 2003

OBJECTIVE

To establish the type and frequency of mutations causing late onset 21‐hydroxylase deficiency and associated clinical and biochemical phenotypes and to compare these findings to those from heterozygotes and homozygotes for classical 21‐hydroxylase deficiency.

DESIGN

Analysis of the 21‐hydroxylase genes by DNA amplification and mutation detection. Measurement of serum 17‐hydroxyprogesterone following stimulation with adrenocorticotrophic hormone.

PATIENTS

Fifteen patients with late onset congenital adrenal hyperplasia, 18 obligate heterozygotes for classical congenital adrenal hyperplasia, 95 patients with classical 21‐hydroxylase deficiency.

MEASUREMENTS

17‐hydroxyprogesterone following adrenal stimulation with adrenocorticotrophic hormone; frequency of 10 common mutations in the 21‐hydroxylase gene.

RESULTS

Of alleles from patients with late onset CAH 46% carried V281L and 10% P30L mutations compared to 2.6% and 1.1% respectively of alleles from patients with the classical disease where the most common mutations are a splice site mutation in intron 2 (34%), deletions (25%) and I172N (15%). Serum 17‐hydroxyprogesterone following Synacthen stimulation reliably differentiated the late onset patients from carriers for the classical disease, whereas there was overlap of the basal 17‐hydroxyprogesterone concen_tration between these two conditions.

CONCLUSIONS

Normal basal serum 17‐hydroxyprogesterone levels cannot exclude late onset CAH, although response to adrenocorticotrophic stimulation clearly distinguished this disorder from carriers of the classical disease. The frequency of mutations causing less severe enzyme deficiency was higher in the late onset patients than in a group of classical patients, although other genes or environmental pressures may determine the age of onset of disease.
Contiguous gene syndrome due to deletion of the first three exons of the Kallmann gene and complete deletion of the steroid sulphatase gene
Authors: Maya‐Núñez Guadalupe , Cuevas‐Covarrubias Sergio , Carlos Zenteno Juan , Ulloa‐Aguirre Alfredo , Kofman‐Alfaro Susana , Pablo Méndez Juan
Published: March 2003

OBJECTIVE

Large terminal or interstitial deletions of the 22.3 region on the short arm of the X chromosome cause contiguous gene syndromes. Kallmann syndrome (hypogonadotrophic hypogonadism with anosmia or hyposmia) associated with X‐linked ichthyosis, due to a contiguous gene syndrome, is an uncommon finding. Genetic defects have been demonstrated in the Xp22.3 region, explaining the presence of one or both entities in affected individuals. In this report we describe the molecular findings of a patient with Kallmann syndrome and X‐linked ichthyosis.

PATIENT

A 20‐year‐old subject with hypogonadism, anosmia and generalized ichthyosis was studied endocrinologically, biochemically and molecularly.

MEASUREMENTS

Levels of LH, FSH, GH, testosterone, oestradiol and cortisol were determined basally and after specific stimulation tests. Enzymatic activity of steroid sulphatase was measured in leucocytes. Polymerase chain reaction of the 14 exons of the Kallmann gene and of the 5′ and 3′ extremes of the steroid sulphatase gene was performed in genomic DNA.

RESULTS

A partial deletion from exon 1 to exon 3 of the Kallmann gene, as well as a complete deletion of the steroid sulphatase gene were observed.

CONCLUSIONS

A patient bearing a contiguous gene syndrome with partial deletion of the Kallmann syndrome gene and complete deletion of the steroid sulphatase gene is described. This is the first time a mutation in the conserved cysteine‐rich N‐terminal region which corresponds to the whey acidic protein motif of the Kallmann gene has been characterized, thus demonstrating the importance of this specific region for the function of the gene.
Variations in erythrocyte antioxidant glutathione peroxidase activity during the menstrual cycle
Authors: Massafra Cosimo , De Felice Claudio , Gioia Dino , Buonocore Giuseppe
Published: March 2003

OBJECTIVE

Antioxidant enzymes are an important part of the defence mechanisms against free radical damage. Little is known, however, about the relationship between sex steroid hormones and cellular antioxidant systems. We have investigated the effect of physiological sex steroid changes on the erythrocyte antioxidant glutathione peroxidase (GSH‐Px) activity during the menstrual cycle in a population of healthy normomenorrhoic women.

DESIGN

Prospective, controlled.

PATIENTS

Ten normally cycling women (age range: 19–28 years; mean: 23.3 years) were recruited for alternate‐day blood sampling from the first day of one menstrual bleed until the first day of the subsequent menstrual phase.

MEASUREMENTS

Plasma was analysed for LH, FSH, oestradiol (E2) and progesterone (P4) concentrations. Erythrocyte GSH‐Px activity was evaluated on the same days in all subjects. Pyruvate‐kinase (PK) activity, as an index of red blood cell population age, was also determined.

RESULTS

All the women examined had a normal ovulatory cycle, as indicated by the hormone plasma pattern. Cycle length was standardized on the basis of the preovulatory E2 peak. Significant cycle phase‐related changes in GSH‐Px (P < 0.03) were observed, with higher GSH‐Px activity levels being found from the later follicular to early luteal phase as compared to early follicular phase (χ2 = 8.53, P < 0.001 and χ2 = 5.54, P < 0.002, respectively). A significant positive correlation was observed between mean E2 and GSH‐Px cycle‐related changes (r = 0.78, P = 0.001). Conversely, no significant cycle phase‐dependent variations were detected in PK.

CONCLUSIONS

The findings support the hypothesis that physiological ovarian E2 production during the menstrual cycle may play an important role in regulating erythrocyte glutathione peroxidase activity.
Expression of an orphan nuclear receptor DAX‐1 in human pituitary adenomas
Authors: Ikuyama Shoichiro , Mu Yi‐Ming , Ohe Kenji , Nakagaki Hiroyuki , Fukushima Takeo , Takayanagi Ryoichi , Nawata Hajime
Published: March 2003

OBJECTIVES

An orphan nuclear receptor, DAX‐1, is known to be involved in the development and differentiation of anterior pituitary cells. The present study aimed to examine 1) whether DAX‐1 is expressed in human pituitary adenomas, and 2) if it is expressed, what types of adenoma express the factor.

MATERIALS AND METHODS

Adenoma tissues examined included 18 clinically non‐functioning adenomas, 14 GH‐secreting adenomas and 7 PRL‐secreting adenomas. The expression of the following genes were tested by reverse transcription‐polymerase chain reaction (RT‐PCR): DAX‐1, Adrenal‐4‐binding protein/steroidogenic factor‐1 (Ad4BP/SF‐1), Pit‐1, LHβ, FSHβ, gonadotrophin‐releasing hormone receptor (GnRH‐R), GH, PRL, and TSHβ, as well as β‐actin as a control.

RESULTS

Eleven clinically non‐functioning adenomas expressed DAX‐1, 10 of which also expressed Ad4BP/SF‐1. Nine out of the 11 DAX‐1‐expressing adenomas also expressed LHβ, FSHβ and GnRH‐R as well, indicating that these adenomas possessed gonadotrophic properties. Nine clinically non‐functioning adenomas expressed Pit‐1 as well as GH, PRL and/or TSHβ, thus having somatomammotrophic or thyrotrophic properties, 3 of which overlapped with the above DAX‐1‐expressing adenomas. One non‐functioning adenoma expressed Ad4BP/SF1 and FSHβ but not DAX‐1, and another one expressed DAX‐1 and Ad4BP/SF‐1 with PRL. On the other hand, all GH‐secreting and PRL‐secreting adenomas expressed Pit‐1 and GH and/or PRL, but neither DAX‐1 nor Ad4BP/SF‐1.

CONCLUSIONS

The results shown here indicate that DAX‐1 is expressed in the majority of human pituitary adenomas of gonadotrophic origin in parallel with Adrenal‐4‐binding protein/steroidogenic factor‐1.
Lack of associations between serum leptin, a polymorphism in the gene for the β3‐adrenergic receptor and glucose tolerance in the Dutch population.
Authors: Janssen J. A. M. J. L. , Koper J. W. , Stolk R. P. , Englaro P. , Uitterlinden A. G. , Huang Q. , Van Leeuwen J. P. T. M. , Blum W. F. , Attanasio A. M. F. , Pols H. A. P. , Grobbee D. E. , De Jong F. H. , Lamberts S. W. J.
Published: March 2003

BACKGROUND

The associations between leptin levels and the prevalence of a polymorphism in the β3‐adrenergic receptor were studied in a cross‐sectional analysis of 600 participants in a population‐based study, which were stratified for glucose tolerance by an oral glucose tolerance test.

METHODS

In a random sample of 600 participants in the Rotterdam study, aged 55–75 years at baseline (309 men, 291 women) the relationships were studied between the presence of Trp64 Arg mutation in the β3‐adrenergic receptor gene and fasting leptin, glucose and insulin (fasting and after an oral glucose load), and other components of the insulin resistance syndrome.

RESULTS

Mean age of the study population was 66.9 years (SD 5.7). Fasting serum leptin levels overall in men and women were 6.1 μg/l (SE 0.2) and 21.7 μg/l (0.9), respectively, (P < 0.001). These differences were independent of age, body mass index and waist to hip ratio.We identified 73/600 persons who were heterozygotes for the Trp64 Arg polymorphism (allelic frequency 6.1%), but failed to find an association between the presence of this polymorphism and leptin or any measured parameter indicative for obesity, impaired glucose tolerance or type 2 diabetes mellitus.

CONCLUSION

Heterozygosity for the Trp64Arg polymorphism of the β3‐adrenergic receptor gene is not accompanied by obesity, impaired glucose tolerance and type 2 diabetes mellitus in the general elderly Dutch population, and is also not associated with changes in circulating leptin levels.
Prevalences of Gsα, ras, p53 mutations and ret/PTC rearrangement in differentiated thyroid tumours in a Korean population
Authors: Park  , Koh  , Kim  , Park  , Gong  , Hong  , Ahn
Published: March 2003

OBJECTIVE

In thyroid tumours, ras, Gsα, p53 mutations and ret/PTC rearrangement have been reported with variable prevalences in different geographical regions. We studied the prevalence of these mutations and rearrangement in thyroid tumours in a Korean population. As MDM2 and Bcl‐1 protein expressions have been suggested to be associated with p53 protein, we also studied possible relationships among them.

PATIENTS AND DESIGN

Eleven cases of adenomatous goitre, eight cases of follicular adenoma, five cases of follicular carcinoma and 37 cases of papillary carcinoma were included in this study. To find mutations and rearrangement, RT–PCR, SSCP and/or direct sequencing, after subcloning if necessary, were used, and immunohistochemical stainings were performed for p53, MDM2 and Bcl‐2 proteins in cases of papillary carcinoma.

RESULTS

We could not find any rearrangement for ret/PTC‐1, ‐2, ‐3 and mutation of Gsα. For the ras oncogene, K and H‐ras mutations were not found, but N‐ras mutations, point mutation of CAA to CGA in codon 61, were detected in one follicular adenoma (12.5%, 1/8) and one follicular carcinoma (33%, 1/3). p53 mutations were detected in only one case of papillary carcinoma (3%, 1/31: exon 8, codon 266 GGA → GAA). In 30 cases of papillary carcinoma without p53 mutation, the prevalences of positive immunohistochemical staining were 13.3% for p53 protein, 53.3% for MDM2 protein and 56.7% for Bcl‐2 protein. While over‐expression of p53 protein was not significantly related to that of MDM2 and Bcl‐2 proteins, over‐expression of MDM2 and Bcl‐2 in papillary carcinoma were associated.

CONCLUSION

ret/PTC rearrangement, Gsα, ras and p53 mutations are relatively rare in differentiated thyroid neoplasms from a Korean population, which may reflect genetic and environmental differences from patients in countries with high prevalences. P53 protein over‐expression was noted in 13.3% of papillary carcinoma cases without p53 mutation and was not significantly related to MDM2 and Bcl‐2 expression.