Architectural and functional alterations of the small intestinal mucosa in classical Whipple's disease.

Classical Whipple's disease (CWD) affects the gastrointestinal tract and rather elicits regulatory than inflammatory immune reactions. Mechanisms of malabsorption, diarrhea, and systemic immune activation are unknown. We here analyzed mucosal architecture, barrier function, and immune activation as potential diarrheal trigger in specimens from 52 CWD patients. Our data demonstrate villus atrophy and crypt hyperplasia associated with epithelial apoptosis and reduced alkaline phosphatase expression in the duodenum of CWD patients. Electrophysiological and flux experiments revealed increased duodenal permeability to small solutes and macromolecules. Duodenal architecture and permeability ameliorated upon antibiotic treatment. Structural correlates for these alterations were concordant changes of membranous claudin-1, claudin-2, claudin-3, and tricellulin expression. Tumor necrosis factor-α and interleukin-13 were identified as probable mediators of epithelial apoptosis, and altered tight junction expression. Increased serum markers of microbial translocation and their decline following treatment corroborated the biological significance of the mucosal barrier defect. Hence, mucosal immune responses in CWD elicit barrier dysfunction. Diarrhea is caused by loss of absorptive capacity and leak flux of ions and water. Downregulation of tricellulin causes increased permeability to macromolecules and subsequent microbial translocation contributes to systemic inflammation. Thus, therapeutic strategies to reconstitute the mucosal barrier and control inflammation could assist symptomatic control of CWD.

-295 T-to-C promoter region IL-16 gene polymorphism is associated with Whipple's disease.

Whipple's disease (WD) is a rare systemic condition caused, in genetically predisposed subjects, by Tropheryma whipplei, a common bacterium widespread in the environment. The relevance of genetic predisposition in WD is shown by the association with HLA alleles DRB1*13 and DQB1*06 and by the demonstration that, in patients with WD, the cytokine genetic profile is skewed toward a Th2 and Treg response. Since IL-16 is involved in hampering the development of a protective macrophagic response against Tropheryma whipplei, we investigated whether the genetic background of IL-16 is different between patients with WD and controls. The -295 T-to-C polymorphism of the promoter region of the IL-16 gene was studied in 90 patients with WD and 152 healthy controls. Levels of serum IL-16 protein were also tested. The frequency of the wild type T allele was significantly higher in patients with WD compared to the controls (155/180 vs. 235/304; p = 0.02 for the Chi(2) test), odds ratio 1.82 [95 % confidence interval (CI) 1.07-3.10]. The TT genotype was found in 65/90 patients with WD and 88/152 controls (p = 0.026). No relationship was found between serum levels of IL-16 and genotypes. Although the functional consequences of this genetic background on levels of IL-16 and on the course of the disease are still unknown, we found, for the first time, that the wild type T allele and the TT genotype of the -295 polymorphism are associated with WD.

[Tropheryma whipplei infection. Colonization, self-limiting infection and Whipple's disease]. / Infektionen mit Tropheryma whipplei. Besiedlung, selbstlimitierte Infektion und Morbus Whipple.

Whipple's disease is a multisystemic infection caused by the ubiquitous bacterium Tropheryma whipplei. Immunological host factors enable classical Whipple's disease; however, T. whipplei can be found in three other clinical conditions: healthy colonization, self-limiting infections, and isolated endocarditis. The genetic predisposition of the host rather than the genotype of the bacterium influences the infection. Modern diagnostic methods elucidate the many facets of Whipple's disease. In particular, isolated T. whipplei-induced infective endocarditis can only be diagnosed after valve resection. The sole treatment of Whipple's disease evaluated prospectively comprises intravenous induction therapy with ceftriaxone or meropenem, followed by continuation therapy with oral TMP-SMX. In the case of Immune reconstitution inflammatory syndrome (IRIS) or inflammatory lesions of the CNS in the setting of Whipple's disease, additional treatment with corticosteroids should be considered to avoid severe tissue damage.

Impairment of the intestinal barrier is evident in untreated but absent in suppressively treated HIV-infected patients.

BACKGROUND AND AIMS: Impairment of the gastrointestinal mucosal barrier contributes to progression of HIV infection. The purpose of this study was to investigate the effect of highly active antiretroviral therapy (HAART) on the HIV-induced intestinal barrier defect and to identify underlying mechanisms. METHODS: Epithelial barrier function was characterised by impedance spectroscopy and [(3)H]mannitol fluxes in duodenal biopsies from 11 untreated and 8 suppressively treated HIV-infected patients, and 9 HIV-seronegative controls. The villus/crypt ratio was determined microscopically. Epithelial apoptoses were analysed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling (TUNEL) and caspase-3 staining. Tight junction protein expression was quantified by densitometric analysis of immunoblots. Mucosal cytokine production was determined by cytometric bead array. RESULTS: Only in untreated but not in treated HIV-infected patients, epithelial resistance was reduced (13 (1) vs 23 (2) ohm cm(2), p<0.01) and mannitol permeability was increased compared with HIV-negative controls (19 (3) vs 9 (1) nm/s, p<0.05). As structural correlates, epithelial apoptoses and expression of the pore-forming claudin-2 were increased while expression of the sealing claudin-1 was reduced in untreated compared with treated patients and HIV-negative controls. Furthermore, villous atrophy was evident and mucosal production of interleukin 2 (IL2), IL4 and tumour necrosis factor alpha (TNFalpha) was increased in untreated but not in treated HIV-infected patients. Incubation with IL2, IL4, TNFalpha and IL13 reduced the transepithelial resistance of rat jejunal mucosa. CONCLUSIONS: Suppressive HAART abrogates HIV-induced intestinal barrier defect and villous atrophy. The HIV-induced barrier defect is due to altered tight junction protein composition and elevated epithelial apoptoses. Mucosal cytokines are mediators of the HIV-induced mucosal barrier defect and villous atrophy.

[Immune reconstitution inflammatory syndrome (IRIS) of the small bowel in an immunocompromised patient suffering from systemic lupus erythematosus and non-tuberculous mycobacteriosis]. / Immunrekonstitutionssyndrom des Dünndarms bei einer immunsupprimierten Patientin mit systemischem Lupus erythematodes und nichttuberkulöser Mykobakteriose.

An overwhelming immune reaction resulting in granulomatous inflammation after infection with opportunistic pathogens is termed immune reconstitution inflammatory syndrome (IRIS). It has mainly been described in patients with human immunodeficiency virus (HIV) infection on highly active antiretroviral therapy (HAART) who show a significant increase of low CD4 T cells (initially <50/microl). IRIS may lead to organ damage and differential diagnosis is often difficult. We report the case of a 38-year-old female patient who developed a Mycobacteria genavense infection of the liver and the bowel after several immunosuppressive therapies for systemic lupus erythematosus. CD4 T cell counts as low as 17/microl were found and immunosuppressive therapy was stopped. Despite several courses of antibiotic treatment and rising CD4 T cell counts, severe malabsorption persisted. Upper endoscopy revealed a continuous inflammation with pseudopolyps of the small bowel and histologically, a granulomatous infiltrate was detected. After exclusion of a persisting infection by Mycobacteria genavense, IRIS of the small bowel was suspected and treatment with prednisolone was started. The clinical and histological picture improved significantly, the number of CD25(+)CD4(+) cells decreased in the lamina propria of the duodenum under treatment with prednisolone and Foxp3+ regulatory T cells (Treg) accumulated around granulomas. This case shows that IRIS is not restricted to HIV patients but may also occur in otherwise immunosuppressed patients. Due to different treatment strategies, distinguishing IRIS from infectious diseases is essential. The role of Treg in IRIS has to be elucidated.

Mechanisms of epithelial translocation of the alpha(2)-gliadin-33mer in coeliac sprue.

BACKGROUND AND AIMS: The alpha(2)-gliadin-33mer has been shown to be important in the pathogenesis of coeliac disease. We aimed to study mechanisms of its epithelial translocation and processing in respect to transcytotic and paracellular pathways. METHODS: Transepithelial passage of a fluorescence-labelled alpha(2)-gliadin-33mer was studied in Caco-2 cells by using reverse-phase high-performance liquid chromatography, mass spectrometry, confocal laser scanning microscopy (LSM) and fluorescence activated cell sorting (FACS). Endocytosis mechanisms were characterised with rab-GFP constructs transiently transfected into Caco-2 cells and in human duodenal biopsy specimens. RESULTS: The alpha(2)-gliadin-33mer dose-dependently crossed the epithelial barrier in the apical-to-basal direction. Degradation analysis revealed translocation of the 33mer polypeptide in the uncleaved as well as in the degraded form. Transcellular passage was identified by confocal LSM, inhibitor experiments and FACS. Rab5 but not rab4 or rab7 vesicles were shown to be part of the transcytotic pathway. After pre-incubation with interferon-gamma, translocation of the 33mer was increased by 40%. In mucosal biopsies of the duodenum, epithelial 33mer uptake was significantly higher in untreated coeliac disease patients than in healthy controls or coeliac disease patients on a gluten-free diet. CONCLUSION: Epithelial translocation of the alpha(2)-gliadin-33mer occurs by transcytosis after partial degradation through a rab5 endocytosis compartment and is regulated by interferon-gamma. Uptake of the 33mer is higher in untreated coeliac disease than in controls and coeliac disease patients on a gluten-free diet.

Association of HLA-DRB1*02 with osteoarthritis in a cohort of 106 patients.

OBJECTIVE: We have previously shown that the inflammatory cytokines tumour necrosis factor alpha (TNF-alpha) and interleukin (IL) 6 or IL-1beta are up-regulated in chondrocytes of patients with osteoarthritis (OA). However, the inflammatory responses associated with OA are of low grade and restricted. To investigate the involvement of the immune system in the pathogenesis of OA, we analysed patients for their HLA-DRB1 haplotypes. METHODS: Combining single-stranded oligo or sequence-specific primer typing procedures, 139 randomly selected controls and 106 OA patients were typed for their HLA-DRB1 alleles. RESULTS: The OA cohort showed statistically significant differences in the frequencies of the DR2 and DR5 alleles compared with the controls. While the frequency of the DR2 allele was elevated among the OA patients, the DR5 allele was negatively associated with the disease. The P values for differences from the controls were 0.0431 for DR2 and 0.0386 for DR5 and the odds ratios for the two alleles were 1.58 and 0.54 respectively. CONCLUSION: The association of DR2 and DR5 with OA hints at linkage disequilibrium between HLA-DRB1 genes and genes involved in the pathogenesis of OA. Alternatively, DR2 has a direct role in restricting immunological responses to the low-grade inflammation characteristic of OA.

Association of genotypes affecting the expression of interleukin-1beta or interleukin-1 receptor antagonist with osteoarthritis.

OBJECTIVE: The majority of cytokines and growth factors known to be involved in cartilage metabolism are synthesized by the chondrocytes themselves. They are up-regulated in osteoarthritic (OA) cartilage, resulting in 2 opposite phenotypes, TNFalpha(high) and TNFalpha(low), that are characterized by an elevated number of tumor necrosis factor alpha (TNFalpha)-positive and interleukin-1beta (IL-1beta)-positive chondrocytes, respectively. To establish a hierarchy among the cytokines and growth factors expressed in articular chondrocytes, this study investigated cytokine genes for known polymorphisms that may contribute to the deregulated expression in OA cartilage. METHODS: Polymerase chain reaction techniques were performed either in a thermal cycler using standard methods or in a light cycler to analyze the frequencies of the TNFalpha (-308), IL-1 receptor antagonist (IL-1Ra) (intron 2), IL-1beta (exon 5), and IL-6 (-174) polymorphisms in 61 OA patients and 254 randomly chosen controls. RESULTS: For the TNFalpha(low) phenotype, a statistically significant association was found with the less frequent allele of IL-1beta, which carries a single-basepair substitution in exon 5 and may contribute to the characteristic increase in IL-beta-positive chondrocytes. In contrast, the TNFalpha(high) phenotype was significantly associated with the less frequent allele of IL-1Ra, which carries two 86-bp repeats in the second intron and is assumed to lead to an elevated expression of the antagonist. CONCLUSION: These results point to an association between the IL-1beta polymorphism and the TNFalpha(high) phenotype and between the IL-1Ra polymorphism and the TNFalpha(low) phenotype found in OA. Both associations suggest that IL-1beta may be more important than TNFalpha for the regulation of cytokine and growth factor expression in articular chondrocytes.

Immunohistological analysis of cytokine expression in human osteoarthritic and healthy cartilage.

OBJECTIVE: To investigate osteoarthritic cartilage in comparison to normal cartilage in humans for the presence of the most relevant cytokines/growth factors known to be important for degradation and formation of new cartilage. METHODS: Cartilage from knee or hip joints was obtained from 10 patients with osteoarthritis (OA) and from 7 age matched control patients with intact cartilage. Additionally, normal cartilage from 2 young patients (12 and 17 years old) was obtained after knee traumas. Immunohistological staining of cartilage sections was performed using antibodies for the following cytokines/growth factors: tumor necrosis factor alpha (TNF-alpha), interleukin 1alpha (IL-1alpha), IL-1beta, interferon-gamma, IL-6, IL-4, IL-10, transforming growth factor beta1 (TGF-beta1), insulin-like growth factor I (IGF-I), IGF-II, platelet derived growth factor AA (PDGF-AA), and PDGF-BB. RESULTS: Immunohistochemical stainings were positive for all cytokines in OA cartilage, while only a faint or no staining was found in healthy cartilage. Activated chondrocytes expressing most of the cytokines were located in the middle and partly in the lower layer of cartilage, with the exception of IGF-I, which was expressed exclusively in the upper cartilage layer close to the surface. More chondrocytes stained positive for TNF-alpha than for IL-1, and expression of the degrading cytokine TNF-alpha was inversely correlated to the expression of the regulatory cytokines IL-4, IL-10, and TGF-beta. CONCLUSION: The most relevant cytokines known to be involved in cartilage metabolism are produced by chondrocytes themselves. They are upregulated in OA cartilage, suggesting that they serve some regulatory function and could be a target for future treatment.

Differential expression of major histocompatibility complex class II genes on murine macrophages associated with T cell cytokine profile and protective/suppressive effects.

Protective/suppressive major histocompatibility complex (MHC) class II alleles have been identified in humans and mice where they exert a disease-protective and immunosuppressive effect. Various modes of action have been proposed, among them differential expression of MHC class II genes in different types of antigen-presenting cells impacting on the T helper type 1 (Th1)-Th2 balance. To test this possibility, the expression of H-2 molecules from the four haplotypes H-2(b), H-2(d), H-2(k), and H-2(q) was determined on bone marrow-derived macrophages (BMDMs) and splenic B cells. The I-Ab and I-Ek molecules, both well characterized as protective/suppressive, are expressed at a high level on almost all CD11b+ BMDMs for 5-8 days, after which expression slowly declines. In contrast, I-Ad, I-Ak, and I-Aq expression is lower, peaks over a shorter period, and declines more rapidly. No differential expression could be detected on B cells. In addition, the differential MHC class II expression found on macrophages skews the cytokine response of T cells as shown by an in vitro restimulation assay with BMDMs as antigen-presenting cells. The results indicate that macrophages of the protective/suppressive haplotypes express MHC class II molecules at a high level and exert Th1 bias, whereas low-level expression favors a Th2 response. We suggest that the extent of expression of the class II gene gates the back signal from T cells and in this way controls the activity of macrophages. This effect mediated by polymorphic nonexon segments of MHC class II genes may play a role in determining disease susceptibility in humans and mice.