Verena Moos

Whipple's disease: new aspects of pathogenesis and treatment

100 years after its first description by George H Whipple, the diagnosis and treatment of Whipples disease is still a subject of controversy. Whipples disease is a chronic multisystemic disease. The infection is very rare, although the causative bacterium, Tropheryma whipplei, is ubiquitously present in the environment. We review the epidemiology of Whipples disease and the recent progress made in the understanding of its pathogenesis and the biology of its agent. The clinical features of Whipples disease are non-specific and sensitive diagnostic methods such as PCR with sequencing of the amplification products and immunohistochemistry to detect T whipplei are still not widely distributed. The best course of treatment is not completely defined, especially in relapsing disease, neurological manifestations, and in cases of immunoreconstitution after initiation of antibiotic treatment. Patients without the classic symptoms of gastrointestinal disease might be misdiagnosed or insufficiently treated, resulting in a potentially fatal outcome or irreversible neurological damage. Thus, we suggest procedures for the improvement of diagnosis and an optimum therapeutic strategy.

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 [3H]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) Ω cm2, 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 α (TNFα) was increased in untreated but not in treated HIV-infected patients. Incubation with IL2, IL4, TNFα 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.

Mechanisms of epithelial translocation of the α2-gliadin-33mer in coeliac sprue

Background and aims: The α2-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 α2-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 α2-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-γ, 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 α2-gliadin-33mer occurs by transcytosis after partial degradation through a rab5 endocytosis compartment and is regulated by interferon-γ. Uptake of the 33mer is higher in untreated coeliac disease than in controls and coeliac disease patients on a gluten-free diet.

Correlation of T cell response and bacterial clearance in human volunteers challenged with Helicobacter pylori revealed by randomised controlled vaccination with Ty21a-based Salmonella vaccines

Background: Helicobacter pylori remains a global health hazard, and vaccination would be ideal for its control. Natural infection appears not to induce protective immunity. Thus, the feasibility of a vaccine for humans is doubtful. Methods: In two prospective, randomised, double-blind, controlled studies (Paul Ehrlich Institute application nos 0802/02 and 1097/01), live vaccines against H pylori were tested in human volunteers seronegative for, and without evidence of, active H pylori infection. Volunteers (n = 58) were immunised orally with Salmonella enterica serovar Typhi Ty21a expressing H pylori urease or HP0231, or solely with Ty21a, and then challenged with 2×105 cagPAI− H pylori. Adverse events, infection, humoral, cellular and mucosal immune response were monitored. Gastric biopsies were taken before and after vaccination, and postchallenge. Infection was terminated with antibiotics. Results: Vaccines were well tolerated. Challenge infection induced transient, mild to moderate dyspeptic symptoms, and histological and transcriptional changes in the mucosa known from chronic infection. Vaccines did not show satisfactory protection. However, 13 of 58 volunteers, 8 vaccinees and 5 controls, became breath test negative and either cleared H pylori (5/13) completely or reduced the H pylori burden (8/13). H pylori-specific T helper cells were detected in 9 of these 13 (69%), but only in 6 of 45 (13%) breath test-positive volunteers (p = 0.0002; Fisher exact test). T cells were either vaccine induced or pre-existing, depending on the volunteer. Conclusion: Challenge infection offers a controlled model for vaccine testing. Importantly, it revealed evidence for T cell-mediated immunity against H pylori infection in humans.

Reduced Peripheral and Mucosal Tropheryma whipplei-Specific Th1 Response in Patients with Whipple’s Disease

Whipple’s disease is a rare infectious disorder caused by Tropheryma whipplei. Major symptoms are arthropathy, weight loss, and diarrhea, but the CNS and other organs may be affected, too. The incidence of Whipple’s disease is very low despite the ubiquitous presence of T. whipplei in the environment. Therefore, it has been suggested that host factors indicated by immune deficiencies are responsible for the development of Whipple’s disease. However, T. whipplei-specific T cell responses could not be studied until now, because cultivation of the bacteria was established only recently. Thus, the availability of T. whipplei Twist-MarseilleT has enabled the first analysis of T. whipplei-specific reactivity of CD4+ T cells. A robust T. whipplei-specific CD4+ Th1 reactivity and activation (expression of CD154) was detected in peripheral and duodenal lymphocytes of all healthy (16 young, 27 age-matched, 11 triathletes) and disease controls (17 patients with tuberculosis) tested. However, 32 Whipple’s disease patients showed reduced or absent T. whipplei-specific Th1 responses, whereas their capacity to react to other common Ags like tetanus toxoid, tuberculin, actinomycetes, Giardia lamblia, or CMV was not reduced compared with controls. Hence, we conclude that an insufficient T. whipplei-specific Th1 response may be responsible for an impaired immunological clearance of T. whipplei in Whipple’s disease patients and may contribute to the fatal natural course of the disease.

Genotyping reveals a wide heterogeneity of Tropheryma whipplei.

Tropheryma whipplei, the causative agent of Whipples disease, is associated with various clinical manifestations as well as an asymptomatic carrier status, and it exhibits genetic heterogeneity. However, relationships that may exist between environmental and clinical strains are unknown. Herein, we developed an efficient genotyping system based on four highly variable genomic sequences (HVGSs) selected on the basis of genome comparison. We analysed 39 samples from 39 patients with Whipples disease and 10 samples from 10 asymptomatic carriers. Twenty-six classic gastrointestinal Whipples disease associated with additional manifestations, six relapses of classic Whipples disease (three gastrointestinal and three neurological relapses), and seven isolated infections due to T. whipplei without digestive involvement (five endocarditis, one spondylodiscitis and one neurological infection) were included in the study. We identified 24 HVGS genotypes among 39 T. whipplei DNA samples from the patients and 10 T. whipplei DNA samples from the asymptomatic carriers. No significant correlation between HVGS genotypes and clinical manifestations of Whipples disease, or asymptomatic carriers, was found for the 49 samples tested. Our observations revealed a high genetic diversity of T. whipplei strains that is apparently independent of geographical distribution and unrelated to bacterial pathogenicity. Genotyping in Whipples disease may, however, be useful in epidemiological studies.

Impaired Immune Functions of Monocytes and Macrophages in Whipple's Disease

BACKGROUND & AIMS Whipples disease is a chronic multisystemic infection caused by Tropheryma whipplei. Host factors likely predispose for the establishment of an infection, and macrophages seem to be involved in the pathogenesis of Whipples disease. However, macrophage activation in Whipples disease has not been studied systematically so far. METHODS Samples from 145 Whipples disease patients and 166 control subjects were investigated. We characterized duodenal macrophages and lymphocytes immunohistochemically and peripheral monocytes by flow cytometry and quantified mucosal and systemic cytokines and chemokines indicative for macrophage activation. In addition, we determined duodenal nitrite production and oxidative burst induced by T whipplei and by other bacteria. RESULTS Reduced numbers of duodenal lymphocytes, increased numbers of CD163(+) and stabilin-1(+), reduced numbers of inducible nitric synthase+ duodenal macrophages, and increased percentages of CD163(+) peripheral monocytes indicated a lack of inflammation and a M2/alternatively activated macrophage phenotype in Whipples disease. Incubation with T whipplei in vitro enhanced the expression of CD163 on monocytes from Whipples disease patients but not from control subjects. Chemokines and cytokines associated with M2/alternative macrophage activation were elevated in the duodenum and the peripheral blood from Whipples disease patients. Functionally, Whipples disease patients showed a reduced duodenal nitrite production and reduced oxidative burst upon incubation with T whipplei compared with healthy subjects. CONCLUSIONS The lack of excessive local inflammation and alternative activation of macrophages, triggered in part by the agent T whipplei itself, may explain the hallmark of Whipples disease: invasion of the intestinal mucosa with macrophages incompetent to degrade T whipplei.

The Immune Reconstitution Inflammatory Syndrome in Whipple Disease: A Cohort Study

BACKGROUND Whipple disease, which is caused by infection with Tropheryma whipplei, can be treated effectively with antimicrobials. Occasionally, inflammation reappears after initial improvement; this is often interpreted as refractory or recurrent disease. However, polymerase chain reaction for T. whipplei in tissue is sometimes negative during reinflammation, indicating absence of vital bacteria, and this reinflammation does not respond to antimicrobials but does respond to steroids. OBJECTIVE To demonstrate that the immune reconstitution inflammatory syndrome (IRIS) occurs in patients treated for Whipple disease. DESIGN Cohort study. (International Standard Randomised Controlled Trial Number Register registration number: ISRCTN45658456) SETTING 2 academic medical centers in Germany. METHODS 142 patients treated for Whipple disease out of a cohort of 187 were observed for reappearance of inflammatory signs after effective antibiotic therapy. Definitions of IRIS in HIV infection, tuberculosis, and leprosy were adapted for application to Whipple disease. RESULTS On the basis of study definitions, IRIS was diagnosed in 15 of 142 patients. Symptoms included fever, arthritis, pleurisy, erythema nodosum, inflammatory orbitopathy, small-bowel perforation, and a hypothalamic syndrome. Two patients died. There was a positive correlation with previous immunosuppressive treatment and a negative correlation with previous diarrhea and weight loss. LIMITATIONS The study was observational and thus has inherent weaknesses, such as incomplete and potentially selective data recording. CONCLUSION The immune reconstitution inflammatory syndrome was diagnosed in about 10% of patients with Whipple disease in the study cohort; the outcome varied from mild to fatal. Patients who had had previous immunosuppressive therapy were at particular risk. An immune reconstitution syndrome should be considered in patients with Whipple disease in whom inflammatory symptoms recur after effective treatment. Early diagnosis and treatment with steroids may be beneficial; prospective studies are needed. PRIMARY FUNDING SOURCE European Commission and Deutsche Forschungsgemeinschaft.

High Frequency of Tropheryma whipplei in Culture-Negative Endocarditis

ABSTRACT “Classical” Whipples disease (cWD) is caused by Tropheryma whipplei and is characterized by arthropathy, weight loss, and diarrhea. T. whipplei infectious endocarditis (TWIE) is rarely reported, either in the context of cWD or as isolated TWIE without signs of systemic infection. The frequency of TWIE is unknown, and systematic studies are lacking. Here, we performed an observational cohort study on the incidence of T. whipplei infection in explanted heart valves in two German university centers. Cardiac valves from 1,135 patients were analyzed for bacterial infection using conventional culture techniques, PCR amplification of the bacterial 16S rRNA gene, and subsequent sequencing. T. whipplei-positive heart valves were confirmed by specific PCR, fluorescence in situ hybridization, immunohistochemistry, histological examination, and culture for T. whipplei. Bacterial endocarditis was diagnosed in 255 patients, with streptococci, staphylococci, and enterococci being the main pathogens. T. whipplei was the fourth most frequent pathogen, found in 16 (6.3%) cases, and clearly outnumbered Bartonella quintana, Coxiella burnetii, and members of the HACEK group (Haemophilus species, Actinobacillus actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae). In this cohort, T. whipplei was the most commonly found pathogen associated with culture-negative infective endocarditis.

Osteopontin as two-sided mediator of intestinal inflammation

Osteopontin (OPN) is characterized as a major amplifier of Th1‐immune responses. However, its role in intestinal inflammation is currently unknown. We found considerably raised OPN levels in blood of wild‐type (WT) mice with dextran sodium sulfate (DSS)‐induced colitis. To identify the role of this mediator in intestinal inflammation, we analysed experimental colitis in OPN‐deficient (OPN−/−) mice. In the acute phase of colitis these mice showed more extensive colonic ulcerations and mucosal destruction than WT mice, which was abrogated by application of soluble OPN. Within the OPN−/– mice, infiltrating macrophages were not activated and showed impaired phagocytosis. Reduced mRNA expression of interleukin (IL)‐1 β and matrix metalloproteinases was found in acute colitis of OPN−/– mice. This was associated with decreased blood levels of IL‐22, a Th17 cytokine that may mediate epithelial regeneration. However, OPN–/– mice showed increased serum levels of tumour necrosis factor (TNF)‐α, which could be due to systemically present lipopolysaccharide translocated to the gut. In contrast to acute colitis, during chronic DSS‐colitis, which is driven by a Th1 response of the lamina propria infiltrates, OPN−/– mice were protected from mucosal inflammation and demonstrated lower serum levels of IL‐12 than WT mice. Furthermore, neutralization of OPN in WT mice abrogated colitis. Lastly, we demonstrate that in patients with active Crohns disease OPN serum concentration correlated significantly with disease activity. Taken together, we postulate a dual function of OPN in intestinal inflammation: During acute inflammation OPN seems to activate innate immunity, reduces tissue damage and initiates mucosal repair whereas during chronic inflammation it promotes the Th1 response and strengthens inflammation.