Thomas Marth

Reciprocal IFN-γ and TGF-β responses regulate the occurrence of mucosal inflammation

Abstract Recent studies of oral tolerance and experimental colitis indicate that the occurrence of gastrointestinal inflammation is determined by a balance between proinflammatory interferon γ responses and anti-inflammatory transforming growth factor β responses. Here, Warren Strober and colleagues discuss the new findings.

Expression of interleukin‐12‐related cytokine transcripts in inflammatory bowel disease: Elevated interleukin‐23p19 and interleukin‐27p28 in Crohn's disease but not in ulcerative colitis

Background: It has been suggested that Crohns disease (CD) is associated with an exaggerated T‐helper 1 cytokine response manifested by increased production of interleukin (IL)‐12. IL‐12 is a heterodimeric protein comprising 2 disulfide‐linked subunits designated p35 and p40. Recently, IL‐12‐related cytokines, IL‐23 and IL‐27, were described. Biologically active IL‐23 is a heterodimer whose p40 subunit is identical to IL‐12p40 whereas its p19 subunit is distantly related to IL‐12p35. IL‐27 consists of EBI3, an IL‐12p40‐related protein, and p28, a newly discovered IL‐12p35‐related polypeptide. Aim: We sought to determine whether mucosal expression of IL‐23p19 and IL‐27p28 transcripts correlate with the inflammatory activity in inflammatory bowel disease (IBD). Patients/Methods: Messenger RNA expression in colonic mucosa from patients with Crohns disease (CD; n = 37) and ulcerative colitis (UC; n = 19), and in non‐IBD control subjects (specific colitis [SC]; n = 16) and normal, nondiseased control patients (n = 12) was measured by reverse‐transcribed real‐time polymerase chain reaction. Results: IL‐23p19 was significantly increased in inflamed mucosa in CD (P = 0.0377) and to a lesser extent also in UC patients but not in SC patients. Elevation of IL‐23p19 transcript levels in CD correlated with the severity of endoscopic lesions. IL‐27p28 transcripts and EBI3 transcripts were significantly elevated only in active CD. Discussion: IL‐23p19, IL‐27p28, and EBI3 transcripts are strongly up‐regulated in CD. The stimulatory effects of these cytokines on naive T cells in addition to a strongly synergistic action with IL‐12 to trigger interferon‐&ggr; production may contribute to the perpetuation of the inflammatory process in patients with CD. Notably, increased expression of IL‐23 and IL‐27 transcripts in CD suggests a T helper 1‐dominated immunologic function in this disease.

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.

Anti–interleukin 12 treatment regulates apoptosis of Th1 T cells in experimental colitis in mice

BACKGROUND & AIMS Trinitrobenzene sulfonic acid (TNBS)-induced colitis is a T-helper 1 (Th1) T cell-mediated inflammation that is rapidly reversed by administration of anti-interleukin (IL) 12. This study sought to define the mechanism of this curative effect. METHODS Cells and tissue from mice with TNBS colitis receiving treatment with anticytokines were analyzed for phenotype, cytokine production, and apoptosis. RESULTS In initial studies, we found that treatment of mice with TNBS-induced colitis with anti-IL-12 was more effective than with anti-interferon (IFN)-gamma, and that anti-IL-12 led to complete normalization of IFN-gamma production by lamina propria T cells ex vivo, whereas anti-IFN-gamma did not. These data suggesting that anti-IL-12 leads to reversal of colitis by elimination of the Th1 T cells were substantiated by studies showing that anti-IL-12 treatment led to increased numbers of apoptotic cells in the lamina propria and spleen by both TUNEL staining of tissues and dispersed spleen cell populations. Finally, we found that the observed apoptosis was mediated by the Fas pathway because (1) MRL/MpJ-lpr(fas) mice lacking Fas function develop colitis that responds poorly to treatment with anti-IL-12; and (2) SJL/J mice with TNBS colitis that received Fas-Fc to block the Fas pathway were resistant to anti-IL-12 treatment. CONCLUSIONS These studies show that a main mechanism of action of anti-IL-12 in TNBS-induced colitis is the induction of Fas-mediated apoptosis of the Th1 T cells, causing inflammation.

Whipple's disease

Whipples disease, or intestinal lipodystrophy, is a systemic infectious disorder affecting mostly middle-aged white men. Patients present with weight loss, arthralgia, diarrhoea, and abdominal pain. The disease is commonly diagnosed by small-bowel biopsy; the appearance of the sample is characterised by inclusions in the lamina propria staining with periodic-acid-Schiff, which represent the causative bacteria. Tropheryma whipplei has been classified as an actinomycete and has been propagated in vitro, which allows the possibility of improving diagnostic strategies, for example through antibody-based detection of the bacillus on duodenal tissue or in circulating monocytes. Cell-mediated immunity in active and inactive Whipples disease has subtle defects that might predispose some individuals to symptomatic infection with this bacillus, which probably occurs ubiquitously. Although most patients respond well to empirical antibiotic treatment, some with relapsing disease have a poor outlook. The recent findings and concerted research might allow development of new strategies for diagnosis, treatment, and monitoring of patients with Whipples disease.

Efficacy of Ceftriaxone or Meropenem as Initial Therapies in Whipple's Disease

BACKGROUND & AIMS Whipples disease is a chronic infection caused by the actinomycete Tropheryma whipplei. We conducted a randomized controlled trial of the efficacy of antimicrobials that are able to cross the blood-brain barrier and to which T whipplei is susceptible. METHODS Patients from central Europe with previously untreated Whipples disease (n = 40) were assigned randomly to groups given daily infusions of either ceftriaxone (1 x 2 g, 20 patients) or meropenem (3 x 1 g, 20 patients) for 14 days, followed by oral trimethoprim-sulfamethoxazole for 12 months. The primary outcome measured was maintenance of remission for 3 years, determined by a composite index of clinical and laboratory data as well as histology. RESULTS All patients were observed for the entire follow-up period (median, 89 mo; range, 71-128 mo); all achieved clinical and laboratory remission. Remission was maintained in all patients during the time of observation, except for 2 who died from unrelated causes. A single patient with asymptomatic cerebrospinal infection who was resistant to both treatments responded to chloroquine and minocycline. The odds ratio for the end point (remission for at least 3 years) was 0.95 (95% confidence interval, 0.05-16.29; P = 1.0). CONCLUSIONS This was a randomized controlled trial to show that treatment with ceftriaxone or meropenem, followed by trimethoprim-sulfamethoxazole, cures patients with Whipples disease. One asymptomatic individual with infection of the cerebrospinal fluid required additional therapy.

Immunosuppressive Therapy in Whipple's Disease Patients is Associated with the Appearance of Gastrointestinal Manifestations

OBJECTIVES:Whipples disease is a rare chronic disorder, which is caused by systemic infection with Tropheryma whipplei. The first symptom of Whipples disease usually is a nondestructive polyarthritis resembling in many aspects seronegative rheumatoid arthritis. This polyarticular inflammatory arthropathy preceding the diagnosis of Whipples disease for several years frequently is treated with nonsteroidal antiinflammatory drugs (NSAIDs) and with immunosuppressive therapy. There is evidence that altered immune functions play a role in the manifestation of the disease and that Whipples disease is associated with opportunistic infections. We therefore asked whether immunosuppressive treatment for arthropathy may alter the course of Whipples disease.PATIENTS AND METHODS:In a series of 27 patients with Whipples disease clinical data were documented and the patients were followed for 3–4 yr. The patients were classified into three groups according to their medication: (i) patients with immunosuppressive therapy preceding the diagnosis, (ii) patients with NSAIDs before diagnosis, and (iii) patients without such therapies.RESULTS:Arthropathies occurred in the mean 8 yr before diagnosis and were the first symptom in 63% of the patients. Gastrointestinal involvement usually became evident later on and frequently led to the diagnosis of Whipples disease. In patients with immunosuppressive treatment, diarrhea occurred in the median 4 months after the initiation of such therapy and diagnosis of Whipples disease was made after another 2 months. In contrast, other medical treatments were not closely followed by the onset of diarrhea.CONCLUSIONS:These results indicate an association between immunosuppressive therapy and the onset of diarrhea in Whipples disease and thus support the concept that immunologic factors play a role in disease pathogenesis. Further investigation on the interaction of the immune system and Tropheryma whipplei infection are required to understand the factors contributing to the clinical manifestation of this rare disorder and possibly to introduce preventive interventions.

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.

Treatment of Refractory Whipple Disease with Interferon-γ

Whipple disease is an infectious, chronic multisystem disorder characterized by diarrhea, malabsorption, arthralgias, and (in later stages) involvement of the central nervous system [1]. The infection is caused by an intracellular bacterium for which the name Tropheryma whippelii has been proposed [2]. The pathogen has only recently been successfully isolated and propagated by using interleukin-4-deactivated macrophages [3]. Before the use of antibiotics, Whipple disease was fatal [4]; even with antimicrobial therapy, some patients have severe relapse [5-7]. The pathogenesis of T. whippelii infection is poorly understood; immune defects involving T cells and macrophages have been described [8-12]. Recently, reduced interleukin-12 and interferon- production in patients with Whipple disease has been detected [13]. These cytokines may be important in this context because of their ability to contain and clear intracellular bacteria [14, 15]. Therefore, we tested the usefulness of antibiotic therapy supplemented by interferon- in treating a patient with a 10-year history of antibiotic-resistant Whipple disease. Case Report In 1985, Whipple disease was diagnosed by periodic acid-Schiff (PAS)-positive macrophages in the duodenal biopsy specimen of a 66-year-old patient. The patient had a 10-year history of diarrhea and weight loss and was continuously treated with antibiotics to eradicate the bacterium. However, the patient had seven relapses despite this therapy. Duodenal biopsy results remained positive on testing for T. whippelii-specific DNA, PAS staining, and electron microscopy. In 1995, although the patient had no neurologic symptoms, involvement of the central nervous system was documented by the presence of T. whippelii-specific DNA and PAS-positive cells in the cerebrospinal fluid (Table 1). At this time, both in vivo and in vitro evidence of deficient T-cell function was detected. Testing of cutaneous delayed hypersensitivity with seven recall antigens (Multitest Merieux, Institut Merieux GmbH, Leimen, Germany) yielded abnormal results; only two responses were observed, and the combined diameter was 8 mm (normal test results yield at least 3 responses with a combined score >10 mm in diameter). The patient had no serologic evidence of HIV or cytomegalovirus infection, and repeated stool tests were negative for pathogenic bacteria and parasites. Table 1. Characteristics of the Patient before and after Treatment with Combination Therapy (Interferon- and Trimethoprim-Sulfamethoxazole)* Because of central nervous system involvement and the refractory course of the disease, the patient was treated with ceftriaxone and chloramphenicol (Table 1) and trimethoprim-sulfamethoxazole supplemented with recombinant human interferon- (Polyferon, Rentschler, Laupheim, Germany) (100 g subcutaneously three times per week). The patients clinical state indicated severe Whipple disease: weight loss, diarrhea, and reduced serum -carotene level (one of the best markers for malabsorption in Whipple disease [1]). Three weeks after initiation of therapy with interferon-, the patient recovered from clinical symptoms (Figure 1). Six months later, T. whippelii-specific DNA and PAS-positive cells were no longer found in the cerebrospinal fluid. However, duodenal biopsy continued to show PAS-positive cells and T. whippelii-specific DNA (Table 1). Figure 1. Effects of interferon- therapy. To deal with persistent subclinical infection, the dose of interferon- was increased to 150 g three times per week. Four months after initiation of high-dose interferon- therapy, cutaneous hypersensitivity testing showed normal responses to three antigens (with a score of 28 mm in diameter); electron microscopy of duodenal mucosa showed no intact bacteria in the remaining PAS-positive macrophages. For the first time, polymerase chain reaction analysis of duodenal biopsy was negative for T. whippelii-specific DNA. Combination therapy was discontinued after 16 months, and the patient remained symptom-free. When he was reevaluated in July 1997, a normal duodenal mucosa was seen during endoscopy, no cells containing sickleform particles were seen in the biopsy specimens, and results of polymerase chain reaction assay remained negative. In December 1997, the dose of trimethoprim-sulfamethoxazole was reduced to one application daily. In March 1998, the patient had no clinical symptoms. Methods Details of the use of the polymerase chain reaction assay to detect T. whippelii-specific DNA have been described previously [16, 17]. T-cell subpopulations were analyzed by flow cytometry, and interferon- production was studied in cultures of phytohemagglutinin-stimulated mononuclear cells from peripheral blood after 48 hours by a commercially available enzyme-linked immunosorbent assay (Laboserv, Staufenberg, Germany). The peripheral CD4+ T-cell count was as low as 126 cells/L before treatment (control [n = 5]: median, 1006 cells/L [range, 620 to 1295 cells/L]). Nine months after initiation of interferon- therapy, the CD4+ T-cell count was 616 cells/L, and 1 year after stopping interferon- therapy, the CD4+ T-cell count increased to 1003 cells/L (Figure 1). Interferon- production increased from 329 ng/L per 105 cells (control [n = 5]: median, 1595 ng/L per 105 cells [range, 817 to 2235 ng/L per 105 cells]) to 1865 ng/L per 105 cells at 9 months. In March 1998, interferon- production still exceeded pretreatment levels (Figure 1). Role of Funding Source The funding source had no role in gathering, analyzing, or interpreting the data or in deciding to submit the paper for publication. Discussion Our findings provide evidence that immunomodulatory therapy with interferon- in addition to antibiotics is beneficial in treating patients infected with the intracellular pathogen T. whippelii. Our patient had chronic Whipple disease that involved the central nervous system and was refractory to therapy with several usually effective antibiotics, including those that cross the blood-brain barrier. Final eradication of the bacterium occurred after the patient received combination therapy and has lasted for 1 year after interferon- therapy was stopped. It is unlikely that the eradication resulted from initial ceftriaxone-chloramphenicol treatment because the patient did not have a clinical response until 3 weeks after initiation of interferon- therapy. In addition, clearance of infection required a course of high-dose interferon-. The decision to include interferon- in this patients therapy was influenced by studies that showed the important role of this cytokine in controlling intracellular infections under experimental conditions and its success in treating persistent intracellular infections [15]. In addition, immunologic studies have shown reduced production of interleukin-12 and interferon- in patients with Whipple disease [13]. Of interest, the patients immunologic status improved during and after interferon- therapy. However, our study does not allow us to determine whether the immunoregulatory defect is a primary or secondary result of the infection. The addition of interferon- may be an option in the treatment of patients with refractory Whipple disease, but further studies are needed to clarify the clinical benefit of this therapy.