Germ-free Environment Research Articles

Role of T cells in ovariectomy induced bone loss—revisited

Role of T cells in ovariectomy induced bone loss—revisited

Does multiple sclerosis originate in the gut?

Autoreactive T and B cells may have important roles in multiple sclerosis, but what triggers autoreactivity is unclear. Here, the authors examined a relapsing–remitting variant of spontaneously developing murine experimental autoimmune encephalomyelitis (EAE). In this multiple sclerosis model, disease is driven by T cells that recognize a myelin autoantigen, and by recruited B cells that produce autoantibodies. Interestingly, relapsing–remitting mice that had been brought up in a germ-free environment, and hence had no commensal gut microbes, did not develop EAE and had impaired B-cell recruitment, suggesting that such microbes may have a role in multiple sclerosis.

Effects of a germ-free environment on gut immune regulation and diabetes progression in non-obese diabetic (NOD) mice

Microbial factors influence the development of diabetes in NOD mice. Studies in germ-free animals have revealed important roles of microbiota in the regulation of Th17 and forkhead box P3 (FOXP3)(+) T regulatory (Treg) activation in the intestine. However, the effects of intestinal microbiota in immune regulation and diabetes development in NOD mice are still poorly understood. A colony of germ-free NOD mice was established to evaluate the effects of intestinal microbiota on regulatory immunity in the gut, and on the development of insulitis and diabetes in NOD mice. Diabetes developed in roughly equal numbers in germ-free and specific pathogen-free NOD mice. Insulitis was accentuated in germ-free NOD mice; yet insulin preservation was unaltered. Germ-free NOD mice showed increased levels of Il17 (also known as Il17a) mRNA in the colon, and of Th17 and Th1 cells in the mesenteric and pancreatic lymph nodes, while Foxp3 mRNA and FOXP3(+) Tregs were reduced. In the islet infiltrates, FOXP3(+)CD4(+) T cells were slightly increased in germ-free mice. B cells appeared less activated in the peritoneum and were less abundant in islet infiltrates. These results indicate that lack of intestinal microbiota promotes an imbalance between Th1, Th17 and Treg differentiation in the intestine. This imbalance is associated with accelerated insulitis, but intact recruitment of FOXP3(+) Tregs into islets, suggesting: (1) a microbial dependence of local induction of Treg in the gut and draining lymph nodes; but (2) a potentially compensatory function of naturally occurring Tregs in the islets, which may help control diabetogenic T cells.

Fas (CD95/APO-1) limits the expansion of T lymphocytes in an environment of limited T-cell antigen receptor/MHC contacts

Fas-deficient mice (Fas(lpr/lpr)) and humans have profoundly dysregulated T lymphocyte homeostasis, which manifests as an accumulation of CD4(+) and CD8(+) T cells as well as an unusual population of CD4(-)CD8(-)TCRαβ(+) T cells. To date, no unifying model has explained both the increased T-cell numbers and the origin of the CD4(-)CD8(-)TCRαβ(+) T cells. As Fas(lpr/lpr) mice raised in a germ-free environment still manifest lymphadenopathy, we considered that this process is primarily driven by recurrent low-avidity TCR signaling in response to self-peptide/MHC as occurs during homeostatic proliferation. In these studies, we developed two independent systems to decrease the number of self-peptide/MHC contacts. First, expression of MHC class I was reduced in OT-I TCR transgenic mice. Although OT-I Fas(lpr/lpr) mice did not develop lymphadenopathy characteristic of Fas(lpr/lpr) mice, in the absence of MHC class I, OT-I Fas(lpr/lpr) T cells accumulated as both CD8(+) and CD4(-)CD8(-) T cells. In the second system, re-expression of β(2)m limited to thymic cortical epithelial cells of Fas(lpr/lpr) β(2)m-deficient mice yielded a model in which polyclonal CD8(+) thymocytes entered a peripheral environment devoid of MHC class I. These mice accumulated significantly greater numbers of CD4(-)CD8(-)TCRαβ(+) T cells than conventional Fas(lpr/lpr) mice. Thus, Fas shapes the peripheral T-cell repertoire by regulating the survival of a subset of T cells proliferating in response to limited self-peptide/MHC contacts.

Negative regulation of Toll‐like receptor signaling plays an essential role in homeostasis of the intestine

A healthy intestinal tract is characterized by controlled homeostasis due to the balanced interaction between commensal bacteria and the host mucosal immune system. Human and animal model studies have supported the hypothesis that breakdown of this homeostasis may underlie the pathogenesis of inflammatory bowel diseases. However, it is not well understood how intestinal microflora stimulate the intestinal mucosal immune system and how such activation is regulated. Using a spontaneous, commensal bacteria-dependent colitis model in IL-10-deficient mice, we investigated the role of TLR and their negative regulation in intestinal homeostasis. In addition to IL-10(-/-) MyD88(-/-) mice, IL-10(-/-) TLR4(-/-) mice exhibited reduced colitis compared to IL-10(-/-) mice, indicating that TLR4 signaling plays an important role in inducing colitis. Interestingly, the expression of IRAK-M, a negative regulator of TLR signaling, is dependent on intestinal commensal flora, as IRAK-M expression was reduced in mice re-derived into a germ-free environment, and introduction of commensal bacteria into germ-free mice induced IRAK-M expression. IL-10(-/-) IRAK-M(-/-) mice exhibited exacerbated colitis with increased inflammatory cytokine gene expression. Therefore, this study indicates that intestinal microflora stimulate the colitogenic immune system through TLR and negative regulation of TLR signaling is essential in maintaining intestinal homeostasis.

Altered Macrophage Function Contributes to Colitis in Mice Defective in the Phosphoinositide-3 Kinase Subunit p110δ

Innate immune responses are crucial for host defense against pathogens but need to be tightly regulated to prevent chronic inflammation. Initial characterization of mice with a targeted inactivating mutation in the p110δ subunit of phosphoinositide 3-kinase (PI3K p110δ(D910A/D910A)) revealed defects in B- and T-cell signaling and chronic colitis. Here, we further characterize features of inflammatory bowel diseases in these mice and investigate underlying innate immune defects. Colons and macrophages from PI3K p110δ(D910A/D910A) mice were evaluated for colonic inflammation and innate immune dysfunction. Colonic p110δ messenger RNA expression was examined in interleukin (IL)-10(-/-) and wild-type germ-free mice during transition to a conventional microbiota. To assess polygenic impact on development of colitis, p110δ(D910A/D910A) mice were backcrossed to IL-10(-/-) mice. A mild spontaneous colitis was shown in PI3K p110δ(D910A/D910A) mice at 8 weeks, with inflammation increasing with age. An inflammatory mucosal and systemic cytokine profile was characterized by expression of IL-12/23. In PI3K p110δ(D910A/D910A) macrophages, augmented toll-like receptor signaling and defective bactericidal activity were observed. Consistent with an important homeostatic role for PI3K p110δ, wild-type mice raised in a germ-free environment markedly up-regulated colonic PI3K p110δ expression with the introduction of the enteric microbiota; however, colitis-prone IL-10(-/-) mice did not. Moreover, PI3K p110δ(D910A/D910A) mice crossed to IL-10(-/-) mice developed severe colitis at an early age. This study describes a novel model of experimental colitis that highlights the importance of PI3K p110δ in maintaining mucosal homeostasis and could provide insight into the pathogenesis of human inflammatory bowel disease.

Immunologic rheumatic disorders

We provide the basics for clinicians who might be called on to consider the diagnosis of diseases such as systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA) in their practice. We will emphasize clinical recognition and first-line laboratory testing. Only characteristics of the classic rheumatic inflammatory diseases (ie, RA, seronegative spondyloarthropathy, SLE, antiphospholipid syndrome, Sjögren syndrome, scleroderma, and polymyositis/dermatomyositis) will be covered. In the past decade, treatment for RA and seronegative spondyloarthropathy has substantially improved. Their treatment has been revolutionized by the use of methotrexate and, more recently, TNF inhibitors, T-cell costimulation modulators, and B-cell depletion. The goal of RA treatment today is to induce a complete remission as early as possible in the disease process, with the mantra being "elimination of synovitis equals elimination of joint destruction." The hope is that if the major mediators of Sjögren syndrome, SLE, or scleroderma can be identified and then blocked, as in the example of TNF inhibitors in patients with RA, more specific treatments will become available. Thus RA has become an excellent model of this evolving paradigm. Through the identification of major mediators in its pathogenesis, novel and highly efficacious therapeutic agents have been developed.

Inflammation and autoimmunity caused by a SHP1 mutation depend on IL-1, MyD88, and a microbial trigger

A recessive phenotype called spin (spontaneous inflammation) was induced by N-ethyl-N-nitrosourea (ENU) mutagenesis in C57BL/6J mice. Homozygotes display chronic inflammatory lesions affecting the feet, salivary glands and lungs, and antichromatin antibodies. They are immunocompetent and show enhanced resistance to infection by Listeria monocytogenes. TLR-induced TNF and IL-1 production are normal in macrophages derived from spin mice. The autoinflammatory phenotype of spin mice is fully suppressed by compound homozygosity for Myd88(poc), Irak4(otiose), and Il1r1-null mutations, but not Ticam1(Lps2), Stat1(m1Btlr), or Tnf-null mutations. Both autoimmune and autoinflammatory phenotypes are suppressed when spin homozygotes are derived into a germ-free environment. The spin phenotype was ascribed to a viable hypomorphic allele of Ptpn6, which encodes the tyrosine phosphatase SHP1, mutated in mice with the classical motheaten alleles me and me-v. Inflammation and autoimmunity caused by SHP1 deficiency are thus conditional. The SHP1-deficient phenotype is driven by microbes, which activate TLR signaling pathways to elicit IL-1 production. IL-1 signaling via MyD88 elicits inflammatory disease.

What is “physiological” intestinal inflammation and how does it differ from “pathological” inflammation?

The gastrointestinal tract is unique when compared to other organs of the body because is in direct contact with the external environment and continuously exposed to antigens derived from foods and the enteric flora. To provide a balanced response (homeostasis) to these massive antigenic challenges and still allow the selective absorption of essential nutrients, the intestine has developed a series of powerful defense mechanisms that comprise both physical and biological controls. Physical controls include peristalsis, mucus secretion, and an uninterrupted monolayer of epithelial cells regulated by tight junctions. Biological controls include secretion of antibacterial molecules like defensins, lactoferrin, and lysozyme, trafficking of immune cells in and out of the gut, and a highly specialized immunological system that utilizes both innate and adaptive immune components. The integrated response of these combined defense systems is reflected by the accumulation of an enormous amount of immune cells that are scattered all along the mucosal surface of the gastrointestinal tract, distributed as either organized structures (Peyer’s patches in the small bowel and lymphoid follicles in the colon) or diffusely in the epithelium and lamina propria (intraepithelial lymphocytes and lamina propria mononuclear cells, respectively).1 Thus, when examined histologically, the intestinal mucosa appears to be “inflamed” compared to other mucosal surfaces that are devoid of immune cells or sparsely populated by them. Hence, the term “physiological” intestinal inflammation has been coined.2 Three obvious questions follow this definition of physiological intestinal inflammation: What are the mechanisms underlying its existence? What is its functional significance? What happens when physiological inflammation goes awry? During the last decade major advances in our understanding of the cellular, molecular, and functional mechanisms of physiological intestinal inflammation have occurred. Most of these advances are due to a substantially improved appreciation of the interactions between the normal commensal flora and the mucosal immune system.3 For a long time it has been known that animals kept in a germ-free environment only develop a very rudimentary mucosal immune system, a phenomenon primarily due to the lack of antigenic exposure to the bacteria that normally reside in the intestinal lumen. We know now that bacterial antigens are sampled by mucosal dendritic cells, critical antigen-presenting cells that initiate and control innate immune responses.4 There is recognition that bacterial antigen is mediated by Toll-like receptors (TLRs), NOD-like receptors (NLRs), and RIG-1-like receptors (RLRs)5,6 that discriminate between molecular patterns expressed by commensal or pathogenic bacteria, with subsequent presentation to and activation of effector cells of the adaptive immune system, primarily T and B cells, and development of an immune response that maintains mucosal immune homeostasis. Anatomically, the establishment of this homeostasis is translated by the presence of physiological intestinal inflammation. The functional significance of developing physiological inflammatory reactions in the gut is that they enable the body to recognize foreign and bacterial commensal antigens, and mount an appropriate immune response that is highly regulated and “controlled.” This way, intestinal tissue damage is prevented. The ability of responding to an antigenic challenge without causing injury or disease is what is defined as “tolerance,” or “oral tolerance” in the case of the gastrointestinal tract, and endows the essential ability of continuously adapt to the surrounding environment by creating a limited degree of inflammation that does not cause disease.7 The balance that controls intestinal homeostasis is delicate but, in most individuals, sturdy enough to withstand the challenges of qualitative or quantitative changes of dietary and microbial antigens, and physiological intestinal inflammation is preserved. If an acute infection occurs, as in salmonellosis or shigellosis, physiological inflammation transforms into pathological inflammation, which, however, is self-limited and followed by complete resolution. In contrast, in individuals destined to develop inflammatory bowel disease (IBD), the transformation of physiological into pathological inflammation never results in complete resolution, and overt chronic intestinal inflammation ensues, as typically observed in patients with Crohn’s disease (CD) or ulcerative colitis (UC).8,9 In individuals with an enhanced genetic risk of developing gut inflammation, like first-degree relatives of CD patients, there is a high prevalence of subclinical intestinal inflammation,10 which could be viewed as an intermediate From the Departments of Pathobiology and Gastroenterology & Hepatology, The Cleveland Clinic Foundation, Ohio, USA. Copyright © 2008 Crohn’s & Colitis Foundation of America, Inc. DOI 10.1002/ibd.20618 Published online in Wiley InterScience (www.interscience.wiley.com).

Mechanisms of Action of Probiotics

At birth, the newborn leaves the germ-free intrauterine environment and enters a highly contaminated extrauterine world, which requires potent host defenses to prevent disease. Intestinal defenses develop during gestation and have the capacity to respond but first must be exposed to colonizing bacteria. I review the importance of bacterial colonization for the appearance of normal mucosal immune function and the clinical consequences of inadequate colonization with regard to development of disease. For example, we now know that an imbalance in T-helper (Th) cells (e.g., Th2 levels greater than Th1 levels) can predispose to autoimmune disease and gut inflammation or disease, such as necrotizing enterocolitis. As we determine the role of bacterial colonization in the gut (bacterial-epithelial "cross talk"), we should have more-appropriate ways to modulate the gut immune responses-for example, by use of probiotics to prevent the expression of these gastrointestinal diseases.

Tooth replantation in germ-free and conventional rats.

The purpose of the present study was to evaluate the influence of bacterial infection on the pulpal and periodontal tissues in replanted teeth using germ-free and conventional rats. Forty maxillary and mandibular first molars from ten 6-week-old germ-free male Wistar rats were used. The animals and all materials were maintained in a germ-free environment inside vinyl isolators throughout the experimental periods. Twenty conventional male Wistar rats served as controls. The first molars were intentionally replanted immediately after extraction. At 3 days, and 1, 2, 4 and 8 weeks after replantation, animals were sacrificed and the replanted teeth were histopathologically evaluated. Diversity of pulp tissue response was notable in conventional rats, which initially showed various degrees of neutrophil infiltration and then displayed different types of response, including revascularization with reparative dentin formation and complete necrosis. Pulpal responses of germ-free rats were less variable, being characterized by an almost complete lack of neutrophil infiltration and a high frequency of bone-like tissue ingrowth. Typical inflammatory resorption was detected only in conventional rats, whereas a higher incidence of ankylosis was notable in germ-free rats. The present results may corroborate the concept that bacterial infection is a major cause of serious healing complications following tooth replantation, such as pulp necrosis and inflammatory root resorption. The difficulty in optimally controlling bacterial infection seems to be highly relevant to the complexity and unpredictability of the outcome of this procedure. It should also be emphasized that extensive mechanical damage to the periodontal tissues may trigger the development of unfavorable healing complications as ankylosis, even under strictly aseptic conditions.

The mitochondrial and kidney disease phenotypes of kd/kd mice under germfree conditions

Interstitial nephritis occurs spontaneously in kd/kd mice, but the mechanisms leading to this disease have not been fully elucidated. The earliest manifestation of a phenotype is the appearance of ultrastructural defects in the mitochondria of mice as young as 42 days of age. To examine the influence of the environment on the phenotype, homozygous B6. kd/kd mice were transferred from specific pathogen-free (SPF) conditions to a germfree (GF) environment, and the development of the disease was observed. The GF state resulted in a highly significant reduction in the frequency of tubulointerstitial nephritis. In addition, GF conditions markedly reduced the appearance of the mitochondrial phenotype, with no sign of mitochondrial abnormalities in GF mice of up to 155 days of age. These results suggest that environmental factors are involved in the progression of all known manifestations of this disease phenotype.

The role of gut-associated lymphoid tissues and mucosal defence

The newborn infant leaves a germ-free intrauterine environment to enter a contaminated extrauterine world and must have adequate intestinal defences to prevent the expression of clinical gastrointestinal disease states. Although the intestinal mucosal immune system is fully developed after a full-term birth, the actual protective function of the gut requires the microbial stimulation of initial bacterial colonization. Breast milk contains prebiotic oligosaccharides, like inulin-type fructans, which are not digested in the small intestine but enter the colon as intact large carbohydrates that are then fermented by the resident bacteria to produce SCFA. The nature of this fermentation and the consequent pH of the intestinal contents dictate proliferation of specific resident bacteria. For example, breast milk-fed infants with prebiotics present in breast milk produce an increased proliferation of bifidobacteria and lactobacilli (probiotics), whereas formula-fed infants produce more enterococci and enterobacteria. Probiotics, stimulated by prebiotic fermentation, are important to the development and sustainment of intestinal defences. For example, probiotics can stimulate the synthesis and secretion of polymeric IgA, the antibody that coats and protects mucosal surfaces against harmful bacterial invasion. In addition, appropriate colonization with probiotics helps to produce a balanced T helper cell response (Th1=Th2=Th3/Tr1) and prevent an imbalance (Th1>Th2 or Th2>Th1) contributing in part to clinical disease (Th2 imbalance contributes to atopic disease and Th1 imbalance contributes to Crohn's disease and Helicobacter pylori-induced gastritis). Furthermore, a series of pattern recognition receptors, toll-like receptors on gut lymphoid and epithelial cells that interact with bacterial molecular patterns (e.g. endotoxin (lipopolysaccharide), flagellin, etc.), help modulate intestinal innate immunity and an appropriate adaptive immune response. Animal and clinical studies have shown that inulin-type fructans will stimulate an increase in probiotics (commensal bacteria) and these bacteria have been shown to modulate the development and persistence of appropriate mucosal immune responses. However, additional studies are needed to show that prebiotics can directly or indirectly stimulate intestinal host defences. If this can be demonstrated, then prebiotics can be used as a dietary supplement to stimulate a balanced and an appropriately effective mucosal immune system in newborns and infants.

Disinfection and the prevention of infectious disease: No adverse effects?

We write in response to the publication by Cozad and Rhona in the American Journal of Infection Control, which we have read with great interest. The review was undertaken to assess what evidence there is to support disinfection as an infection control measure. The authors reach the conclusion that the use of disinfection is beneficial in preventing infectious disease and thus results in a public health benefit. However, we believe the scientific approach used in the work and, correspondingly, the authors’ findings deserve comment. The work was financed by the Consumer Specialty Product Association, a ‘‘premier trade association representing the interests of the consumer specialty products industry—a dynamic industry that provides households institutions and industrial customers with products that help provide a cleaner and healthier environment.’’ The association’s product range includes among other things items designed to control or eliminate microbes in any environment. Moreover, both the authors are members of Scientific and Regulatory Consultants, Inc, whose staff offers ‘‘expertise gained from over 50 years of combined service to the antimicrobial industry.’’ Thus, the work is not without bias, takes a one-sided approach, and disregards the known adverse effects of using surface disinfection in infection control. What do we know about the role of the hospital environment as a reservoir for infectious diseases? According to current scientific knowledge, microbial contamination of the patient’s inanimate environment seems to be only a minor causative factor within the complex nature of nosocomial infection. Maki and

Plant development in the absence of epiphytic microorganisms.

Microorganisms (bacteria, fungi) are common residents of the roots, stems and leaves of higher plants. In order to explore the dependency of plant development on the presence of epiphytic microorganisms, the achenes (seeds) of sunflower (Helianthus annuus L.) were sterilized and germinated under aseptic conditions. The sterility of the seedlings was determined with the agar impression method. In seedlings from non-sterile seeds (control) that were likewise raised in a germ-free environment, all plant organs investigated (stem, cotyledons and primary leaves) were contaminated with bacteria. Hypocotyl elongation was not affected by epiphytic microorganisms. However, the growth rates of the cotyledons and primary leaves were higher in sterile seedlings compared with the control. The implications of this differential inhibition of organ development by epiphytic bacteria that are transmitted via the outer surface of the seed coat are discussed. We conclude that epiphytes in the above-ground phytosphere are not necessary for the development of the sunflower seedling.

Role of gut flora on intestinal group II phospholipase A2 activity and intestinal injury in shock.

We previously showed that group II phospholipase A2 (PLA2-II), a secretory, bactericidal, and proinflammatory protein in intestinal crypts, is upregulated after lipopolysaccharide (LPS) and platelet-activating factor (PAF) challenge. Here we examined whether germ-free environment (GF) or antibiotic treatment (ABX) affects the pathophysiological responses and intestinal PLA2-II activity after PAF (1.5 microg/kg) or LPS (8 mg/kg) injection. We found that LPS and PAF induced hypotension and mild intestinal injury in conventionally fed (CN) rats; these changes were milder in ABX rats, whereas GF rats showed no intestinal injury. PLA2-II enzyme activity was detected in normal rat small intestine; the basal level was not diminished in ABX or GF rats. PAF and LPS caused an increase in PLA2-II activity, which was abrogated in GF and ABX rats. Recolonization of GF rats by enteral contamination restituted their PLA2-II response to PAF and LPS and susceptibility to bowel injury. We conclude that PAF- and LPS-induced increases in PLA2-II activity are dependent on gut bacteria, and ABX and GF rats are less susceptible to LPS-induced injury than CN rats.

The Bacterial Flora in Inflammatory Bowel Disease: Current Insights in Pathogenesis and the Influence of Antibiotics and Probiotics

The pathogenesis of inflammatory bowel disease (IBD) remains unknown, although in recent years more data have become available. The contribution of genetic and environmental factors is evident, and the luminal bacterial flora plays a major role in the initiation and perpetuation of chronic IBD. Animal models of IBD have shown that colitis does not occur in a germ-free environment. In human IBD, inflammation is present in parts of the gut containing the highest bacterial concentrations. Moreover, the terminal ileum, caecum and rectum are areas of relative stasis, providing prolonged mucosal contact with luminal contents. Enhanced mucosal permeability may play a pivotal role in maintaining a chronic inflammatory state, due to a genetic predisposition or as a result of direct contact with bacteria or their products. A defective epithelial barrier may cause a loss of tolerance to the normal enteric flora. Furthermore, an increased mucosal absorption of viable bacteria and bacterial products is found in IBD. Serum and secreted antibodies are increased and mucosal T-lymphocytes that recognize luminal bacteria are present. However, there is evidence that the immune system reacts over aggressively towards the normal luminal flora rather than the flora being altered in IBD. Several approaches have been used in attempts to discover a specific microbial agent in the cause of IBD. These include demonstration of the presence of organisms or specific antigens in affected tissues, culture of microbes from the affected tissues, demonstration of serological responses to several agents, and localization and detection of individual pathogen-specific nucleic acid sequences in affected tissue by in situ hybridization and polymerase chain reaction. So far, no specific micro-organism has been directly associated with the pathogenesis of IBD. Analysis of the luminal enteric flora, however, has revealed differences in the composition of this flora compared to healthy controls. In Crohn disease, concentrations of Bacteroides, Eubacteria and Peptostreptococcus are increased, whereas Bifidobacteria numbers are significantly reduced. Furthermore, in ulcerative colitis, concentrations of facultative anaerobic bacteria are increased. The arrival of new molecular techniques qualifying and quantifying the complex intestinal flora has induced a revival of interest in this microflora. Therapeutic approaches geared towards changing the environment at the mucosal border have been attempted by the use of elemental diets, total parenteral nutrition, surgical diversion of the faecal stream and antibiotics. Over the past few years, the use of probiotics in IBD and other intestinal disorders has gained attention. Strengthened by promising experimental data and commercial interests, research in this field is rapidly expanding. Manipulation of the colonic bacteria with antibiotic drugs and probiotic agents may prove to be more effective and better tolerated than immunosuppressants in the future.

The Role of Environmental Antigens in the Spontaneous Development of Autoimmunity in MRL-lprMice

AbstractIt has been proposed that the “normal” stimulation of the immune system that occurs from interactions with environmental stimuli, whether infectious or dietary, is necessary for the initiation and/or continuation of autoimmunity. We tested this hypothesis by deriving a group of MRL-lpr mice into a germfree (GF) environment. At 5 mo of age, no differences between GF and conventional MRL-lpr mice were noted in lymphoproliferation, flow cytometric analysis of lymph node cells (LN), or histologic analysis of the kidneys. Autoantibody levels were comparably elevated in both groups. A second experiment tested the role of residual environmental stimuli by contrasting GF mice fed either a low m.w., ultrafiltered Ag-free (GF-AF) diet or an autoclaved natural ingredient diet (GF-NI). At 4 mo of age, both groups showed extensive lymphoproliferation and aberrant T cell formation, although the GF-AF mice had ∼50% smaller LNs compared with sex-matched GF-NI controls. Autoantibody formation was present in both groups. Histologic analysis of the kidneys revealed that GF-AF mice had much lower levels of nephritis, while immunofluorescence analysis demonstrated no difference in Ig deposits but did reveal a paucity of C3 deposition in the kidneys of GF-AF mice.These data do not support a role for infectious agents in the induction of lymphoproliferation and B cell autoimmunity in MRL-lpr mice. Furthermore, they suggest that autoantibodies do not originate from B cells that were initially committed to exogenous Ags. They do suggest a possible contributory role for dietary exposure in the extent of lymphoproliferation and development of nephritis in this strain.

Reactive arthritis.

Reactive arthritis is one of the spondyloarthropathy family of clinical syndromes. The clinical features are those shared by other members of the spondyloarthritis family, though it is distinguished by a clear relationship with a precipitating infection. Susceptibility to reactive arthritis is closely linked with the class 1 HLA allele B27; it is likely that all sub-types pre-dispose to this condition. The link between HLA B27 and infection is mirrored by the development of arthritis in HLA B27-transgenic rats. In this model, arthritis does not develop in animals maintained in a germ-free environment. Infections of the gastrointestinal, genitourinary and respiratory tract appear to provoke reactive arthritis and a wide range of pathogens has now been implicated. Although mechanistic parallels may exist, reactive arthritis is distinguished from Lyme disease, rheumatic fever and Whipple's disease by virtue of the distinct clinical features and the link with HLA B27. As in these conditions both antigens and DNA of several micro-organisms have been detected in joint material from patients with reactive arthritis. The role of such disseminated microbial elements in the provocation or maintenance of arthritis remains unclear. HLA B27-restricted T-cell responses to microbial antigens have been demonstrated and these may be important in disease pathogenesis. The importance of dissemination of bacteria from sites of mucosal infection and their deposition in joints has yet to be fully understood. The role of antibiotic therapy in the treatment of reactive arthritis is being explored; in some circumstances, both the anti-inflammatory and anti-microbial effects of certain antibiotics appear to be valuable. The term reactive arthritis should be seen as a transitory one, reflecting a concept which may itself be on the verge of replacement, as our understanding of the condition develops. Nevertheless it appropriately describes arthritis that is associated with demonstrable infection at a distant site without traditional evidence of sepsis at the affected joint(s). Although several forms of disease could be described as "reactive", particularly acute rheumatic fever, post-meningococcal septicaemia arthritis and Lyme disease, in clinical practice the term is restricted to an acute spondyloarthritis, usually, but not exclusively, linked to acute genitourinary or gastrointestinal infection. A proportion of patients fulfil criteria for Reiter's Syndrome [1].

Autoimmunity, immunodeficiency and mucosal infections: chronic intestinal inflammation as a sensitive indicator of immunoregulatory defects in response to normal luminal microflora.

Despite the fact that target antigens and the genetic basis of several autoimmune diseases are now better understood, the initial events leading to a loss of tolerance towards self-components remain unknown. One of the most attractive explanations for autoimmune phenomena involves various infections as possible natural events capable of initiating the process in genetically predisposed individuals. The most accepted explanation of how infection causes autoimmunity is based on the concept of "molecular mimicry" (similarity between the epitopes of an autoantigen and the epitopes in the environmental antigen). Infectious stimuli may also participate in the development of autoimmunity by inducing an increased expression of stress proteins (hsp), chaperones and transplantation antigens, which leads to abnormal processing and presentation of self antigens. Superantigens are considered to be one of the most effective bacterial components to induce inflammatory reactions and to take part in the development and course of autoimmune mechanisms. It has long been known that defects in the host defense mechanism render the individual susceptible to infections caused by certain microorganisms. Impaired exclusion of microbial antigens can lead to chronic immunological activation which can affect the tolerance to self components. Defects in certain components of the immune system are associated with a higher risk of a development of autoimmune disease. The use of animal models for the studies of human diseases with immunological pathogenesis has provided new insights into the influence of immunoregulatory factors and the lymphocyte subsets involved in the development of disease. One of the most striking conclusion arising from work with genetically engineered immunodeficient mouse models is the existence of a high level of redundancy of the components of the immune system. However, when genes encoding molecules involved in T cell immunoregulatory functions are deleted, spontaneous chronic inflammation of the gut mucosa (similar to human inflammatory bowel disease) develops. Surprisingly, when such immunocompromised animals were placed into germfree environment, intestinal inflammation did not develop. Impairment of the mucosal immune response to the normal bacterial flora has been proposed to play a crucial role in the pathogenesis of chronic intestinal inflammation. The use of immunodeficient models colonized with defined microflora for the analysis of immune reactivity will shed light on the mode of action of different immunologically important molecules responsible for the delicate balance between luminal commensals, nonspecific and specific components of the mucosal immune system.