Gut-associated Lymphoid System Research Articles

98 Gut Microbiome: Implications on gut Health

Abstract The rumen and the hindgut, which includes cecum and colon, of cattle are inhabited by a diverse microbial community, which is composed mainly of bacteria, but also includes archaea, protozoa, fungi and viruses. Most reside in the lumen, but also colonize the epithelial surface and their compositions are a critical component of the host health. Recent advances in sequencing techniques and biological computational tools have further underscored the complexity of the microbial community. The vast repertoire of the gut microbiome provides the host complementary genetic resources to harvest energy, provide proteins and vitamins, contribute to the development of gut epithelium and gut-associated lymphoid system, and to the overall gut health. The gut epithelium-vascular interface allows secretions, and absorption and metabolism of fermentation products and serves as a selective barrier to prevent translocation and systemic dissemination of microbes, microbial toxins, and immunogens. The barrier function includes protection form mechanical damage caused by feedstuffs, chemical damage from acidity, toxins and microbial invasion, especially pathogens. The mechanisms employed likely differ between the two fermentative regions of the gut, given the dramatic difference in epithelial structures. Rumen is lined by stratified squamous epithelium compared a single layer of columnar epithelium interspersed with mucus-producing goblet cells in the hindgut. The mucus layer is the first barrier the pathogen has to overcome for colonization and translocation. An imbalance in the composition of gut microbiota, called dysbiosis, leads to gut functional disorders. The dysbiosis can range from a change in one to a few species to the perturbation of entire microbial community. The assessment of gut microbial dysbiosis is a challenge because of the complexity and huge variations in the community composition, including animal-to-animal. The gut barrier function determines whether the dysbiotic changes are contained within the lumen or disseminated systemically to affect the health and productivity.

Critical review of the safety assessment of nano-structured silica additives in food.

The development of nano-materials is viewed as one of the most important technological advances of the 21st century and new applications of nano-sized particles in the production, processing, packaging or storage of food are expected to emerge soon. This trend of growing commercialization of engineered nano-particles as part of modern diet will substantially increase oral exposure. Contrary to the proven benefits of nano-materials, however, possible adverse health effects have generally received less attention. This problem is very well illustrated by nano-structured synthetic amorphous silica (SAS), which is a common food additive since several decades although the relevant risk assessment has never been satisfactorily completed. A no observed adverse effect level of 2500 mg SAS particles/kg body weight per day was derived from the only available long-term administration study in rodents. However, extrapolation to a safe daily intake for humans is problematic due to limitations of this chronic animal study and knowledge gaps as to possible local intestinal effects of SAS particles, primarily on the gut-associated lymphoid system. This uncertainty is aggravated by digestion experiments indicating that dietary SAS particles preserve their nano-sized structure when reaching the intestinal lumen. An important aspect is whether food-borne particles like SAS alter the function of dendritic cells that, embedded in the intestinal mucosa, act as first-line sentinels of foreign materials. We conclude that nano-particles do not represent a completely new threat and that most potential risks can be assessed following procedures established for conventional chemical hazards. However, specific properties of food-borne nano-particles should be further examined and, for that purpose, in vitro tests with decision-making cells of the immune system are needed to complement existing in vivo studies.

Orthotopic Small Bowel Transplantation in Rats

Small bowel transplantation has become an accepted clinical option for patients with short gut syndrome and failure of parenteral nutrition (irreversible intestinal failure). In specialized centers improved operative and managing strategies have led to excellent short- and intermediate term patient and graft survival while providing high quality of life (1,3). Unlike in the more common transplantation of other solid organs (i.e. heart, liver) many underlying mechanisms of graft function and immunologic alterations induced by intestinal transplantation are not entirely known(6,7). Episodes of acute rejection, sepsis and chronic graft failure are the main obstacles still contributing to less favorable long term outcome and hindering a more widespread employment of the procedure despite a growing number of patients on home parenteral nutrition who would potentially benefit from such a transplant. The small intestine contains a large number of passenger leucocytes commonly referred to as part of the gut associated lymphoid system (GALT) this being part of the reason for the high immunogenity of the intestinal graft. The presence and close proximity of many commensals and pathogens in the gut explains the severity of sepsis episodes once graft mucosal integrity is compromised (for example by rejection). To advance the field of intestinal- and multiorgan transplantation more data generated from reliable and feasible animal models is needed. The model provided herein combines both reliability and feasibility once established in a standardized manner and can provide valuable insight in the underlying complex molecular, cellular and functional mechanisms that are triggered by intestinal transplantation. We have successfully used and refined the described procedure over more than 5 years in our laboratory (8-11). The JoVE video-based format is especially useful to demonstrate the complex procedure and avoid initial pitfalls for groups planning to establish an orthotopic rodent model investigating intestinal transplantation.

Orthotopic Small Bowel Transplantation in Rats

Small bowel transplantation has become an accepted clinical option for patients with short gut syndrome and failure of parenteral nutrition (irreversible intestinal failure). In specialized centers improved operative and managing strategies have led to excellent short- and intermediate term patient and graft survival while providing high quality of life 1,3. Unlike in the more common transplantation of other solid organs (i.e. heart, liver) many underlying mechanisms of graft function and immunologic alterations induced by intestinal transplantation are not entirely known6,7. Episodes of acute rejection, sepsis and chronic graft failure are the main obstacles still contributing to less favorable long term outcome and hindering a more widespread employment of the procedure despite a growing number of patients on home parenteral nutrition who would potentially benefit from such a transplant. The small intestine contains a large number of passenger leucocytes commonly referred to as part of the gut associated lymphoid system (GALT) this being part of the reason for the high immunogenity of the intestinal graft. The presence and close proximity of many commensals and pathogens in the gut explains the severity of sepsis episodes once graft mucosal integrity is compromised (for example by rejection). To advance the field of intestinal- and multiorgan transplantation more data generated from reliable and feasible animal models is needed. The model provided herein combines both reliability and feasibility once established in a standardized manner and can provide valuable insight in the underlying complex molecular, cellular and functional mechanisms that are triggered by intestinal transplantation. We have successfully used and refined the described procedure over more than 5 years in our laboratory 8-11. The JoVE video-based format is especially useful to demonstrate the complex procedure and avoid initial pitfalls for groups planning to establish an orthotopic rodent model investigating intestinal transplantation.

Immunostimulatory activity ofBacillusspores

Bacillus species, typically Bacillus subtilis, are being used as probiotics and mounting evidence indicates that Bacillus species are important for development of a robust gut-associated lymphoid system (GALT). We used a number of gut isolates of Bacillus incorporating three species, B. subtilis, Bacillus licheniformis and Bacillus flexus to evaluate the nature of interaction between spores and the GALT. In mice orally administered with spores, evidence of cell proliferation was determined in the germinal centers of Peyer's patches. Stimulation of antigen-presenting cells and T lymphocytes was also markedly enhanced. Cytokines were shown to be induced in spleens and mesenteric lymph nodes of mice including the proinflammatory cytokines, tumour necrosis factor-alpha and IL-6. We also demonstrated that vegetative cells of B. subtilis can stimulate expression of the toll-like receptor (TLR) genes for TLR2 and TLR4. However, we were able to show that spores could not stimulate either and must, by default, interact with another TLR and by this mechanism help activate innate immunity.

Anatomical evidence for ileal Peyer’s patches innervation by enteric nervous system: a potential route for prion neuroinvasion?

We have examined the innervation of the gut-associated lymphoid system of the sheep ileum, with a view to identifying potential sites for neuroinvasion by pathogens, such as prions (PrP(Sc)). Special attention has been paid to the follicles of Peyer's patches (PPs), which are major sites of PrP(Sc) accumulation during infection. Evidence exists that the enteric nervous system, together with the parasympathetic and sympathetic pathways projecting to the intestine, are important for PrP(Sc) entry into the central nervous system. Thus, PrP(Sc) might move from PPs to the neurons and nerve fibres that innervate them. We investigated, by immunohistochemistry and retrograde tracing (DiI) from the follicles, the distribution and phenotype of enteric neurons innervating the follicles. Antibodies against protein gene product 9.5, tyrosine hydroxylase, dopamine beta hydroxylase, choline acetyltransferase, calbindin (CALB), calcitonin gene-related peptide (CGRP), and nitric oxide synthase were used to characterise the neurons. Immunoreactivity for each of these was observed in fibres around and inside PP follicles. CGRP-immunoreactive fibres were mainly seen at the follicular dome. Retrograde tracing revealed submucosal neurons that contributed to the innervation of PPs, including Dogiel type II neurons and neurons immunoreactive for CALB and CGRP. The major source of the adrenergic fibres are the sympathetic ganglia. Our results thus suggest that enteric and sympathetic neurons are involved during the first stage of neuroinvasion, with neurons connecting to them acting as potential carriers of PrP(Sc) to the central nervous system.

Enteral virus infections in early childhood and an enhanced type 1 diabetes-associated antibody response to dietary insulin

Enteral virus infections may trigger the development of β-cell-specific autoimmunity by interacting with the gut-associated lymphoid system. We analyzed the effect of three different virus infections on immunization to dietary insulin in children carrying increased genetic risk for type 1 diabetes. Forty-six of 238 children developed multiple diabetes-associated autoantibodies and 31 clinical diabetes (median follow-up time 75 months). Insulin-binding antibodies were measured with EIA method (median follow-up time 24 months). Antibodies to enteroviruses, rotavirus and adenovirus were measured with EIA in samples drawn at birth and the ages of 3 and 6 months. Nineteen enterovirus, 14 rotavirus and 8 adenovirus infections were diagnosed. At the ages of 6, 12, and 18 months, the concentrations of insulin-binding antibodies were higher in children with postnatal entero-, rota- and/or adenovirus infections than in children without these infections. Children who subsequently developed ICA or IA-2 antibodies or clinical type 1 diabetes had higher concentrations of insulin-binding antibodies than children who remained autoantibody negative. Our data suggest that enteral virus infections can enhance immune response to insulin, induced primarily by bovine insulin in cow's milk. An enhanced antibody response to dietary insulin preceded the development of β-cell specific autoimmunity and type 1 diabetes.

Gut-Associated Lymphoid Tissue in Microtus montanus from Natural Populations

Impaired immunocompetence may contribute to the multiannual fluctuations ("cycles") in population density of arvicoline rodents (Mihok et al. 1985, Pinter 1988, Lochmiller et al. 1993). In a variety of species environmental variables influence at least one component of the immune system, the gut-associated lymphoid system, GALT (Lopez et al. 1985, Li et al. 1995). Preliminary laboratory observations suggest that the development of GALT in Microtus montanus can be influenced by such factors as age and diet (Smith and Pinter 2000). However, these observations are from captive-bred voles, maintained on a synthetic laboratory diet, under strictly controlled laboratory conditions. The complexity and abundance of variables under natural conditions must influence the development and function of GALT- yet, nothing is known about it from Microtus montanus under natural conditions. The purpose of this study was essentially twofold: (1) to describe the morphometries of GALT in wild-caught M. montanus, and (2) to characterize the morphometric changes that occur in GALT of these animals as a function of their age, sex, and the season of the year.

Bronchus-associated lymphoid tissue (BALT) in the lungs of children who had died from sudden infant death syndrome and other causes.

Bronchus-associated lymphoid tissue (BALT) is well characterised in rabbits and rats. In humans, however, it does not seem to be present in the healthy adult lung, although it can develop after certain microbial stimulation. In the present study a consecutive series of lungs from 88 children who had died of sudden infant death syndrome (SIDS) and 34 control cases of comparable age were examined for the presence of BALT. BALT was present in 36.4% of the patients who had died of SIDS and in 44.1% of the control cases. The probability of finding BALT increased with age, with similar kinetics in both groups. Future studies need to define when and at what rate BALT disappears as children get older. In young children BALT may act as an entry site for antigens to initiate an immune response, as is well documented for the gut-associated lymphoid system.

Gut intraepithelial lymphocytes and gastrointestinal diseases

Intraepithelial lymphocytes interspersed between intestinal epithelial cells form a large population of T cells, mainly CD8+, close to the intestinal lumen. Together with lamina propria T cells and plasma cells, they represent the effector limb of the gut-associated lymphoid system. Recent data highlighting their original properties and their interactions with epithelial cells are reviewed; their contributions to immune defenses and gastrointestinal diseases in humans are discussed. Current Opinion in Gastroenterology 1993, 9:953-961

Anti-tumor effects of the oral administration of the streptococcal preparation OK-432 (PICIBANIL) — the inhibition of carcinogenesis and growth in rats with ENNG-induced gastrointestinal tumors

We examined whether the Streptococcal preparation OK-432, an immunopotentiating agent, increases immunocompetence of the gut-associated lymphoid system (GALS), inhibits gastrointestinal carcinogenesis, and has an anti-tumor effect. 14C-labelled OK-432 was orally and intraperitoneally administered to rats, and the distribution of the agent in various organs then serially evaluated. The concentration of OK-432 in Peyer's patches and mesenteric lymph nodes was higher after oral administration than after intraperitoneal administration, and showed a biphasic pattern peaking at 30 minutes and 5 hours following administration, in the Peyer's patches. With regard to immunocompetence, PHA- and PWM-stimulated blastogenesis of lymphocytes derived from the mesenteric lymph nodes and peripheral blood enhanced, and the helper/suppressor T-cell ratio was elevated after the oral administration of OK-432. Moreover, chemotactic activity of peritoneal macrophages was also increased. ENNG-induced gastrointestinal carcinogenesis was observed in 60 per cent of the rats orally administered OK-432 as compared with 88 per cent of the controls. The 13-month survival rate of the rats with gastrointestinal cancer was 50 per cent in those administered OK-432 as compared with 25 per cent in those administered OK-432 as compared with 25 per cent in the controls. When administered orally, the agent prevented reduction in immuno-competence in the course of carcinogenesis, suppressed carcinogenesis, and prolonged the survival of animals with cancer without any of the side effects associated with injection. The oral administration of OK-432 is thus considered to be an effective non-specific immunotherapy against gastro-intestinal malignancies.

Immunoprophylaxis of Chlamydia trachomatis lymphogranuloma venereum pneumonitis in mice by oral immunization.

Groups of BALB/c mice were orally immunized with chlamydiae and challenged intranasally to determine whether oral immunization offers protection against pulmonary disease and to characterize the nature and kinetics of the chlamydial antibody response in the lung and other mucosal sites. Protection by oral immunization from chlamydial lung disease was demonstrated by lack of replication of the organism and the lack of chlamydial antigen in lung tissue. The chlamydial immunoglobulin A (IgA) antibody response was present at all body sites, reaching peak levels in the lung as well as in the serum. Classical IgA booster effect kinetics was observed after intranasal challenge, especially in the lung. Specific IgG antibody was detected at all body sites but at lower levels. Furthermore, animals immunized orally had no pneumonic process, as determined by histopathology. These studies also suggest that passively acquired specific serum IgG antibody may not significantly influence the course of mucosal replication of the organism. These observations indicate that oral immunization activating the gut-associated lymphoid tissue system gave total protection against chlamydial lung disease, suggesting migration of immunologically competent cells from the intestine to the lung.

Antibodies to Human T-Lymphotropic Virus Type III (HTLV-III) in Saliva of Acquired Immunodeficiency Syndrome (AIDS) Patients and in Persons at Risk for AIDS

Whole saliva samples collected from available people at risk in Boston for infection with human T-lymphotropic virus type III (HTLV-III/LAV), from late 1984 through early 1985, were analyzed for the presence of antibodies to viral proteins. Fourteen of 20 (70%) acquired immunodeficiency syndrome (AIDS) patients and 14 of 15 (93%) AIDS-related complex (ARC) patients had salivary antibodies that reacted with the virus-encoded glycoproteins gp160 and gp120 of HTLV-III infected cells. All of the AIDS and ARC patients had serum antibodies to the same antigens. Of 20 sex partners of AIDS/ARC patients, nine (45%) showed anti-HTLV-III antibodies, and four of 18 (22%) healthy homosexual males also were positive for such antibodies. Serum and salivary antibody status were the same in these groups. A minority of those patients positive for salivary antibodies to env gene-encoded gp160 and gp120 also had salivary antibodies to gag gene-encoded proteins of 55,000, 24,000, and/or 17,000 daltons. Immunoglobulin A (IgA) class antibodies comprised the majority of the salivary antibody response. The spectrum of HTLV-III proteins detected by the salivary and serum antibodies was similar. The possibility that secretory IgA from the gut-associated lymphoid system may play a role to restrict salivary transmission of HTLV-III should be considered.

Antibodies to human T-lymphotropic virus type III (HTLV-III) in saliva of acquired immunodeficiency syndrome (AIDS) patients and in persons at risk for AIDS

Whole saliva samples collected from available people at risk in Boston for infection with human T-lymphotropic virus type III (HTLV-III/LAV), from late 1984 through early 1985, were analyzed for the presence of antibodies to viral proteins. Fourteen of 20 (70%) acquired immunodeficiency syndrome (AIDS) patients and 14 of 15 (93%) AIDS- related complex (ARC) patients had salivary antibodies that reacted with the virus-encoded glycoproteins gp160 and gp120 of HTLV-III infected cells. All of the AIDS and ARC patients had serum antibodies to the same antigens. Of 20 sex partners of AIDS/ARC patients, nine (45%) showed anti-HTLV-III antibodies, and four of 18 (22%) healthy homosexual males also were positive for such antibodies. Serum and salivary antibody status were the same in these groups. A minority of those patients positive for salivary antibodies to env gene-encoded gp160 and gp120 also had salivary antibodies to gag gene-encoded proteins of 55,000, 24,000, and/or 17,000 daltons. Immunoglobulin A (IgA) class antibodies comprised the majority of the salivary antibody response. The spectrum of HTLV-III proteins detected by the salivary and serum antibodies was similar. The possibility that secretory IgA from the gut-associated lymphoid system may play a role to restrict salivary transmission of HTLV-III should be considered.

Lymphomas of the mucosa-associated lymphoid tissue. Signet ring cell lymphomas presenting in mucosal lymphoid organs.

Four cases of lymphoma are reported which were postulate to have developed from neoplastic transformation of the lymphoid cells of the mucosa-associated lymphoid tissue (MALT). This contention is primarily based on two observations: the peculiar "homing" tendency of these lymphomas and the immunoglobulin isotype. Both properties are characteristic of the lymphoid cells of the MALT. These lymphomas can also be identified by their histologic structure (signet ring cell lymphomas) and their presentation in an MALT organ. These lymphomas are probably separable from other lymphomas proposed to arise from lymphocytes of the gut-associated lymphoid system (GALT).

Interaction between Cells of Peyer's Patches and Cells of Bone Marrow Origin in the Immune Response

Abstract There has been a good deal of interest in Peyer's patches as a part of the gut-associated lymphoid system, but their actual function has not been defined. Much of the early work on Peyer's patches was concerned with defining their role as the mammalian equivalent of the bursa of Fabricius, populated primarily by thymus-independent, bursal equivalent (B)3 lymphocytes (1–5). More recently, however, several studies have provided evidence that Peyer's patches also contain functional T lymphocytes (6–11). We reported previously that although Peyer's patches of mice do not respond to systemic antigenic stimulation by producing antibody in situ. Peyer's patch cells could act synergistically with spleen cells to produce a 5-fold increase in the number of antibody-forming cells to sheep red blood cells (SRBC) (12). In this communication we report that Peyer's patch cells are able to cooperate with bone marrow cells to produce antibody-forming cells of IgM, IgG, and IgA classes.

The gut-associated lymphoid system: nature and properties of the large dividing cells.

AbstractThe nature of the lymphoid cells and blasts (i.e. cells labeled in vitro with [3H]‐thymidine) of the Peyer's patches (PP), mesenteric lymph nodes (MLN) and thoracic duct lymph (TDL) has been studied by combined immunofluorescence and radioautography, using antisera directed against T (MSLA) or B (MBLA) cell antigenic determinants, or against various Ig chains. The migration pattern of these various cell populations after transfer to syngeneic mice or rats was determined by radioactivity counting and radioautography and the nature of the homed cells was studied by combined immunofluorescence and radioautography. B and T blasts from MLN and TDL have, in contrast to blasts from the peripheral lymph nodes, a marked tendancy to home in the gut, and migrate as well in a graft of fetal intestine as in the recipient's own gut, indicating that this selective accumulation is not the result of a mechanism recognizing antigen absorbed from the gut lumen.The gut‐homing B blasts are cells bearing surface IgA (sIgA) and containing intracellular IgA, which transform into IgA plasam cells in the lamina propria. Evidence is discussed that the progenitors of the intestinal IgA plasma cells are proliferating sIgA cells from the PP geerminal centers, which migrate via lymphatics to MLN and TDL and mature during this migration, acquiring intracellular IgA chains as well as a gut‐homing mechanism which might be related to this increased IgA synthesis.The gut‐homing T blasts migrate both in the lamina propria and the intestinal epithelium. Most, if not all, the intraepithelial lymphocytes of the gut mucosa appear to be T lymphocytes derived from rapidly dividing cells. Evidence is presented for the existence in the lymphoid system of two types of T blasts, one that is circulating and has a marked tendancy to home to the gut, and a second that does not home to the gut and may be sessile.

Gut-associated lymphoblasts and intestinal IgA plasma cells.

The term “gut-associated lymphoid system” (GALS) can he applied, in the mouse and in the rat, to the lymphoid cells present in 1) the Peyers’ patches (PP) ; 2) the intestinal mucosa, where these cells consist of disseminated lymphocytes and IgA secreting plasma cells; 3) the mesenteric lymph nodes (MLN), into which the lymphatic system of the gut drains, and 4) the thoracic duct lymph (TDL), which receives the efferent MLN lymph. It has been shown in the rat that the TDL and MLN “lymphoblasts” (i.e. large dividing cells which are labeled after in vitro incubation with 3H thymidine) display, after transfer into syngeneic recipients, a strong tendency to migrate to the intestinal mucosa (1,2,3), the PP, and to some extent the MLN (2). This migration pattern is not observed with the blasts obtained from immunized peripheral lymph nodes (PLN); these blasts do not migrate to the gut and home more in the PLN than in the MLN (2). Preferential migration is characteristic of only the GALS blasts, since it is not found in transfer experiments when the total lymphoid population from TDL or MLN is labeled (2). Since these observations indicate that some of the GALS blasts have special properties, it was decided to investigate further the nature and properties of the various GALS cells in the mouse and in the rat.