This chapter presents a short overview on interactions between maturing lymphocytes and the epithelial structures of the bursa and the thymus because these interactions form the basis for collaboration of mature lymphocytes. The first cells, representing macrophage/dendritic cell lineage, are responsible for the formation of the epithelial buds which, during the subsequent phase, are colonized by lymphoid precursors. The functional capacity of these cells has not been analyzed in detail, but the results demonstrate that chicken T-cell progenitors are able to differentiate in a xenogeneic thymus. The chicken major histocompatibility complex (MHC), originally described as a blood group system, consists of three loci: B-F (class I), B-L (class II), and B-G (class IV). The chapter carries out a series of experiments aiming at clarifying the mechanisms of tolerance induction in B-cell-chimeric chickens reconstituted with class 1 or class II incompatible bursa cells.

Red cell antigen k occurs in the same frequency and strength as in adults in fetuses of 9 weeks or more and antigens Fy a , Fy b and Vel in fetuses aged at least twelve weeks. Antigen K was encountered twice, in fetuses of 14 and 16 weeks. Lu b appears weaker in fetuses than in adults; it was not detected in a fetus of 9 weeks although both parents were Lu(a–b+). Xg a was found to be yet more poorly developed before birth; it was demonstrated in only four out of 23 fetuses. Its frequency in fullterm newborns was, however, the same as in adults, but with lower scores.

Serum group‐specific components and transferrins of 24 pairs of mother‐fetal samples were studied. The results indicate that the human fetus is able to synthesize its own group‐specific components as early as the 10th to 13th week of gestation. A transferrin pattern different from the mother's could be demonstrated in the sera of two fetuses of about 9 and 21 weeks. Other fetuses showed transferrin qualitatively identical with the mother's. The concentration of fetal transferrins was greatly reduced.

OBJECTIVE:To compare the composition of intestinal microbiota of patients with early rheumatoid arthritis (RA) or fibromyalgia (FM), fecal samples were collected from 51 patients with RA and 50 with FM.METHODS:RA patients fulfilled the RA criteria of the American College of Rheumatology, and duration of their disease was < or = 6 months. Only nonhospitalized patients from outpatient care were included. Patients having extreme diets or previous disease modifying antirheumatic drug or glucocorticoid medication were excluded, as were those taking antibiotics or having gastroenteritis for at least 2 months prior to sampling. Fecal bacterial composition was analyzed with a method based on flow cytometry, 16S rRNA hybridization, and DNA-staining. A set of 8 oligonucleotide probes was used.RESULTS:In comparison to patients with FM, the RA patients had significantly less bifidobacteria and bacteria of the Bacteroides-Porphyromonas-Prevotella group, Bacteroides fragilis subgroup, and Eubacterium rectale--Clostridium coccoides group. Results from the 8 probes showed a significant overall difference between the 2 patient groups, indicating widespread microbial differences.CONCLUSION:These findings support the hypothesis that intestinal microbes participate in the etiopathogenesis of RA.

Abstract Many diverse concepts and speculations related to the etiopathogenesis of rheumatoid arthritis have been proposed, most of which implicate both genetic and environmental factors. The disease is known to be associated with certain HLA-DRB1 alleles, encoding a common sequence of five amino acids in the hypervariable region of the HLA-DR chain, the so-called susceptibility epitope. This epitope is found in 80–90% of Caucasoid patients with rheumatoid arthritis and in 40–50% of non-rheumatoid subjects. Disease heritability is estimated to be about 60%, of which HLA accounts for less than a half, and multiple loci outside the HLA region are responsible for the rest . Several alternative possibilities have been proposed for the participation of the susceptibility epitope , the most common being that it specifies HLA-DR molecules binding arthritogenic peptides. Recently it has become apparent that bacterial colonization of the host could be determined by genetic factors, including HLA, leaving a novel role also for the susceptibility epitope .

This review is concentrated on specifically diagnosable viral diseases causing recent onset polyarthritis. A suspicion of viral etiology of arthritis should arise when there are symptoms of infection such as fever, skin manifestations, neurologic signs, or when several cases are occurring at the same time, suggesting an outbreak. In addition to the history and clinical examination, serology is a key element in the diagnosis. Some of these infections are known to occur at certain geographic areas, and the local clinicians are aware of this possibility. HIV and HCV infections occur worldwide, and they should be kept in mind when treating patients with recent-onset arthritis.

Hybridization of bacteria with fluorescent probes targeting 16S rRNA and inspection of hybridized bacteria with fluorescence microscopy (microscopy-FISH, i.e. fluorescence in situ hybridization) have constituted an accessible method for the analysis of mixed bacterial samples such as feces. However, microscopy-FISH is a slow method and prone to errors. Flow cytometry (FCM) enables analysis of bacteria more rapidly, accurately and reliably than microscopy. In this study, a FCM method for the analysis of 16S rRNA-hybridized and DNA-stained fecal bacteria was developed. The results of FCM-FISH were comparable to those of microscopy-FISH, and the coefficients of variation of the FCM analyses were extraordinarily low. In previous FCM-FISH studies, the Eub 338 probe, which is supposed to hybridize all bacteria, has been used to detect all bacteria present in the sample. We found that Eub 338 did not bind to all bacteria, which could be detected by DNA-staining; while SYTOX® Orange DNA-stain detected all bacterial species tested and produced high fluorescence intensities enabling clear separation of bacteria from non-bacterial material. Thus, DNA-staining is a method of choice for the detection of all bacteria in FCM-FISH. We conclude that FCM of 16S rRNA-hybridized and DNA-stained bacteria is a rapid and reliable method for the analysis of mixed bacterial samples including feces.

The gastrointestinal tracts of developed animals are colonized by an extremely complex and diverse microbial ecosystem. The host and its microbiota are in close interaction with each other, and the host’s genetic characteristics have been suggested to have an influence on the composition of fecal bacteria. However, different sections of gastrointestinal tract harbor microbes typical of each particular section and knowledge of the effect of the host’s genotype on the microbiota in the different parts of the gastrointestinal tract is limited. In this study, mice from two inbred strains, C57BL/6J and BALB/c, were raised in identical conditions. Bacterial samples were collected from four parts of the gastrointestinal tract and analyzed for bacterial fatty acids using gas-liquid chromatography (GLC). Significant differences between the microbiota in the feces, in the cecum, in the small bowel and in the stomach were observed. Cecal samples produced more diverse bacterial fatty acid profiles than any of the other samples, revealing a higher bacterial density and a higher number of bacterial species. Further, a significant difference between the two strains of mice was observed throughout the gastrointestinal tract. These findings indicate that the host’s genotype has an influence on the gastrointestinal microbiota as a whole, and provide further evidence that the cecum is the most species-rich region of the murine gut.

Moshira El-Abasy, Maki Motobu, Kikuyasu Nakamura, Kenji Koge, Takashi Onodera, Olli Vainio, Paavo Toivanen, Yoshikazu Hirota* Department of Molecular Immunology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan National Institute of Animal Health, National Agricultural Research Organization, Department of Safety Research, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan Chigasaki Laboratory, Shin Mitsui Sugar Co., Ltd., 1-2-14 Honson, Chigasaki, Kanagawa 253-0042, Japan Department of Medical Microbiology, Turku University, SF-20520 Turku 52, Finland

Effects of oral administration of sugar cane extract (SCE) on immunosuppression in chickens treated with cyclophosphamide (CPA) were evaluated. Three-week-old inbred chickens were inoculated into the crop with SCE (500 mg/kg/day) for three consecutive days before or after injection of CPA 12 or 20 mg/chicken. At the last day of SCE or CPA treatment, all chickens were immunized intravenously with sheep red blood cells (SRBC) and Brucella abortus (BA). Chickens administered SCE showed a significant increase in body weight, gain in body weight/day, relative weight of the bursa of Fabricius and antibody responses to SRBC and BA than untreated control chickens. Chickens injected with CPA alone showed significantly decreased body weight, gain in body weight/day, relative weight of the bursa and antibody responses to SRBC and BA, showing immunosuppression in the bursa-dependent immune system. All chickens administered SCE before or after the treatment with CPA showed significantly higher values in body weight, gain in body weight/day, relative bursal weight and antibody responses to both antigens, when compared to chickens treated with CPA alone. In histological examination, chickens administered SCE showed a typical bursa with well constituted follicles, although chickens treated with CPA alone showed a severely atrophied bursa with rudimentary follicles and enormous proliferation of interfollicular connective tissue. Chickens treated with SCE and CPA showed a well-reconstituted bursa with almost normal structure. These results suggest that SCE has functionally and morphologically reconstituting effects on the bursa-dependent immune system in immunosuppressed chickens induced by injection of CPA.

The gastrointestinal tract and the microbes colonizing it form a complex ecosystem that has various effects on the well-being of the host. In addition to acute infections, the composition of the gastrointestinal microbiota has been suspected to influence the etiopathogenesis of many chronic diseases, such as rheumatoid arthritis and inflammatory bowel diseases. It has been suggested that the bacterial colonization of the gastrointestinal tract is genetically determined. Using gas–liquid chromatography of bacterial cellular fatty acids we show in this study that modulation of the microbiota by a course of antibiotics is followed by regeneration of the murine intestinal flora depending on the genotype of the host. The mice used in our study were acclimatized to identical living conditions before treatment with ciprofloxacin and clindamycin for 1 week via drinking water. Within a few days of finishing the antibiotic course, the cellular fatty acid profiles of fecal samples resembled those of the pre-course community, showing a considerable indigenous recovery potential. Colonization of the gastrointestinal tract appeared to be genetically regulated since differences in communities between the mouse strains were observed. Our results are in harmony with earlier observations, indicating that the gut community is not established by chance and that it is influenced by host-derived factors.

A series of observations have led to the hypothesis that normal intestinal microbiota in patients with rheumatoid arthritis may harbour, for genetic reasons, bacteria with cell walls capable of inducing arthritis. Differences occur between bacterial species, and even between strains of a single species, because some cell walls induce experimental chronic arthritis, whereas some others induce only a transient acute arthritis or no arthritis at all. In susceptible subjects, with continuous seeding of bacterial products from the gut, the synovial inflammation is followed by erosion, exposition of cartilage antigens, and self perpetuating chronic arthritis.

It has been suggested that the sympathetic nervous system communicates with lymphocytes expressing cell surface receptors for neurotransmitters such as norepinephrine (NE), on the basis of the finding that neurotransmitters modify immune responses in mammalian species. We confirmed that chicken lymphocytes in the brusa of Fabricius, thymus and spleen expressed beta-adrenergic receptor (beta-AR) mRNA from embryonic day (E) 10 and that intracellular cAMP level was elevated by NE, suggesting that lymphocytes express functional beta-AR on their surface at an early embryonal stage. To clarify whether the nervous system is involved in the development of the immune system, the effects of 6-hydroxydopamine (6-OHDA), one of sympathectomizing agents, on chicken lymphocytes was investigated. A single injection of 6-OHDA at a dose of 400 microg into a chicken embryo was carried out at E7 or 14 (as referred to E7 group and E14 group, respectively). NE level and the relative proportion of Bu-1a(+), CD4(+) and CD8(+) cells in the spleen of 3-week-old chickens were not altered by 6-OHDA treatment. However, the proliferative responses and expression of IL-2 mRNA in spleen cells cultured with pokeweed mitogen were reduced in E7 group compared with those of control. Furthermore, in CD8(+) spleen cells of E14 group of 3-week-old chickens, the expression of beta-AR mRNA and the relative increase of intracellular cAMP stimulated with NE were significantly decreased. These results suggest that the sympathetic nervous system affects the development of the immune system.

OBJECTIVE:To study the presence of bacterial components in the synovial tissue (ST) of patients with advanced rheumatoid arthritis (RA).METHODS:ST was collected during joint surgery from 41 RA patients. Tissue from 39 patients with osteoarthritis (OA), 4 patients with undifferentiated inflammatory arthritis (UA), and 3 cases of accidental deaths served as controls. The pan-bacterial polymerase chain reaction (PCR) with primers for the 23S ribosomal RNA (rRNA) and 16S rRNA genes was used to detect bacterial DNA. In addition, synovial fluid (SF) samples from patients with chlamydial reactive arthritis (ReA) were also examined by the same method. The positive controls, bacterial DNA or ST spiked with different living bacteria, were analyzed alongside clinical samples. Most of the ST samples were also analyzed by gas chromatography-mass spectrometry (GC-MS) for determining the presence of bacteria-derived muramic acid. Strict precautions were followed in the clinics and the laboratory to prevent contamination.RESULTS:In GC-MS analysis, muramic acid was observed in the ST from 4 of 35 RA patients and from 2 of 14 OA patients, but not in ST from 2 patients with UA and 3 cadavers. Bacterial DNA was not detected by either one of the PCR primers used in ST from 42 patients with RA and 39 patients with OA. However, 5 of 15 SF samples from ReA patients were PCR positive. The sensitivity of GC-MS to detect muramic acid was 2 pg/injected amount (227 pg muramic acid/mg ST), and that of the pan-bacterial PCR was 2-20 bacteria colony forming units/reaction.CONCLUSION:These results indicate that a bacterial component, muramic acid, is detectable by GC-MS in ST from a few patients with advanced RA or OA. However, no bacterial DNA was detectable by PCR.

BACKGROUND:Bacterial cell wall (CW) arthritis develops in susceptible strains of rats after a single intraperitoneal injection of the CW from certain bacterial species, both pathogenic and non-pathogenic. For the development of chronic bacterial CW arthritis, the structure of the bacterial peptidoglycan (PG) has been found to be decisive. OBJECTIVE:To define the role of PG subtypes in the pathogenesis of chronic bacterial CW arthritis. METHOD:Arthritis was induced with CWs of Lactobacillus plantarum, L casei B, L casei C, and L fermentum. Gas chromatography-mass spectrometry was used to measure the presence of CW derived muramic acid in the liver and to determine PG subtypes. CWs were also tested for their resistance to lysozyme in vitro. RESULTS:These results and those published previously indicate that PGs of CWs which induce chronic arthritis, no matter whether they were derived from strains of Streptococcus, Bifidobacterium, Collinsella, or Lactobacillus, all have lysine as the third amino acid of the PG stem peptide, representing PG subtypes A3alpha and A4alpha. Those strains which induce only transient acute arthritis or no arthritis at all do not have lysine in this position, resulting in different PG subtypes. CONCLUSIONS:In vivo degradation of only those PGs with the subtypes A3alpha and A4alpha leads to the occurrence of large CW fragments, which persist in tissue and have good proinflammatory ability. CWs with other PG subtypes, even if they are lysozyme resistant, do not cause chronic arthritis, because the released fragments are not phlogistic. It is emphasised that a variety of microbial components not causing inflammation have been found in animal and human synovial tissue.

It is largely unknown how bacterial cell walls (BCW) modulate human immune responses. In the present work the effect of Gram-positive BCW on lymphocyte proliferation responses towards several microbial antigens (Ag) or mitogens was studied. Gram-positive BCW were derived from four indigenous bacterial strains and from one pathogen ( Streptococcus pyogenes ). All BCW preparations used non-specifically suppressed the proliferation responses of peripheral blood mononuclear cells (PBMC) against bacterial and viral Ag, but not against mitogens. Both lymphocytes and macrophages or their secreted products mediated the suppressive effects of BCW, which were not IL-10 dependent. Furthermore, the expression of HLA-DR and CD86 on monocytes/macrophages was downregulated by BCW. Unlike in LPS-induced suppression, the CD14 pathway was not used by BCW of Lactobacillus casei ( L.c .). The observed results indicate that Gram-positive BCW suppress antigen-specific lymphocyte proliferation through several mechanisms. This non-specific immunosuppression might be a general function of BCW in the bacteria-host interaction, being of importance for bacterial survival and pathogenicity.

Lipopolysaccharide (LPS) is a glycolipid present in the outer membrane of all Gram-negative bacteria, and it is one of the signature molecules recognized by the receptors of the innate immune system. In addition to its lipid A portion (the endotoxin), its O-chain polysaccharide (the O-antigen) plays a critical role in the bacterium-host interplay and, in a number of bacterial pathogens, it is a virulence factor. We present evidence that, in Yersinia enterocolitica serotype O:8, a complex signalling network regulates O-antigen expression in response to temperature. Northern blotting and reporter fusion analyses indicated that temperature regulates the O-antigen expression at the transcriptional level. Promoter cloning showed that the O-antigen gene cluster contains two transcriptional units under the control of promoters P(wb1) and P(wb2). The activity of both promoters is under temperature regulation and is repressed in bacteria grown at 37 degrees C. We demonstrate that the RosA/RosB efflux pump/potassium antiporter system and Wzz, the O-antigen chain length determinant, are indirectly involved in the regulation mainly affecting the activity of promoter P(wb2). The rosAB transcription, under the control of P(ros), is activated at 37 degrees C, and P(wb2) is repressed through the signals generated by the RosAB system activation, i.e. decreased [K+] and increased [H+]. The wzz transcription is under the control of P(wb2), and we show that, at 37 degrees C, overexpression of Wzz downregulates slightly the P(wb1) and P(wb2) activities and more strongly the P(ros) activity, with the net result that more O-antigen is produced. Finally, we demonstrate that overexpression of Wzz causes membrane stress that activates the CpxAR two-component signal transduction system.

It is largely unknown how bacterial cell walls (BCW) modulate human immune responses. In the present work the effect of Gram‐positive BCW on lymphocyte proliferation responses towards several microbial antigens (Ag) or mitogens was studied. Gram‐positive BCW were derived from four indigenous bacterial strains and from one pathogen (Streptococcus pyogenes). All BCW preparations used non‐specifically suppressed the proliferation responses of peripheral blood mononuclear cells (PBMC) against bacterial and viral Ag, but not against mitogens. Both lymphocytes and macrophages or their secreted products mediated the suppressive effects of BCW, which were not IL‐10 dependent. Furthermore, the expression of HLA‐DR and CD86 on monocytes/macrophages was downregulated by BCW. Unlike in LPS‐induced suppression, the CD14 pathway was not used by BCW of Lactobacillus casei (L.c.). The observed results indicate that Gram‐positive BCW suppress antigen‐specific lymphocyte proliferation through several mechanisms. This non‐specific immunosuppression might be a general function of BCW in the bacteria‐host interaction, being of importance for bacterial survival and pathogenicity.