Immunological response of human leucocytes after exposure to lipopolysaccharides from Porphyromonas gingivalis.

Porphyromonas gingivalis (P. gingivalis) is a gram-negative bacterium and an important etiologic agent of periodontitis. P. gingivalis releases outer membrane vesicles containing lipopolysaccharides (LPS), which can penetrate periodontal tissues. Once in the periodontal tissues and in contact with immune cells, it may participate in the destructive innate host response associated with the disease. The exact mechanism of P. gingivalis LPS in the disease process is not clear, but it is known to affect a variety of immune responses. OBJECTIVES: To investigate how LPS from P. gingivalis affect neutrophil extracellular trap (NET) formation, cell death and production of cytokines from human neutrophils and peripheral mononuclear blood mononuclear cells (PBMCs). MATERIALS AND METHODS: Isolated neutrophils and PBMCs were cultured with LPS from P. gingivalis or Escherichia coli (E. coli) (control). The NET formation was measured using Sytox green stain. Cell death of neutrophils and PBMCs was analyzed using flow cytometry or Sytox green stain. Cytokine production was measured using enzyme-linked immunosorbent assay (ELISA) kit or Bio-Plex assay. RESULTS: Exposure to LPS from P. gingivalis and E. coli caused significantly lower cell death in neutrophils. NETs were formed after exposure to the two different LPS. In PBMCs, exposure to P. gingivalis and E. coli LPS caused increased levels of IL-1ß and IL-6 compared to unstimulated controls. Increased cell death in PBMCs after exposure to LPS from E. coli in comparison to LPS from P. gingivalis and unstimulated controls was also observed. CONCLUSIONS: LPS from P. gingivalis has the ability to affect both human neutrophils and PBMCs with regard to cytokine production, cell death and production of NETs. LPS from P. gingivalis could be involved in the pathogenesis of periodontitis, and our results may contribute information regarding possible markers for diagnosis and targets for treatment of periodontal disease.

The effects of the dental methacrylates TEGDMA, Bis-GMA, and UDMA on neutrophils in vitro.

OBJECTIVES: The prevalent usage of methacrylates in modern dentistry demands good knowledge of their biological impacts. While there have been several studies demonstrating the effects of different methacrylic monomers on mononuclear white blood cells, very little is known about the effects caused by these monomers on neutrophilic granulocytes. The objective of this study was to add novel knowledge about how neutrophils are affected by exposure to triethylene glycol dimethacrylate (TEGDMA), urethane dimethacrylate (UDMA), and bisphenol A glycol dimethacrylate (Bis-GMA) alone or in combinations. MATERIALS AND METHODS: Isolated neutrophils were cultured in the presence or absence of methacrylates. The IL-8 release was measured using a DuoSet ELISA development kit. Apoptosis and necrosis were analyzed using flow cytometry. The formation of neutrophil extracellular traps (NETs) was investigated using Sytox green DNA staining combined with microscopically examination of released DNA and myeloperoxidase (MPO). RESULTS: The release of IL-8 was significantly increased after exposure to TEGDMA, Bis-GMA, UDMA, or TEGDMA in combination with Bis-GMA or UDMA compared to the unstimulated controls. Exposure to TEGDMA, UDMA, and Bis-GMA for 24 hr separately or in combination did not affect apoptosis or necrosis of the exposed neutrophils. NET structures were formed by neutrophils after exposure to the different combinations of the methacrylates. CONCLUSION: The combination of TEGDMA and Bis-GMA had a synergistic proinflammatory effect on neutrophils by increasing the release of IL-8 and the formation of NET structures. The changes in the normal functions of neutrophils caused by methacrylate exposure may lead to altered inflammatory response and relate to previously reported adverse immune reactions caused by these substances.

Inflammatory cytokine release from human peripheral blood mononuclear cells exposed to polyetheretherketone and titanium-6 aluminum-4 vanadium in vitro.

Objective To investigate the cytokine expression profiles of blood cells exposed to polyetheretherketone and titanium-6 aluminum-4 vanadium materials in vitro. Materials and methods Coin-shaped samples composed of titanium-6 aluminum-4 vanadium, polyetheretherketone, and blasted polyetheretherketone were manufactured. The surfaces of the coins were characterized using optical interferometry, scanning electron microscopy, and contact angle measurements. Peripheral blood mononuclear cells collected from 10 blood donors were cultured for one, three, and six days in the presence or absence of the coins, and then assayed for cytokine production. Quantification of the peripheral blood mononuclear cells attached to the coins was performed using confocal microscopy after immunofluorescence staining. Results The machined titanium-6 aluminum-4 vanadium coins had a smoother surface topography compared to the machined polyetheretherketone and blasted polyetheretherketone. The highest mean contact angle was noted for the blasted polyetheretherketone, followed by the machined polyetheretherketone and titanium-6 aluminum-4 vanadium. The peripheral blood mononuclear cells produced significantly more proinflammatory cytokines when exposed to the polyetheretherketone surface compared to the titanium-6 aluminum-4 vanadium surface, while the blasted polyetheretherketone induced the highest level of proinflammatory cytokine release from the peripheral blood mononuclear cells. Significantly more cells attached to both polyetheretherketone surfaces, as compared to the titanium-6 aluminum-4 vanadium surface. Conclusion Polyetheretherketone induces a stronger inflammatory response from peripheral blood mononuclear cells than does titanium-6 aluminum-4 vanadium. Surface topography has an impact on cytokine release from peripheral blood mononuclear cells.

The Immunomodulatory Properties of 2-Hydroxyethyl Methacrylate are Mediated by the NLRP3 Inflammasome.

PURPOSE: The methacrylate monomer 2-hydroxyethyl methacrylate (HEMA), commonly used in dentistry, has multiple effects on the immune system. This study examined whether HEMA affects the immune system by inducing formation of the NLRP3 inflammasome. MATERIALS AND METHODS: Human peripheral blood mononuclear cells (PBMCs) and the human monocyte cell line THP1 were cultured with or without 1000 µM HEMA. To block NLRP3 inflammasome activation, 130 mM KCl was also added to some of the cultures. For the in vivo studies, two different experimental setups were used. In the first experimental setup, mice were injected subcutaneously at the base of the tail with 20 µmol HEMA with or without 100 mM KCl. After 3 weeks, the animals were given an identical booster injection. Two weeks after the last injection, the mice were sacrificed and splenectomized. In the second experimental setup, HEMA (20 µmol), with or without 100 mM KCl, was injected subcutaneously into the tails of BALB/c mice. The mice were given two similar injections at 3-week intervals to allow evaluation of the local inflammation induced by HEMA. After the last inoculation, the injection site was examined daily for 4 days, after which the mice were sacrificed. RESULTS: Cultures of PBMCs and THP1 cells exposed to HEMA in vitro produced more IL-1ß and IL-18 than did control cells. Increased extracellular concentration of KCl inhibited the secretion of IL-1ß. HEMA exposure did not induce cytokine production in variants of the THP1 cell line unable to form the NLRP3 inflammasome. For the first experimental setup, the level of unstimulated basic splenocyte proliferation in vitro was significantly higher in cultures from mice exposed in vivo to HEMA only than in cultures from mice injected with HEMA plus KCl. In the second experimental setup of the in vivo studies, the HEMA-treated mice developed more pronounced inflammation at the site of injection compared to the group of mice given HEMA plus KCl. CONCLUSION: HEMA affects the immune system by inducing formation of the NLRP3 inflammasome.

Sublingual administration of 2-hydroxyethyl methacrylate enhances antibody responses to co-administered ovalbumin and Streptococcus mutans.

OBJECTIVE: The oral mucosa of patients undergoing dental procedures is often exposed to residual monomers leaking from incompletely cured acrylic resins. We investigated whether 2-hydroxyethyl methacrylate (HEMA) monomers applied to the sublingual mucosa in mice modulate the antibody responses towards co-administered ovalbumin (OVA) or live oral bacteria. MATERIAL AND METHODS: OVA, live mouse oral commensal Lactobacillus murinus or live human oral commensal Streptococcus mutans were administered sublingually with or without HEMA to BALB/c mice on four weekly occasions. One week after the last administration, the experiment was terminated and serum antibody levels were analyzed using ELISA. RESULTS: Significantly increased IgG and IgE anti-OVA antibody activity was found in the sera from mice immunized with OVA together with HEMA, as compared to mice immunized with OVA alone. Likewise, S. mutans together with HEMA induced an IgG anti-S. mutans antibody response that was significantly higher than the antibody response detected after application of S. mutans alone. No IgG anti-L. murinus antibody response was detected in mice immunized with L. murinus together with HEMA, as compared to the background activity. CONCLUSIONS: We report that HEMA monomers have adjuvant properties when sublingually administered in combination with OVA or S. mutans.

Triethylene glycol dimethacrylate: adjuvant properties and effect on cytokine production.

Objective: Leakage of monomers from dental fillings due to incomplete curing is very common. The objective of the present study was to examine the cytokine profile in cells exposed to triethyleneglycol dimethacrylate (TEGDMA) and the adjuvant properties of TEGDMA. Materials and methods: Human peripheral blood mononuclear cells were exposed to TEGDMA (500 and 1000 µM) for 24 h in vitro. Bio-Plex Pro™ assays were used for analysis and detection of cytokines. In vivo, BALB/c mice were immunized subcutaneously in the base of the tail with TEGDMA in combination with ovalbumin (OVA). Results: The cytokine levels of IL-8, IL-18, GRO-α and MCP-1 were significantly increased for both concentrations. IL-1ß, IL-6 and TNF-α was only significantly increased in cultures exposed to 500 µM TEGDMA. The concentration of TNF-α was significantly decreased in cultures exposed to 1000 µM TEGDMA. Animals immunized with OVA co-administrated with TEGDMA had a significantly higher IgE and IgG anti-OVA antibody levels in blood than animals immunized with OVA only. Conclusions: TEGDMA affects production of proinflammatory cytokines IL-1ß, IL-6, IL-8, IL-18 and TNF-α. This inflammatogenic capacity renders TEGDMAs adjuvant properties, which may interfere with the homeostasis between the immune system and the indigenous microflora in the oral cavity.

Hydrogen sulfide exposure induces NLRP3 inflammasome-dependent IL-1ß and IL-18 secretion in human mononuclear leukocytes in vitro.

The aim was to investigate if hydrogen sulfide (H2S) induces the formation of the NLRP3 inflammasome and subsequent IL-1ß and IL-18 secretion in human peripheral blood mononuclear cells (PBMCs) and in the human monocyte cell line THP1. Bacterial production of H2S has been suggested to participate in the inflammatory host response in periodontitis pathogenesis. H2S is a toxic gas with pro-inflammatory properties. It is produced by bacterial degradation of sulfur-containing amino acids, for example, cysteine. We hypothesize that H2S affects the inflammatory host response by inducing formation of the NLRP3 inflammasome and thereby causes the secretion of IL-1ß and IL-18. PBMCs from eight healthy blood donors, the human monocyte cell line THP1 Null, and two variants of the THP1 cell line unable to form the NLRP3 inflammasome were cultured in the presence or absence of 1 mM sodium hydrosulfide (NaHS) in 24-well plates at 37°C for 24 hr. Supernatants were collected and the IL-1ß and IL-18 concentrations were measured with DuoSet ELISA Development kit. PBMCs exposed to NaHS produced more IL-1ß and IL-18 than unexposed control cells (p = .023 and p = .008, respectively). An increase of extracellular potassium ions (K+) inhibited the secretion of IL-1ß and IL-18 (p = .008). Further, NaHS triggered the secretion of IL-1ß and IL-18 in human THP1-Null monocytes (p = .0006 and p = .002, respectively), while the NaHS-dependent secretion was reduced in the monocyte cell lines unable to form the NLRP3 inflammasome. Hence, the results suggest that NaHS induces the formation of the NLRP3 inflammasome and thus the secretion of IL-1ß and IL-18. Enhanced NLRP3 inflammasome-dependent secretion of IL-1ß and IL-18 in human mononuclear leukocytes exposed to NaHS in vitro is reported. This may be a mode for H2S to contribute to the inflammatory host response and pathogenesis of periodontal disease.

Effects of the methacrylate/acrylate monomers HEMA, TEGDMA, DEGDA, and EMA on the immune system.

Incomplete curing of dental fillings may lead to leakage of methacrylate/acrylate monomers, which may come in contact with different cells of the immune system in oral tissues. Very little is known about the different immunologic effects caused by these methacrylates/acrylates. The objective of the present study was to study if and how the methacrylate/acrylate monomers ethyl methacrylate (EMA) and diethylene glycol diacrylate (DEGDA) affect the immune system in vivo and in vitro in comparison to 2-hydroxyethyl methacrylate (HEMA) and triethylene glycol dimethacrylate (TEGDMA). Human peripheral blood mononuclear cells were exposed to the different monomers (500 and 1000 µM) for 24 hr in vitro. BioPlex Pro™ assays were used for cytokine analysis. In vivo, BALB/c mice were immunized subcutaneously at the base of the tail with HEMA, TEGDMA, EMA, or DEGDA in combination with ovalbumin (OVA) in order to study adjuvant properties of the 4 monomers. Peripheral blood mononuclear cells exposed to DEGDA had viability less than 50% of the cells. A pattern was observed where the levels of most cytokines were elevated after exposure to HEMA or TEGDMA. Since that, many cells died after DEGDA-exposure, the only observed cytokine secretion was a significantly increased production of interleukin-18. In the in vivo experiments, all mice immunized with DEGDA died after the booster injection. Mice receiving OVA in combination with HEMA, TEGDMA, or EMA developed a higher immunoglobulin G anti-OVA antibody levels compared to the group immunized with OVA alone. We could not demonstrate any significant difference in antibody levels among the mice receiving the various methacrylate/acrylate monomers. The different monomers affected the production, increase and decrease, of different cytokines in vitro but resulted also in vivo in increased antibody production and T-cell activity.

Subgingival bacterial clusters and serum antibody response as markers of extent and severity of periodontitis in adult Chinese.

This study evaluated the associations between clinical, microbiological, and antibody activity manifestations of periodontitis in 123 adult rural Chinese subjects with no dental intervention. All participants were registered for full-mouth clinical attachment level (CAL) and pocket probing depth (PD) measurements, and microbial samples were taken from four sites and analyzed for 18 different bacterial species using the 'checkerboard'. Serum from each individual was analyzed to determine the antibody activity against the same 18 species. Exploratory factor analysis disclosed two microbial factors - Factor 1, consisting of seven species associated with periodontal health ('early colonizers'); and Factor 2, consisting of eight species associated with periodontitis ('putative periodontopathogens') - which explained 87% of the variation among the microbial variables. Factor 2 was consistently associated with disease-severity measures, whereas the 'early colonizer' factor was not. The antibody response showed weak or no correlations with bacterial load or with disease severity. We conclude that the bacteria investigated are resident in the subgingival plaque; that their load and proportions in the pocket may be ecologically driven; and that the antibody response is based on bacterial carrier state rather than on disease. The different antibody-response pattern found between the individuals may suggest that each individual could be classified as a good or a weak immune responder.

Inflammatory cytokine release is affected by surface morphology and chemistry of titanium implants.

To investigate in vitro cellular cytokine expression in relation to commercially pure titanium discs, comparing a native surface to a fluorinated oxide nanotube surface. Control samples pure titanium discs with a homogenous wave of the margins and grooves and an often smeared-out surface structure. Test samples pure titanium discs with a fluorinated titanium oxide chemistry and surface morphology with nanopore/tube geometry characterized by ordered structures of nanotubes with a diameter of ≈ 120 nm, a spacing of ≈ 30 nm, and a wall thickness of ≈ 10 nm. Cross-section view showed vertically aligned nanotubes with similar lengths of ≈ 700 nm. Peripheral blood mononuclear leucocytes were cultured for 1, 3, and 6 days according to standard procedures. BioPlex Pro™ assays were used for analysis and detection of cytokines. Selected inflammatory cytokines are reported. A pronounced difference in production of the inflammatogenic cytokines was observed. Leucocytes exposed to control coins produced significantly more TNF-α, IL-1ß, and IL-6 than the test nanotube coins. The effect on the TH2 cytokine IL-4 was less pronounced at day 6 compared to days 1 and 3, and slightly higher expressed on the control coins. The morphology and surface chemistry of the titanium surface have a profound impact on basic cytokine production in vitro. Within the limitations of the present study, it seems that the fluorinated oxide nanotube surface results in a lower inflammatory response compared to a rather flat surface that seems to favour inflammation.

2-Hydroxyethyl methacrylate (HEMA) promotes IgG but not IgM antibody production in vivo in mice.

Individuals working in a dental clinic are exposed to 2-hydroxyethyl methacrylate (HEMA). HEMA has been found to have several effects on the immune system, including acting as an adjuvant in mice and stimulating the production of human IgG1 in vitro. In this study we continued to explore the immunomodulatory properties of HEMA in mice. Mice were co-injected subcutaneously with the following: HEMA + ovalbumin (OVA) in bicarbonate buffer, OVA in bicarbonate buffer, HEMA in bicarbonate buffer, or bicarbonate buffer alone. Mice immunized with OVA were killed 2 wk after a booster injection. Mice exposed to HEMA only were killed 6 d after the last injection with HEMA. Serum and spleens were collected. The activities of anti-OVA IgG and anti-OVA IgM were determined using ELISAs, as was the in vitro production of tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) by splenocytes after 2 d of incubation. Splenocyte proliferation was analyzed using [(3) H]thymidine decomposition. Mice exposed twice to HEMA in vivo had a higher baseline and a higher concanavalin A-stimulated proliferation of splenocytes, and produced less TNF-α in relation to IL-6, compared with controls. Immunization of mice with OVA/HEMA resulted in a higher anti-OVA IgG activity, relative to anti-OVA IgM activity, compared with controls. In conclusion, HEMA has selective effects on cytokine and antibody production in mice.

Effects on mouse immunity of long-term exposure in vivo to minute amounts of HEMA.

2-Hydroxyethyl methacrylate (HEMA) leaks from cured restorations over time. Hence, HEMA can come into contact with cells of the immune system that are present in the oral mucosa and in the dental pulp. In this study, our aim was to develop a model of long-term exposure to minute amounts of HEMA and to record the immunological effects in mice. Osmotic pumps filled with either HEMA (8.2 M or 183 µM) or 0.9% NaCl (control) were implanted subcutaneously into the backs of mice and left in situ for 40 d, during which time the animals were immunized with ovalbumin (OVA). After 40 d, spleens and serum were collected. Splenocyte proliferation in vitro was analyzed by measuring the decomposition of [(3)H]thymidine. Splenocyte cytokine production and serum anti-OVA IgG, IgM and IgA activity were analyzed using ELISAs. Mice exposed to both the higher and the lower HEMA concentrations gained significantly less weight and produced significantly reduced amounts of interleukin-2 (IL-2) in vitro compared with control mice. Mice exposed to the lower HEMA concentration had a significantly reduced concanavalin A-stimulated splenocyte proliferation in vitro and blood anti-OVA IgA activity. In conclusion, long-term exposure to minute amounts of HEMA in vivo affects the general health of mice and suppresses certain immunological functions.

Salivary resistin reflects local inflammation in Sjögren's syndrome.

OBJECTIVE: To assess the role of resistin in primary Sjögren's syndrome (pSS) and its relation to local inflammation. METHODS: Blood and saliva were collected from 37 patients with pSS (duration of symptoms 12.6+/-1 yrs) and 32 healthy controls. Expression of resistin in salivary glands was visualized immunohistologically, and levels of resistin were detected by ELISA. Levels of resistin were evaluated at baseline and following oral dehydroepiandrosterone (DHEA) treatment (50 mg/day). The effect of DHEA treatment on the secretion of resistin was assessed in vitro in human leukocytes after challenge with insulin and lipopolysaccharide. RESULTS: Levels of resistin in saliva were significantly higher in patients with pSS than in controls, while circulating levels of resistin were similar in both groups. Resistin was expressed in the epithelial cells of striated ducts and in the lymphocytic foci. Resistin levels in saliva were related to the intensity of inflammation in the minor salivary glands of pSS patients. No changes of the levels of resistin in blood or saliva were observed during DHEA treatment. Exposure of naive leukocytes to DHEA in vitro induced significant expression of resistin compared to nonstimulated peripheral blood mononuclear cells (p=0.031). CONCLUSION: We showed that levels of resistin are upregulated locally in the salivary glands of patients with pSS; and that the levels of resistin correspond to the intensity of lymphocytic inflammation in patients with pSS. We suggest that resistin is expressed in the salivary glands of patients with pSS and may be a driving factor of local inflammation.

Effect of 2-hydroxyethyl-methacrylate (HEMA) on the phagocytic and respiratory burst activity of human neutrophils and monocytes.

Neutrophils and monocytes/macrophages (MØ), found in oral mucosa and gingival sulcus, phagocytose and kill bacteria using products produced during a respiratory burst. 2-Hydroxyethyl-methacrylate (HEMA) is a major component released from resin glass ionomer and dental adhesives. Hence, in pulp and gingiva, phagocytes can come into contact with unpolymerized HEMA monomers. The aim of this study was to examine the effects of exposure to HEMA on neutrophil and monocyte bactericidal function. Blood collected from five female volunteers was exposed in vitro to HEMA for 2 h and then phagocytosis, respiratory burst, and cellular integrity were measured using flow cytometry. Respiratory burst was quantified by measuring fluorescent rhodamine 123 generated via oxidation of dihydrorhodamine 123. Cellular membrane integrity was evaluated by staining with propidium iodide. The respiratory burst activity of the neutrophils was significantly decreased by exposure to 7.5 and 15 mM HEMA. No significant effect of HEMA was seen on the number of granulocytes or monocytes capable of performing respiratory burst. Furthermore, there was no significant effect of HEMA on the phagocytic activity of the monocytes or the granulocytes. In conclusion, HEMA did not affect the phagocytosis activity of neutrophils; however, the ability of the cells to kill internalized prey was significantly reduced.

Application of HEMA on intact mouse skin--effects on the immune system.

Previously, we have shown that 2-hydroxyethylmethacrylate (HEMA) can bind to protein and that autoantibodies were induced in mice by immunization with a self-protein in vitro conjugated with HEMA. The present study aimed to develop a model for HEMA-induced sensitivity by the application of the substance on intact skin. Female BALB/c mice were painted on the dorsum of each ear with 50% HEMA in vehicle twice a week for 6 weeks. The anti-CD3epsilon-stimulated lymph node production and the spontaneous spleen-cell production of the cytokines interleukin (IL)-2, IL-4, IL-6, IL-10 and interferon-gamma were assessed by enzyme-linked immunosorbent assay. In another experiment, the cytokines were followed after subcutaneous HEMA injections. Animals painted with HEMA had a significantly higher IL-6 production by anti-CD3epsilon-stimulated lymph node cells and significantly suppressed IL-10 production by spleen cells compared to vehicle-treated mice. This correlated to some extent with the spontaneous spleen-cell production induced by subcutaneously administered HEMA. An injection of 20 micromol of HEMA induced an increased production of IL-6, while injection of 40 micromol depressed both IL-6 and IL-10 production. Although there was no sign of inflammation on the ears, findings suggest that HEMA had penetrated the skin and induced a reaction in the immune system.

Inflammatogenic and adjuvant properties of HEMA in mice.

2-Hydroxyethylmethacrylate (HEMA), a common constituent in dental materials, is known to cause hypersensitivity reactions. While the means by which this small molecule causes adverse responses has not been ascertained, we have previously demonstrated that it binds to protein and in mice induces the production of autoantibodies to HEMA-conjugated self-protein. The present study explores the inflammatory and adjuvant properties of HEMA in response to the subcutaneous injection of HEMA and a protein. Ovalbumin (OVA) was used as a 'reporter antigen', and mouse serum albumin (MSA), conjugated in vitro with HEMA (MSA(H)) to a low degree (0.5 molecules of HEMA per molecule of MSA on average), was used to mimic a possible in vivo situation. Inflammatory responses at injection sites were scored by using an ordinal scale, and immunoglobulin (Ig)G1, IgG2a, and IgE activities to OVA or MSA were assessed by enzyme-linked immunosorbent assay (ELISA). Injections of 20 micromol HEMA induced overt inflammatory skin responses, the severity of which was influenced by the co-administered substances. A significantly higher IgG1 and IgE response to OVA was induced by the presence of HEMA. Interestingly, injections with low conjugated MSA(H) only induced the production of autoantibodies if free HEMA was included at the time of immunization. These findings suggest that HEMA is an inflammatogenic substance with adjuvant properties.

Bystander suppression of collagen-induced arthritis in mice fed ovalbumin.

We wanted to assess whether B-cell and/or T-cell responses to collagen and thereby the course of collagen-induced arthritis could be suppressed by regulatory mechanisms associated with oral tolerance to an unrelated protein. DBA/1 mice were fed ovalbumin (OVA)-containing pellets ad libitum for 1 week and subsequently coimmunized twice, with a mixture of bovine collagen type II (BCII) and OVA in Freund's complete adjuvant. Mice fed OVA before coimmunization with BCII and OVA had significantly lower arthritic scores than mice immunized with BCII only. Their body weight increased during the study period and their anti-BCII antibody activity was significantly IgG2a lower. The frequency of spleen cells producing IgG anti-BCII antibody was also reduced. Coimmunization per se slightly ameliorated the development of arthritis, resulting in an early, transient reduction. It resulted in significantly higher IgG1 anti-BCII antibody activity and increased splenocyte secretion of IFN-gamma and IL-10 in response to BCII. Our findings demonstrate that OVA-specific regulatory events induced by feeding OVA, i.e. bystander suppression, reduced the severity of arthritis in animals immunized with BCII and OVA. Anti-BCII specific antibody responses and cytokine secretion by types 1 and 2 T helper cells were also decreased.

Vitamin A deficiency leads to severe functional disturbance of the intestinal epithelium enzymes associated with diarrhoea and increased bacterial translocation in gnotobiotic rats.

A disturbance of the integrity of the intestinal epithelium with an increased risk for bacterial translocation is one of the suggested factors underlying the increased incidence of infections and septicaemia during vitamin A deficiency. In the present study the effects of vitamin A deficiency on the enzymic activity of enterocytes in response to bacterial colonization with a non-pathogenic Escherichia coli strain were studied in monocolonized and conventional Wistar rats. The monocolonized, but not the conventional, vitamin A-deficient rats had markedly reduced weight compared to their pair-fed controls and presented neurological symptoms, such as hind leg weakness, tremor and slow gait. Moreover, only in the monocolonized vitamin A-deficient rats were severe diarrhoea and bacterial translocation to extraintestinal sites-mainly kidneys-detected. Measurements of enterocyte brush-border enzyme activities revealed that lactase, sucrase, gamma-glutamyltranspeptidase (GGT) and dipeptidyl peptidase IV (DPP IV) were significantly reduced in the monocolonized vitamin A-deficient rats compared to the pair-fed controls, indicating a severe functional disturbance of the enterocytes. In conventional vitamin A-deficient rats only sucrase activity was markedly lower than in the respective controls. Our observation, that the deficient vitamin A status led to a strong reduction of enterocyte enzymic activities, associated with diarrhoea and increased bacterial translocation, mainly in the gnotobiotic rats, suggests that the composition of the bacterial flora, i.e. the colonization state, has a strong influence on triggering the severity of the functional disturbances of the intestinal epithelium, and adds to the clinical manifestations of vitamin A deficiency.