The multicomponent phytopharmaceutical SKI306X inhibits in vitro cartilage degradation and the production of inflammatory mediators.

Clinical studies have demonstrated that SKI306X, a purified preparation of three medicinal plants, relieves joint pain and improves functionality in osteoarthritis patients. To study the biological action of SKI306X, bovine cartilage explants and human peripheral blood mononuclear cells (PBMC) were stimulated with IL-1 beta and lipopolysaccharide (LPS) respectively, in the presence or absence of SKI306X and its individual composites. All tested compounds inhibited dose-dependently IL-1 beta-induced proteoglycan release and nitric oxide production by cartilage, indicating cartilage protective activity. SKI306X and two of its compounds inhibited PGE(2), TNF-alpha and IL-1 beta production by LPS-stimulated PBMC, indicating anti-inflammatory activity. These results demonstrate that the biological effect of SKI306X is at least bipartite: (1) cartilage protective and (2) anti-inflammatory. The observed anti-inflammatory effects may provide an explanation for the outcome of the clinical studies. Long-term clinical trails are necessary to elucidate whether the in vitro cartilage protective activity results in disease-modifying effects.

No effects of probiotics on atopic dermatitis in infancy: a randomized placebo-controlled trial.

BACKGROUND: Studies have been performed suggesting that administration of probiotics may have therapeutic and/or preventive benefits in the development of sensitization and atopic disease, particularly in infants with atopic dermatitis (AD). OBJECTIVE: The purpose of this study was to evaluate the clinical and immunological effects of supplementation of a hydrolysed formula with two probiotic strains of bacteria on symptoms of AD in infancy. METHODS: We conducted a randomized, double-blind, placebo-controlled study. After 4-6 weeks of baseline and double-blind, placebo-controlled challenges for diagnosis of cow's milk allergy (CMA), infants less than 5 months old with AD received a hydrolysed whey-based formula as placebo (n = 17), or supplemented with either Lactobacillus rhamnosus (n = 17) or Lactobacillus GG (n = 16) for 3 months. Before, during and after intervention, the clinical severity of AD was evaluated using SCORing index Atopic Dermatitis (SCORAD). Allergic sensitization was evaluated by measurement of total IgE and a panel of food-specific IgEs as well as skin prick testing for cow's milk. Inflammatory parameters were blood eosinophils, eosinophil protein X in urine, fecal alpha-1-antitrypsin and production of IL-4, IL-5 and IFN-gamma by peripheral blood mononuclear cells after polyclonal stimulation. RESULTS: No statistically significant effects of probiotic supplementation on SCORAD, sensitization, inflammatory parameters or cytokine production between groups were found. Only four infants were diagnosed with CMA. CONCLUSION: We found no clinical or immunological effect of the probiotic bacteria used in infants with AD. Our results indicate that oral supplementation with these probiotic bacterial strains will not have a significant impact on the symptoms of infantile AD.

New cohorts of naive T cells exacerbate ongoing allergy but can be suppressed by regulatory T cells.

Although as pretreatment oral tolerance is a potent means to achieve systemic suppression, its application in ongoing disease is controversial. Here we propose that availability of naive T cells may critically determine whether immunological tolerance is achieved during ongoing antigenic reactivity. Infusion of naive antigen-specific T cells into mice directly prior to eliciting a secondary Th2 response induces these naive cells to actively engage in the antigenic response despite presence of established memory. Naive antigen-specific T-cells divided faster, produced more interleukin (IL)-2, IL-4 and IL-5 and enhanced immunoglobulin E (IgE) release during a secondary Th2 response, compared with naive T cells that were infused prior to a primary response. Despite such contribution by new cohorts of naive T cells co-infusion of mucosal Tr together with naive T cells could suppress enhanced IgE release during a secondary Th2 response. We conclude that naive T cells contribute to a secondary Th2 response and although they can still be suppressed in the presence of sufficient numbers of mucosal Tr, they may interfere with potential therapeutic application of mucosal tolerance.

Differences in N-acetylmuramyl-L-alanine amidase and lysozyme in serum and cerebrospinal fluid of patients with bacterial meningitis.

N-acetylmuramyl-L-alanine amidase (NAMLAA) specifically degrades peptidoglycan, a major component of bacterial cell walls. Lysozyme degrades peptidoglycan differently by hydrolyzing the aminosugar backbone of peptidoglycan. In another study, it was shown that the two enzymes act synergistically to inactivate the inflammatory properties of peptidoglycan. The presence of lysozyme and NAMLAA was determined in serum and cerebrospinal fluid (CSF) of patients with bacterial meningitis. High concentrations of lysozyme were found in CSF while, surprisingly, NAMLAA was not present. To explain this phenomenon, the degranulation pattern of neutrophils in CSF was compared with that of neutrophils from blood. Specific granules contain lysozyme and the azurophil granules contain both lysozyme and NAMLAA. CD66b expression on the cell surface, indicative for fusion of the specific granules with the cell membrane, was higher in CSF than in blood, while the marker for the azurophil granules was lower.

Expression and intracellular localization of the human N-acetylmuramyl-L-alanine amidase, a bacterial cell wall-degrading enzyme.

N-acetylmuramyl-L-alanine amidase (NAMLAA) specifically degrades peptidoglycan, which is a major component of bacterial cell walls with strong inflammatory properties. For instance, peptidoglycan is capable of stimulating peripheral blood cells to release pro-inflammatory cytokines and is capable of inducing chronic arthritis in an animal model. In a previous study we found that degradation of peptidoglycan by purified NAMLAA reduced its inflammatory effects. To determine where NAMLAA is located in tissues, monoclonal antibodies against purified NAMLAA were produced for use in immunohistochemistry, immunoelectron microscopy, flow cytometric analysis, and Western blotting. The immunohistochemical studies showed NAMLAA-positive cells in human spleen, liver, arthritic synovial tissues, and lymph nodes. In flow cytometric studies of blood and bone marrow, neutrophilic and eosinophilic granulocytes proved to be positive. Monocytes were negative, although they do contain lysozyme, the other important peptidoglycan-degrading enzyme. However, mature macrophages obtained by bronchoalveolar lavage and subsequent selection based on autofluorescence did possess NAMLAA. In immunocytochemical staining of blood smears, thrombocytes were also positive for NAMLAA. Western blot analysis and immunoelectron microscopy of neutrophils and eosinophils showed that NAMLAA is located in azurophilic granules of neutrophils and in secretory vesicles and crystalloid-containing granules of eosinophils. Flow cytometric analysis of blood and bone marrow from different French-American-British-classified acute myeloid leukemia (AML) patients showed that AML-M2 myeloblasts were the first in the granulocyte maturation lineage that were positive for NAMLAA. The more immature AML, such as AML-M0 and AML-M1, did not express NAMLAA. CD15- and CD13-negative megakaryoblasts, corresponding to AML-M7, were also positive for NAMLAA. The expression pattern of NAMLAA in the myeloid lineage suggests that the monoclonal antibody AAA4, recognizing NAMLAA, is useful for discrimination between AML in the monocyte lineage and in the granulocyte lineage.

Inflammatory properties of peptidoglycan are decreased after degradation by human N-acetylmuramyl-L-alanine amidase.

Human N-acetylmuramyl-L-alanine amidase (EC 3.5.1.28) degrades peptidoglycan, a major component of bacterial cell walls with potent pro-inflammatory cytokine-inducing properties. We postulate that degradation of peptidoglycan by N-acetylmuramyl-L-alanine amidase is important for the inactivation of inflammatory peptidoglycan products in human tissues. The inflammatory activities of peptidoglycan digested by lysozyme and/or amidase were investigated using two properties of peptidoglycan: its capacity to induce the release of the inflammatory cytokines IL-1, IL-6 and TNF-alpha in vivo and in vitro and its capacity to induce arthritis in Lewis rats. The results show that after subsequent treatment with both lysozyme and amidase, the peptidoglycan products were unable to induce arthritis in Lewis rats. The production of pro-inflammatory cytokines in mice after intravenous injection of cell wall fragments was lower after in vitro degradation of the cell wall fragments by amidase. These in vivo results were confirmed with whole blood assays in which the production of pro-inflammatory cytokines was measured after stimulation with lysozyme- and amidase-treated peptidoglycan. The results show that human N-acetylmuramyl-L-alanine amidase possesses an enzymatic activity capable of inactivating inflammatory peptidoglycan by lowering its cytokine-inducing properties.

Purification and characterization of N-acetylmuramyl-L-alanine amidase from human plasma using monoclonal antibodies.

N-Acetylmuramyl-L-alanine amidase (EC 3.5.1.28) cleaves the amide bond between N-acetyl muramic acid and L-alanine in the peptide side chain of different peptidoglycan products. The enzyme was purified from human plasma using a three-step column chromatography procedure. Monoclonal antibodies were produced against the purified human enzyme. By coupling of a high affinity monoclonal antibody to sepharose beads an immunoadsorbent column was prepared. Using this second purification method it was possible to purify large amounts of the amidase from human plasma in a single step. SDS-PAGE showed one single band of 70 kDa and two-dimensional electrophoresis showed the presence of multiple isomeric forms of the protein with pI between 6.5 and 7.9. Two different methods were used for determination of substrate specificity, a HPLC method separating peptidoglycan monomers from the reaction products after incubation with amidase and a colorimetric method when high molecular weight peptidoglycan was used as a substrate for amidase. It is shown that the disaccharide tetra peptide, disaccharide penta peptide and the anhydro disaccharide tetrapeptide are good substrates for the amidase and that muramyl dipeptide and disaccharide dipeptide are not a substrate for the amidase. Using one of the monoclonal antibodies against the amidase it was shown in FACScan analysis that N-acetylmuramyl-L-alanine amidase is present in granulocytes but not in monocytes from unstimulated peripheral blood of a healthy donor. The presence of N-acetylmuramyl-L-alanine amidase in granulocytes is a novel finding and perhaps important for the inactivation of biologically active peptidoglycan products still present after hydrolysis by lysozyme.

Presence of bacterial flora-derived antigen in synovial tissue macrophages and dendritic cells.

In previous studies using and animal model human bacterial flora-derived peptidoglycan Polysaccharides were shown to be arthropathic after a single subcutaneous injection. A prerequisite for proof of the hypothesis that bacterial products from the normal resident flora are involved in the immune reaction of human chronic polyarthritis of unknown aetiology is the presence of these antigens in synovial tissue. 2E9, a monoclonal antibody we developed against intestinal peptidoglycan polysaccharides was used in a histochemical study in rats and stained macrophages in the spleen red pulp. In this study human synovial tissues from 10 rheumatoid arthritis (RA) and 20 non-RA patients were stained with 2E9. We found that eight out of 10 RA patients had 2E9-positive macrophages and dendritic cells in their synovia. A significant difference was observed with the control group in which seven out of 20 were positive. No positive cells or staining of the matrix was found in the cartilage of six RA patients. These results show that exogenous bacterial antigens are present in synovial tissue macrophages and dendritic cells. It was concluded that the unknown antigen in the immune reaction in RA is not necessarily endogenous.

Detection of muramic acid in a carbohydrate fraction of human spleen.

In previous studies, we showed that peptidoglycan polysaccharides from anaerobic bacteria normally present in the human gut induced severe chronic joint inflammation in rats. Our hypothesis is that peptidoglycan from the gut flora is involved in perpetuation of idiopathic inflammation. However, in the literature, the presence of peptidoglycan or subunits like muramyl peptides in blood or tissues is still a matter of debate. We were able to stain red pulp macrophages in all six available human spleens by immunohistochemical techniques using a monoclonal antibody against gut flora-derived antigens. Therefore, these human spleens were extracted, and after removal of most of the protein, the carbohydrate fraction was investigated for the presence of muramic acid, an amino sugar characteristic for peptidoglycan. Using three different methods for detection of muramic acid, we found a mean of 3.3 mumol of muramic acid with high-pressure liquid chromatography, 1.9 mumol with a colorimetric method for detection of lactate, and 0.8 mumol with an enzymatic method for detection of D-lactate per spleen (D-lactate is a specific group of the muramic acid molecule). It is concluded that peptidoglycan is present in human spleen not as small muramyl peptides as were previously searched for by other investigators but as larger macromolecules probably stored in spleen macrophages.