The importance of international collaboration for rare diseases research: a European perspective.

Over the last two decades, important contributions were made at national, European and international levels to foster collaboration into rare diseases research. The European Union (EU) has put much effort into funding rare diseases research, encouraging national funding organizations to collaborate together in the E-Rare program, setting up European Reference Networks for rare diseases and complex conditions, and initiating the International Rare Diseases Research Consortium (IRDiRC) together with the National Institutes of Health in the USA. Co-ordination of the activities of funding agencies, academic researchers, companies, regulatory bodies, and patient advocacy organizations and partnerships with, for example, the European Research Infrastructures maximizes the collective impact of global investments in rare diseases research. This contributes to accelerating progress, for example, in faster diagnosis through enhanced discovery of causative genes, better understanding of natural history of rare diseases through creation of common registries and databases and boosting of innovative therapeutic approaches. Several examples of funded pre-clinical and clinical gene therapy projects show that integration of multinational and multidisciplinary expertize generates new knowledge and can result in multicentre gene therapy trials. International collaboration in rare diseases research is key to improve the life of people living with a rare disease.

CD200R1 regulates the severity of arthritis but has minimal impact on the adaptive immune response.

CD200R1 is a member of the immunoglobulin supergene family that is thought to play an inhibitory role in immunity. Previous studies have established the anti-arthritic effect of CD200Fc, an agonist of CD200R1. However, the physiological role played by CD200R1 in arthritis remains to be established. The aims of this study are to assess the contribution of endogenous CD200R1 in regulating the severity of arthritis and to determine its role in shaping the immune response to type II collagen within the context of collagen-induced arthritis, an animal model of rheumatoid arthritis. Arthritis was induced in DBA/1 mice by immunization with type II collagen and the kinetics of expression of CD200R1 and CD200 were monitored by quantitative reverse transcription-polymerase chain reaction. Next, a comparison was made between CD200R1(-/-) and wild-type mice in terms of the progression of collagen-induced arthritis, as well as the B and T cell responses to type II collagen. The expression of both CD200R1 and CD200 was increased after immunization and reached maximal levels at the height of the inflammatory response. In addition, the severity of arthritis was increased significantly in CD200R1(-/-) mice compared to wild-type mice. However, little or no differences were observed between CD200R1(-/-) and wild-type mice in terms of the T or B cell responses to type II collagen. It was concluded that the CD200R1/CD200 pathway is up-regulated in arthritis and plays a significant physiological role in regulating the severity of disease. In contrast, CD200R1 plays a minimal role in shaping the immune response to collagen in this model.

DNA mismatch repair enzyme expression in synovial tissue.

BACKGROUND: Oxidative stress in RA synovial tissue can cause DNA damage and suppress the DNA mismatch repair (MMR) system in cultured synoviocytes. This mechanism includes two enzyme complexes, hMutSalpha (hMSH2/hMSH6) and hMutSbeta (hMSH2/hMSH3). OBJECTIVE: To examine the expression and distribution of MMR enzymes in synovial tissues from patients with arthritis and from normal subjects. METHODS: Synovial tissues from patients with RA, osteoarthritis (OA), or normal subjects were analysed by immunohistochemistry using monoclonal antibodies to hMSH2, hMSH3, and hMSH6. MMR protein expression was evaluated by computer assisted digital image analysis. RESULTS: hMSH2, hMSH3, and hMSH6 were found in most synovial tissues evaluated, with greater levels in the intimal lining than sublining regions. In RA and OA, sublining perivascular staining for hMSH6 and hMSH3 was also prominent. Significantly higher sublining expression of hMSH2, hMSH3, and hMSH6 was seen in RA and OA than in normal synovium. Double label immunohistochemistry demonstrated that the main cells expressing MMR enzymes were CD68(+) and CD68(-) cells in the intimal lining. CONCLUSIONS: DNA MMR enzyme expression is greatest in the synovial intimal lining layer, where maximal oxidative stress in RA occurs. Although MMR enzyme expression is greater in RA than in normal tissue, this compensatory response cannot overcome the genotoxic environment, and DNA damage accumulates.

Role of peptidoglycan subtypes in the pathogenesis of bacterial cell wall arthritis.

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.

Enzyme degradation and proinflammatory activity in arthritogenic and nonarthritogenic Eubacterium aerofaciens cell walls.

Two almost-identical strains of Eubacterium aerofaciens isolated from the normal human gut flora were used. The cell wall (CW) of one strain with a peptidoglycan (PG) type A4alpha induces chronic arthritis in the rat after a single intraperitoneal injection, whereas CW of the other with PG type A4beta induces only a transient acute arthritis. The CW of the arthritogenic E. aerofaciens was a twofold-more-potent stimulator of the proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and monocyte chemoattractant protein 1 (MCP-1) than the nonarthritogenic CW. After degradation with mutanolysin, the capacity of the arthritogenic PG to stimulate production of TNF-alpha and MCP-1 was significantly increased, whereas that of the nonarthritogenic PG was significantly decreased. In other words, after enzyme degradation the arthritogenic PG had a four- to fivefold-stronger stimulatory capacity than that of the enzyme-treated nonarthritogenic PG. These findings indicate that the arthritogenicity of CW or a PG is not dependent on the enzyme resistance alone but also on how the PG fragments released by enzyme degradation stimulate the production of proinflammatory cytokines.

Experimental chronic arthritis and granulomatous inflammation induced by bifidobacterium cell walls.

Effects of cell walls (CWs) from two almost identical strains of Bifidobacterium adolescentis were studied in rats, using three different doses. A single i.p. injection of both CWs triggered a long-lasting arthritis with CW degradation products present in the joint tissue. Histologically, the arthritis was characterized by inflammatory cells, synovial hyperplasia, pannus formation and bone erosion, closely resembling human rheumatoid arthritis (RA). In addition, CWs of the other strain induced a remarkable granuloma formation in the spleen and liver. Both CWs have the same peptidoglycan (PG) type A4alpha/beta, but differ from each other in three aspects. CW of the granuloma inducing strain: firstly has more lysine and less ornithine in PG stem peptides; secondly is more resistant to lysozyme degradation, and thirdly is better retained in the spleen. All these in comparison to the other strain used. Such characteristics are associated with the capacity to induce chronic arthritis, but it remains open how crucial they are for the granuloma formation.

Characterisation of Eubacterium cell wall: peptidoglycan structure determines arthritogenicity.

OBJECTIVE: To elucidate factors involved in the arthritogenicity of bacterial cell walls. METHODS: For characterisation of an arthritogenic Eubacterium aerofaciens cell wall, peptidoglycan-polysaccharide (PG-PS) polymers were isolated by removing cell wall associated proteins (CWPs), PG and PS moieties were separated, and an attempt was made to de-O-acetylate PG-PS. The cell wall of E limosum was used as a non-arthritogenic control. The chemical composition of these cell wall preparations was analysed by gas chromatography-mass spectrometry. Also, their ability to resist lysozyme degradation and to sustain experimental chronic arthritis was tested. RESULTS: The observations made with the cell wall of E aerofaciens, an anaerobic habitant of the human intestine, were compared with those reported from a pathogenic Streptococcus, showing that in both strains a complex consisting of PG-PS is required for the induction of chronic arthritis. The PS moiety most probably protects PG from enzyme degradation, allowing prolonged tissue persistence and leading to the chronic synovial inflammation. CWPs attached to PG-PS are not necessary for this function. O-Acetylation of PG, which is required for arthritogenicity of the streptococcal cell wall, seems not to be present in the arthritogenic E aerofaciens PG or only occurs to a small degree; attempts to de-O-acylate the E aerofaciens cell wall did not affect its arthritogenicity or lysozyme resistance. CONCLUSION: The results obtained indicate that the source of bacterial cell wall plays no part in the chemical or structural requirements for PG to induce chronic cell wall arthritis in the rats; the chemical structure of the PG moiety is decisive.

Cytokine production in arthritis susceptible and resistant rats: a study with arthritogenic and non-arthritogenic Lactobacillus cell walls.

The basis of the different susceptibility to bacterial cell wall-induced arthritis between Lewis and Fischer rats is unclear. Likewise, it is not known why cell walls of some species of Lactobacillus are arthritogenic and those of others are not. With these two questions in mind, we investigated the role of anti-inflammatory (interleukin (IL)-10, IL-4) and proinflammatory (tumour necrosis factor (TNF)-alpha, IL-1 beta) cytokines in Lewis and Fischer rats injected intraperitoneally with cell walls from arthritogenic or nonarthritogenic species of Lactobacillus. Cytokine levels in the serum and in vitro production by peritoneal macrophages and splenocytes were studied. The results obtained indicate that the differences in the production of IL-10, IL-4, TNF-alpha or IL-1 beta do not explain the difference in the arthritis susceptibility between Lewis and Fischer rats. Likewise, the arthritogenicity of different Lactobacillus cell walls appears not to be dependent on their capacity to stimulate cytokine production.

What determines arthritogenicity of bacterial cell wall? A study on Eubacterium cell wall-induced arthritis.

OBJECTIVE: To study what determines the arthritogenicity of the bacterial cell wall (CW) using Eubacterium CW-induced arthritis in the rat. METHODS: Eubacterium aerofaciens, previously reported as arthritogenic, and E. limosum and E. alactolyticum, known as non-arthritogenic, were used. Gas chromatography-mass spectrometry (GC-MS) was applied to analyse the chemical composition of the bacterial cell wall. Cellular immune response was measured by concanavalin A (Con A) stimulation and FACScan analysis. Also, serum antibodies against the injected cell wall were determined. RESULTS: Unexpectedly, from the two strains of E. aerofaciens used only one proved to be arthritogenic (with a CW inducing chronic arthritis after a single intraperitoneal injection), even though these two strains were 100% identical by 16S rDNA analysis. CW of the other E. aerofaciens strain induced only transient acute arthritis; CW of E. limosum and E. alactolyticum induced weak signs of acute arthritis. Based on the GC-MS analysis and on the results published previously, putative structures of peptidoglycan (PG) in the four CW preparations are presented. It is apparent that the presence of lysine in position 3 of the PG stem peptide contributes to arthritogenicity but is alone not decisive. Both strains of E. aerofaciens were immunosuppressive, when tested by Con A response at 2 weeks after CW injection. Such an immunosuppression was not observed after injection of CW from E. limosum or E. alactolyticum. FACScan analysis for six T cell markers and studies on serum antibody responses did not reveal any differences in the effect of the four bacterial strains used. CONCLUSIONS: The results obtained suggest that the chemical structure of PG present in the bacterial CW is decisive in determining arthritogenicity/non-arthritogenicity. Therefore, from two bacterial strains belonging to normal human intestinal flora and 100% identical by 16S rDNA analysis, one proved to be arthritogenic and the other non-arthritogenic.

Bacterial cell wall-induced arthritis: chemical composition and tissue distribution of four Lactobacillus strains.

To study what determines the arthritogenicity of bacterial cell walls, cell wall-induced arthritis in the rat was applied, using four strains of Lactobacillus. Three of the strains used proved to induce chronic arthritis in the rat; all were Lactobacillus casei. The cell wall of Lactobacillus fermentum did not induce chronic arthritis. All arthritogenic bacterial cell walls had the same peptidoglycan structure, whereas that of L. fermentum was different. Likewise, all arthritogenic cell walls were resistant to lysozyme degradation, whereas the L. fermentum cell wall was lysozyme sensitive. Muramic acid was observed in the liver, spleen, and lymph nodes in considerably larger amounts after injection of an arthritogenic L. casei cell wall than following injection of a nonarthritogenic L. fermentum cell wall. The L. casei cell wall also persisted in the tissues longer than the L. fermentum cell wall. The present results, taken together with those published previously, underline the possibility that the chemical structure of peptidoglycan is important in determining the arthritogenicity of the bacterial cell wall.

Tissue distribution and persistence of arthritogenic and non-arthritogenic Eubacterium cell walls.

OBJECTIVE: To study the tissue distribution and persistence of arthritogenic and non-arthritogenic Eubacterium cell walls (CWs), using arthritogenic Eubacterium aerofaciens and non-arthritogenic Eubacterium limosum. METHODS: Eubacterium aerofaciens or Eubacterium limosum CW was injected into Lewis rats intraperitoneally. Inflammatory changes in the synovium and periarticular tissues were graded histologically. On days 14, 28 and 56 after the injection, the presence of CW in the liver, spleen, mesenteric lymph nodes and synovium was studied by indirect immunofluorescence. In parallel, CW-derived muramic acid in the liver and spleen was measured by gas chromatography-mass spectrometry. In addition, serum TNF-alpha, IL-1 beta and IL-10 concentrations were determined by ELISA. RESULTS: Systemic injection of Eubacterium aerofaciens CW, but not of Eubacterium limosum CW, resulted in chronic arthritis. Both E. aerofaciens and E. limosum CWs were observed in the liver and spleen at all of the time points studied. In addition, Eubacterium limosum CW was present in non-arthritic synovium on day 14. It was not, however, detected in the synovium or lymph nodes on days 28 and 56, in clear contrast to the rats injected with E. aerofaciens CW. According to the analysis by gas chromatography-mass spectrometry, non-arthritogenic E. limosum CW had accumulated in the liver cells on days 14 and 28 after the injection to a greater extent than arthritogenic E. aerofaciens CW, leading to a lesser distribution in the other organs. A weak trend was observed suggesting that the production of TNF-alpha and IL-1 beta, but not of IL-10, is stimulated better by arthritogenic CW than by non-arthritogenic CW. CONCLUSION: Our results indicate that non-arthritogenic CWs are handled by the rat's defence mechanisms in a different way than arthritogenic CWs. The tissue distribution and persistence of CWs play a role in arthritogenicity, but additional factors must exist to determine why the CWs of certain bacteria are arthritogenic and those of others are not.