The role of interleukin-6 signalling and its therapeutic blockage in skewing the T cell balance in rheumatoid arthritis.

Therapeutic blockage of cytokine signalling in autoimmune diseases has improved our understanding of the role of these cytokines in triggering, shaping and perpetuating autoimmune responses. In rheumatoid arthritis (RA), immunopathology is driven by a predominance of arthritogenic T helper cells secreting interferon-γ [T helper type 1 (Th1)] and interleukin (IL)-17 (Th17) over regulatory T cells (Treg ). The pleiotropic cytokine IL-6 is crucial to the differentiation of Th17 cells and the balance between pathogenic Th17 and protective Treg . Targeting the IL-6 receptor (IL-6R) by humanized antibodies improves signs and symptoms of RA, and has provided new insights into the mechanisms of inflammation and immune regulation. Here we review current evidence on the role of IL-6 in the pathogenesis of RA and the molecular consequences of IL-6R blockage in disease, with special focus on the Th17/Treg balance and plasticity.

Presencia de linfocitos T reguladores en periodontitis crónica / Presence of T regulatory cells in chronic periodontitis

La enfermedad periodontal requiere de un hospedero susceptible para su desarrollo y progresión. Dentro de las características del hospedero se encuentra la respuesta T reguladora, que otorga tolerancia frente a antígenos propios, participa durante las enfermedades infecciosas limitando el daño tisular, sin disminuir la respuesta antibacteriana. El presente estudio tiene por objetivo determinar la presencia, reclutamiento y función de Tregs en pacientes con periodontitis crónica. En 10 biopsias de tejido periodontal sano y con periodontits crónica se realizó inmunohistoquímica para marcadores (CD4, CD25, Foxp3), quimioquinas (CCL17, CCL22) y citoquinas (TGF-B, IL-10) de Tregs. Además de Western-Blot para detectar las citoquinas. Los resultados obtenidos sugieren una posible asociación entre células Tregs y la infección periodontal, ya que se confirma su reclutamiento y presencia. Sin embargo, son necesarios más estudios del posible desbalance con su contraparte pro-inflamatoria Th17, que expliquen en parte la compleja etiopatogenia de la enfermedad periodontal.

Periodontal disease requires a susceptible host to initiation, development and progression. T regulatory response is one of these inmunoregulatory characteristics of the susceptible host, which provide tolerance, tissular protection during infection without impairing the control of periodontopathogens. The aim of this study is to determinate the presence, homing and function of T regulatory cells (Tregs) in patients with chronic periodontitis. Ten biopsies were taken from pockets, the presence of Tregs markers (CD4, CD25, Foxp3), chemokines (CCL17, CCL22) and cytokines (TGF-p, IL-10) were determinate by immunohistochemistry. Cytokines also were detected with Western-Blot. Our results suggest a possible association between Tregs and periodontal infection, confirming homing and presence of Tregs. However, further studies are required to determine the possible imbalance with pro-inflammatory part Th17, that might explain the complex etiopathogenesis of periodontal disease.

Modulation of established murine collagen-induced arthritis by a single inoculation of short-term lipopolysaccharide-stimulated dendritic cells.

BACKGROUND: The use of regulatory or immature dendritic cells (DCs) as tools for modulating experimental rheumatoid arthritis is very recent. Tumour necrosis factor (TNF)-stimulated DCs have been shown to restore tolerance in experimental autoimmune encephalomyelitis and collagen-induced arthritis (CIA). OBJECTIVE: We investigated the capacity of short-term lipopolysaccharide (LPS)-stimulated DCs pulsed with type II collagen (CII) to induce tolerance against established CIA. METHODS: Bone marrow-derived DCs were generated in the presence of granulocyte monocyte colony-stimulating factor (GM-CSF). After CIA induction, mice were injected at day 35 with a single dose of 4- or 24-h LPS-stimulated DCs that had been loaded with CII (4hLPS/CII/DCs or 24hLPS/CII/DCs). Arthritis progression was monitored by clinical and histological evaluations. RESULTS: Flow cytometry of 4hLPS/CII/DCs showed intermediate CD40 and CD86 expression, lower than that of 24hLPS/CII/DCs (fully mature) and higher than that of CII/DCs (immature). A functional assay showed that 4hLPS/CII/DCs display increased endocytosis ability with respect to 24hLPS/CII/DCs, indicating a semimature state. The single inoculation of 4hLPS/CII/DCs in mice with established CIA reduced disease severity significantly over time. Histological evaluation of mice treated with 4hLPS/CII/DCs revealed diminished inflammatory synovitis, cartilage damage and fibrosis. Co-cultures of DCs with splenocytes from CIA mice showed that collagen-specific interferon (IFN)gamma production was dramatically inhibited by 4hLPS/CII/DCs. 4hLPS/CII/DCs were high IL10 producers, which could explain the inhibition of arthritis progression in mice receiving this treatment because neither antibodies nor regulatory CD4+CD25+Foxp3+ T lymphocytes were demonstrated to be involved. CONCLUSION: Short-term LPS-modulated DCs inoculation interferes with CIA progression when loaded with CII.

Basal anti-cyclic citrullinated peptide (anti-CCP) antibody levels and a decrease in anti-CCP titres are associated with clinical response to adalimumab in rheumatoid arthritis.

OBJECTIVE: To investigate the effect of adalimumab treatment on anti-cyclic citrullinated peptide antibodies (anti-CCP) in patients with rheumatoid arthritis (RA). METHODS: 70 RA patients who failed treatment with disease modifying antirheumatic drugs (DMARDs) received 40 mg adalimumab subcutaneously every other week during 24 weeks. Serum samples were collected at baseline and at weeks 8, 16 and 24 before the corresponding adalimumab dose. The serum anti-CCP levels were tested by enzyme linked immunosorbent assay. RESULTS: At baseline, 52 of the 70 patients (74.3%) were positive for anti-CCP antibodies. 60 % of the anti CCP positive patients and 44.4% of the anti CCP negative patients were ACR 20 responders at week 24 (p<0.049). The serum levels of anti-CCP antibodies decreased significantly after 24 weeks of adalimumab treatment only in those patients who met ACR 20 response criteria at week 24 (p<0.00044). Differences between baseline anti-CCP titers and those at 8, 16 and 24 weeks were all statistically significant (p<0.014, 0.003 and 0.019 respectively). No statistically significant changes in the anti-CCP levels were observed in patients who did not meet the ACR 20 response criteria. CONCLUSION: Basal anti-CCP antibodies levels correlate with clinical response to adalimumab. A decrease in anti-CCP levels on time was observed in patients showing also clinical improvement, suggesting that serum anti-CCP antibodies determination may be useful in assessing treatment efficacy in RA patients.

Tumour necrosis factor (TNF)alpha -308 G/G promoter polymorphism and TNFalpha levels correlate with a better response to adalimumab in patients with rheumatoid arthritis.

OBJECTIVE: To investigate the influence of -308 tumour necrosis factor-alpha (TNFalpha) promoter polymorphism and circulating TNFalpha levels in the clinical response to adalimumab treatment in patients with rheumatoid arthritis (RA). METHODS: Eighty-one patients with active RA were genotyped for the -308 TNFalpha polymorphism by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis and subdivided into two groups for each polymorphism (G/A and G/G genotype). All received 40 mg of adalimumab subcutaneously every other week. We compared the groups' clinical responses to adalimumab at 8, 16, and 24 weeks using the Disease Activity Score in 28 joints (DAS28). RESULTS: Both groups showed a significant improvement from baseline. A significant difference between groups was found at week 24. We found that 88.2% of G/G versus 68.4% of G/A for the -308 polymorphism were DAS28 responders (p = 0.05). The score improvement at week 24 was 2.5 +/- 1.3 in the G/G group and 1.8 +/- 1.3 in the G/A group for the -308 polymorphism (p = 0.04). The median of serum TNFalpha levels of the G/A group were lower than those of the G/G group, and statistically different at weeks 8 and 24 (p < 0.039 and p < 0.043). When comparing baseline levels to those achieved at 8, 16, and 24 weeks for the whole group, only responder patients showed a statistically significant overall increase in TNFalpha over time (p < 0.000001). CONCLUSION: A relationship between DAS28 improvement, the -308 G/G polymorphism, and increased circulating TNFalpha levels was found in Chilean RA patients treated with adalimumab.

Tumour necrosis factor-alpha (TNF-alpha) levels and influence of -308 TNF-alpha promoter polymorphism on the responsiveness to infliximab in patients with rheumatoid arthritis.

OBJECTIVE: To investigate the influence of -308 tumour necrosis factor-alpha (TNF-alpha) promoter polymorphism and circulating TNF-alpha levels in the clinical response to the infliximab treatment in patients with rheumatoid arthritis (RA). METHODS: One hundred and thirty-two RA patients were genotyped for TNF-alpha promoter by polymerase-chain reaction restriction fragment-length polymorphism (PCR-RFLP) analysis. Ten patients with the -308 TNF-alpha gene promoter genotype G/A, and 10 with the G/G genotype were selected and received 3 mg/kg of infliximab at Weeks 0, 2, 6, and 14. RESULTS: Both groups showed a significant improvement with treatment in all variables studied. Total mean TNF-alpha levels increased significantly with respect to basal levels in most of patients after treatment [probability (p)=0.04]. Only patients from G/A showed a statistically significant correlation between ACR 50 and the increase of TNF-alpha levels (p<0.03). CONCLUSION: A relationship was detected between ACR criteria of improvement and increased circulating TNF-alpha levels in RA patients subjected to anti-TNF-alpha therapy.

The -308 polymorphism in the tumour necrosis factor (TNF) gene promoter region and ex vivo lipopolysaccharide-induced TNF expression and cytotoxic activity in Chilean patients with rheumatoid arthritis.

OBJECTIVE: To investigate the association of the -308 polymorphism in the promoter region of the tumour necrosis factor (TNF) gene with susceptibility to the development of RA. We also explored the expression and cytotoxicity of TNF in relation to the -308 polymorphism. METHODS: We recruited 92 RA patients and 42 healthy control subjects. Genotyping for the TNF promoter was performed by polymerase chain reaction-restriction fragment length polymorphism analysis. To study the overexpression of TNF we used a whole-blood culture system. TNF cytotoxicity was assessed in the L929 cell line. RESULTS: The TNF2 allele was found in 23% of RA patients and 10% of controls. Although both groups showed high variability in serum TNF concentration, in the lipopolysaccharide-induced TNF level and in the cytotoxicity of the cytokine in the L929 cell line, these differences were not associated with the -308 TNF polymorphism. CONCLUSION: No associations were found between the -308 TNF promoter polymorphism, serum and ex vivo TNF levels and the cytotoxic activity of TNF in RA patients.

Genetic polymorphism at position-308 in the promoter region of the tumor necrosis factor (TNF): implications of its allelic distribution on susceptibility or resistance to diseases in the Chilean population.

Several single-nucleotide polymorphisms have been identified in the human TNF gene promoter. The polymorphism at position-308 (TNF-308), which involves substituting G for A and designing the TNF2 allele, leads to a higher rate of TNF gene transcription than the wild-type TNF1 allele in in vitro expression studies. It has also been linked to increased susceptibility to a variety of illnesses. Using PCR-RFLP analysis we detected significant differences in the TNF-308 genotypes of Chilean and other populations. We conclude that there is a gradient in the distribution of the TNF2 allele according to ethnicity; we have also hypothesized that populations bearing a higher proportion of the TNF2 allele may have an increased predisposition toward or incidence of several chronic metabolic, degenerative, inflammatory and autoimmune diseases.

Daily production of human tumor necrosis factor in lipopolysaccharide (LPS)-stimulated ex vivo blood culture assays.

Tumor necrosis factor (TNF) is a pleiotropic cytokine with immunological and neuroendocrine activities. A useful tool for studying TNF is the measurement of its in vitro and/or ex vivo over-expression, induced by a variety of stimuli on isolated peripheral mononuclear cells or whole blood, respectively. The capacity to over-express TNF, in ex vivo LPS-stimulated whole blood from 18 normal individuals, showed inter-individual variations ranging from high (3 ng/ml) to low (0.7 ng/ml) producers. Although at a lower level, a similar situation was observed in the spontaneous production of the cytokine. In order to detect cyclic effects in these variations, blood samples were taken at 08:00, 12:00, 16:00 and 20:00 hours, from nine healthy volunteers, and cultured in the ex vivo system. TNF and cortisol were measured by immunometric assays. Both, LPS-stimulated whole blood and plasma showed important, individual variations in TNF levels. Although cortisol levels presented a normal circadian cycle, these individual patterns in TNF production were basically conserved during the day (p > 0.05), and no correlation was observed between the levels of the hormone and those of the cytokine. When total TNF levels were determined at 20:00 hours, a moderate, temporary variation pattern of the cytokine production was found. These results suggest that cortisol does not play a predominant role in determining the ex vivo capacity of blood to produce TNF. Presumably, the variable capacity to produce the cytokine may have a strong genetic component.

Tc45, a dimorphic Trypanosoma cruzi immunogen with variable chromosomal localization, is calreticulin.

We demonstrate that Tc45, a polypeptide described as an immunogenetically restricted Trypanosoma cruzi antigen in mice, is calreticulin, a dimorphic molecule encoded by genes with variable chromosomal distribution. Previously we showed that IgG from A.SW (H2s) mice immunized with T. cruzi trypomastigotes or epimastigotes and sera from infected humans recognize Tc45, a 45 kD parasite polypeptide. Herein we describe the cloning, sequencing, and expression of the Tc45 gene. A 98% homology in the deduced amino acid sequence was found with a T. cruzi calreticulin-like molecule and 41% with Leishmania donovani and human calreticulin. In the T. cruzi CL Brener clone and in the Tulahuén strain, the gene is located in two and four chromosomes, respectively. Calreticulin was detected in several T. cruzi clones, in the Tulahuén strain, and in T. rangeli, displaying alternative 43 and 46 kD forms.

Immunodiagnosis of fasciolosis in horses and pigs using western blots.

Crude and partially purified somatic (S) and excretory-secretory (ES) antigens of Fasciola hepatica were subjected to Western blot analysis in order to identify polypeptides that would enable specific and sensitive immunodiagnosis of horse and pig fasciolosis to be undertaken. Sera from 20 horses and 20 pigs with natural infections of F. hepatica and the same number of uninfected hosts of each species were tested, together with sera from 2 pigs with Cysticercus cellulosae infections. Using crude S antigens, sera from infected horses and pigs reacted specifically with a wide range of polypeptides of 14-19, 22-30, 35-37 and 42 kDa. Likewise, specific reactivity between polypeptides of 14-17, 22-30 and 40-42 kDa in crude ES antigens and sera from infected horses and pigs was obtained. Against the criteria of high sensitivity and specificity, the 22-30-kDa polypeptides would appear to be the most suitable candidate antigens for use in the immunodiagnosis of fasciolosis in horses and pigs.

Immunomodulation of LPS ability to induce the local Shwartzman reaction.

Immunologically, the septic shock is a natural model of immunomediated vascular pathology where the interaction between cytokines and the endothelium mediates the syndrome and lethality. Tumour necrosis factor (TNF), a non-species-specific cytokine, has outstanding pleiotropic activities as an important mediator of the septic shock syndrome. In rabbits, passive immunization with anti-lipopolysaccharide (LPS) polyclonal antibodies prior to the intravenous (i.v.) injection of LPS inhibits the haemorrhagic necrotic lesion characteristic of the local Shwartzman reaction (an excellent localized in vivo correlate of the septic shock). Paradoxically, tested in an ex vivo assay (short-term whole human blood culture, stimulated with LPS), these antibodies mediated an increase in TNF production by mononuclear phagocytes and, in the rabbit model, they induced an increase in body temperature, as compared with the pre-immune reagent. Although anchoring of immune complexes containing LPS to receptors (Fc or C4b-C3b) on circulating monocytes may facilitate the access of LPS to these cells, access to localized, LPS-sensitized macrophages may be impaired. Consequently inhibition of the local Shwartzman reaction and increased TNF production in the ex vivo system were observed. Concordantly, the higher temperature in the passively immunized animals may be a consequence of a higher, immune complex-induced, systemic TNF production. These experimental results suggest that the use of anti-LPS immunoglobulins, as a potential immunotherapy for septic shock syndrome in vertebrates, may lead to increased TNF production, with adverse effects such as the pyrogenic.

Purification and preliminary sequencing of Tc-45, an immunodominant Trypanosoma cruzi antigen: absence of homology with cruzipain, cruzain, and a 46-kilodalton protein.

In spite of being separated by more than 20 million years of evolution, the murine and human immune systems share extensive similarities. Thus, experimental results obtained with the murine model may have predictive value for human Chagas' disease. Challenge of the H-2 congenic mouse stains A.SW (H-2s) and A.CA (H-2f) with Trypanosoma cruzi yields different results. The A.CA animals die approximately 12 days postinfection, while A.SW mice survive indefinitely. A 45-kD protein (Tc45), an antigen differentially recognized by the A.SW strain, is present in cultured epimastigotes and blood trypomastigotes. We describe here its purification from epimastigotes. The presence of Tc45 was monitored and a single band was detected. Since the molecular weights of Tc45, cruzipain, cruzain, and a 46-kD parasite polypeptide are similar, it was important to determine if these molecules are related. A complete lack of homology was observed when the sequence of cruzain, cruzipain, and the 46-kD polypeptide were compared with the preliminary sequence of Tc45.

Humoral immune response anti K99 pilus from enterotoxigenic Escherichia coli in experimentally inoculated calves

The bovine model is extremely interesting to study several basic aspects of mucosal local immunity. Many reports have shown that, in young calves, the infectivity of enterotoxigenic Escherichia coli may be inhibited by passively administered antibodies anti K99 pilus. We have measured, by immunoradiometric assays, the IgG response anti K99 pilus in the serum of calves, deprived of colostrum and orally inoculated with enteropathogenic K99+ E. coli. Although variable levels of IgG anti K99 pilus were detected, their protective value could not be ascertained in vivo due to the acute development of the infection. In an effort to correlate the presence of serum antibodies anti K99 pilus with their protective capacity, an ex-vivo assay to monitor the interaction of radiolabeled K99 pilus with the bovine mucosa was standardized. Paradoxically, although K99 pilus, purified by standard procedures, was recognized by polyclonal rabbit and calf antisera, its interaction with the bovine intestinal mucosa, quantitated in the ex-vivo system, was not inhibited by these reagents, indicating that the antibodies did not effectively block those K99 pilus domains involved in the interaction with mucosal receptors

An immunogenetically defined and immunodominant Trypanosoma cruzi antigen.

Two strains of mice, A. SW (H-2s) and A.CA (H-2f), were immunized with live trypomastigotes or epimastigotes of the Tulahuen strain of Trypanosoma cruzi or with their sonicates. By immunowestern blotting, sera from A.SW mice, but not from A.CA, recognized, in an immunodominant fashion, a 45 kDal polypeptide (Tc45) present in both epimastigotes and trypomastigotes. Since A.SW and A.CA strains are congenic for the major histocompatibility H-2 complex, recognition of Tc45 seems to be controlled by this genetic region or by gene(s) located in its immediate vicinity. Subcellular fractionation revealed that Tc45 is mainly present at the cytoplasmic compartment.