Novel path to IL-6 trans-signaling through thrombin-induced soluble IL-6 receptor release by platelets.

Interleukin (IL)-6 is a multifunctional cytokine with a critical role in inflammatory, immunoregulatory and haemopoietic responses. Its receptor consists of an ubiquitously expressed membrane transducing element (gp130) and of the specific element IL-6R-alpha (gp80), present only on hepatocytes and some leukocyte subsets. IL-6R-alpha also exists as soluble protein (sIL-6R) that, in the presence of IL-6, forms a complex able to bind gp130 and, thanks to the mechanism called trans-signaling, transduces IL-6 effect through tyrosine phosphorylation and activation of the signal transducer and transcription activator (STAT)-3. The aim of this study was to analyze the bidirectional relationships between platelet aggregation and IL-6-dependent effects. While platelets do not produce IL-6, we found that resting platelets express gp130, but not gp80, on their membranes. Upon activation by thrombin or calcium ionophore A23187, but not by ADP, the IL-6R-alpha is released in soluble form, while cangrelor, the specific inhibitor of P2Y12 receptor, can partially inhibit sIL-6R release. This sIL-6R is biologically active and, in the presence of IL-6, can trigger IL-6 trans-signaling, inducing an autocrine activation loop (as measured by an increase in gp80 and gp130 content) and STAT3 phosphorylation. On the other hand, IL-6 trans-signaling has no effect on platelet degranulation or aggregation by itself, nor on thrombin-induced platelet aggregation. Our data add an important piece to the puzzle of thrombosis and inflammation: in the presence of IL-6, which can be produced by stressed endothelial cells, the platelet-derived IL-6 trans-signaling could be crucial for the evolution of inflammation within a damaged vessel.

Skeletal muscle cells: from local inflammatory response to active immunity.

The skeletal muscles are the major living component of the human body. They are constituted by stable cells, the myofibres, and by adult multipotent stem cells, the satellite cells, which can multiply to regenerate and repair the damaged tissues. Injections of DNA in muscle cells have been used to produce recombinant proteins with opposite goals: somatic reparation of genetic defects, which needs to elicit no inflammatory or immune response, and DNA vaccination, which needs a robust immune response. Because of possible therapeutical interventions, a growing body of information is being produced dealing with every aspect of the myofibres during inflammatory and autoimmune responses: skeletal muscle-antigen presenting cell (APC) interaction and intrinsic APC capabilities of myoblasts and myocytes, the response to released cytokines and their endogenous production, the regulation of Toll-like receptors and major histocompatibility complex expression. According to these data, the muscle tissue is now emerging no longer as a passive bystander, but more as an active player that, when correctly manipulated, can drive tolerance or immunization to these de novo produced proteins. In the present review, we summarize the recent developments on the control of muscle immune function.

Neurophysiological and mitochondrial abnormalities in MuSK antibody seropositive myasthenia gravis compared to other immunological subtypes.

OBJECTIVE: To compare the electrophysiological and histopathological features of immunological myasthenia gravis (MG) subtypes. METHODS: Fifty MG patients underwent clinical examination, MuSK-Ab and AChR-Ab analysis. The majority underwent quantitative and single-fiber electromyography (QEMG, SFEMG), repetitive nerve stimulation and deltoid muscle biopsy. From muscle specimens with histological mitochondrial dysfunction, we amplified mitochondrial DNA (mtDNA). In specimens with mtDNA deletions, the nuclear gene POLG1 was sequenced. RESULTS: Five AChR-Ab seropositive [AChR(+)] and 5 seronegative [AChR(-)] patients were MuSK-Ab seropositive [MuSK(+)]. Five of 7 neurophysiologically examined MuSK(+) patients (71%) had proximal myopathic pattern, compared to 7 of 31 MuSK(-)/AChR(+) patients (23%) (P=0.012). SFEMG was abnormal in all examined MuSK(+) patients. All 7 biopsied MuSK(+) and 32 MuSK(-) patients (89%) had cytochrome c oxidase (COX) negative fibers. Three of five MuSK(+) and 13 of 20 MuSK(-) patients analyzed had multiple mtDNA deletions but no POLG1 mutations. CONCLUSIONS: Similar degree of SFEMG abnormalities was present in proximal muscles among MuSK(+) and AChR(+) patients. Proximal myopathy was over-represented in MuSK(+) patients; however, both MuSK(+) and MuSK(-) patients had mild myopathy with frequent mitochondrial abnormalities. SIGNIFICANCE: The weakness in MuSK(+) patients is most likely due to disturbed neuromuscular transmission. The frequently encountered mitochondrial dysfunction in MG warrants further study.

Effect of transforming growth factor-beta1 on interleukin-6 secretion in human myoblasts.

Transforming growth factor-beta (TGF-beta) is involved in several autoimmune neurological diseases. It is still unclear whether its local action can be pro-inflammatory or anti-inflammatory in the muscle tissue, because of the few reports on this subject. We have previously shown that human myoblasts secrete interleukin-6 (IL-6) when stimulated with inflammatory cytokine such as interleukin-1beta (IL-1beta) or tumor necrosis factor alpha. In the present report, we show that TGF-beta1 can induce IL-6 production; moreover, costimulation or short term pre-incubation with TGF-beta1 increases IL-1beta effect, while a longer incubation inhibits its action.

Constitutive and cytokine-induced expression of MHC and intercellular adhesion molecule-1 (ICAM-1) on human myoblasts.

We studied the expression of MHC-I and MHC-II molecules and ICAM-1 in cultured human myoblasts in response to IL-1beta, IL-4, IL-6, IFN-gamma and LPS. IFN-gamma, LPS and IL-4 greatly increase MHC-I molecule expression. MHC-II molecule expression is induced only by IFN-gamma. Membrane ICAM-1 and mRNA expression are absent under basal conditions, but can be induced by IFN-gamma, IL-1beta, IL-4, LPS and IL-6 with different efficiencies and time-courses. Soluble ICAM-1 secretion can be induced to a different extent by all cytokines. Our study shows that the expression of adhesion-related molecules in muscle is finely regulated by these cytokines.

Effect of pro-inflammatory/anti-inflammatory agents on cytokine secretion by peripheral blood mononuclear cells in rheumatoid arthritis and systemic lupus erythematosus.

We studied a well-selected population of patients with active rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE) without immunosuppressive therapy. Control and patient peripheral blood mononuclear cells (PBMC) were incubated with IL-1beta, IL-10, TGF-beta or LPS for 20 h and the in vitro basal and stimulated secretions of IL-6, TNF-alpha, IL-1beta and IL-1ra were measured by ELISA. We found that in the SLE patients the basal secretion of IL-6 was significantly lower and that of IL-1ra significantly higher than in control subjects, while in the RA group the basal IL-1ra secretion was higher than in healthy subjects. SLE and RA PBMC responded to LPS and IL-1beta reaching higher cytokine secretion values than controls. The in vitro response of SLE and RA PBMC to TGFbeta was normal, while that to IL-10 was defective: IL-10 was able to stimulate the production of IL-6 and IL-1ra in PBMC from normal subjects, but it was unable to enhance IL-6 secretion in RA cells and it was also completely ineffective in inducing IL-1ra secretion in both SLE and RA PBMC. Our work add new data useful for the evaluation of IL-10 and IL-1ra as therapeutic agents in rheumatic diseases.

Myasthenia gravis, thymectomy, and antiacetycholine receptor antibody.

The antiacetylcholine receptor antibody was titered in the serum of 63 patients with myasthenia gravis (MG) and 20 control healthy subjects. The titer was significantly high in 92% of MG patients in contrast with none of the controls and no correlation was found with the thymus pathology and the severity of the disease. The titer decreased after thymectomy almost steadily with the improvement of the myasthenic signs. The role of the antibody in the pathogenesis of the disease is discussed.

When is there a full recovery for a myasthenia gravis patient?

A myasthenia gravis (MG) patient who seems to have recovered can later have recurrence of myasthenic signs. Clearly clinical remission does not always correspond to the normalization of all the factors involved in the pathogenesis of the disease. In ten patients who had apparently recovered from MG, electromyographic tests of repetitive supramaximal stimulation were performed and the anti-acetylcholine receptor (anti-AChR) antibody was assessed. In two of the ten patients all these tests were normal, thus showing lack of electromyographic myasthenic fatigability and the absence of circulating anti-AChR antibodies. Our hypothesis is that for these two subjects the risk of a recurrence of MG is lower than for the others.

The association of silent thyroiditis with active systemic lupus erythematosus.

Autoimmune thyroid disorders have been shown to occur in patients with connective tissue diseases. Hypothyroidism and thyrotoxicosis have been recognized in systemic lupus erythematosus (SLE). Moreover, a high prevalence of antithyroid antibodies has been found in patients with SLE. We studied thyroid function in a group of SLE female patients without a history or clinical diagnosis of thyroid disease and then correlated the prevalence of abnormal function test results with the laboratory indexes of active disease and with the presence of antithyroid antibodies. The SLE patients had significantly lower T4 levels than the controls. Basal TSH and TSH concentrations after TRH stimulation were significantly higher in patients with active SLE in comparison to both patients with inactive SLE and to controls. 45.5% of patients with active SLE presented antithyroid antibodies. Antithyroglobulin and antimicrosomal antibodies were not found in patients with inactive SLE nor in controls. Our results confirm the existence of a mild hypothyroidism in SLE that is clinically silent. The altered thyroid function appears to be dependent on the activity of the systemic autoimmune process.

Anticardiolipin antibodies in patients with primary antiphospholipid syndrome: a correlation between IgG titre and antibody-induced cell dysfunctions in neuronal cell cultures.

Anticardiolipin antibodies (aCL) of the immunoglobulin (Ig) G isotype have been significantly associated with neurological manifestations of antiphospholipid syndrome (APS). In a previous study we described the direct pathogenic effects of IgG aCL on living neurons in culture. Therefore, we studied the IgG aCL titre as a factor influencing the extent of this effect. Seventeen patients with a history of primary antiphospholipid syndrome were grouped according to their IgG aCL titre into low positive (GPL < or = 40), high positive (40< GPL <100) and very high positive (GPL >100). IgG from these patients were incubated with cerebellar neurons in primary culture for 24h and the effect was evaluated by using the tetrazolium salt (MTT) assay. We found that almost all patients' IgGs reduced cell viability in vitro, but the differences in the extent of the effect were statistically significant only for patients with >40 GPL. Our results reinforce the causal association between increasing level of IgG aCL and clinical features of aPS.

ACTH, cortisol and prolactin in active rheumatoid arthritis.

Prolactin (PRL) and glucocorticoids are hormones involved in the regulation of the immune system. Rheumatoid arthritis (RA) is an inflammatory condition that presents a diurnal rhythm of disease activity. ACTH, PRL, cortisol, IL-1 beta and TNF-alpha circadian rhythms have been studied in active RA (aRA) to evaluate a possible relationship between the neuroendocrine system and immunological activity in rheumatoid patients. ACTH, PRL, cortisol, PRL/cortisol ratio and IL-1 beta and TNF-alpha levels were determined in aRA patients and in control subjects at 6.00, 10.00, 14.00, 18.00, 22.00 and 02.00 h. In aRA patients we observed lower ACTH and cortisol levels at 22.00 h and 2.00 h, respectively and higher PRL and PRL/cortisol ratio at 2.00 h when compared to controls. IL-1 beta and TNF-alpha reached their highest serum levels in aRA patients at 2.00 and 6.00 h. This study provides evidence that in aRA there could be a temporary and probably causal relationship between diurnal disease activity, hormonal disequilibrium and cytokine secretion. An imbalance in favour of proinflammatory hormones (PRL and cytokines) as opposed to levels of anti-inflammatory hormones could be responsible for the diurnal rhythm of activity disease observed in aRA patients.

[Keratoconjunctivitis sicca in rheumatoid arthritis: correlation with the autoantibody pattern]. / Cheratocongiuntivite secca nell'artrite reumatoide: correlazione con il pattern autoanticorpale.

In order to evaluate the autoantibody pattern of subjects affected by rheumatoid arthritis (RA) with clinical features of keratoconjunctivitis, we studied 32 out- and in-patients (26 women, 6 men, average age 52 years, average disease duration 5.5 years) at the Division of Rheumatology, Catholic University of Rome. We found keratoconjunctivitis sicca and xerostomia in 22 (68.75%) patients with RA. Rheumatoid factor was present in 17 (53.1%) patients, antinuclear antibodies (ANA) were observed in 15 (48.4%) patients, and anti-rheumatoid arthritis nuclear antigens (RANA) in 22 (68.7%) patients; anti-SSA antibodies were confirmed in 3 (9.4%) patients and anti-SSB antibodies in 2 (6.2%) patients. None of the patients evidenced anti-U1RNP. Although keratoconjunctivitis sicca and xerostomia correlated significantly with the presence of rheumatoid factor, we found no relationship between these two conditions and ANA or anti-RANA antibodies. The high frequency of keratoconjunctivitis sicca and xerostomia in our RA patients is the expression of extra-articular involvement in this disease and is correlated with the presence of rheumatoid factor. ANA and anti-RANA antibodies may represent aspecific polyclonal activation in RA.

[Antiphospholipid antibodies in systemic lupus erythematosus: study of a caseload of 120 patients]. / Anticorpi antifosfolipidi nel lupus eritematoso sistemico: studio di una casistica di 120 pazienti.

In order to evaluate the presence of antiphospholipid antibodies (aPL) in patients with systemic lupus erythematosus (SLE), we measured IgG and IgM anticardiolipin antibodies (aCL) in a group of out- and in-patients of the Rheumatology Division at the Catholic University of Rome. Lupus anticoagulant and VDRL were also measured in 30 patients. One hundred thirteen women and 7 men (mean age 38.5 years, mean disease duration 4.27 years) were studied. The control group consisted of 60 age and sex matched healthy subjects. aCL IgG and IgM were positive in 48 (40%) and 49 (40.8%) patients respectively. aCL IgG correlated positively with the activated partial thromboplastin time. aCL IgM correlated positively with immunoglobulins of the G and M classes and with circulating immune complexes, and negatively with the C4 fraction. aCL antibodies did not correlate with spontaneous abortion or neurologic and psychiatric disturbances, although in women with a history of abortion, aCL IgG concentration was correlated with the number of spontaneous abortions. The presence of aPL does not seem to indicate a subgroup of SLE patients. The occurrence of abortion and/or neurologic and psychiatric disturbances in SLE seems to point to a complex pathogenesis with aPL as one of the causative agents.

[Immunogenetic and functional study of first-degree relatives of patients with type I diabetes mellitus for identifying prediabetic subjects]. / Studio immunogenetico e funzionale dei consanguinei di I grado di pazienti affetti da diabete di tipo I per l'identificazione dei soggetti prediabetici.

Insulin dependent diabetes mellitus is an autoimmune disease with HLA-related genetic susceptibility. There is a latent phase before overt disease. During this phase islet cell antibodies (ICA) as a marker of humoral autoimmunity are detected and i.v. glucose tolerance test (IVGTT) is decreased. The aim of our work was to correlate IVGTT, HLA typing and ICA testing in all siblings of IDDM patients in order to identify high risk subjects (HRS). IVGTT showed significantly lower insulin levels in siblings vs controls (P less than 0.0001). This phenomenon could be caused by HLA unrelated genetic predisposition to low insulin secretion. Insulin level values of ICA+ siblings were lower than those of ICA- siblings, even if the difference was not significant.

[A case of transitory Fanconi syndrome associated with acute renal insufficiency and hypoplasia of the bone marrow]. / Su un caso di sindrome di Fanconi transitoria associata ad insufficienza renale acuta e ad ipoplasia del midollo osseo.

The authors report a case of an eight-years old child, who presented with transient Fanconi syndrome, mild renal failure and hypoplastic bone marrow. No recognized etiology of the Fanconi syndrome was demonstrated in the patient. Laboratory data and clinical course are consistent with the hypothesis of a tubulo-interstitial lesion caused, directly or through an abnormal immune response, by an unknown etiologic agent.

[Epidemiologic research for thalassemia and hemoglobinopathy traits in the territory of a local health unit in Liguria]. / Indagine epidemiologica per traits talassemici ed emoglobinopatie nel territorio di una U.S.L. della Liguria.

The authors investigated the incidence of thalassemia traits and hemoglobinopathies in western Liguria, where up to 70% of people comes from other italian regions, particularly from the South. The authors screened 442 primary school pupils in Albenga and Andora (Savona). Laboratory investigations permitted to detect 19 thalassemia trait carrier subjects (4.30% of the total examined): 12 of them were diagnosed heterozygous for beta-thalassemia, 6 for alpha-thalassemia, and 1 for Hb S. Authors would underline that more than half of the screening positive subjects resulted carrier of beta-thalassemia or Hb S trait, both potentially able to give origin to severe diseases: homozygous beta-thalassemia, sickle cell anemia, and beta-thalassemia/Hb S double heterozygosity.

Viral-load and B-lymphocyte monitoring of EBV reactivation after allogeneic hemopoietic SCT in children.

EBV-associated post transplant lymphoproliferative disease (EBV-PTLD) is a life-threatening complication that may occur after hemopoietic SCT. We prospectively screened 80 children on a weekly basis using nested quantitative PCR to evaluate EBV genome copies. EBV viral load <1000 copies per 10(5) PBMC was observed in 63% of transplants, whereas it was between 1000 and 9999 copies per 10(5) PBMC in 13%, and between 10 000 and 19 999 in 10%, with no significant increase in percentage of CD20+ lymphocytes. Viral load reached > or = 20 000 copies per 10(5) PBMC in 14% of patients, and rituximab was administered to 75% of them. None of the patients except one developed a lymphoproliferative disease. Our study found that only 13% of unrelated donor HSCT recipients had a very high risk of EBV-PTLD defined as > or = 20 000 geq per 10(5) PBMC associated with an increase in CD20+ lymphocyte. We suggest that rituximab could be administered in the presence of very high levels of EBV-DNA viral load or in the presence of mid levels of EBV-DNA viral load associated with an increase in the percentage of CD20+ lymphocytes. Through this approach, we significantly reduced the number of patients treated with rituximab, and consequently the acute and chronic adverse events related to this treatment.