Splicing machinery is profoundly altered in systemic lupus erythematosus and antiphospholipid syndrome and directly linked to key clinical features.

OBJECTIVES: To characterize the splicing machinery (SM) of leukocytes from primary antiphospholipid syndrome (APS), systemic lupus erythematosus (SLE) and antiphospholipid syndrome with lupus (APS + SLE) patients, and to assess its clinical involvement. METHODS: Monocytes, lymphocytes and neutrophils from 80 patients (22 APS, 35 SLE and 23 APS + SLE) and 50 HD were purified, and 45 selected SM components were evaluated by qPCR-microfluidic array. Relationship with clinical features and underlying regulatory mechanisms were assessed. RESULTS: APS, SLE and APS + SLE leukocytes displayed significant and specific alterations in SM-components (SMC), associated with clinical features [autoimmune profiles, disease activity, lupus nephritis (LN), and CV-risk markers]. A remarkable relationship among dysregulated SMC in monocytes and the presence of LN in SLE was highlighted, revealing a novel pathological mechanism, which was further explored. Immunohistology analysis of renal biopsies highlighted the pathological role of the myeloid compartment in LN. Transcriptomic analysis of monocytes from SLE-LN(+) vs SLE-LN(-) identified 271 genes differentially expressed, mainly involved in inflammation and IFN-signaling. Levels of IFN-related genes correlated with those of SMC in SLE-LN(+). These results were validated in two external SLE-LN(+) datasets of whole-blood and kidney biopsies. In vitro, SLE-LN(+)-serum promoted a concomitant dysregulation of both, the IFN signature and several SMC, further reversed by JAKinibs treatment. Interestingly, IFNs, key inflammatory cytokines in SLE pathology, also altered SMC. Lastly, the over/down-expression of selected SMC in SLE-monocytes reduced the release of inflammatory cytokines and their adhesion capacity. CONCLUSION: Overall, we have identified, for the first time, a specific alteration of SMC in leukocytes from APS, SLE and APS + SLE patients that would be responsible for the development of distinctive clinical profiles.

Salutogenic health promotion program for migrant women at risk of social exclusion.

BACKGROUND: Migrant women at risk of social exclusion often experience health inequities based on gender, country of origin or socioeconomic status. Traditional health promotion programs designed for this population have focused on covering their basic needs or modifying lifestyle behaviors. The salutogenic model of health could offer a new perspective enabling health promotion programs to reduce the impact of health inequities. This study evaluated the effectiveness of a salutogenic health promotion program focused on the empowerment of migrant women at risk of social exclusion. METHODS: A four-session salutogenic health promotion program was conducted over a period of 6 months. In a quasi-experimental pre-test post-test design, an ad hoc questionnaire was administered to 26 women to collect sociodemographic data, together with 5 validated instruments: Antonovsky's Sense of Coherence (SOC-13), Duke-UNC-11 (perceived social support), Quality of Life Short Form-36 (SF-36), Rosenberg's Self-Esteem Scale, and the Cohen et al. Perceived Stress Scale (PSS-10). Descriptive analysis and multiple linear regression models were performed. Statistical tests were considered significant with a two-tailed p value < 0.05. RESULTS: Participants had a low initial SOC-13 score (60.36; SD 8.16), which did not show significant change after the health promotion program. Perceived social support (37.07; SD 6.28) and mental quality of life also remained unchanged, while physical quality of life increased from 50.84 (SD 4.60) to 53.08 (SD 5.31) (p = 0.049). Self-esteem showed an increasing trend from 30.14 (SD 4.21) to 31.92 (SD 4.38) (p = 0.120). Perceived stress decreased from 20.57 (SD 2.91) to 18.38 (SD 3.78) (p = 0.016). A greater effect was observed at the end of the program in women with lower initial scores for SOC-13 and quality of life and higher initial scores of perceived stress. CONCLUSIONS: The health promotion program reduced perceived stress, increased physical quality of life and showed a trend toward increased self-esteem, especially among migrant women with multiple vulnerability factors. The salutogenic model of health should be considered as a good practice to apply in health promotion programs and to be included in national policies to reduce health inequity in migrant populations.

Defective glucose and lipid metabolism in rheumatoid arthritis is determined by chronic inflammation in metabolic tissues.

BACKGROUND: Rheumatoid arthritis (RA) patients are at increased risk of insulin resistance (IR); however, the specific mechanisms mediating this association are currently unknown. OBJECTIVE: To investigate whether the inflammatory activity associated with RA accounts for the observed defective glucose metabolism and lipid metabolism in these patients. METHODS: We followed two main strategies: (i) extensive metabolic profiling of a RA cohort of 100 patients and 50 healthy control subjects and (ii) mechanistic studies carried out in both a collagen-induced arthritis mouse model and 3T3-L1 adipocytes treated with conditioned serum from RA patients. RESULTS: Following the exclusion of obese and diabetic subjects, data from RA patients demonstrated a strong link between the degree of systemic inflammation and the development of IR. These results were strengthened by the observation that induction of arthritis in mice resulted in a global inflammatory state characterized by defective carbohydrate and lipid metabolism in different tissues. Adipose tissue was most susceptible to the RA-induced metabolic alterations. These metabolic effects were confirmed in adipocytes treated with serum from RA patients. CONCLUSIONS: Our results show that the metabolic disturbances associated with RA depend on the degree of inflammation and identify inflammation of adipose tissue as the initial target leading to IR and the associated molecular disorders of carbohydrate and lipid homeostasis. Thus, we anticipate that therapeutic strategies based on tighter control of inflammation and flares could provide promising approaches to normalize and/or prevent metabolic alterations associated with RA.

Diagnostic potential of NETosis-derived products for disease activity, atherosclerosis and therapeutic effectiveness in Rheumatoid Arthritis patients.

OBJECTIVES: 1) To assess the association of NETosis and NETosis-derived products with the activity of the disease and the development of cardiovascular disease in RA; 2) To evaluate the involvement of NETosis on the effects of biologic therapies such as anti-TNF alpha (Infliximab) and anti-IL6R drugs (Tocilizumab). METHODS: One hundred and six RA patients and 40 healthy donors were evaluated for the occurrence of NETosis. Carotid-intimae media thickness was analyzed as early atherosclerosis marker. Inflammatory and oxidative stress mediators were quantified in plasma and neutrophils. Two additional cohorts of 75 RA patients, treated either with Infliximab (n = 55) or Tocilizumab (n = 20) for six months, were evaluated. RESULTS: NETosis was found increased in RA patients, beside myeloperoxidase and neutrophil elastase protein levels. Cell-free nucleosomes plasma levels were elevated, and strongly correlated with the activity of the disease and the positivity for autoantibodies, alongside inflammatory and oxidative profiles in plasma and neutrophils. Moreover, ROC analyses showed that cell-free nucleosomes levels could identify RA patients showing early atherosclerosis with high specificity. RA patients treated either with IFX or TCZ for six months exhibited decreased generation of NETs. Concomitantly, clinical parameters and serum markers of inflammation were found reduced. Mechanistic in vitro analyses showed that inhibition of NETs extrusion by either DNase, IFX or TCZ, further abridged the endothelial dysfunction and the activation of immune cells, thus influencing the global activity of the vascular system. CONCLUSIONS: NETosis-derived products may have diagnostic potential for disease activity and atherosclerosis, as well as for the assessment of therapeutic effectiveness in RA.

[Vogt-Koyanagi-Harada disease. Characteristics of a series of Andalusian patients]. / Enfermedad de Vogt-Koyanagi-Harada. Características de un grupo de pacientes andaluces.

BACKGROUND: To describe the characteristics of a series of patients diagnosed of Vogt-Koyanagi-Harada disease (VKHD) and controlled by an Uveitis Unit (composed of ophthalmologists and internists) in our population. PATIENTS AND METHOD: Retrospective descriptive study of 11 patients with VKHD (5 males and 6 women; median age at diagnosis 32.6 years old) followed-up between 1980 and 2003. RESULTS: All patients suffered panuveitis and/or exudative retinal detachment. Extraocular signs were present in all cases: neurological in 63.7% (aseptic meningitis and/or focal symptoms), cutaneous in 81.8% (vitiligo, whiteness, poliosis, alopecia), neurosensorial hypoacusis (50% of patients with audiometry), and general symptoms in 25%. They all received systemic corticosteroids. Cyclosporine was added in 5 patients (45.4%) with posterior uveitis, and azathioprine in 2 of them for iridocyclitis. The final visual acuity was 0.5 or better in 81.8% of cases, but 2 patients had an unfavourable evolution (one, who had cataract and band keratopathy in the left eye and severe visual worsening, needed right vitrectomy, and the other suffered severe ocular hypotension). Complications developed in 2 other patients: cataract and glaucoma in one, and synechiae without glaucoma in the other. CONCLUSIONS: We found less frequency of exudative retinal detachment and greater frequency of cutaneous signs than the communicated. A significant percentage of cases needed immunosuppressive agents. Final visual acuity was good in the majority of patients.

[Partial purification and use of Trypanosoma cruzi glycosidic fractions for Chagas disease diagnosis]. / Purificación parcial y empleo de fracciones glicosídicas de Trypanosoma cruzi en el diagnóstico de la enfermedad de Chagas.

Protein and glycoprotein fractions are used for Chagas' disease diagnosis, but they are degraded by parasite endogenous proteases. Therefore protease inhibitors are used for their conservation increasing the antigen cost. The possibility of using protease-resistant glycosidic fractions could solve this problem allowing to obtain stable antigens, with a high sensitivity at a lower cost. This proposal is reinforced by the existence of anti-galactosyl antibodies against T. cruzi oligosaccharic fractions in sera from chagasic patients. The aim of this work was to obtain T. cruzi glycosidic fractions and to evaluate their possible use for Chagas' disease serologic diagnosis. Total protein and glycoproteins from the four stages of T. cruzi were obtained. The glycosidic fractions were obtained by exhaustive proteolysis with Proteinase K. Protein, glycoprotein and glycosidic profiles were analysed by SDS-PAGE followed by staining proteins (Coomassie/silver) or glycoproteins and glycosidic fractions (APABGP). Antigenicity of the different fractions was determined by Western Blot and luminography, using control hyperimmune serum anti-epimastigote and a "pool" of chagasic human sera. Finally, the capacity of the glycosidic fractions to discriminate chagasic and non-chagasic human sera and the sensitivity of the fractions respect to control serum, were determined by ELISA. Main findings were: a) there are peptides, glycopeptides and glycosidic fractions that are common and specific to parasite stages; b) there are more antigenic glycoproteins in epimastigotes and metacyclics than in trypomastigotes and amastigotes; c) there is a correlation between the glycosidic fraction-pattern and the host type; d) glycosidic fractions can be used as antigens to discriminate, by ELISA, chagasic and non chagasic patients, but show lower titers with respect to total proteic and glycoprotein antigens. It is concluded that it is possible to develop a diagnostic kit for Chagas' disease employing glycosidic fractions, eliminating the disadvantages of the current kits.

Oral administration of a turmeric extract inhibits LDL oxidation and has hypocholesterolemic effects in rabbits with experimental atherosclerosis.

The oxidation of low-density lipoproteins (LDL) plays an important role in the development of atherosclerosis. Curcumin is a yellow pigment obtained from rhizomes of Curcuma longa and is commonly used as a spice and food colouring. Curcumin and turmeric extracts have several pharmacological effects including antitumour, anti-inflammatory, antioxidant and antiinfectious activities although the precise mechanisms involved remain to be elicited. We evaluated the effect of an ethanol-aqueous extract obtained from rhizomes of C. longa on LDL oxidation susceptibility and plasma lipids in atherosclerotic rabbits. A total of 18 rabbits were fed for 7 weeks on a diet containing 95.7% standard chow, 3% lard and 1. 3% cholesterol, to induce atherosclerosis. The rabbits were divided into groups, two of which were also orally treated with turmeric extract at doses of 1.66 (group A) and 3.2 (group B) mg/kg body weight, respectively. A third group (group C) acted as a control. Plasma and LDL lipid composition, plasma alpha-tocopherol, plasma retinol, LDL TBARS, LDL lipid hydroperoxides and analysis of aortic atherosclerotic lesions were assayed. The low but not the high dosage decreased the susceptibility of LDL to lipid peroxidation. Both doses had lower levels of total plasma cholesterol than the control group. Moreover, the lower dosage had lower levels of cholesterol, phospholipids and triglycerides in LDL than the 3.2-mg dosage. In conclusion, the use of this extract could be useful in the management of cardiovascular disease in which atherosclerosis is important.

[The use of interferon alfa-2r in the treatment of autoimmune uveitis (primary or associated with Behçet's disease)]. / Utilización de interferón alfa 2r en el tratamiento de las uveítis autoinmunes (primarias o asociadas a enfermedad de Behçet).

BACKGROUND: The improvement in ocular inflammation in patients with cyclosporine A resistant Behçet disease (CyA) during therapy of severe aphthosis with recombinant interferon-alpha 2b (IFN alpha) prompted its evaluation for treatment of refractory autoimmune uveitis. METHOD: IFN alpha was used in 18 patients with posterior uveitis, nine with Behçet disease and nine with primary uveitis (mean evolution time 3.8 years) previously treated with corticosteroids (18), CyA (16) and azathioprine (1). The dose was 5 MU/day (4 weeks) and then twice weekly (12 weeks) maintaining the previous therapy. Evaluations were made at the start of the study, two weeks and two months and a cross-sectional study in February 1995. RESULTS: At the early phase both vitreal cellularity (p = 0.01) and macular edema (p = 0.003) improved; at the late phase improvements were noted in vitreal cellularity (p < 0.0001), macular (p < 0.0001) and papillar edema (p = 0.04) and visual acuity (p = 0.006). In February 1995 (mean evolution time 30 months), ten patients (56%) remain without treatment with inactive disease, six (33%) with CyA and two (11%) with CyA and IFN alpha. Improvements in vitreal cellularity (p = 0.0001), macular edema (p = 0.0001) and visual acuity (p = 0.013) were still present although macular ischemia was more severe than at the beginning (p = 0.035). The most important adverse reactions (late reactions) included depression (three cases) and thyroid changes (two cases). CONCLUSIONS: IFN alpha is an important therapeutic alternative for posterior uveitis refractory to corticosteroid therapy (included CyA).

Asp537, Asp812 are essential and Lys631, His811 are catalytically significant in bacteriophage T7 RNA polymerase activity.

To define catalytically essential residues of bacteriophage T7 RNA polymerase, we have generated five mutants of the polymerase, D537N, K631M, Y639F, H811Q and D812N, by site-directed mutagenesis and purified them to homogeneity. The choice of specific amino acids for mutagenesis was based upon photoaffinity-labeling studies with 8-azido-ATP and homology comparisons with the Klenow fragment and other DNA/RNA polymerases. Secondary structural analysis by circular dichroism indicates that the protein folding is intact in these mutants. The mutants D537N and D812N are totally inactive. The mutant K631M has 1% activity, confined to short oligonucleotide synthesis. The mutant H811Q has 25% activity for synthesis of both short and long oligonucleotides. The mutant Y639F retains full enzymatic activity although individual kinetic parameters are somewhat different. Kinetic parameters, (kcat)app and (Km)app for the nucleotides, reveal that the mutation of Lys to Met has a much more drastic effect on (kcat)app than on (Km)app, indicating the involvement of K631 primarily in phosphodiester bond formation. The mutation of His to Gln has effects on both (kcat)app and (Km)app; namely, three- to fivefold reduction in (kcat)app and two- to threefold increase in (Km)app, implying that His811 may be involved in both nucleotide binding and phosphodiester bond formation. The ability of the mutant T7 RNA polymerases to bind template has not been greatly impaired. We have shown that amino acids D537 and D812 are essential, that amino acids K631 and H811 play significant roles in catalysis, and that the active site of T7 RNA polymerase is composed of different regions of the polypeptide chain. Possible roles for these catalytically significant residues in the polymerase mechanism are discussed.

Heterogeneous forms of polymerase proteins exist in influenza A virus-infected cells.

In influenza virus-infected cells a virus coded polymerase that consists of three polypeptide subunits, namely PB1, PB2 and PA, mediates both transcription and replication. Radioimmunoprecipitation with monospecific antisera to each of the polymerase proteins revealed additional forms of PB1 and PA proteins in infected cells. PA antiserum detected two additional proteins of 62k and 60k and PB1 antiserum recognized two additional proteins of 85k and 70k. Further investigation was carried out on the 62k PA and 85k PB1 related proteins. Limited proteolysis peptide mapping showed that these proteins are subsets of their normal counter-parts. These new forms of polymerase proteins are designated as "b" forms (PAb and PB1b) to distinguish them from the previously recognized forms designated as "a" forms (PAa and PB1a). Both PAb and PB1b proteins were found in cells infected with all the influenza type A viruses tested indicating that they are evolutionarily conserved. Pulse chase experiments showed that the "b" forms are not derived from "a" forms. This suggested that "b" forms are translated independently. The "b" forms were not detected in purified virus but were found to be associated with intracellular RNP templates, suggesting a role for these proteins in intracellular virus replication events.