Araceli Páez

17β-estradiol inhibits the adhesion of leukocytes in TNF-α stimulated human endothelial cells by blocking IL-8 and MCP-1 secretion, but not its transcription

Inflammation, and especially mononuclear cell adhesion to endothelium, is an important physiopathological component of atherosclerosis. Since coronary heart disease in women of reproductive age and/or with estrogen replacement therapy is reduced, our aim was to determine if 17β-estradiol had a regulatory effect on the adhesion of lymphocytes to the endothelium. We performed U-937 cells adhesion assays in TNF-α-stimulated HUVECs, and we also quantitated IL-8 and MCP-1 in culture supernatants, in the presence or not of 17β-estradiol. The presence of α- and β-estrogen receptors was determined by Western blot and RT-PCR, respectively, whereas the transcription of both chemokines was evaluated by RT-PCR. The results showed a 35% decrease in the adhesion of U-937 monocyte cells to TNF-α-stimulated HUVECs, and a 54% and 65% inhibition of TNF-α-induced IL-8 and MCP-1 secretion by physiological and physiologically high doses of 17β-estradiol. The hormone did not affect the transcription of both chemokine genes. Tamoxifen reverted the inhibitory effect induced by 17βestradiol. In conclusion, 17β-estradiol modifies the adhesion of leukocytes to endothelial cells by inhibiting the secretion, but not the gene transcription, of proinflammatory chemokines.

Statin-induced inhibition of MCF-7 breast cancer cell proliferation is related to cell cycle arrest and apoptotic and necrotic cell death mediated by an enhanced oxidative stress.

Statins have antiproliferative and anti-tumoral effects in MCF-7 cells. We determined the effect of statins upon MCF-7 cell cycle, toxicity, cell death, reactive oxygen species (ROS) production and mitochondrial membrane potential. Fluvastatin, simvastatin and atorvastatin inhibited cell proliferation. Antiproliferation was associated with a decrease in the DNA synthesis and a cell cycle arrest in the G1 and G2/M phases. A loss in the mitochondrial membrane potential was observed with fluvastatin. Statins induced increase in ROS production that was associated with cell death, which was abrogated by the antioxidant NAC. Our results suggest that the cytotoxic effect observed is mediated by an oxidative stress.

HUVECs from newborns with a strong family history of diabetes show diminished ROS synthesis in the presence of high glucose concentrations.

A family history of type 2 diabetes mellitus (DM) increases the probability to develop DM and endothelial dysfunction. The probable mechanism involves augmented reactive oxygen species (ROS) synthesis. The aim of this study was to evaluate the synthesis of ROS in human umbilical vein endothelial cells (HUVECs) obtained from healthy newborns with (experimental) and without (control) a strong family history of type 2 DM, exposed to different glucose concentrations.

The altered expression of inflammation-related molecules and secretion of IL-6 and IL-8 by HUVEC from newborns with maternal inactive systemic lupus erythematosus is modified by estrogens.

Systemic lupus erythematosus (SLE) predominantly affects women, especially those in reproductive age. Genetic contributions to disease susceptibility as well as immune dysregulation, particularly persistent inflammatory responses, are considered essential features. Our aim was to determine whether human umbilical vein endothelial cells (HUVEC) isolated from healthy newborns to women with inactive SLE show inflammation-related abnormalities that might lead to an early development of SLE in the offsprings. HUVEC isolated from six women with inactive SLE were stimulated with 2.5 ng/mL of TNF-α and/or physiological and pharmacological doses of 17-β estradiol (E2). Then the expression of VCAM-1, ICAM-1, E-selectin, toll-like receptor-9 (TLR-9), heat shock protein 70 (HSP70) and HSP90 were measured. The concentrations of IL-6, IL-8, and IL-10 were also determined in maternal serum and in TNF-α stimulated and non-stimulated HUVEC culture supernatant. HUVEC from children with no family history of autoimmune disease served as controls. Our results showed that in HUVEC from SLE+ mothers, a constitutively low expression of adhesion molecules was enhanced by TNF-α treatment. The E2 (1 ng/mL) increased the expression of adhesion molecules but had no effect upon TNF-α-treated cells. IL-6 was constitutively higher in SLE+ HUVEC, whereas IL-8 was lower; E2 treatment diminished the latter. The E2 had no effect upon IL-6 and IL-8 secretions in TNF-α-treated cells. SLE+ HUVEC showed a disordered cytoskeleton and overexpressed HSP70, HSP90, and TLR-9. Our results indicate that endothelial cells of newborns to SLE+ mothers are in a proinflammatory condition which can be upregulated by estrogens.

Secretion of IFN-γ by bovine peripheral blood mononuclear cells stimulated with Mycobacterium bovis protein fractions obtained by isoelectric-focusing

Due to the complexity and variety of biological effects found in Mycobacterium bovis (M. bovis) proteins analyzed solely on a molecular weight (MW) basis, we approached the purification of M. bovis proteins through their isoelectric point (pI). Twenty M. bovis culture filtrate protein extract (CFPE) isoelectric focused (IEF) protein fractions, confined between pI3 and 10, were isolated. The MW of the major proteins isolated in the various fractions correlated with protein already reported 14-, 18-, 20-, 25-, 31-, 38-, 45-, 64-, 67- and 70 kDa by SDS-PAGE. Since several different pI fractions showed proteins of the same MW we tested the ability of all IEF fractions to stimulate interferon-gamma (IFN-gamma) production by peripheral blood mononuclear cells (PBMC) isolated from cattle with well defined M. bovis tuberculosis (TB) infection. In animals with few lesions IFN-gamma inductive IEF fractions were in the acid range. As the number of lesions increased, neutral fractions were also inductive. Some fractions with relatively few proteins induced as much IFN-gamma production as others with abundant proteins. None of the 20 IEF fractions enhanced IFN-gamma production by anergic cells. We conclude that IFN-gamma production in diseased animals is induced mainly by acidic mycobacterial proteins and that the response towards these proteins is enhanced as the disease progresses, what coincides with higher PPD reactivity. However, the IFN-gamma production in anergic status was severely affected. We found that this cytokine production is spontaneous and antigen-independent.

IDENTIFICATION OF A 20-KDA PROTEIN WITH CALCIUM UPTAKE TRANSPORT ACTIVITY.RECONSTITUTION IN A MEMBRANE MODEL

This paper presents results of experiments designed to further purify the membrane system involved in mitochondrial calcium transport. A partially purified extract, which transported calcium with a specific activity of 1194 nmol45Ca2+/mg protein/5 min, was used to obtain mouse hyperimmune serum. This serum inhibited calcium uptake both in mitoplasts and in vesicles reconstituted with mitochondrial proteins containing cytochrome oxidase. Western blot analysis of the semipurified fraction showed that the serum recognized specifically two antigens of 75 and 20 kDa. Both antibodies were purified by elution from the nitrocellulose sheets and their inhibition capacity was analyzed. The antibody that recognized the 20-kDa protein produced a higher degree of inhibition than the other one.

Collagen degrading activity associated with Mycobacterium species.

BACKGROUND The mechanism of Mycobacterium tuberculosispenetration into tissues is poorly understood but it is reasonable to assume that there is a contribution from proteases capable of disrupting the extracellular matrix of the pulmonary epithelium and the blood vessels. A study was undertaken to identify and characterise collagen degrading activity of M tuberculosis. METHODS Culture filtrate protein extract (CFPE) was obtained from reference mycobacterial strains and mycobacteria isolated from patients with tuberculosis. The collagen degrading activity of CFPE was determined according to the method of Johnson-Wint using 3H-type I collagen. The enzyme was identified by the Birkedal-Hansen and Taylor method and its molecular mass determined by SDS-PAGE and Sephacryl S-300 gel filtration chromatography using an electroelution purified enzyme. RESULTS CFPE fromMycobacterium tuberculosis strain H37Rv showed collagenolytic activity that was four times higher than that of the avirulent strain H37Ra. The 75 kDa enzyme responsible was divalent cation dependent. Other mycobacterial species and those isolated from patients with tuberculosis also had collagen degrading activity. CONCLUSIONS Mycobacteriumspecies possess a metalloprotease with collagen degrading activity. The highest enzymatic activity was found in the virulent reference strain H37Rv.

Purification of a N-acetyl-d-galactosamine specific lectin from the orchid Laelia autumnalis

From the pseudobulbs of the orchid L. autumnalis a lectin was purified on immobilized porcine mucin with A + H blood group substance. This lectin is a dimeric glycoprotein of M(r) 12,000 with an Sw,20 of 2.2, showing haemagglutinating activity directed mainly to human A1 desialylated erythrocytes. The lectin possesses sugar specificity for N-acetyl-D-galactosamine and also shows high specificity for glycoproteins containing the T (galactose beta 1,3GA1NAc alpha 1,0 Ser/Thr) or the Tn antigen (GalNAc alpha 1,0 Ser/Thr).

Type Iii Interferons in Systemic Lupus Erythematosus: Association Between Interferon λ3, Disease Activity, and Anti-ro/ssa Antibodies

Objective The aim of this study was to assess associations between serum type III (&lgr;) interferons (IFN-&lgr;) and disease activity in systemic lupus erythematosus (SLE). Methods Serum levels of IFN-&lgr;1, IFN-&lgr;2, and IFN-&lgr;3 were measured in 93 SLE patients and 67 healthy individuals. The associations with overall disease activity, organ-specific damage, and SLE-related antibodies were assessed. Results Median IFN-&lgr;1 levels were 0 pg/mL (range, 0–510 pg/mL) and 0 pg/mL (0–171 pg/mL; P = 0.814) in SLE patients and control subjects, respectively. These figures were 0 pg/mL (0–28 pg/mL) and 0 pg/mL (0–43 pg/mL; P = 0.659) for IFN-&lgr;2, as well as 83 pg/mL (0–965 pg/mL) and 42 pg/mL (0–520 pg/mL; P = 0.002) for IFN-&lgr;3, respectively. According to the Systemic Lupus Erythematosus Disease Activity Index categories, IFN-&lgr;3 levels were 44 pg/mL (0–158 pg/mL) in quiescent, 117 pg/mL (0–344 pg/mL) in mild, 79 pg/mL (0–965 pg/mL) in moderate, and 78 pg/mL (0–329 pg/mL) in severe disease, with the highest levels found in patients with serosal or cutaneous involvement. In line with this, IFN-&lgr;3 levels were inversely correlated with C3 (&rgr; = −0.44; 95% confidence interval, −0.62 to −0.20; P = 0.0003) and C4 (&rgr; = −0.40; 95% confidence interval, −0.59 to −0.15; P = 0.0001) complement proteins. In addition, higher IFN-&lgr;3 levels were found in patients positive for anti-Ro/SSA antibodies than in those negative for that antibody (122 pg/mL [0–965 pg/mL] vs. 0 pg/mL [0–165 pg/mL]; P = 0.001). The concentration of IFN-&lgr;3 also was higher in patients receiving glucocorticoids (104 pg/mL [0–965 pg/mL] vs. 30 pg/mL [0–165 pg/mL]; P = 0.009), and a dose-related effect was observed. Conclusions Interferon &lgr;3, a subtype of type III IFNs, is associated with the extent of lupus activity, in particular with active serosal and cutaneous disease. This association could be mechanistically related to anti-Ro/SSA antibodies.OBJECTIVE The aim of this study was to assess associations between serum type III (λ) interferons (IFN-λ) and disease activity in systemic lupus erythematosus (SLE). METHODS Serum levels of IFN-λ1, IFN-λ2, and IFN-λ3 were measured in 93 SLE patients and 67 healthy individuals. The associations with overall disease activity, organ-specific damage, and SLE-related antibodies were assessed. RESULTS Median IFN-λ1 levels were 0 pg/mL (range, 0-510 pg/mL) and 0 pg/mL (0-171 pg/mL; P = 0.814) in SLE patients and control subjects, respectively. These figures were 0 pg/mL (0-28 pg/mL) and 0 pg/mL (0-43 pg/mL; P = 0.659) for IFN-λ2, as well as 83 pg/mL (0-965 pg/mL) and 42 pg/mL (0-520 pg/mL; P = 0.002) for IFN-λ3, respectively. According to the Systemic Lupus Erythematosus Disease Activity Index categories, IFN-λ3 levels were 44 pg/mL (0-158 pg/mL) in quiescent, 117 pg/mL (0-344 pg/mL) in mild, 79 pg/mL (0-965 pg/mL) in moderate, and 78 pg/mL (0-329 pg/mL) in severe disease, with the highest levels found in patients with serosal or cutaneous involvement. In line with this, IFN-λ3 levels were inversely correlated with C3 (ρ = -0.44; 95% confidence interval, -0.62 to -0.20; P = 0.0003) and C4 (ρ = -0.40; 95% confidence interval, -0.59 to -0.15; P = 0.0001) complement proteins. In addition, higher IFN-λ3 levels were found in patients positive for anti-Ro/SSA antibodies than in those negative for that antibody (122 pg/mL [0-965 pg/mL] vs. 0 pg/mL [0-165 pg/mL]; P = 0.001). The concentration of IFN-λ3 also was higher in patients receiving glucocorticoids (104 pg/mL [0-965 pg/mL] vs. 30 pg/mL [0-165 pg/mL]; P = 0.009), and a dose-related effect was observed. CONCLUSIONS Interferon λ3, a subtype of type III IFNs, is associated with the extent of lupus activity, in particular with active serosal and cutaneous disease. This association could be mechanistically related to anti-Ro/SSA antibodies.

Association of Nail Dystrophy With Accrued Damage and Capillaroscopic Abnormalities in Systemic Lupus Erythematosus.

BackgroundThere are some nail abnormalities described in systemic lupus erythematosus (SLE). ObjectivesThe aim of this study was to evaluate the association between nail dystrophy (ND) and disease activity, accrued organ damage, capillaroscopic abnormalities, autoantibodies, and some markers of endothelial cell activation in patients with SLE. MethodsThis was a cross-sectional study of SLE patients from a rheumatology clinic in a tertiary care hospital. Patients were allocated in groups, according to the presence or absence of ND. Demographics, clinical data, disease activity, accrued damage, serology, nailfold capillaroscopy characteristics, serum levels of anti–endothelial cell antibodies, and plasma levels of endothelin 1 were compared between groups. Disease activity was assessed by the Systemic Lupus Erythematosus Disease Activity Index 2000 index and accrued organ damage by the Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index. ResultsSixty-one patients were included; 50 patients (82%) were female. Thirty-two patients (52.5%) showed ND, and 29 did not. Besides a more frequent use of cyclophosphamide (46.9% vs 20.7%; P = 0.03) in the ND group, clinical features were similar. A greater organ damage was found in patients with ND (median Systemic Lupus International Collaborating Clinics/American College of Rheumatology index = 0.5, minimum = 0, maximum = 6) than in patients without ND (0, 0, 3, respectively; P = 0.04); specifically, only the skin domain was associated with ND (P = 0.04). Onycholysis (40.6%) and longitudinal ridging (25%) were the most frequent nail changes. Nailfold capillaroscopy changes were more frequent in ND patients (40.6%) than in control subjects (13.8%) (P = 0.02). The most frequent nailfold capillaroscopy findings in the ND group were enlarged capillaries (40.6%) and microhemorrhages (12.5%). There was no association between ND and the autoantibody profile, plasma endothelin 1, or serum anti–endothelial cell antibodies. ConclusionsNail dystrophy was associated with higher accrued organ damage and the presence of capillaroscopic abnormalities. This suggests that ND might be related to chronic microvascular damage.