Myostatin mediates abdominal aortic atherosclerosis progression by inducing vascular smooth muscle cell dysfunction and monocyte recruitment.

Myostatin (Mstn) is a skeletal muscle growth inhibitor involved in metabolic disorders and heart fibrosis. In this study we sought to verify whether Mstn is also operative in atherosclerosis of abdominal aorta. In human specimens, Mstn expression was almost absent in normal vessels, became detectable in the media of non-progressive lesions and increased with the severity of the damage. In progressive atherosclerotic lesions, Mstn was present in the media, neointima, plaque shoulder and in infiltrating macrophages. Mstn co-localized with α-smooth muscle actin (α-SMA) staining and with some CD45+ cells, indicating Mstn expression in VSMCs and bloodstream-derived leukocytes. In vitro, Mstn was tested in VSMCs and monocytes. In A7r5 VSMCs, Mstn downregulated proliferation and Smoothelin mRNA, induced cytoskeletal rearrangement, increased migratory rate and MCP-1/CCR2 expression. In monocytes (THP-1 cells and human monocytes), Mstn acted as a chemoattractant and increased the MCP-1-dependent chemotaxis, F-actin, α-SMA, MCP-1 and CCR2 expression; in turn, MCP-1 increased Mstn mRNA. Mstn induced JNK phosphorylation both in VSMCs and monocytes. Our results indicate that Mstn is overexpressed in abdominal aortic wall deterioration, affects VSMCs and monocyte biology and sustains a chronic inflammatory milieu. These findings propose to consider Mstn as a new playmaker in atherosclerosis progression.

Endothelin receptor antagonists: effects on extracellular matrix synthesis in primary cultures of skin fibroblasts from systemic sclerosis patients.

Endothelin-1 (ET-1) seems to enhance the pro-fibrotic protein synthesis by skin fibroblasts and its effects are mediated by endothelin-A and B (ETA and ETB) receptors. This study aimed to investigate the effects of ETA and ETB receptor antagonists (ETARA-sitaxsentan and ETA/BRA-bosentan) on type I collagen (COL-1), fibronectin (FN) and fibrillin-1 (FBL-1) synthesis in primary cultures of skin fibroblasts from systemic sclerosis patients. Primary cultures of fibroblasts were obtained from skin biopsies of 6 female systemic sclerosis patients and were treated with ET-1 (100 nM) for 24 and 48 hrs with or without pre-treatment (1 hr) with ETARA (2 µM) or ETA/BRA (10 µM). Primary culture of human scleroderma skin fibroblasts not treated with ET-1 or ET receptor antagonists (ETARA and ETA/BRA) were used as controls. COL-1, FN and FBL-1 synthesis was evaluated by immunocytochemistry and Western blot analysis. Immunocytochemistry and Western blot analysis showed that ET-1 significantly increased COL-1 and FN synthesis at 24 and 48 hrs and FBL-1 synthesis at 48 hrs vs untreated cells. ETARA significantly contrasted the ET-1-mediated increase in COL-1 and FN at 24 hrs as well as COL-1 and FBL-1 at 48 hrs, but not FN synthesis vs ET-1-treated fibroblasts. Conversely, ETA/BRA significantly antagonized the ET-1-mediated overproduction of COL-1 and FN both at 24 and 48 hrs and the FBL-1 synthesis at 48 hrs vs ET-1-treated cells. The single ETARA treatment seems to contrast significantly the increase in COL-1 synthesis, whereas the dual ETA/BRA treatment seems active in significantly antagonizing both COL-1 and FN overproduction induced by ET-1. In conclusion, ET-1 antagonism might have positive effects in contrasting the profibrotic activity of systemic sclerosis skin fibroblasts.

[CTLA4-Ig interferes and downregulates the proinflammatory activities of rheumatoid synovial macrophages in monoculture]. / CTLA4-Ig influenza e inibisce le attività proinfiammatorie di macrofagi sinoviali reumatoidi in monocoltura.

OBJECTIVE: CTLA4-Ig, a biologic agent employed in rheumatoid arthritis (RA) treatment, downregulates the immune response and exerts anti-inflammatory effects acting on different cells including dendritic/T cells interaction and directly on osteoclasts. We investigated the anti-inflammatory effects of CTLA4-Ig in primary monocultures of RA synovial macrophages (SM). METHODS: SM were obtained, from 8 RA patients (7 F, 1 M; DAS28>5.2) who underwent therapeutic arthroscopic synoviectomy and were cultured in the absence and in the presence of CTLA4-Ig at the concentration of [500 microg/ml], the most reliable dose related to the previous pharmacological clinical and experimental experiences. Inflammatory cytokine (IL-6, TNFalpha, IL-1beta) expression was evaluated by immunocytochemistry (ICC with relative image analysis), western blot (WB), and quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: ICC analysis revealed that CTLA4-Ig treatment significantly downregulated cytokine expression (p<0.001 for IL-6, TNFalpha and IL-1beta) when compared to untreated RA SM. WB and qRT-PCR confirmed partially the data. CONCLUSIONS: CTLA4-Ig was found to exert a direct and significant anti-inflammatory effect on primary monocultures of RA SM, suggesting a therapeutic power in different phases of the disease activity.

Evaluation of metabolic acidosis in patients with a kidney graft: comparison of the bicarbonate-based and strong ion-based methods.

BACKGROUND: According to the traditional bicarbonate-based approach, metabolic acidosis is highly prevalent in kidney transplant recipients. However, the bicarbonate-based approach has been questioned by intensivists using strong ion difference-based methods. METHODS: We compared the results obtained by the strong ion-based with the traditional approach based on bicarbonate among a cohort of 83 kidney transplant recipients. RESULTS: Fifty-five percent of the patients were acidotic based on venous bicarbonate (<23 mmol/L) and 49% by the use of the effective strong ion difference (SID(effective)) (<37 mmol/L). Bicarbonate and SID(effective) were linearly correlated (r=0.94; P<.0001), with a slope close to 1. A greater percentage of patients presented with an increase in unexplained anions by the strong ion gap (SIG) than by the anion gap corrected (AG(corrected)) method (42 vs 32%, respectively). AG(corrected) and SIG were directly related (r=0.919; P<.0001), but the best fit of the relationship was polynomial with a progressively greater effect on SIG with increased AG(corrected), suggesting that as anions progressively accumulate, their detection by SIG increases. By multiple regression analysis, plasma chloride, potassium, uric acid, and phosphate predicted blood bicarbonate. Analogously, chloride, potassium and uric acid predicted SID(effective). Age was a predictor of changes in AG(corrected), whereas age and plasma urea predicted SIG. CONCLUSIONS: The use of the SID yielded results that were similar to the traditional bicarbonate-based approach. Conversely, SIG appeared to be more sensitive than AG for detection of anion accumulation among patients with a kidney graft.

Endothelin and sex hormones modulate the fibronectin synthesis by cultured human skin scleroderma fibroblasts.

OBJECTIVE: To evaluate the influence of endothelin-1 (ET-1) and sex hormones on cell proliferation and extracellular matrix (ECM) synthesis (ie, fibronectin, laminin) by cultured normal and scleroderma (SSc) human skin fibroblasts (FBs). METHODS: Primary cultures of FBs were treated with ET-1 and sex hormones (17beta-oestradiol or testosterone) for 24 h. Cell growth was analysed by methiltetrazolium salt test, ECM synthesis was evaluated by immunocytochemistry and western blot, both at 24 h. RESULTS: In normal FBs, ET-1 and 17beta-oestradiol, as well as their combination, increased cell growth (p<0.001, p<0.001, p<0.01 vs untreated cells (control), respectively) and fibronectin synthesis (p<0.05, p<0.05, p<0.01 vs control, respectively). By contrast, testosterone either alone or in combination with ET-1 did not influence cell proliferation, but decreased fibronectin synthesis (p<0.05, testosterone vs control). In SSc FBs, ET-1 and 17beta-oestradiol alone or their combination induced an increased fibronectin synthesis (p<0.05, p<0.05, p<0.01 vs control, respectively). Unexpectedly, testosterone induced an increase of fibronectin synthesis (p<0.05 vs control). CONCLUSIONS: ET-1 and 17beta-oestradiol seem to exert a profibrotic effect in normal and SSc culture FBs and might suggest their synergistic effect in the pathogenesis of the fibrotic process in SSc.

Hydroxylated estrogen metabolites influence the proliferation of cultured human monocytes: possible role in synovial tissue hyperplasia.

INTRODUCTION: 17Beta-estradiol, estrone, and several of their hydroxylated metabolites, have been found to be significantly increased in synovial fluid of rheumatoid arthritis (RA) patients. In this study, we investigated whether the estrogen metabolites are able to exert direct effects on monocyte cell proliferation, which is important in RA synovial tissue activation and growth. METHODS: Human monocytes (THP-1) were treated with the following estrogen metabolites at different concentrations (from 10-8M, 10-9M, 10-10M to 10-11M) for 24, 48 and 72 hours: 16-hydroxyestrone (16OH-E1), 16-hydroxyestradiol (16OH-E2), 4-hydroxyestrone (4OH-E1), 4-hydroxyestradiol (4OH-E2), 2-hydroxyestrone (2OH-E1) and 2-hydroxyestradiol (2OH-E2). Monocytes were activated with interferon-gamma (INF-gamma). Cell cultures were also performed in presence of tamoxifen (10-7M) to evaluate whether the estrogen metabolites act through the estrogen receptors (ER). Cell growth was detected by MTT test and cell viability through the LDH release assay. RESULTS: 4OH-E1 and 2OH-E1 significantly increased cell growth at low concentration (10-10M), whereas they significantly reduced cell proliferation at high concentrations (10-9M). 16OH-E2 and 4OH-E2 induced opposite effects: cell proliferation at high concentration and antiproliferative action at low doses. On the contrary, 16OH-E1 and 2OH-E2 were found to be estrogen metabolites that induced cell proliferative effects for most of the tested doses. Tamoxifen caused the loss of effects on cell proliferation for almost all the metabolites. CONCLUSION: This study first demonstrates that different downstream estrogen metabolites interfere with monocyte proliferation and generally might modulate the immune response. Therefore, since estrogen metabolite/ratios are altered in the synovial fluid of RA patients, they might play important roles at least in RA synovial tissue hyperplasia.

Apoptosis in the kidneys of patients with type II diabetic nephropathy.

The occurrence and extent of apoptosis in the kidneys of patients with diabetic nephropathy is largely unknown. We evaluated apoptosis in renal biopsies obtained from patients with early or advanced type II diabetic nephropathy. Apoptosis was about 6- and 3-fold higher, respectively, in glomeruli and tubules in kidneys of patients with early nephropathy than in the normal kidney and this was not further increased in advanced diabetic nephropathy. Glomerular apoptosis was related directly to hemoglobin A1(c) and systolic blood pressure, whereas tubular cell apoptosis correlated to diabetes duration and low-density lipoprotein-cholesterol. Fas, Fas ligand, and p38 mitogen-activated protein kinase expressions were enhanced in glomeruli and tubules; however, this did not correlate with apoptosis. In patients with proteinuria, apoptosis was associated with the subsequent loss of kidney function. When these parameters were subjected to multivariate analysis, only glomerular apoptosis retained a significant independent predictive value. Our findings suggest that apoptosis might be a clinically relevant mechanism of glomerular and tubular cell loss in proteinuric type II diabetic patients.

Peripheral tissue release of interleukin-6 in patients with chronic kidney diseases: effects of end-stage renal disease and microinflammatory state.

To examine if uremia influences muscle interleukin-6 (IL-6) metabolism we studied the exchange of IL-6 across the forearm in 16 patients with chronic kidney disease (CKD) (stages 3 and 4), in 15 hemodialysis (HD)-treated end-stage renal disease (ESRD) patients (n=15), and in six healthy controls. In addition, we performed an analysis of both IL-6 protein and IL-6 mRNA expression in muscle of CKD (stage 4) patients showing evidence of inflammation and in controls. A release of IL-6 from the forearm was observed in patients with elevated IL-6 plasma levels. Arterial IL-6 was directly related to released IL-6 (r=0.69; P<0.004) in HD patients. Both IL-6 protein and IL-6 mRNA expression were increased in muscle of inflamed CKD patients vs controls (P<0.05). Although muscle net protein balance was similar in all patients, it was significantly more negative in HD patients with high than in those with low IL-6 plasma levels (P<0.05). In addition, net protein balance was related to the forearm release of IL-6 in HD patients only (r=0.47; P<0.038). These data demonstrate that IL-6 expression is upregulated in muscle, and that muscle tissue, by releasing this cytokine, may contribute to the inflammatory response in HD patients. The release of IL-6 from peripheral tissues is associated with an increase in muscle protein loss in HD patients, suggesting that muscle release of IL-6 is linked to protein catabolism in these patients. The release of IL-6 from peripheral tissues may act as a signal for the inflammatory response and contribute to functional dysregulation in uremia.

Serum cytokines and steroidal hormones in polymyalgia rheumatica and elderly-onset rheumatoid arthritis.

BACKGROUND: Polymyalgia rheumatica (PMR) may create some difficulties in the differential diagnosis of elderly-onset rheumatoid arthritis (EORA) and of EORA with PMR-like onset (EORA/PMR). AIM: To investigate possible differences between three groups of patients, with regard to serum levels of inflammatory cytokines and steroidal hormones at baseline and after 1 month of treatment with glucocorticoids (prednisone 7.5-12.5 mg/day). PATIENTS AND METHODS: 14 patients with PMR, 15 with EORA and 14 with EORA/PMR, as well as 15 healthy, matched controls were analysed. Tumour necrosis factor alpha (TNFalpha), interleukin (IL)6, IL1 receptor antagonist (IL1Ra), cortisol, dehydroepiandrosterone sulphate (DHEAS) and 17-hydroxy-progesterone (PRG) were evaluated. RESULTS: Serum levels of both TNFalpha and IL6 were significantly higher in all three groups of patients than in controls (p<0.01). Serum IL6 levels were significantly higher in patients with both PMR and EORA/PMR than in patients with EORA (p<0.05). IL1Ra serum levels were significantly higher in patients with EORA than in controls (p<0.001) and in patients with PMR and EORA/PMR (p<0.05). DHEAS was significantly lower in patients with EORA/PMR than in those with EORA (p<0.05). PRG was significantly higher in all patient groups (p<0.05). After glucocorticoid treatment, serum TNFalpha and IL6 levels significantly decreased in all patient groups; IL1Ra significantly increased in patients with PMR and in those with EORA/PMR; cortisol, DHEAS, and PRG significantly decreased in patients with PMR and in those with EORA/PMR (p<0.05). CONCLUSIONS: Different cytokine and steroidal hormone patterns suggest that patients with PMR and those with EORA/PMR seem to be have a more intensive inflammatory reaction and are more efficient responders to glucocorticoid treatment than patients with EORA.

Anti-inflammatory effects of leflunomide in combination with methotrexate on co-culture of T lymphocytes and synovial macrophages from rheumatoid arthritis patients.

OBJECTIVES: To investigate the anti-inflammatory effects of the active leflunomide metabolite A771726 (Lef-M) in combination with methotrexate (MTX) on synovial macrophages (SM) from rheumatoid arthritis (RA) patients co-cultured with an activated T cell line (Jurkat cell line). METHODS: Pro-inflammatory cytokines (TNFalpha, IL1beta, IL6), adhesion molecule ICAM-1, cyclooxygenase isoenzymes (COX1, COX2), and the nuclear factor kappaB (NF-kappaB) complex were analysed on SM co-cultured with a T cell line, as intracellular protein expression by immunocytochemistry (ICC) and western blot analysis, as extracellular protein expression by ELISA assay, and as mRNA expression by reverse transcriptase-multiplex PCR (RT-MPCR) after treatment with Lef-M (1, 10, 30 micromol/l) alone or in combination with MTX (50 ng/ml). RESULTS: The most significant intracellular decrease in cytokines was observed by ICC in SM treated with the combination of Lef-M (1, 10, 30 micromol/l) and MTX (50 ng/ml) versus untreated SM (TNFalpha 29%, 37%, 49%, IL1beta 56%, 43%, 50%, and IL6 59%, 62%, 71%, respectively). Furthermore, a significant decrease was confirmed concerning cytokine levels evaluated by ELISA in the medium of SM treated with the combination Lef-M+MTX (TNFalpha 40%, 41%, 44%; IL1beta 10%, 20%, 60%; IL6 37%, 41%, 49%, respectively). Western blot and RT-PCR analysis confirmed these results. Concordant decreased expression was observed for ICAM-1, COX1, COX2, and the NF-kappaB complex after Lef-M+MTX treatment. CONCLUSIONS: The combination of MTX and Lef-M shows additive inhibitory effects on the production of inflammatory mediators from SM co-cultured with a T cell line. These observations might support the positive results obtained in RA clinical studies by combination therapy.

Altered circadian rhythms in rheumatoid arthritis patients play a role in the disease's symptoms.

The circadian changes in the metabolism or nocturnal secretion of endogenous corticosteroids (reduction) observed in rheumatoid arthritis (RA) patients are responsible, in part, for the time-dependent changes that are observed in the inflammatory response and related early morning clinical symptoms of the disease. Melatonin (MLT), another circadian nocturnal hormone that is the secretory product of the pineal gland, has been implicated in the time-dependent RA inflammatory reaction with effects that are opposite to those of corticosteroids. As a consequence, altered functioning of the HPA axis (early morning reduced corticosteroid production) and of the pineal gland (night increased MLT production) found in RA patients, seem to be important factors in the appearance and perpetuation of the clinical circadian symptoms of the disease. Consistently, human proinflammatory Th1-type cytokine production (related to MLT stimulation) exhibits a diurnal rhythmicity with peak levels during the night and early morning, at a time when plasma cortisol (inducing the Th2-type cytokine production) is lowest and MLT is highest. Reduced daily light exposure as observed in northern Europe (Estonia), at least during the winter, might explain the higher and more prolonged serum MLT concentrations that were observed in northern RA patients, as well as some epidemiological features versus southern Europe patients.

[Effects of estrogen peripheral metabolism in rheumatoid arthritis]. / Effetti del metabolismo periferico degli estrogeni nell'artrite reumatoide.

It is well known that the immune reactivity is modulated by gender. In fact, women show a more effective immune response as well as a more frequent development of autoimmune diseases. In particular, 17 beta-estradiol (E2) in patients with systemic inflammatory diseases leads to an higher production of IgG and IgM in peripheral blood mononucleated cells (PBMC) and the secretion of metalloproteinases and IL-6 by synovial fibroblasts. The effect of E2 seems to be partially related to its concentration. In fact, at the physiological concentration, E2 seems to exert a pro-inflammatory effect, while at pharmacological concentrations shows anti-inflammatory effects. Steroid hormones can be converted in downstream hormones along defined pathways. The conversion of dehydroepiandrosterone (DHEA) in peripheral macrophages leads to the androgen production. Subsequently the enzyme aromatase converts androgens in estrogens, and its activity is increased by some inflammatory cytokines such as IL-1beta, IL-6 and TNF-alpha. In the synovial fluids of rheumatoid arthritis (RA) patients the levels of estrogens result significantly increased compared with controls, showing the consequence of this unbalanced steroid metabolism. Furthermore, the metabolism of estrogens leads to some downstream hydroxylated metabolites, that are not waste products, but still active molecules in the inflammatory response. In fact, it has been found that synovial fluids of RA patients present a different ratio of 16-hydroxylated estrogen metabolites/ 2-hydroxylated metabolites, confirming that also the unbalanced metabolism of estrogens and not only the estrogen concentration seems to be related to the development and worsening of rheumatoid arthritis.

Circadian melatonin and cortisol levels in rheumatoid arthritis patients in winter time: a north and south Europe comparison.

BACKGROUND: Altered functioning of the hypothalamic-pituitary-adrenal axis and altered melatonin production might modulate the circadian symptoms in patients with rheumatoid arthritis. OBJECTIVE: To investigate the influence of different winter photoperiods on the circadian rhythms of serum melatonin, cortisol, tumour necrosis factor alpha (TNFalpha), and interleukin 6 (IL6) in patients with rheumatoid arthritis from a north Europe country (Estonia) and a south Europe country (Italy). METHODS: The patients from Estonia (n = 19) and Italy (n = 7) had similar disease severity and duration and were compared with healthy age and sex matched controls in the two countries. Blood samples were collected during the period January to February at 8 pm, 10 pm, midnight, 2 am, 4 am, 6 am, 8 am, and 3 pm. Melatonin was measured by radioimmunoassay using (125)I-melatonin. Serum cortisol, TNFalpha, and IL6 cytokines were assayed by standard methods. RESULTS: Higher circadian melatonin concentrations from 10 pm and an earlier peak were observed in Estonian patients than in their age and sex matched controls (p<0.01). Starting from midnight, melatonin concentrations were significantly higher in the Estonian patients than in the Italian patients. No significant differences were observed for serum cortisol. Serum TNFalpha was higher (p<0.05) in Estonian patients than in their controls and was correlated with the melatonin levels. CONCLUSIONS: In a north European country (Estonia), the circadian rhythm of serum concentrations of melatonin and TNFalpha in patients with rheumatoid arthritis were significantly higher than in matched controls or in rheumatoid patients from a south Europe country (Italy).

Sex hormones influence on the immune system: basic and clinical aspects in autoimmunity.

Sex hormones seem to play an important role as modulators of the autoimmune disease onset/perpetuation. Generally, steroid hormones are implicated in the immune response, with estrogens as enhancers at least of the humoral immunity and androgens and progesterone (and glucocorticoids) as natural immunosuppressors. Synovial fluid levels (SF) of proinflammatory estrogens relative to androgens are significantly elevated in both male and female rheumatoid arthritis (RA) patients, as compared to controls, which is most probably due to increase of local enzymatic aromatase activity. Serum levels of estrogens have been found altered in RA patients, particularly estradiol in man. Thus, available steroid prehormones are rapidly converted to proinflammatory estrogens in the synovial tissue in the presence of inflammatory cytokines (i.e., TNFalpha, IL-1, IL-6). The increased estrogen concentrations observed in RA SF of both sexes are characterized mainly by the hydroxylated forms, in particular, 16alpha-hydroxyestrone, showing a mitogenic tumor growth stimulating role. Altered serum hydroxylated estrogens have been found also in serum of systemic lupus erythematosus (SLE) patients. As a matter of fact, our recent studies indicate that 17-beta estradiol (E2) clearly enhanced the expression of markers of cell growth and proliferation, whereas testosterone (T) induced an increase of markers indicating DNA damage and apoptosis. In particular, our data further shows that the enhancing role of estrogens on immune/inflammatory response is exerted by activating the NFkB complex pathway. In conclusion, locally increased estrogens (i.e., synovial tissue in RA or skin in SLE) might exert activating effects on cell proliferation, including macrophages and fibroblasts, suggesting new roles for estrogens in autoimmunity.

Anti-inflammatory effects of leflunomide on cultured synovial macrophages from patients with rheumatoid arthritis.

BACKGROUND: Leflunomide and its active metabolite A77 1726 reversibly inhibits the enzyme dihydro-orotate dehydrogenase, the rate limiting step in de novo synthesis of pyrimidines and progression of the cell cycle in different cell lines, mainly activated T lymphocytes. OBJECTIVE: To analyse in vitro the possible anti-inflammatory effects exerted by A77 1726, on cultured macrophages, obtained from the synovial tissues of patients with rheumatoid arthritis (RA). METHODS: The effects of different doses of A77 1726 on intracytoplasmic expression and extracellular concentration of inflammatory cytokines (tumour necrosis factor alpha (TNFalpha), interleukin (IL) 1beta, IL6), as well as the influence on production and expression of intercellular adhesion molecule-1 (ICAM-1) and cyclo-oxygenase 2 (COX-2) by primary cultures of synovial macrophages from patients with RA, were evaluated by immunocytochemistry and western blot analysis. The observations were made at four and 24 hours. RESULTS: A progressive and significant time and dose dependent decrease of the number of positive macrophages for intracellular TNFalpha and IL1beta, treated with different doses of A77 1726, was found in comparison with untreated cells. The extracellular concentration of TNFalpha was found to be significantly decreased in media containing cultured macrophages at 24 hours for all tested doses of A77 1726. At 24 hours, a significant time and dose dependent decrease of ICAM-1 and COX-2 expression by cultured macrophages after A77 1726 treatment was found. CONCLUSIONS: In conclusion, the mechanism of antiproliferative activity exerted by leflunomide on activated T lymphocytes seems to be the same mechanism (alteration of the cell cycle progression) which interferes with the functions of other activated cells-namely, the monocytes/macrophages, which are strongly involved in the inflammatory reaction in RA synovial tissue. The positive clinical results seem to confirm that leflunomide exerts an anti-inflammatory action on phagocytic cells in short and long term treatment of RA.

New roles for estrogens in rheumatoid arthritis.

Sex hormones appear to play an important role as modulators of autoimmune disease onset/perpetuation. Steroid hormones are implicated in the immune response, with estrogens as enhancers at least of humoral immunity, and androgens and progesterone (and glucocorticoids) as natural immune suppressors. Serum levels of estrogens have been found to be normal in rheumatoid arthritis (RA) patients. Synovial fluid levels (SF) of proinflammatory estrogens relative to androgens are significantly elevated in both male and female RA patients as compared to controls, which is most probably due to an increase in local aromatase activity. Thus, available steroid pre-hormones are rapidly converted to proinflammatory estrogens in the synovial tissue in the presence of inflammatory cytokines (i.e. TNF alpha, IL-1, IL-6). The increased estrogen concentrations observed in RA SF of both sexes are characterized mainly by the hydroxylated forms, in particular 16 alpha-hydroxyestrone, showing a mitogenic stimulating role. Indeed, recent studies by us indicate that 17-beta estradiol (E2) clearly enhanced the expression of markers of cell growth and proliferation, whereas testosterone (T) induced an increase in markers indicating DNA damage and apoptosis. In particular, our data further shows that the enhancing role of estrogens on the immune/inflammatory response is exerted by activating the NFkB complex. In conclusion, locally increased estrogens may exert activating effects on synovial cell proliferation, including macrophages and fibroblasts, suggesting new roles for estrogens in RA.

Melatonin serum levels in rheumatoid arthritis.

The pineal hormone melatonin (MLT) exerts a variety of effects on the immune system. MLT activates immune cells and enhances inflammatory cytokine and nitric oxide production. Cytokines are strongly involved in the synovial immune and inflammatory response in rheumatoid arthritis (RA) and reach the peak of concentration in the early morning, when MLT serum level is higher. Nocturnal MLT serum levels were evaluated in 10 RA patients and in 6 healthy controls. Blood samples were obtained at 8 and 12 p.m., as well as at 2, 4, 6, and 8 a.m. MLT serum levels at 8 p.m. and 8 a.m. were found to be higher in RA patients than in controls (p < 0.05). In both RA patients and healthy subjects, MLT progressively increased from 8 p.m. to the first hours of the morning, when the peak level was reached (p < 0.02). However, MLT serum level reached the peak at least two hours before in RA patients than in controls (p < 0.05). Subsequently, in RA patients, MLT concentration showed a plateau level lasting two to three hours, an effect not observed in healthy controls. After 2 a.m., MLT levels decreased similarly in both RA patients and healthy subjects. Several clinical symptoms of RA, such as morning gelling, stiffness, and swelling, which are more evident in the early morning, might be related to the neuroimmunomodulatory effects exerted by MLT on synovitis and might be explained by the imbalance between cortisol serum levels (lower in RA patients) and MLT serum levels (higher in RA patients).

Identification of an import signal for, and the nuclear localization of, human lactoferrin.

Many different unique functions have been attributed to lactoferrin (Lf), including DNA and RNA binding, and transport into the nucleus, where Lf binds to specific sequences and activates transcription. A pentapeptide, Gly-Arg-Arg-Arg-Arg, corresponding to a region of the N-terminal portion of human Lf rich in basic amino acids, was synthesized and its intracellular localization was investigated. Peptide internalization was assayed using the rhodaminated form of the same molecule. This N-terminal peptide sequence is able to be internalized within less than 10 min at concentration as low as 1 microM, and its intracellular localization is nuclear, mainly nucleolar. Similar behaviour was observed using peptides composed of either all l or d amino acids, the last one being a retro-inverse peptide. The internalization process does not involve an endocytotic pathway, since no inhibition of the uptake was observed at 4 degrees C. The kinetics of peptide internalization was also evaluated. The internalization properties of such a short Lf pentapeptide have been assayed for its ability to transport peptide nucleic acids (PNAs) inside cells in order to improve their efficacy. The abundant transmembrane transport and nuclear localization of the proposed peptide, deriving from hLf and, for the first time, identified as a nuclear localization signal, could be used as an alternative strategy to tackle the unsolved problem of intracellular accumulation of antisense and antigene drugs and for the development of new pharmacological tools.

Apoptosis induced by serum withdrawal in human mesangial cells. Role of IGFBP-3.

Apoptosis has been reported to occur both during the course of kidney development and the progression of kidney injury to scarring. Insulin-like growth factor binding protein-3 (IGFBP-3), a component of the IGF system, has been shown to induce apoptosis in cancer cell lines. However, if IGFBP-3 has similar effects in human mesangial cells (HMC) remains unknown. The purpose of this study was to examine the expression of IGFBP-3 and its possible effect on the induction of apoptosis in HMC during serum deprivation. We have observed that IGFBP-3 accumulates progressively in HMC in which serum has been withdrawn. In these cells, an increase of IGFBP-3 is observed before the production of apoptosis suggesting a link between these phenomena. Furthermore, the addition of IGFBP-3 in physiological amounts (from 100 to 400 ng/ml) to culture medium devoid of growth factors accelerates and increases the apoptotic process with a dose-dependent effect. These findings suggest that IGFBP-3 is a mediator of cell death in human mesangial cells when the availability of growth factors is curtailed. These data also suggest that IGFBP-3 could contribute to apoptotic processes observed in human disease.