Urinary high-mobility group box-1 associates specifically with lupus nephritis class V.

INTRODUCTION: High-mobility group box 1 protein (HMGB-1) has been implicated in the pathogenesis of lupus nephritis (LN). There is increased HMGB-1 expression in the kidneys and increased levels are observed in serum and urine of patients with LN. This study was performed to determine whether the increased urinary HMGB-1 was specific for active lupus or secondary to renal damage. METHODS: Urine from 61 lupus patients (32 had active LN and 29 had systemic lupus erythematosus (SLE) with no evidence of LN) and 14 control proteinuric patients (all with hypertension and eight also with diabetes) were included in this study. HMGB-1 was detected by Western blot. Urine protein was normalized to urine creatinine to account for volume of the specimen. RESULTS: Median normalized urine HMGB-1 levels were significantly elevated in LN patients compared to lupus patients without kidney disease (53.81 vs 9.46, p < 0.001). A difference in median levels was seen between LN classes, with a significant difference between proliferative and membranous disease (33.4 vs 138.8, p = 0.003). Urine protein to urine creatinine ratio (P/C) correlated with urinary HMGB-1 (r = 0.52, p < 0.001), but across the classes this was true only for membranous disease (r = 0.71, p = 0.022, proliferative, p = 0.63; mixed, p = 0.34). CONCLUSIONS: HMGB-1 is elevated in the urine of patients with active LN. Levels are associated with LN class, and higher levels of urinary HMGB-1 are seen in patients with class V when compared to both proliferative and mixed classes. Therefore, urinary HMGB-1 may be suggestive of membranous LN and warrants further evaluation in a large lupus cohort.

Differential regulation of cell death programs in males and females by Poly (ADP-Ribose) Polymerase-1 and 17ß estradiol.

Cell death can be divided into the anti-inflammatory process of apoptosis and the pro-inflammatory process of necrosis. Necrosis, as apoptosis, is a regulated form of cell death, and Poly-(ADP-Ribose) Polymerase-1 (PARP-1) and Receptor-Interacting Protein (RIP) 1/3 are major mediators. We previously showed that absence or inhibition of PARP-1 protects mice from nephritis, however only the male mice. We therefore hypothesized that there is an inherent difference in the cell death program between the sexes. We show here that in an immune-mediated nephritis model, female mice show increased apoptosis compared to male mice. Treatment of the male mice with estrogens induced apoptosis to levels similar to that in female mice and inhibited necrosis. Although PARP-1 was activated in both male and female mice, PARP-1 inhibition reduced necrosis only in the male mice. We also show that deletion of RIP-3 did not have a sex bias. We demonstrate here that male and female mice are prone to different types of cell death. Our data also suggest that estrogens and PARP-1 are two of the mediators of the sex-bias in cell death. We therefore propose that targeting cell death based on sex will lead to tailored and better treatments for each gender.

Fas, ceramide and serum withdrawal induce apoptosis via a common pathway in a type II Jurkat cell line.

Ceramide is a key mediator of apoptosis, yet its role in Fas-mediated apoptosis is controversial. Some reports have indicated that ceramide is either a primary signaling molecule in Fas-induced cell death, or that it functions upstream of Fas by increasing FasL expression. Other studies have suggested that ceramide is not relevant to Fas-induced cell death. We have approached this problem by studying ceramide-induced apoptosis in unique Jurkat cell clones selected for resistance to membrane-bound FasL-induced death. Resistance of the mutant Jurkat cells was specific for FasL killing, since the mutant clones were sensitive to other apoptotic stimuli such as cycloheximide and staurosporine. We tested the effects of serum withdrawal, one of the strongest inducers of ceramide, and of exogenous ceramide on apoptosis of both wild-type and FasL-resistant clones. Wild-type Jurkat cells were remarkably sensitive to serum withdrawal and to exogenous ceramide. In contrast all FasL-resistant mutant clones were resistant to these apoptosis-inducing conditions. In contrast to previous work, we did not detect an increase in FasL in either wild-type or mutant clones. Moreover activation of stress-activated protein kinases (JNK/SAPKs) after serum withdrawal and exogenous ceramide treatment was detected only in the wild-type and not in the resistant clones. Because of the parallel resistance of the mutant clones to Fas and to ceramide-induced apoptosis, our data support the notion that ceramide is a second messenger for the Fas/FasL pathway and that serum withdrawal, through production of ceramide, shares a common step with the Fas-mediated apoptotic pathway. Finally, our data suggest that activation of JNK/SAPKs is a common mediator of the three pathways tested.

Phagocytosis and clearance of apoptotic cells is mediated by MER.

Apoptosis is fundamental to the development and maintenance of animal tissues and the immune system. Rapid clearance of apoptotic cells by macrophages is important to inhibit inflammation and autoimmune responses against intracellular antigens. Here we report a new function for Mer, a member of the Axl/Mer/Tyro3 receptor tyrosine kinase family. mer(kd) mice with a cytoplasmic truncation of Mer had macrophages deficient in the clearance of apoptotic thymocytes. This was corrected in chimaeric mice reconstituted with bone marrow from wild-type animals. Primary macrophages isolated from mer(kd) mice showed that the phagocytic deficiency was restricted to apoptotic cells and was independent of Fc receptor-mediated phagocytosis or ingestion of other particles. The inability to clear apoptotic cells adequately may be linked to an increased number of nuclear autoantibodies in mer(kd) mice. Thus, the Mer receptor tyrosine kinase seems to be critical for the engulfment and efficient clearance of apoptotic cells. This has implications for inflammation and autoimmune diseases such as systemic lupus erythematosus.

Spontaneous and induced apoptosis in systemic lupus erythematosus: multiple assays fail to reveal consistent abnormalities.

The immunologic basis of systemic lupus erythematosus (SLE) is multifactorial and still elusive. Recent advances in the field of apoptosis have suggested new paradigms for the development of lupus autoimmunity. In the present studies we examined the possibility that individual populations of T and B cells are abnormally resistant to apoptosis or that they stand out in over- or underexpressing Fas. Fas was generally overexpressed in cells freshly isolated from SLE patients but the apoptotic response to FasL was normal. We did not find increased spontaneous ongoing apoptosis in SLE lymphocytes. Normal cleavage of PARP similarly implied that the final biochemical pathway of apoptosis is relatively intact in SLE. Finally we placed special emphasis on the response of SLE patient cells to UV irradiation, especially cells from photosensitive patients, and found no difference in Fas expression. In conclusion our results indicate that SLE patients do not suffer from a major apoptotic abnormality. The results also raise questions concerning the dynamic expression of Fas and the significance of ongoing apoptosis as a risk for autoimmune disease.

Apoptosis provoked by the oxidative stress inducer menadione (Vitamin K(3)) is mediated by the Fas/Fas ligand system.

Menadione, or vitamin K(3) (VK(3)), a potent oxidative stress inducer, has been recently used as an effective and remarkably safe cytotoxic drug for treatment of several human tumors. VK(3) induces apoptotic cell death through a poorly understood mechanism. Here we show for the first time that VK(3)-induced apoptosis requires the Fas/FasL system. Spleen cells from both Fas- and FasL-deficient mice (C57BL/6-lpr and C57BL/6-gld, respectively) had much lower levels of VK(3) apoptosis in vitro compared to cells from control C57BL/6 mice. VK(3) cytotoxicity toward mouse splenocytes was also blocked with a Fas-Fc fusion protein. VK(3) induced apoptosis in Jurkat cells, coincident with an increase in both Fas and FasL expression. A FasL-resistant variant of these Jurkat cells was also resistant to VK(3)-induced apoptosis. Furthermore, because VK(3) effects were inhibited by glutathione, a potent antioxidant, oxidative stress was linked to the Fas/FasL system. Moreover, since the Jurkat cell lines were p53 null, the activation of Fas/FasL system after oxidative stress apparently acted through a p53-independent pathway. The therapeutic relevance of the K vitamins has been growing in recent years; our findings offer new insight for improving and expanding their applications.

Serum zinc and copper in active rheumatoid arthritis: correlation with interleukin 1 beta and tumour necrosis factor alpha.

Serum zinc and copper levels and serum interleukin 1 beta (IL1 beta) and tumour necrosis factor alpha (TNF alpha) levels were evaluated in 57 female patients with active rheumatoid arthritis (RA) to investigate a possible role of IL1 beta and TNF alpha on zinc and copper homeostasis in RA. Serum zinc levels were significantly lower and serum copper levels significantly higher in RA patients when compared with osteoarthritis or asymmetrical psoriatic oligoarthritis patients and with normal controls. No differences were observed in serum IgM rheumatoid factor positive and serum IgM rheumatoid factor negative patients as regards serum zinc and copper concentration. In RA patients the erythrocyte sedimentation rate and acute-phase proteins correlated negatively with serum zinc and positively with serum copper. IL1 beta and TNF alpha were found to correlate negatively with zinc and positively with copper in RA patients. Lower levels of zinc may be due to an accumulation of zinc-containing proteins in the liver and in the inflamed joints in RA. Elevated serum copper levels seem to be linked to the increased synthesis of ceruloplasmin by the liver.

bca: an activation-related B-cell gene.

We have identified a novel activation related B-cell gene (bca) through differential hybridization screening of a murine B cell cDNA library. The deduced amino acid sequence predicted a protein of 482 amino acids with strong sequence similarity to the SH2 and SH3 domains present within the non-catalytic regions of several protein tyrosine kinases. Northern analysis of RNA from several murine B-cell lines revealed a transcript of 1.8 kb, which was not detected in T-cell and non-lymphoid cell lines. bca was transcribed at low levels in resting spleen cells from a variety of normal mouse strains and was strongly expressed in kidney RNA. bca expression was markedly increased in RNA prepared from mitogen activated B cells, and in freshly isolated spleen and lymph node cells of MRL/lpr and NZB autoimmune strains. The unique sequence of bca, which bears no obvious similarity to any specific class of proteins containing SH2 and SH3 domains, suggests that this gene encodes a novel protein potentially involved in B-cell signal transduction.

Fas/Fas ligand interactions are involved in ultraviolet-B-induced human lymphocyte apoptosis.

We wondered whether the apoptosis known to occur after UV-B irradiation might involve the Fas/Fas ligand (FasL) signaling pathway. We exposed PBLs from normal individuals, and also the Jurkat (E6-1) and U937 cell lines, to graded doses of UV-B irradiation and observed a prompt and marked increase in Fas expression at doses as low as 0.5 mJ/cm2. Increased Fas expression did not require new protein synthesis, since cycloheximide-treated cells also showed an increase in Fas after UV-B. UV-B-irradiated cells cultured in the presence of zinc showed inhibition of apoptosis coincident with a marked increase in Fas+ cells, apparently indicating the accumulation of Fas-bearing cells unable to undergo apoptosis. After UV-B irradiation, PBLs showed increased expression of Fas ligand; the E6-1 lymphocytic cell line also released soluble FasL. UV-B induced apoptosis could be partially blocked by neutralizing FasL Abs, and a FasL-resistant variant of E6-1 cell line showed reduced apoptosis after UV-B irradiation, implying that the increase in Fas expression signified a role for Fas in UV-induced apoptosis. UV-induced Fas expression may serve to target stress-injured cells for removal by FasL-bearing cells or by FasL produced by the cells themselves in response to the stimuli, and may represent a general function of the Fas/FasL pathway in facilitating the apoptosis and elimination of undesirable or harmful cells.

[Quantitative ultrasonography in the evaluation of postmenopausal osteoporosis. Comparison with dual energy x-ray absorptiometry]. / Ultrasonografia quantitativa nello studio dell'osteoporosi postmenopausale. Confronto con l'assorbimetria a raggi X a doppia energia.

In order to compare the capacities of quantitative ultrasonography and dual energy X-ray absorptiometry to measure bone mineral density (BMD) in postmenopausal women, 45 postmenopausal patients were studied. They were divided into two groups on the basis of the results of absorptiometry: Group A (20 patients) had BMD values within normal limits, and Group B (25 patients) had BMD values lower than 1 standard deviation (SD) of the average for an age and sex matched population. Ultrasonography of the non-dominant heel was carried out on both groups to determine broadband ultrasound attenuation (BUA), speed of sound (SOS) and bone velocity (BV). A negative correlation between age and BMD, BV, SOS, and BUA was observed in all subjects. A positive correlation between BMD and BV and between BMD and SOS was found. In Group A, age correlated negatively with BMD, BUA, SOS and BV, and BMD correlated positively with BV. In Group B, age correlated negatively with BMD, and BMD correlated positively with BV and SOS. BV values were significantly elevated in Group B. The study evidenced a relationship between ultrasonography and absorptiometry in determining bone density in postmenopausal women. As ultrasonography and absorptiometry provided comparable information on bone density, we point out that ultrasonography can be used as a simple and sensitive indicator for postmenopausal osteoporosis.

Posture in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a connective autoimmune disease that may involve many organ systems and neural function impairment. Postural apparatus dysfunction in SLE may develop in the case of central and peripheral neural involvement. We studied the presence of postural abnormalities in SLE patients with different degrees of disease activity. Twenty-eight subjects (18 SLE patients and 10 normal controls) underwent postural evaluation by means of Static Computerised Posturography. Disease severity or specific clinical problems did not form selection criteria. Lupus activity was assessed using the Systemic Lupus Activity Measure (SLAM). A statistically significant (p < 0.05) increase in the parameters of trace length, trace surface, trace velocity, standard deviation in velocity, and Fast Fourier trX and Y (FET), were found in both the closed and opened eyes tests, when lupus patients were compared to controls subjects. No significant pattern variation in posture was observed between more active and less active SLE patients. Postural control alterations in SLE may be considered a complication of the chronic autoimmune inflammatory process, independent of disease activity as evaluated by the SLAM index. The site of the neuropathy remains uncertain, although we believe it to be peripheral rather than central in origin due to the absence of symptoms or clinical signs of CNS involvement or vascular degeneration.

Platelet lactate dehydrogenase activity in systemic lupus erythematosus: correlation with anticardiolipin antibodies.

OBJECTIVE: To evaluate lactate dehydrogenase (LDH) activity in platelet subpopulations in systemic lupus erythematosus (SLE) and to correlate platelet LDH activity with concentrations of anticardiolipin antibody (aCL). METHODS: Twelve female patients with SLE and 12 age matched female control subjects were studied. Platelets were separated on the Percoll gradient, their density values controlled by density marker beads. LDH activity was measured after platelet lysis, expressed as nU/fl. ELISA were used to measure levels of IgG and IgM aCL. RESULTS: A significant increase of LDH activity with a significant correlation to IgG and IgM aCL were found in small, light platelets with a volume < 5 mu 3 compared to large, dense platelets and to controls. LDH activity did not correlate with immunoglobulin classes, anti-DNA antibodies, and complement fractions in small and large SLE platelets. CONCLUSION: Our data suggest a possible chronic activation of subpopulations of small platelets in patients with SLE independent of thrombotic process. Low levels of aCL can mediate small platelet activation. Quantitative and qualitative analysis of the small, light platelets can serve a clinical diagnostic purpose as an in vivo platelet activation index in SLE.

[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.