Nafamostat Mesilate Attenuates Ischemia-Reperfusion-Induced Renal Injury.

BACKGROUND: It has been reported that nafamostat mesilate (NM) inhibits inflammatory injury via inhibition of complement activation in ischemic heart, liver, and intestine. However, it is unclear if NM also inhibits apoptosis in ischemia-reperfusion (IR)-injured kidney. We therefore investigated whether NM attenuates IR renal injury that involves inhibition of apoptosis. METHODS: HK-2 cells and male C57BL/6 mice were used for this study. C57Bl/6 mice were divided into 4 groups: sham, NM (2 mg/kg) + sham, IR injury (IR injury; reperfusion 27 minutes after clamping of both the renal artery and vein), and NM + IR injury. Kidneys were harvested 24 hours after IR injury, and functional and molecular parameters were evaluated. For in vitro studies, HK-2 cells were incubated for 6 hours with mineral paraffin oil to induce hypoxic injury, and then treated with various doses of NM to evaluate the antiapoptotic effects. RESULTS: Blood urea nitrogen, serum creatinine levels, and renal tissue injury scores in NM + IR-injured mice were significantly lower than those of control IR mice (all P < .01). NM significantly improved cell survival in hypoxic HK-2 cells (P < .01), significantly decreased renal Bax expression (P < .05), and increased renal Bcl-2 protein levels in IR kidneys and hypoxic HK-2 cells compared with those of the sham and control groups. The numbers of terminal deoxynucleotide transferase-mediated dUTP nick-end labeling- and 8-oxo-2'-deoxyguanosine-positive cells were significantly lower in NM + IR-injured kidneys compared with those in control IR-injured mice (P < .05); NM treatment decreased the expression of inducible and endothelial nitric oxide synthase in IR-injured mice (P < .05). CONCLUSIONS: NM ameliorates IR renal injury via inhibition of apoptosis by, at least in part, lowering nitric oxide overproduction, reducing Bax, and increasing Bcl-2.

Beneficial Effects of Necrosis Modulator, Indole Derivative NecroX-7, on Renal Ischemia-Reperfusion Injury in Rats.

Renal ischemia-reperfusion injury (IRI) is involved in multiple diseases, such as kidney transplantation or contrast-induced nephropathy, and leads to acute kidney injury. However, there are no pharmacological agents available to prevent IRI. In this study, we investigated the effects of necroX-7 against renal IRI in a rat model. Seven-week-old male Sprague-Dawley rats were divided into four groups: saline-treated sham or IRI group, necroX-7-treated sham or IRI group. All animals had right nephrectomy and IRI was followed by reperfusion after clamping the left renal vessels for 35 minutes. NecroX-7 or saline was intravenously injected at 5 minutes before reperfusion. The effects of necroX-7 on IRI were evaluated using biochemical, histological, and molecular markers. The serum creatinine level was increased after IRI compared with sham. The necroX-7 significantly decreased creatinine level compared with the saline in IRI (1.36 ± 0.11 vs 2.35 ± 0.42 mg/dL; P < .05). An immunohistochemical study revealed that necroX-7 improved renal tubular injury, and attenuated 8-OHdG-positive cells (P < .001) and high-mobility group Box 1 protein (HMGB1) expression compared with saline treatment in IRI (P < .001). NecroX-7 significantly reduced monocyte chemoattractant protein 1 (MCP-1), tumor necrosis factor (TNF)-α, and interleukin (IL)-1ß in IRI (necroX-7-treated IRI vs saline-treated IRI rats; 1.73 ± 0.42 vs 7.23 ± 0.54-fold for MCP-1, P < .05; 0.79 ± 0.59 vs 3.72 ± 0.37-fold for TNF-α, P < .05; 0.50 ± 0.36 vs 2.43 ± 0.41-fold for IL-1ß, P < .001). In conclusion, necroX-7 improved renal dysfunction after IRI. These effects of necroX-7 occurred with the suppression of reactive oxygen species, HMGB1, and inflammatory responses. We suggest that necroX-7 has potential therapeutic benefits in renal IRI.

Clinical significance of subclinical varicocelectomy in male infertility: systematic review and meta-analysis.

Recent meta-analysis by the Cochrane collaboration concluded that treatment of varicocele may improve an infertile couple's chance of pregnancy. However, there has been no consensus on the management of subclinical varicocele. Therefore, we determine the impact of varicocele treatment on semen parameters and pregnancy rate in men with subclinical varicocele. The randomised controlled trials that assessed the presence and/or treatment of subclinical varicocele were included for systematic review and meta-analysis. Random effect model was used to calculate the weighted mean difference of semen parameters and odds ratio of pregnancy rates. Seven trials with 548 participants, 276 in subclinical varicocelectomy and 272 in no-treatment or clomiphene citrate subjects, were included. Although there was also no statistically significant difference in pregnancy rate (OR 1.29, 95% CI 0.99-1.67), surgical treatment resulted in statistically significant improvements on forward progressive sperm motility (MD 3.94, 95% CI 1.24-6.65). However, the evidence is not enough to allow final conclusions because the quality of included studies is very low and further research is needed.

Ray amputation for the treatment of foot macrodactyly in children.

Macrodactyly of the foot is a rare but disabling condition. We present the results of surgery on 18 feet of 16 patients, who underwent ray amputation and were followed-up for more than two years at a mean of 80 months (25 to 198). We radiologically measured the intermetatarsal width and forefoot area pre-operatively and at six weeks and two years after surgery. We also evaluated the clinical results using the Oxford Ankle Foot Questionnaire for children (OxAFQ-C) and the Questionnaire for Foot Macrodactyly. The intermetatarsal width and forefoot area ratios were significantly decreased after surgery. The mean OxAFQ-C score was 42 (16 to 57) pre-operatively, improving to 47 (5 to 60) at two years post-operatively (p = 0.021). The mean questionnaire for Foot Macrodactyly score two years after surgery was 8 (6 to 10). Ray amputation gave a measurable reduction in foot size with excellent functional results. For patients with metatarsal involvement, a motionless toe, or involvement of multiple digits, ray amputation is a clinically effective option which is acceptable to patients.

A genetic variation in microRNA target site of KRT81 gene is associated with survival in early-stage non-small-cell lung cancer.

BACKGROUND: MicroRNAs (miRNAs) have a key role in carcinogenesis through negative regulation of their target genes. Therefore, genetic variations in miRNAs or their target sites may affect miRNA-mRNA interactions, thereby result in altered expression of target genes. This study was conducted to investigate the associations between single-nucleotide polymorphisms (SNP) located in the miRNA target sites (poly-miRTSs) and survival of patients with early-stage non-small-cell lung cancer (NSCLC). METHODS: Using public SNP database and miRNA target sites prediction program, 354 poly-miRTSs were selected for genotyping. Among these, 154 SNPs applicable to Sequenom's MassARRAY platform were investigated in 357 patients. A replication study was carried out on an independent patient population (n = 479). Renilla luciferase assay and reverse transcription-polymerase chain reaction were conducted to examine functional relevance of potentially functional poly-miRTSs. RESULTS: Of the 154 SNPs analyzed in a discovery set, 14 SNPs were significantly associated with survival outcomes. Among these, KRT81 rs3660G>C was found to be associated with survival outcomes in the validation cohort. In the combined analysis, patients with the rs3660 GC + CC genotype had a significantly better overall survival compared with those with GG genotype [adjusted hazard ratio (aHR) for OS, 0.65; 95% confidence interval (CI) 0.50-0.85; P = 0.001]. An increased expression of the reporter gene for the C allele of rs3660 compared with the G allele was observed by luciferase assay. Consistently, the C allele was associated with higher relative expression level of KRT81 in tumor tissues. CONCLUSION: The rs3660G>C affects KRT81 expression and thus influences survival in early-stage NSCLC. The analysis of the rs3660G>C polymorphism may be useful to identify patients at high risk of a poor disease outcome.

Clinical impact of microscopic extrathyroidal extension in patients with papillary thyroid microcarcinoma treated with hemithyroidectomy.

BACKGROUND: Pathologically confirmed microscopic extrathyroidal extension (ETE) is often identified after hemithyroidectomy in patients with papillary thyroid microcarcinoma (PTMC). Without the presence of microscopic ETE, these patients would be optimal candidates for hemithyroidectomy. AIM: The present study aimed at evaluating the clinical impact of microscopic ETE on the recurrence of PTMC treated with hemithyroidectomy. SUBJECTS AND METHODS: We compared the clinicopathological characteristics and 5-year outcomes for 262 PTMC patients without ETE and 86 with microscopic ETE who were treated with hemithyroidectomy between January 2004 and December 2010. RESULTS: The mean tumour size was larger (0.67 vs. 0.54 cm, p < 0.001) and the proportion of tumours measuring ≥0.5 cm was higher (84.9 vs. 66.8 %, p = 0.001) in patients with microscopic ETE as compared with patients without ETE. Occult multifocal disease was more frequent in patients with microscopic ETE than in those without ETE (14.0 vs. 6.5 %, p = 0.030). However, the recurrence rate was not different between the two groups during the mean 55.8-month follow-up period. In addition, univariate and multivariate analyses revealed no meaningful association between recurrence and microscopic ETE in patients with PTMC treated with hemithyroidectomy. CONCLUSIONS: Although microscopic ETE was associated with large tumour size and multifocal disease, its clinical impact on disease recurrence was not significant in PTMC patients treated with hemithyroidectomy. Therefore, microscopic ETE identified after hemithyroidectomy would not be an absolute indication for completion thyroidectomy in patients with PTMC.

Dysregulation of miR-106a and miR-591 confers paclitaxel resistance to ovarian cancer.

BACKGROUND: MicroRNAs are noncoding regulatory RNAs strongly implicated in carcinogenesis, cell survival, and chemosensitivity. Here, microRNAs associated with chemoresistance in ovarian carcinoma, the most lethal of gynaecological malignancies, were identified and their functional effects in chemoresistant ovarian cancer cells were assessed. METHODS: MicroRNA expression in paclitaxel (PTX)-resistant SKpac sublines was compared with that of the PTX-sensitive, parental SKOV3 ovarian cancer cell line using microarray and qRT-PCR. The function of differentially expressed microRNAs in chemoresistant ovarian cancer was further evaluated by apoptosis, cell proliferation, and migration assays. RESULTS: Upregulation of miR-106a and downregulation of miR-591 were associated with PTX resistance in ovarian cancer cells and human tumour samples. Transfection with anti-miR-106a or pre-miR-591 resensitized PTX-resistant SKpac cells to PTX by enhancing apoptosis (23 and 42% increase), and inhibited their cell migration (43 and 56% decrease) and proliferation (64 and 65% decrease). Furthermore, ZEB1 was identified as a novel target gene of miR-591, and BCL10 and caspase-7 were target genes of miR-106a, as identified by immunoblotting and luciferase assay. CONCLUSION: MiR-106a and miR-591 have important roles in conferring PTX resistance to ovarian cancer cells. Modulation of these microRNAs resensitizes PTX-resistant cancer cells by targeting BCL10, caspase-7, and ZEB1.

agr Dysfunction and persistent methicillin-resistant Staphylococcus aureus bacteremia in patients with removed eradicable foci.

PURPOSE: Persistent Staphylococcus aureus bacteremia (SAB) has been observed in patients with eradicated foci, but there are few studies of the risk factors and clinical outcomes of persistent bacteremia. This study determined the risk factors for persistent methicillin-resistant S. aureus (MRSA) bacteremia in patients without retained eradicable foci, including genotypic characteristics. METHODS: All adult SAB patients were investigated between 2008 and 2010. Persistent bacteremia was defined as bacteremia lasting >7 days after treatment and patients were monitored prospectively. The study included patients without retained eradicable foci, e.g., removed prosthetic devices and intravenous catheters removed after diagnosis, and those without metastatic infections. RESULTS: Persistent bacteremia occurred in 36 % (31/87) SAB patients with eradicated foci. There were no significant differences in successful defervescence (2.0 vs. 2.0 days, P = 0.55) and total length of hospital stay after bacteremia in the persistent bacteremia group and resolved bacteremia group (P = 0.32). The difference in MRSA bacteremia-related 30-day mortality with persistent bacteremia and resolved bacteremia was not significant (P = 0.12). However, agr dysfunction was higher in persistent bacteremia patients (94 %) than those with resolved bacteremia (75 %, P = 0.03). Multivariate analysis using a logistic regression model found that only agr dysfunction [odds ratio (OR) 4.83, 95 % confidence interval (CI) 1.02-22.89, P = 0.04] was an independent risk factor for persistent bacteremia. CONCLUSIONS: This study suggests that persistent bacteremia with eradicated foci might not adversely affect the outcome for MRSA bacteremia patients. agr dysfunction in S. aureus was significantly associated with persistent bacteremia.

cAMP response element binding protein H mediates fenofibrate-induced suppression of hepatic lipogenesis.

AIMS/HYPOTHESIS: Fenofibrate is a drug used to treat hyperlipidaemia that works by inhibiting hepatic triacylglycerol synthesis. Sterol regulatory element binding protein-1c (SREBP-1c) is a major regulator of the expression of genes involved in hepatic triacylglycerol synthesis. In addition, endoplasmic reticulum (ER)-bound transcription factor families are involved in the control of various metabolic pathways. Here, we show a novel function for an ER-bound transcription factor, cAMP response element binding protein H (CREBH), in fenofibrate-mediated inhibition of hepatic lipogenesis. METHODS: The effects of fenofibrate and adenovirus-mediated Crebh (also known as Creb313) overexpression (Ad-Crebh) on hepatic SREBP-1c production and lipogenesis in vitro and in vivo were investigated. We also examined whether downregulation of endogenous hepatic Crebh by small interfering (si)RNA restores the fenofibrate effect on hepatic lipogenesis and SREBP-1c production. Finally, we examined the mechanism by which CREBH inhibits hepatic SREBP-1c production. RESULTS: Fasting and fenofibrate treatment induced CREBH production and decreased SREBP-1c levels. Indeed, Ad-Crebh inhibited insulin- and liver X receptor agonist TO901317-induced Srebp-1c (also known as Srebf1) mRNA expression in cultured hepatocytes. Moreover, increased production of CREBH in the liver of mice following tail-vein injection of Ad-Crebh inhibited high-fat diet-induced hepatic steatosis through inhibition of Srebp-1c expression. The inhibition of endogenous Crebh expression by siRNA restored fenofibrate-induced suppression of Srebp-1c expression and hepatic lipid accumulation both in vitro and in vivo. CONCLUSIONS/INTERPRETATION: These results show that fenofibrate decreases hepatic lipid synthesis through induction of CREBH. This study suggests CREBH as a novel negative regulator of SREBP-1c production and hepatic lipogenesis.

Characteristics of positive surgical margins in robotic-assisted radical prostatectomy, open retropubic radical prostatectomy, and laparoscopic radical prostatectomy: a comparative histopathologic study from a single academic center.

Studies detailing differences in positive surgical margin among open retropubic radical prostatectomy, laparoscopic radical prostatectomy, and robotic-assisted laparoscopic radical prostatectomy are lacking. A retrospective review of all prostatectomies with positive surgical margin performed at our center in 2007 disclosed 99 cases, 6 (5%) of which were reinterpreted cases as having negative margins. Ninety-three cases were, therefore, included, corresponding to 37 retropubic radical prostatectomies, 19 laparoscopic radical prostatectomies, and 37 robotic-assisted laparoscopic radical prostatectomies. The relationship of positive surgical margin characteristics to clinicopathologic parameters and biochemical recurrence was assessed. The most commonly found positive surgical margin site was the apex/distal third in all groups (62% retropubic prostatectomies, 79% laparoscopic prostatectomies, 60% robotic-assisted prostatectomies). Total linear length of positive surgical margin sites was significantly correlated with preoperative prostate-specific antigen, preoperative prostate-specific antigen density, pT stage, and tumor volume (P ≤ .001). We found no significant differences among the 3 groups with respect to total linear length, number of foci, laterality, or location of positive surgical margin. The rate of biochemical recurrence was also comparable in the 3 groups. On univariate analyses, biochemical recurrence was significantly associated with preoperative prostate-specific antigen values, preoperative prostate-specific antigen density, Gleason score, number of positive surgical margins, and total linear length of positive surgical margin (P ≤ .02). Only preoperative prostate-specific antigen density and number of positive surgical margin foci were statistically significant (P ≤ .03) independent predictors of biochemical recurrence. We found no significant difference in positive surgical margin characteristics or biochemical recurrence among the 3 radical prostatectomy modalities. Preoperative prostate-specific antigen density and number of positive surgical margin foci were the only independent predictors of biochemical recurrence.

Relationship between ED and depression among middle-aged and elderly men in Korea: Hallym aging study.

The aim of this study was to investigate the relationship between ED and depression. The survey was conducted among persons enrolled in panel study about Quality of Life of Korean Elderly Project at the Institute for Aging studies. Subjects were 203 men aged 45-74 years (mean age 65.5 years). ED was assessed by International Index of Erectile Function 5 (IIEF-5) score (Korean version), and depression was assessed by the Geriatric Depression Scale (GDS, Korean version). The baseline questionnaires included demographic and health history information. The age-adjusted prevalence of current depression by GDS (≥18), of ED by IIEF-5 score (<18), and of concomitant ED and depression were 12.2%, 28.2% and 11.0%, respectively. GDS increased according to severity of ED, adjusted for age, marital status, education, smoking, alcohol, hypertension, regular exercise, total cholesterol level, fasting blood sugar, body mass index (P<0.001, by analysis of covariance). ED was strongly associated with depression symptoms after controlling for potential confounding factors using logistic regression. Compared with GDS <8, odds ratios and 95% confidence intervals for 12-17 GDS and 18 or more GDS were 3.38 (1.30-8.77) and 6.56 (2.18-19.81), respectively. ED is significantly associated with highly depressive symptoms, regardless of age, health habit or concomitant comorbidity. Our results demonstrate that multidisciplinary approaches are important for the successful treatment of ED.

Pretreatment with the tumor nerosis factor-alpha blocker etanercept attenuated ischemia-reperfusion renal injury.

INTRODUCTION: Tumor necrosis factor (TNF)-alpha mediates inflammation and apoptosis in ischemia-reperfusion (IR) injury of the kidneys. Etanercept, a soluble TNF-alpha receptor, has shown anti-inflammatory and anti-apoptotic effects in several animal models of renal injury, including chronic insufficiency and unilateral ureteral obstruction. We evaluated the protective effect of etanercept against experimental renal IR injury. METHODS: Male Sprague-Dawley (SD) rats were divided into 4 groups: saline-treated sham rats, etanercept-treated sham rats, saline-treated IR rats, and etanercept-treated IR rats. Renal messenger RNA (mRNA) levels of TNF-alpha and monocyte chemotactic protein-1 (MCP-1) were measured by real-time polymerase chain reaction (PCR) at 24 hours after IR injury. The protein levels of renal Bcl-2 associated X (Bax), B-cell lymphoma 2 (Bcl), extracellular signal-regulated kinase (ERK), and caspase-3 activation were evaluated using Western blot analysis. The degree of apoptosis of renal tubular cells was determined using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays. RESULTS: At 24 hours after IR injury, the serum levels of blood urea nitrogen (BUN) and creatinine were significantly lower among etanercept-treated than saline-treated IR rats. Renal mRNA levels of TNF-alpha and MCP-1 in saline-treated IR rats were significantly higher than the levels in saline-treated sham rats, and TNF-alpha and MCP-1 mRNA levels in etanercept-treated IR rats were significantly lower than those in saline-treated IR rats. Etanercept pretreatment of IR-injured rats significantly increased EKR phosphorylation and reduced the renal Bcl-2/Bax ratio, the renal caspase-3 activation, and the number of TUNEL-positive apoptotic cells. CONCLUSION: Etanercept improved resistance to renal injury during IR by enhancing the activation of ERK and increasing the Bcl-2/Bax ratio.

Characterization of the TSU-PR1 cell line by chromosome painting and flow cytometry.

TSU-PR1 was originally reported as a prostatic carcinoma cell line derived from a lymph node metastasis. Recently, however, this cell line was reported to be derived from T24 bladder carcinoma cells, and thus further definition of its origin is needed. Conventional cytogenetic study of TSU-PR1 showed aneuploidy, ranging from 65 to 86 chromosome with a modal number of 80, and with 10 marker chromosomes, thus conventional cytogenetics cannot be used to determine which chromosomes or regions of chromosomes are critical in cancer development and progression of this cell line. The present study was conducted to characterize genetic changes of the cell line using comparative genomic hybridization (CGH), fluorescence in situ hybridization (FISH), and flow cytometry. CGH results showed that green-to-red fluorescence ratios were within the range of 0.85-1.15, except for a few chromosomes, which reflected near tetraploidy in TSU-PR1. Flow cytometric analysis of TSU-PR1 revealed a DNA index of 3.46n, which is close to the 3.48n calculated from a modal number of 80. The copy numbers of chromosomes 4, 6, 7, 17, and 20 determined by the DNA index and the CGH analyses were 2.85 +/- 0.09, 3.22 +/- 0.77, 3.01 +/- 0.26, 4.05 +/- 0.44, and 4.99 +/- 0.48, respectively. These numbers are also in accordance with the chromosome copy numbers determined with FISH: 2.98 +/- 0.23, 2.91 +/- 0.44, 2.74 +/- 0.44, 3.93 +/- 0.38, and 5.05 +/- 0.78 for chromosomes 4, 6, 7, 17, and 20, respectively (P > 0.05).

Sclerotherapy of peritoneal inclusion cysts: preliminary results in seven patients.

OBJECTIVE: To evaluate the technical feasibility and the clinical effectiveness of sclerotherapy for the treatment of peritoneal inclusion cysts (PICs). MATERIALS AND METHODS: Between June 1996 and February 2001, eight PICs in seven female patients aged 28-43 (mean, 36) years were instilled with sclerosant (povidone-iodine in three, ethanol in three, both povidone-iodine and ethanol in one). All seven patients subsequently experienced less abdominal pain. After drainage via an 8.5-Fr pigtail catheter inserted in the PICs (transabdominally in six cases, transvaginally in one), sclerosant equivalent in volume to about one-third that of drained fluid was introduced daily until the drained volume was less than 5ml. Follow-up by means of clinical procedures and ultrasound was performed every three months, at which time the success rate, possible complications and recurrence were determined. RESULTS: Sclerotherapy was technically successful in all seven patients, though immediately after the procedure, minor complications were noted in three patients (mild pain in two, mild fever in one). During the follow-up of 4-60 (mean, 24.7) months, sclerotherapy proved successful and without long-term complications in all seven patients: lower abdominal pain disappeared and the diameter of the cysts decreased more than 50%, with complete regression in four cases. During the follow-up period there was no recurrence. CONCLUSION: Sclerotherapy following catheter insertion is technically feasible and effective for the treatment of PICs.

Murine pro-tumor necrosis factor expressed in Saccharomyces cerevisiae HF7c localizes to membrane/particulate.

Tumor necrosis factor (TNF) is a cytokine that is produced by immune cells in response to bacterial and viral stimuli and plays important roles in various inflammatory diseases. TNF is produced as a membrane-bound precursor, which is then cleaved to release soluble mature protein. We expressed murine pro-TNF in Saccharomyces cerevisiae and examined processing and cellular localization of the recombinant protein. Yeast cells were transformed with an expression construct carrying the pro-TNF gene under the control of alcohol dehydrogenase promoter. Immunoblotting analysis of cell homogenate revealed expression of 26 kD pro-TNF in transformed cells. Upon centrifugation, pro-TNF transformed cells fractionated into the membrane/particulate. In a clone that expresses a high level of pro-TNF, mature 17 kD TNF was detected in the culture medium, although the amount was far smaller than that of cell-associated pro-TNF. Flow cytometric analysis of yeast spheroplasts demonstrated the presence of TNF on the cell surface. Our results show that pro-TNF expressed in yeast mainly resides in the cellular membrane with an orientation similar to that of pro-TNF produced in mammalian cells. Our data suggest that the transformed yeast cells can be used for the genetic analysis of pro-TNF processing machinery in immune cells.

Thiol-reactive metal compounds inhibit NF-kappa B activation by blocking I kappa B kinase.

Gold compounds are used in the treatment of rheumatoid arthritis. NF-kappa B is a transcription factor implicated in the expression of many inflammatory genes. NF-kappa B is activated by signal-induced phosphorylation and subsequent degradation of inhibitory I kappa B (inhibitory protein that dissociates from NF-kappa B) proteins, and a multisubunit I kappa B kinase (IKK) has been identified previously. We tested the effect of various gold compounds on the activation of NF-kappa B and IKK in LPS-stimulated RAW 264.7 mouse macrophages. A lipophilic gold compound, auranofin, suppressed the LPS-induced increase of nuclear kappa B-binding activity, degradation of I kappa B proteins, and IKK activation. Auranofin also blocked IKK activation induced by TNF and PMA/ionomycin, suggesting that the target of auranofin action is common among these diverse signal pathways. In vitro IKK activity was suppressed by addition of hydrophilic gold compounds, such as aurothiomalate, aurothioglucose, and AuCl3. Other thiol-reactive metal ions such as zinc and copper also inhibited IKK activity in vitro, and induction of IKK in LPS-stimulated macrophages. In vitro IKK activity required the presence of reducing agent and was blocked by addition of thiol group-reactive agents. Two catalytic subunits of IKK complex, IKK alpha and IKK beta, were both inhibited by these thiol-modifying agents, suggesting the presence of a cysteine sulfhydryl group in these subunits, which is critical for enzyme activity. The antiinflammatory activity of gold compounds in the treatment of rheumatoid arthritis may depend on modification of this thiol group by gold.

Nuclear factor kappaB (NF-kappaB) pathway as a therapeutic target in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by persistent joint swelling and progressive destruction of cartilage and bone. Current RA treatments are largely empirical in origin and their precise mechanism of action is uncertain. Increasing evidence shows that chronic inflammatory diseases such as RA are caused by prolonged production of proinflammatory cytokines including tumor necrosis factor (TNF) and interleukin 1 (IL-1). The nuclear factor kappaB (NF-kappaB) plays an essential role in transcriptional activation of TNF and IL-1. NF-kappaB is induced by many stimuli including TNF and IL-1, forming a positive regulatory cycle that may amplify and maintain RA disease process. NF-kappaB and enzymes involved in its activation can be a target for anti-inflammatory treatment. Aspirin and sodium salicylate inhibit activation of NF-KB by blocking IkappaB kinase, a key enzyme in NF-kappaB activation. Glucocorticoids suppress expression of inflammatory genes by binding glucocorticoid receptor with NF-kappaB, and increasing expression of inhibitory protein of NF-kappaB, IkappaBalpha. Sulfasalazine and gold compounds also inhibit NF-kappaB activation. Continuing advances in our understanding of action mechanism of antirheumatic agents will benefit the future development of RA regimens with greater efficacy and less toxicity.

Chloroquine inhibits processing of tumor necrosis factor in lipopolysaccharide-stimulated RAW 264.7 macrophages.

TNF, a potent immunoregulatory cytokine, is associated with inflammatory diseases such as rheumatoid arthritis, multiple sclerosis, and cerebral malaria when produced in excess. Antimalarial agents such as chloroquine and hydroxychloroquine have been used to treat some rheumatic diseases. Chloroquine was reported to inhibit production of TNF, although the underlying mechanism is poorly understood. In RAW 264.7 cells stimulated with LPS, addition of chloroquine at nontoxic concentrations did not inhibit induction of TNF mRNA and NF-kappaB activity. In the same cells, synthesis and steady state level of 26-kDa pro-TNF were also not significantly reduced by addition of chloroquine, while only small amount of 17-kDa mature TNF was detected in the medium. A pulse-chase experiment of pro-TNF produced in chloroquine-treated cells showed significant inhibition of processing of prohormone. Hydroxychloroquine showed similar inhibitory effect, whereas other lysosomal inhibitors such as ammonium chloride and methylamine had no effect on the production of TNF. Our results suggest that chloroquine inhibits production of TNF at the step of processing of membrane-bound pro-TNF to make soluble mature protein in a lysosome-independent manner.

Dietary antioxidant inhibits lipoprotein oxidation and renal injury in experimental focal segmental glomerulosclerosis.

Lipid peroxidation may be involved in the pathogenesis of focal segmental glomerulosclerosis (FSGS). In the present study we examined whether lipid-soluble antioxidants, probucol and vitamin E, could inhibit renal injury in rats with chronic puromycin aminonucleoside (PA) nephrosis and dietary hypercholesterolemia by protecting lipoproteins from oxidation. Male Sprague-Dawley rats received six intraperitoneal injections of PA over a 10 week period and were fed a high cholesterol (HC) diet (PA-HC) or the same diet supplemented with either 1% probucol or vitamin E (100 IU/kg) for 32 weeks. For comparison, a group of rats received PA injections and a normal diet (PA-normal) with or without probucol or vitamin E. Another group rats received saline injections instead of PA and were fed a HC diet (Sal-HC) with or without probucol or vitamin E. At the end of the experiment, proteinuria, FSGS and tubulointerstitial lesions were present in the untreated rats with PA-HC or PA-normal. The magnitude of these lesions was significantly greater in the PA-HC rats than the PA-normal. In contrast to the PA-HC group with hypercholesterolemia, the PA-normal group did not show hypercholesterolemia from week 16 onwards. The rats with PA-HC alone showed significantly higher renal cortical malondialdehyde (MDA) levels and greater susceptibility of plasma very low density lipoprotein (VLDL) + low density lipoprotein (LDL) to the copper-mediated oxidation than the rats with PA-normal or Sal-HC alone. The administration of probucol or vitamin E in the rats with PA-HC significantly reduced the susceptibility of plasma VLDL + LDL to in vitro oxidation, renal cortical MDA level, proteinuria, mesangial volume density and magnitude of FSGS and interstitial lesions. Immunohistochemical staining of renal tissue showed focal segmental distribution of oxidized LDL (Ox-LDL) in the glomeruli of rats with PA-HC. Administration of probucol or vitamin E reduced the intensity of Ox-LDL staining. The staining with ED1 demonstrated that infiltrating glomerular macrophages were significantly more prevalent in the untreated rats with PA-HC than PA-normal or Sal-HC. Treatment with probucol or vitamin E significantly reduced the number of glomerular macrophages in the rats with PA-HC. These results suggest that alimentary hypercholesterolemia aggravates the renal damage in association with increased renal lipid peroxides in chronic PA nephrosis, and that dietary probucol or vitamin E attenuates renal injury in rats with PA-HC possibly by making lipoproteins resistant to oxidation and by inhibiting intraglomerular macrophage infiltration.