Anti-Lipoprotein Lipase Antibodies in Patients with Hypertriglyceridemia without Associated Autoimmune Disease

Background: Anti-lipoprotein lipase antibodies have been described in rare cases of patients with hypertriglyceridemia. However, no systematic study evaluating these antibodies in patients with this lipid abnormality has been undertaken. Objectives: To analyze the correlation of anti-lipoprotein lipase (anti-LPL) antibodies with other laboratory findings in patients with hypertriglyceridemia but no autoimmune disease. Methods: We evaluated 44 hypertriglyceridemic patients without autoimmune disease. Clinical and laboratory evaluations included analyses of comorbidities, fasting lipid profile and anti-LPL antibodies. Results: Mean patient age was 55 +/- 10 years; 46% of the patients were female and 64% were Caucasian. The mean disease duration was 94.4 months and mean body mass index 28.7 +/- 3.6 kg/m(2); 34.0% were diabetic, 25.0% were obese, 72.7% had systemic arterial hypertension, 75% were sedentary, 15.9% were smokers, 56.8% had a family history of dyslipidemia, 45.5% had a family history of coronary insufficiency, 20.5% had acute myocardial infarction, 9.0% had undergone revascularization and 11.0% angioplasty, 79.5% were being treated with statins and 43.2% were taking fibrates. Median triglyceride levels were 254 mg/dl (range 100-3781 mg/dl), and total cholesterol level was 233 +/- 111 mg/dl. High-density lipoprotein was 42.6 +/- 15.4 mg/dl, low-density lipoprotein 110.7 +/- 42.4 mg/dl and very low-density lipoprotein 48 +/- 15 mg/dl. Anti-LPL antibodies were identified in 2 patients (4.5%), both of whom had a family history of dyslipidemia, coronary insufficiency and acute myocardial infarction; one had undergone myocardial revascularization and percutaneous transluminal coronary angioplasty, and both were using fibrates and had normal triglyceride levels. Conclusions: Our findings demonstrate a correlation between the immune response and dyslipoproteinemia in hypertriglyceridemic patients, suggesting that autoimmune disease contributes to the dyslipidemia process.

Oral ingestion of a hydrolyzed gelatin meal in subjects with normal weight and in obese patients: Postprandial effect on circulating gut peptides, glucose and insulin

Gut hormones Ighrelin, peptide YY (PYY) and ghrcagon-like peptide-1 (GLP-1)] are an important group of hormones that target appetite control. They are released from endocrine L cells of the small bowel in proportion to the volume, components and calories in a meal. In the current study, 20 g of gelatin (flavored and sweetened) were given to obese patients (n=12) and lean subjects (n=10). Subsequently, plasma samples were collected at-30-minute intervals rip to 180 minutes and glucose, insulin, PYY, GLP-1 and ghrelin were assayed using specific and sensitive immunofluorometric and radioimmunoassays. As expected, obese patients had normal serum glucose levels, higher serum insulin, and lower plasma concentration of ghrelin at all times compared to lean subjects. GLP-1 plasma levels were significantly elevated at 60 minutes, peaking at 120 minutes in obese patients and lean subjects. As a consequence, there was a significant rise in serum insulin levels with a significantly higher peak level at 60 min (obese) and 30 min (lean). There were no significant changes in PYY plasma concentrations and no correlation was found between body mass index and concentrations of ghrelin, PYY and GLP-1 in the group of obese patients. In conclusion, a single gelatin meal induces a rise in plasma GLP-1 followed by an increase in serum levels of insulin. These findings may be applied to maximize satiety in obese patients as a means of improving adherence to calorie-controlled diets as well as provide better control of diabetic patients.

Cardiovascular, metabolic and hormonal responses to the progressive exercise performed to exhaustion in patients with type 2 diabetes treated with metformin or glyburide

Objectives: To evaluate the effects of Metformin and Glyburide on cardiovascular, metabolic and hormonal parameters during progressive exercise performed to exhaustion in the post-prandial state in women with type 2 diabetes (T2DM). Design and Methods: Ten T2DM patients treated with Metformin (M group), 10 with Glyburide (G group) and 10 age-paired healthy subjects exercised on a bicycle ergometer up to exercise peak. Cardiovascular and blood metabolic and hormonal parameters were measured at times -60 min, 0 min, exercise end, and at 10 and 20 minutes of recovery phase. Thirty minutes before the exercise, a standard breakfast was provided to all participants. The diabetic patients took Metformin or Glyburide before or with meal. Results: Peak oxygen uptake (VO2) was lower in patients with diabetes. Plasma glucose levels remained unchanged, but were higher in both diabetic groups. Patients with diabetes also presented lower insulin levels after meals and higher glucagon levels at exercise peak than C group. Serum cortisol levels were higher in G than M group at exercise end and recovery phase. Lactate levels were higher in M than G group at fasting and in C group at exercise peak. Nor epinephrine, GH and FFA responses were similar in all 3 groups. Conclusion: Progressive exercise performed to exhaustion, in the post-prandial state did not worsen glucose control during and after exercise. The administration of the usual dose of Glyburide or Metformin to T2DM patients did not influence the cardiovascular, metabolic and hormonal response to exercise.

Abnormal chronotropic reserve and heart rate recovery in patients with SLE: a case-control study

Abnormal heart-rate (HR) response during or after a graded exercise test has been recognized as a strong and an independent predictor of all-cause mortality in healthy and diseased subjects. The purpose of the present study was to evaluate the HR response during exercise in women with systemic lupus erythematosus (SLE). In this case-control study, 22 women with SLE (age 29.5 perpendicular to 1.1 years) were compared with 20 gender-, BMI-, and age-matched healthy subjects (age 26.5 +/- 1.4 years). A treadmill cardiorespiratory test was performed and HR response during exercise was evaluated by the chronotropic reserve (CR). HR recovery (Delta HRR) was defined as the difference between HR at peak exercise and at both first (Delta HRR1) and second (Delta HRR2) minutes after exercising. SLE patients presented lower peak VO(2) when compared with healthy subjects (27.6 perpendicular to 0.9 vs. 36.7 perpendicular to 1.1 ml/kg/min, p = 0.001, respectively). Additionally, SLE patients demonstrated lower CR (71.8 +/- 2.4 vs. 98.2 +/- 2.6%, p = 0.001), Delta HRR1 (22.1 +/- 2.5 vs. 32.4 +/- 2.2%, p = 0.004) and Delta HRR2 (39.1 +/- 2.9 vs. 50.8 +/- 2.5%, p = 0.001) than their healthy peers. In conclusion, SLE patients presented abnormal HR response to exercise, characterized by chronotropic incompetence and delayed Delta HRR. Lupus (2011) 20, 717-720.

Worse renal outcome of lupus nephritis in male patients: a case-control study

Background: Progression and long-term renal outcome of lupus nephritis (LN) in male patients is a controversial subject in the literature. The aim of this study was to evaluate the influence of male gender on the renal outcome of LN. Methods: All male (M) LN patients who fulfilled American College of Rheumatology lupus criteria and who were referred for a kidney biopsy from 1999 to 2009 were enrolled in the study. Subjects with end-stage renal disease at baseline, or follow-up time below 6 months, were excluded. Cases were randomly matched to female (F) patients according to the class of LN, baseline estimated glomerular filtration rate (eGFR, Modification of Diet in Renal Disease simplified formula) and follow-up time. Treatment was decided by the clinical staff based on usual literature protocols. The primary endpoint was doubling of serum creatinine and/or end-stage renal disease. The secondary endpoint was defined as a variation of glomerular filtration rate (GFR) per year (Delta GFR/y index), calculated as the difference between final and initial eGFR adjusted by follow-up time for each patient. Results: We included 93 patients (31 M : 62 F). At baseline, M and F patients were not statistically different regarding WHO LN class (II 9.7%, IV 71%, V 19.3%), eGFR (M 62.4 +/- 36.4 ml/min/1.73 m(2) versus F 59.9 +/- 32.7 ml/min/1.73 m(2)), follow-up time (M 44.2 +/- 27.3 months versus F 39.9 +/- 27.9 months), and 24-hour proteinuria (M 5.3 +/- 4.6 g/day versus F 5.2 +/- 3.0 g/day), as well as age, albumin, C3, antinuclear antibody, anti-DNA antibody and haematuria. There was no difference in the primary outcome (M 19% versus F 13%, log-rank p = 0.62). However, male gender was significantly associated with a worse renal function progression, as measured by Delta GFR/y index (beta coefficient for male gender -12.4, 95% confidence interval -22.8 to -2.1, p = 0.02). The multivariate linear regression model showed that male gender remained statistically associated with a worse renal outcome even after adjustment for eGFR, proteinuria, albumin and C3 complement at baseline. Conclusion: In our study, male gender presented a worse evolution of LN (measured by an under GFR recovering) when compared with female patients with similar baseline features and treatment. Factors that influence the progression of LN in men and sex-specific treatment protocols should be further addressed in new studies. Lupus (2011) 20, 561-567.

PPAR gamma expression is increased in systemic lupus erythematosus patients and represses CD40/CD40L signaling pathway

Systemic lupus erythematosus (SLE) is a heterogeneous disease involving several immune cell types and pro-inflammatory signals, including the one triggered by binding of CD40L to the receptor CD40. Peroxisome-proliferator activated receptor gamma (PPAR gamma) is a transcription factor with anti-inflammatory properties. Here we investigated whether CD40 and PPAR gamma could exert opposite effects in the immune response and the possible implications for SLE. Increased PPAR gamma mRNA levels were detected by real-time PCR in patients with active SLE, compared to patients with inactive SLE PPAR gamma/GAPDH mRNA = 2.21 +/- 0.49 vs. 0.57 +/- 0.14, respectively (p < 0.05) or patients with infectious diseases and healthy subjects (p < 0.05). This finding was independent of the corticosteroid therapy. We further explored these observations in human THP1 and in SLE patient-derived macrophages, where activation of CD40 by CD40L promoted augmented PPAR gamma gene transcription compared to non-stimulated cells (PPAR gamma/GAPDH mRNA = 1.14 +/- 0.38 vs. 0.14 +/- 0.01, respectively; p < 0.05). This phenomenon occurred specifically upon CD40 activation, since lipopolysaccharide treatment did not induce a similar response. In addition, increased activity of PPAR gamma was also detected after CD40 activation, since higher PPAR gamma-dependent transcription of CD36 transcription was observed. Furthermore, CD40L-stimulated transcription of CD80 gene was elevated in cells treated with PPAR gamma-specific small interfering RNA (small interfering RNA, siRNA) compared to cells treated with CD40L alone (CD80/GAPDH mRNA = 0.11 +/- 0.04 vs. 0.05 +/- 0.02, respectively; p < 0.05), suggesting a regulatory role for PPAR gamma on the CD40/CD40L pathway. Altogether, our findings outline a novel mechanism through which PPAR gamma regulates the inflammatory signal initiated by activation of CD40, with important implications for the understanding of immunological mechanisms underlying SLE and the development of new treatment strategies. Lupus (2011) 20, 575-587.

Creatine supplementation does not impair kidney function in type 2 diabetic patients: a randomized, double-blind, placebo-controlled, clinical trial

Creatine supplementation may have a therapeutic role in diabetes, but it is uncertain whether this supplement is safe for kidney function. The aim of this study was to investigate the effects of creatine supplementation on kidney function in type 2 diabetic patients. A randomized, double-blind, placebo-controlled trial was performed. The patients were randomly allocated to receive either creatine or placebo for 12 weeks. All the patients underwent exercise training throughout the trial. Subjects were assessed at baseline and after the intervention. Blood samples and 24-h urine samples were obtained for kidney function assessments. Additionally, (51)Cr-EDTA clearance was performed. To ensure the compliance with creatine intake, we also assessed muscle phosphorylcreatine content. The creatine group presented higher muscle phosphorylcreatine content when compared to placebo group (CR Pre 44 +/- A 10, Post 70 +/- A 18 mmol/kg/wt; PL Pre 52 +/- A 13, Post 46 +/- A 13 mmol/kg/wt; p = 0.03; estimated difference between means 23.6; 95% confidence interval 1.42-45.8). No significant differences were observed for (51)Cr-EDTA clearance (CR Pre 90.4 +/- A 16.9, Post 96.1 +/- A 15.0 mL/min/1.73 m(2); PL Pre 97.9 +/- A 21.6, Post 96.4 +/- A 26.8 mL/min/1.73 m(2); p = 0.58; estimated difference between means -0.3; 95% confidence interval -24.9 to 24.2). Creatinine clearance, serum and urinary urea, electrolytes, proteinuria, and albuminuria were unchanged. CR supplementation does not affect kidney function in type 2 diabetic patients, opening a window of opportunities to explore its promising therapeutic role in this population. ClinicalTrials.gov registration number: NCT00992043.

Penile anthropometry in systemic lupus erythematosus patients

The aim of this study was to evaluate penile anthropometry in systemic lupus erythematosus (SLE) patients compared with healthy controls and the possible relevant pubertal, clinical, hormonal and treatment factors that could influence penile dimensions. Twenty-five consecutive SLE patients were assessed by urological examination, sexual function, testicular ultrasound, hormones, sperm analysis, genetic analysis, clinical features and treatment. The control group included 25 age-matched healthy males. SLE patients had a lower median penis length and circumference [8 (7.5-10) vs. 10 (8-13) cm, p = 0.0001; 8 (7-10) vs. 10 (7-11) cm, p = 0.001; respectively], lower median testicular volume by right and left Prader [15 (10-25) vs. 20 (12-25) ml, p = 0.003; 15 (10-25) vs. 20 (12-25) ml, p = 0.006; respectively], higher median of follicle-stimulating hormone [5.8 (2.1-25) vs. 3.3 (1.9-9) IU/l, p = 0.002] and lower morning total testosterone levels (28% vs. 0%, p = 0.009) compared with controls. In spite of that, erectile dysfunction was not observed in patients or controls. Analyses of lupus patients revealed that the median penis circumference was lower in patients with disease onset before first ejaculation compared with those with disease onset after first ejaculation [7.8 (7-10) vs. 9.0 (7.5-10) cm, p = 0.026]. No differences were observed in the median penile anthropometry regarding sexual dysfunction (p = 0.610), lower morning total testosterone levels (p = 0.662), oligo/azoospermia (p = 0.705), SLE Disease Activity Index >= 4 (p = 0.562), Systemic Lupus International Collaborating Clinics/ACR Damage Index >= 1 (p = 0.478), prednisone cumulative dose (p = 0.789) and intravenous cyclophosphamide therapy (p = 0.754). Klinefelters syndrome (46XY/47XXY) was diagnosed in one (4%) SLE patient with decreased penile size whereas Y-chromosomal microdeletions was absent in all of them. In conclusion, we have identified reduced penile dimensions in SLE patients with no deleterious effect in erectile function. Disease onset before first ejaculation seems to affect penis development in pre-pubertal lupus. Lupus (2011) 20, 512-518.

Outpatient treatment with intravenous antimicrobial therapy and oral levofloxacin in patients with febrile neutropenia and hematological malignancies

The purpose of this study was to evaluate outcomes such as success of the initial therapy, failure of outpatient treatment, and death in outpatient treatment during intravenous antimicrobial therapy in patients with febrile neutropenia (FN) and hematological malignancies. In addition, clinical and laboratory data and the Multinational Association for Supportive Care of Cancer index (MASCC) were compared with failure of outpatient treatment and death. In a retrospective study, we evaluated FN following chemotherapy events that were treated initially with cefepime, with or without teicoplanin and replaced by levofloxacin after 48 h of defervescence in patients with good general conditions and ANC > 500/mm(3). Of the 178 FN episodes occurred in 126 patients, we observed success of the initial therapy in 63.5% of the events, failure of outpatient treatment in 20.8%, and death in 6.2%. The success rate of oral levofloxacin after defervescence was 99% (95 out of 96). Using multivariate analysis, significant risks of failure of outpatient treatment were found to be smoking (odds ratio (OR) 3.14, confidence interval (CI) 1.14-8.66; p = 0.027) and serum creatinine levels > 1.2 mg/dL (OR 7.97, CI 2.19-28.95; p = 0.002). With regard to death, the risk found was oxygen saturation by pulse oximetry < 95% (OR 5.8, IC 1.50-22.56; p = 0.011). Using the MASCC index, 165 events were classified as low risk and 13 as high risk. Failure of outpatient treatment was reported in seven (53.8%) high-risk and 30 (18.2%) low-risk episodes (p = 0.006). In addition, death occurred in seven (4.2%) low-risk and four (30.8%) high-risk events (p = 0.004). Ours results show that MASCC index was able to identify patients with high risk. In addition, non-smoking, serum creatinine levels a parts per thousand currency sign1.2 mg/dL, and oxygen saturation by pulse oximetry a parts per thousand yen95% were protection factors.

FGF-23 as a Predictor of Renal Outcome in Diabetic Nephropathy

Background and objectives Fibroblast growth factor 23 (FGF-23) has emerged as a new factor in mineral metabolism in chronic kidney disease (CKD). An important regulator of phosphorus homeostasis, FGF-23 has been shown to independently predict CKD progression in nondiabetic renal disease. We analyzed the relation between FGF-23 and renal outcome in diabetic nephropathy (DN). Design, setting, participants, & measurements DN patients participating in a clinical trial (enalapril+placebo versus enalapril+losartan) had baseline data collected and were followed until June 2009 or until the primary outcome was reached. Four patients were lost to follow-up. The composite primary outcome was defined as death, doubling of serum creatinine, and/or dialysis need. Results At baseline, serum FGF-23 showed a significant association with serum creatinine, intact parathyroid hormone, proteirturia, urinary fractional excretion of phosphate, male sex, and race. Interestingly, FGF-23 was not related to calcium, phosphorus, 25OH-vitamin D, or 24-hour urinary phosphorus. Mean follow-up time was 30.7 +/- 10 months. Cox regression showed that FGF-23 was an independent predictor of the primary outcome, even after adjustment for creatinine clearance and intact parathyroid hormone (10 pg/ml FGF-23 increase = hazard ratio, 1.09; 95% CI, 1.01 to 1.16, P = 0.02). Finally, Kaplan-Meier analysis showed a significantly higher risk of the primary outcome in patients with FGF-23 values of >70 pg/ml. Conclusions FGF-23 is a significant independent predictor of renal outcome in patients with macroalbuminuric DN. Further studies should clarify whether this relation is causal and whether FGF-23 should be a new therapeutic target for CKD prevention. Clin J Am Soc Nephrol 6: 241-247, 2011. doi: 10.2215/CJN.04250510

Primary antiphospholipid syndrome in premenopausal women: low vitamin D, high fat mass and maintained bone mineral mass

The aim of this study was to analyze vitamin D levels and their association with bone mineral density and body composition in primary antiphospholipid syndrome. For this cross-sectional study 23 premenopausal women with primary antiphospholipid syndrome (Sapporo criteria) and 23 age- and race-matched healthy controls were enrolled. Demographic, anthropometric, clinical and laboratorial data were collected using clinical interview and chart review. Serum 25-hydroxyvitamin D levels, parathormone, calcium and 24-hour urinary calcium were evaluated in all subjects. Bone mineral density and body composition were studied by dual X-ray absorptiometry. The mean age of patients and controls was 33 years. Weight (75.61 [20.73] vs. 63.14 [7.34] kg, p=0.009), body mass index (29.57 [7.17] vs. 25.35 [3.37] kg, p=0.014) and caloric ingestion (2493 [1005.6] vs. 1990 [384.1] kcal/day, p=0.03) were higher in PAPS than controls. All PAPS were under oral anticoagulant with INR within therapeutic range. Interestingly, biochemical bone parameters revealed lower levels of 25-hydroxyvitamin D [21.64 (11.26) vs. 28.59 (10.67) mg/dl, p=0.039], serum calcium [9.04 (0.46) vs. 9.3 (0.46) mg/dl, p=0.013] and 24-hour urinary calcium [106.55 (83.71) vs. 172.92 (119.05) mg/d, p=0.027] in patients than in controls. Supporting these findings, parathormone levels were higher in primary antiphospholipid syndrome than in controls [64.82 (37.83) vs. 44.53 (19.62) pg/ml, p=0.028]. The analysis of osteoporosis risk factors revealed that the two groups were comparable (p>0.05). Lumbar spine, femoral neck, total femur and whole body bone mineral density were similar in both groups (p>0.05). Higher fat mass [28.51 (12.93) vs. 20.01 (4.68) kg, p=0.005] and higher percentage of fat [36.08 (7.37) vs. 31.23 (4.64)%, p=0.010] were observed in PAPS in comparison with controls; although no difference was seen regarding lean mass. In summary, low vitamin D in primary antiphospholipid syndrome could be secondary to higher weight and fat mass herein observed most likely due to adipocyte sequestration. This weight gain may also justify the maintenance of bone mineral density even with altered biochemical bone parameters. Lupus (2010) 19, 1302-1306.

Male gender results in more severe lupus nephritis

Gender may produce different characteristics in the manifestation of systemic lupus erythematosus (SLE). The present study investigated the influence of gender on clinical, laboratory, autoantibodies and histopathological classes of lupus nephritis (LN). As much as 81 patients diagnosed with SLE (ACR criteria) and active nephritis, who underwent renal biopsy between 1999 and 2004, and who had frozen serum samples and clinical data available from the time of biopsy, were selected for this study. The presence of anti-P and antichromatin antibodies was measured using ELISA, and anti-dsDNA was measured using indirect immunofluorescence. All of the renal biopsies were reviewed in a blinded manner by the same expert renal pathologist. The charts were extensively reviewed for demographic and renal features obtained at the time of the biopsy. Of the 81 patients (13.6%), 11 were male SLE patients. Both male and female lupus patients were of similar age and race, and had similar durations of lupus and renal disease. The female patients had more cutaneous (95.7 vs. 45.5%, P = 0.0001) and haematological (52.9 vs. 18.2%, P = 0.04) involvements than the male SLE patients. In addition, the articular data, central nervous system analyses, serositis findings and SLEDAI scores were similar in both experimental groups. Positivity for anti-dsDNA, anti-ribosomal P and antichromatin did not differ between the two groups, and both groups showed similarly low C3 or C4 serum levels. Our analysis indicated that no histopathological class of LN was predominant in both males and females. Interestingly, the serum creatinine levels were higher in the male SLE patients compared to the female SLE group (3.16 +/- A 2.49 vs. 1.99 +/- A 1.54 mg/dL, P = 0.03), with an increased frequency of high creatinine (81.8 vs. 47.1%, P = 0.04) as well as renal activity index (7.6 +/- A 3.5 vs. 4.8 +/- A 3.5, P = 0.02). In addition, whilst the mean levels of proteinuria, cylindruria and serum albumin were markedly altered, they were comparable between both lupus men and women. Moreover, the frequencies of dialysis, renal transplantation and death were similar between the two groups. These data suggest that male patients had a more severe LN compared to women diagnosed with this renal abnormality.

Clues for Previously Undiagnosed Connective Tissue Disease in Patients With Trigeminal Neuralgia

Background: Connective tissue diseases (CTD) may be associated with idiopathic trigeminal neuralgia (TN). The prevalence and diagnostic implications of this association are, however, not well established. Objectives: The objective of this study was to evaluate, in TN patients, if rheumatologic clinical and laboratory findings could contribute to the early diagnosis of rheumatic diseases. Methods: Forty-six consecutive TN patients, 67% female, mean disease duration 8.78 +/- 7.25 years, and 47 controls were initially interviewed using a standard questionnaire based on common signs/symptoms of systemic lupus erythematosus, Sjogren syndrome, mixed CTD, and systemic sclerosis. Autoantibodies were detected by standard techniques. Those with rheumatologic complaints or positive autoantibodies were referred to the Rheumatology Outpatient Clinic for a more detailed evaluation. Secondary causes of TN were excluded. Results: The frequency of Raynaud phenomenon (P = 0.026) and ANA reactivity (P = 0.04) were significantly higher in TN patients compared with controls. Fourteen TN patients were ANA positive. Seven of them reported concomitant rheumatic complaints, and interestingly, diffuse CTD was diagnosed in 4 (57%) of these patients: 1 systemic lupus erythematosus; 2 Sjogren syndrome; and 1 undifferentiated disease with scleritis and positive parotid scintigraphy. In all cases, TN preceded by at least 10 months the rheumatologic signs/symptoms. Moreover, these 4 TN patients with CTD had a higher frequency of sicca symptoms (P = 0.001) and higher titers of ANA (>= 1:320) (P = 0.006) than the remaining 42 TN patients without CTD diagnoses. Sixteen patients had isolated laboratory or clinical abnormalities, and none of them had CTD diagnoses. Conclusions: The concomitant presence of sicca symptoms and high titer ANA are clues for the early investigation of rheumatic diseases in TN patients.

LOSS OF CD40 ENDOGENOUS S-NITROSYLATION DURING INFLAMMATORY RESPONSE IN ENDOTOXEMIC MICE AND PATIENTS WITH SEPSIS

Signal transduction through the surface molecule CD40 is critical for cellular activation in immunoinflammatory states such as sepsis. The mechanisms regulating this pathway are not completely understood. Because CD40 displays potentially regulatory cysteine residues and CD40 is probably exposed to NO in the inflammatory milieu, we hypothesized that S-nitrosylation, the interaction of NO with cysteines residues, acts as a post-translational modification on CD40, coregulating the signaling activity and, therefore, the level of cellular activation. As assessed by the biotin switch and the reduction/chemiluminescence S-nitrosylation detection techniques, CD40 was found to be S-nitrosylated endogenously and upon exposure to NO donors in both human and murine macrophages. S-nitrosylation of CD40 was associated with milder activation by its ligand (CD40L), leading to reduced in vitro cytokine (IL-1 beta, IL-12, and TNF-alpha) production, which was reversed in the presence of inhibitors of NO synthesis. S-nitrosylated CD40 was found in resting RAW 246.7 macrophages and BALB/c mice peritoneal macrophages, turning into the denitrosylated state upon in vitro or systemic exposure, respectively, to LPS. Moreover, monocytes from patients with sepsis displayed denitrosylated CD40 in contrast to the CD40 S-nitrosylation measured in healthy individuals. Finally, in an attempt to explain how S-nitrosylation regulates CD40 activation, we demonstrate that NO affects the redistribution of CD40 on the cell surface, which is a requirement for optimal signal transduction. Our results support a novel post-translational regulatory mechanism in which the CD40 signal may be, at least in part, dependent on cellular activation-induced receptor denitrosylation.

Measurement of total phospholipids in urine of patients treated with gentamicin

Aims The excretion of phospholipids in urine may be a marker of the early renal toxicity of the aminoglycoside antibiotics. Urinary phospholipids are formed in myeloid bodies which develop in the lysosomes of proximal tubules during treatment with the aminoglycosides, and overflow into the urine. Methods Published assays were modified in order to measure the total phospholipid concentrations in human urine. Phospholipids were extracted from freeze-dried urine samples, digested in concentrated sulphuric acid, and the inorganic phosphorus content determined by complexing with ammonium molybdate and measuring the absorbance at 820 nm. Ten septicaemic patients treated with gentamicin for 5-7 days had significantly higher urine phospholipid concentrations than 10 healthy untreated control subjects (P<0.0001). There was a negative Linear relationship between phospholipid excretion and creatinine clearance (r(2) = 0.71). Results In 34 patients with acute pyelonephritis, increased phospholipid concentrations were observed prior to treatment compared with healthy controls (P<0.001) and did not alter during treatment with gentamicin. However, the phospholipid concentrations decreased significantly after treatment was completed (P<0.03). Conclusions These studies suggest that urinary phospholipids may indicate early aminoglycoside toxicity but with poor specificity, as many of the infections being treated may themselves be associated with phospholipiduria.