Characterization of two different Ca2+ entry pathways dependent on depletion of internal Ca2+ pools in rat aorta

Ryanodine (10 μM), thapsigargin (1 μM) and cyclopiazonic acid (10 μM) produced a slow, sustained contractile response in rat aorta that only can be observed in Ca²⁺-containing solution. In Ca²⁺-free medium, no response to the drugs was obtained, which suggests that the contraction elicited in presence of Ca²⁺ is mainly due to the contribution of extracellular influx. This Ca²⁺ entry does not depend on the opening of dihydropyridine-dependent Ca²⁺-channels for nimodipine does not affect this. Noradrenaline (1 μM) induced a biphasic response in Ca²⁺-free medium that was mediated by two different Ca²⁺ compartments, one of which is common to caffeine (10 mM), and is also depleted by ryanodine (10 μM), thapsigargin (1 μM) and cyclopiazonic acid (10 μM). This compartment loses its Ca²⁺ content after long exposure (65 min) to Ca²⁺-free EDTA-containing solution and its refilling was also affected by the three agents tested. The other compartment depleted by noradrenaline, but not by caffeine, was also insensitive to ryanodine, thapsigargin and cyclopiazonic acid, and did not lose its Ca²⁺ after 65 min in Ca²⁺-free medium. Contractions induced by noradrenaline (1 μM) or caffeine (10 mM) in Ca²⁺-free medium were not affected by ryanodine, thapsigargin and cyclopiazonic acid when these agents were added 1 min before or during the response to each agonist. After depletion of internal Ca²⁺ stores sensitive to noradrenaline, an increase in the resting tone (IRT) of rat aorta was observed when Ca²⁺ was added again in absence of the agonist. This IRT was not affected by treatment with ryanodine, thapsigargin and cyclopiazonic acid, and represents a Ca²⁺ entry pathway dependent on the depletion of the noradrenaline-sensitive Ca²⁺ compartment. In conclusion, we can differentiate two Ca²⁺ entry pathways in rat aorta that depend on the previous depletion of two internal Ca²⁺ compartments: One corresponds to the classic capacitative Ca²⁺ entry model and is promoted by depletion of the internal pool sensitive to noradreanline, caffeine, ryanodine, thapsigargin and cyclopiazonic acid, the other is dependent only on depletion of an α₁-adrenoceptor-sensitive Ca²⁺ pool. Received: 25 June 1997 / Accepted: 17 September 1997     

Urine Metabolite Profiles Predictive of Human Kidney Allograft Status

Noninvasive diagnosis and prognostication of acute cellular rejection in the kidney allograft may help realize the full benefits of kidney transplantation. To investigate whether urine metabolites predict kidney allograft status, we determined levels of 749 metabolites in 1516 urine samples from 241 kidney graft recipients enrolled in the prospective multicenter Clinical Trials in Organ Transplantation-04 study. A metabolite signature of the ratio of 3-sialyllactose to xanthosine in biopsy specimen-matched urine supernatants best discriminated acute cellular rejection biopsy specimens from specimens without rejection. For clinical application, we developed a high-throughput mass spectrometry-based assay that enabled absolute and rapid quantification of the 3-sialyllactose-to-xanthosine ratio in urine samples. A composite signature of ratios of 3-sialyllactose to xanthosine and quinolinate to X-16397 and our previously reported urinary cell mRNA signature of 18S ribosomal RNA, CD3ε mRNA, and interferon-inducible protein-10 mRNA outperformed the metabolite signatures and the mRNA signature. The area under the receiver operating characteristics curve for the composite metabolite–mRNA signature was 0.93, and the signature was diagnostic of acute cellular rejection with a specificity of 84% and a sensitivity of 90%. The composite signature, developed using solely biopsy specimen-matched urine samples, predicted future acute cellular rejection when applied to pristine samples taken days to weeks before biopsy. We conclude that metabolite profiling of urine offers a noninvasive means of diagnosing and prognosticating acute cellular rejection in the human kidney allograft, and that the combined metabolite and mRNA signature is diagnostic and prognostic of acute cellular rejection with very high accuracy.     

Night shift work and chronic lymphocytic leukemia in the MCC‐Spain case–control study

Chronic lymphocytic leukemia (CLL) has few known modifiable risk factors. Recently, circadian disruption has been proposed as a potential contributor to lymphoid neoplasms' etiology. Serum melatonin levels have been found to be significantly lower in CLL subjects compared with healthy controls, and also, CLL prognosis has been related to alterations in the circadian molecular signaling. We performed the first investigation of an association between night shift work and CLL in 321 incident CLL cases and 1728 population‐based controls in five areas of Spain. Participants were interviewed face‐to‐face by trained interviewers to collect information on sociodemographic factors, familial, medical and occupational history, including work shifts and other lifestyle factors. We used logistic regression models adjusted for potential confounders to estimate odds ratios (OR) and 95% confidence intervals (CI). Seventy‐nine cases (25%) and 339 controls (20%) had performed night work. Overall, working in night shifts was not associated with CLL (OR = 1.06; 95% CI = 0.78–1.45, compared with day work). However, long‐term night shift (>20 years) was positively associated with CLL (OR_{(tertile 3 vs. day‐work)} = 1.77; 95% = 1.14–2.74), although no linear trend was observed (P trend = 0.18). This association was observed among those with rotating (OR_{(tertile 3 vs. day‐work)} = 2.29; 95% CI = 1.33–3.92; P trend = 0.07), but not permanent night shifts (OR_{(tertile 3 vs. day‐work)} = 1.16; 95% CI = 0.60–2.25; P trend = 0.86). The association between CLL and long‐term rotating night shift warrants further investigation.     

Risk factors,clinical presentation and prognosis of mixed candidaemia: a population‐based surveillance in Spain

The low incidence of mixed candidaemia (MC) may have precluded a better knowledge of its clinical presentation. The aim of the study was to analyse the risk factors, clinical presentation and prognosis of MC episodes. A comparison between MC and monomicrobial candidaemia within a prospective programme on candidaemia was performed in 29 hospitals between April 2010 and May 2011. In fifteen episodes of candidaemia corresponding to 15 patients, out of 752, two species of Candida (1.9%) were isolated. MC was more frequent in patients with HIV infection (12%, P = 0.038) and those admitted due to extensive burns (23%, P = 0.012). The Candida species most frequently identified in MC were C. albicans 12 patients (40%), C. glabrata seven patients (23.3%) and C. parapsilosis six patients (20%). Early mortality was higher (nine patients, 60%) in patients with MC than in patients with MMC (223 patients, 30.3%, P = 0.046). In conclusion, MC was was independently associated with increased mortality even after considering other prognostic factors. MC is an infrequent event that is more common in HIV infection and in patients suffering from burns, and is associated with increased mortality.     

PILAR is a novel modulator of human T-cell expansion

Robust T-cell responses without autoimmunity are only possible through a fine balance between activating and inhibitory signals. We have identified a novel modulator of T-cell expansion named proliferation-induced lymphocyte-associated receptor (PILAR). Surface PILAR is markedly up-regulated on CD4 and, to a lesser extent, on CD8 T cells on T-cell receptor engagement. In absence of CD28 costimulation, PILAR signaling through CD161 supports CD3 antibody-dependent and antigen-specificT-cell proliferation by increasing the expression of antiapoptotic Bcl-xL and induces secretion of T helper type 1 cytokines. These effects are abrogated by PILAR blockade with specific antibodies, which decrease surface levels of CD28. In contrast, PILAR induces apoptotic death on naive and early activated T cells if CD161 engagement is blocked. PILAR is expressed by approximately 7% to 10% of CD4 T cells in 2 samples of inflammatory synovial fluid, suggesting a potential role in the pathogenesis of joint inflammation. In addition, in the ovarian cancer microenvironment, effector T cells express PILAR, but not CD161, although expression of both can be augmented ex vivo. Our results indicate that PILAR plays a central role in modulating the extent of T-cell expansion. Manipulation of PILAR signaling may be important for treatment of autoimmune diseases and cancer.     

Organic anion transporter 1B1: an important factor in hepatic thyroid hormone and estrogen transport and metabolism

Sulfation is an important pathway in the metabolism of thyroid hormone and estrogens. Sulfation of estrogens is reversible by estrogen sulfatase, but sulfation of thyroid hormone accelerates its degradation by the type 1 deiodinase in liver. Organic anion transporters (OATPs) are capable of transporting iodothyronine sulfates such as T4 sulfate (T4S), T3S, and rT3S or estrogen sulfates like estrone sulfate (E1S), but the major hepatic transporter for these conjugates has not been identified. A possible candidate is OATP1B1 because model substrates for this transporter include the bilirubin mimic bromosulfophthalein (BSP) and E1S, and it is highly and specifically expressed in liver. Therefore, OATP1B1-transfected COS1 cells were studied by analysis of BSP, E1S, and iodothyronine sulfate uptake and metabolism. Two Caucasian populations (155 blood donors and 1012 participants of the Rotterdam Scan Study) were genotyped for the OATP1B1-Val174Ala polymorphism and associated with bilirubin, E1S, and T4S levels. OATP1B1-transfected cells strongly induced uptake of BSP, E1S, T4S, T3S, and rT3S compared with mock-transfected cells. Metabolism of iodothyronine sulfates by cotransfected type 1 deiodinase was greatly augmented in the presence of OATP1B1. OATP1B1-Val174 showed a 40% higher induction of transport and metabolism of these substrates than OATP1B1-Ala174. Carriers of the OATP1B1-Ala174 allele had higher serum bilirubin, E1S, and T4S levels. In conclusion, OATP1B1 is an important factor in hepatic transport and metabolism of bilirubin, E1S, and iodothyronine sulfates. OATP1B1-Ala174 displays decreased transport activity and thereby gives rise to higher bilirubin, E1S, and T4S levels in carriers of this polymorphism.