Reducing glucocorticoid dosage improves serum osteocalcin in patients with rheumatoid arthritis-results from the TOMORROW study.

UNLABELLED: Decreasing the daily dose of glucocorticoids improved bone metabolic marker levels in patients with rheumatoid arthritis. However, changes in disease activity did not influence bone metabolism. Bone metabolism might thus remain uncontrolled even if disease activity is under good control. Decreasing glucocorticoid dosage appears important for improving bone metabolism. INTRODUCTION: Patients with rheumatoid arthritis (RA) develop osteoporosis more frequently than healthy individuals. Bone resorption is increased and bone formation is inhibited in patients with RA, and glucocorticoid negatively affects bone metabolism. We aimed to investigate factors influencing bone metabolic markers in patients with RA. METHODS: We started the 10-year prospective cohort Total Management of Risk Factors in Rheumatoid Arthritis Patients to Lower Morbidity and Mortality (TOMORROW) study in 2010. We compared changes in urinary cross-linked N-telopeptide of type I collagen (uNTx) and serum osteocalcin (OC), as markers of bone resorption and formation, respectively, in 202 RA patients and age- and sex-matched volunteers between 2010 and 2011. We also investigated factors influencing ΔuNTx and ΔOC in the RA group using multivariate analysis. RESULTS: Values of ΔuNTx were significantly lower in patients with RA than in healthy controls (-0.51 vs. 7.41 nmol bone collagen equivalents (BCE)/mmol creatinine (Cr); p = 0.0013), whereas ΔOC values were significantly higher in RA patients (0.94 vs. 0.37 ng/ml; p = 0.0065). Changes in prednisolone dosage correlated negatively with ΔOC (ß = -0.229, p = 0.001), whereas changes in disease activity score, bisphosphonate therapy, and period of biologics therapy did not correlate significantly with ΔOC. No significant correlation was seen between ΔuNTx and change in prednisolone dosage. CONCLUSIONS: Decreased glucocorticoid dosage improved bone metabolic markers in RA, but disease activity, bisphosphonate therapy, and period of biologics therapy did not influence levels of bone metabolic markers. Decreasing glucocorticoid dosage appears important for improving bone metabolic marker profiles in patients with RA.

Temporal and spatial association of Streptococcus suis infection in humans and porcine reproductive and respiratory syndrome outbreaks in pigs in northern Vietnam.

Porcine reproductive and respiratory syndrome (PRRS) outbreaks in pigs are associated with increased susceptibility of pigs to secondary bacterial infections, including Streptococcus suis - an important zoonotic pathogen causing bacterial meningitis in humans. This case-control study examined the association between human S. suis infection and PRRS outbreaks in pigs in northern Vietnam. We included 90 S. suis case-patients and 183 non-S. suis sepsis controls from a referral hospital in Hanoi in 2010, a period of major PRRS epizootics in Vietnam. PRRS exposure was determined using data from the National Centre of Veterinary Diagnosis. By univariate analysis, significantly more S. suis patients were reported residing in or adjacent to a PRRS district compared to controls [odds ratio (OR) 2·82, 95% confidence interval (CI) 1·35-5·89 and OR 3·15, 95% CI 1·62-6·15, respectively]. Only residency in adjacent districts remained significantly associated with risk of S. suis infection after adjusting for sex, occupation, and eating practices. SaTScan analysis showed a possible cluster of S. suis infection in humans around PRRS confirmed locations during the March-August period. The findings indicate an epidemiological association between PRRS in pigs and S. suis infections in humans. Effective strategies to strengthen control of PRRS in pigs may help reduce transmission of S. suis infection to humans.

Detection of HPAI H5N1 viruses in ducks sampled from live bird markets in Vietnam.

In Vietnam, highly pathogenic avian influenza (HPAI) H5N1 infections in poultry often occur without concomitant clinical signs and outbreaks are not consistently reported. Live bird markets represent a convenient site for surveillance that does not rely on farmers' notifications. Two H5N1 surveys were conducted at live bird markets/slaughter points in 39 districts (five provinces) in the Red River, Mekong delta, and central Vietnam during January and May 2011. Oropharyngeal and rectal swab samples from 12 480 ducks were tested for H5N1 by reverse transcription-polymerase chain reaction in pools of five. Traders and stallholders were interviewed using standardized questionnaires; 3·3% of pools tested positive. The highest prevalence (6·6%) corresponded to the Mekong delta, and no H5N1 was detected in the two Red River provinces. The surveys identified key risk behaviours of traders and stallholders. It is recommended that market surveys are implemented over time as a tool to evaluate progress in HPAI control in Vietnam.

[Metastatic ectopic cervical thymoma with prolonged survival; report of a case].

A 60-year-old woman presented with a palpable and painful nodule of her neck. Physical examination revealed that the anterior neck mass was enlarged without other positive findings. The tumor was not diagnosed with a fine needle aspiration biopsy, so that the excision of the tumor was underwent. The diagnosis at permanent section analysis revealed a non-invasive thymoma. The patient did not receive adjunctive postoperative therapy. For the duration of 6.5-year follow-up, metastasis of the left lobe of thyroid and right upper lobe of lung in twice have been sequentially detected and resected, and now she has been healthy with no known recurrence of the tumor. The recurrence of a non-invasive cervical ectopic thymoma has been reported as extremely rare, and this case indicates that the surgical control for recurrence lesion of cervical ectopic thymoma is effective when the tumor is resectable.

Concentrations of an activator protein for sphingolipid hydrolysis in liver and brain samples from patients with lysosomal storage diseases.

The hydrolysis of sphingolipids by lysosomal enzymes requires the presence of additional proteins, which have been called activator proteins. The number of activator proteins, their specificity, exact mechanism of action, and response to a storage process all remain to be determined. In this study, antibodies to an activator protein known to bind sphingolipids and activate the enzymatic hydrolysis of GM1 ganglioside and sulfatide were used to estimate the concentration of this activator protein in small samples of liver and brain from patients with lysosomal storage diseases. By using rocket immunoelectrophoresis, the concentration of cross-reacting material (CRM) was determined. Control livers had an average of 0.95 +/- 0.18 (mean +/- 1 SD) microgram CRM/mg protein in the extracts, and control brains had an average of 0.25 +/- 0.14 microgram CRM/mg protein. Extremely high levels of CRM were found in extracts of livers from patients with type 1 GM1 gangliosidosis (15.1 and 16.9), and type A Niemann-Pick disease (10.7). Extracts of brain samples revealed a large amount of CRM in type 1 GM1 gangliosidosis (14.8), Tay-Sachs disease (5.3 and 8.7), and Sandhoff disease (13.5). Significantly elevated CRM was also measured in brain samples from patients with type 2 GM1 gangliosidosis, type A Niemann-Pick disease, metachromatic leukodystrophy, and Krabbe disease. The highest levels are found in those genetic diseases where the lipids stored, primarily or secondarily to the genetic defect, bind to this activator protein. This activator protein may have an important function in regulating intralysosomal lipid catabolism, and changes in its concentration in certain genetic diseases may be the cause of clinical, biochemical, and pathological heterogeneity found in the patients.

Metabolism of cerebroside sulfate and subcellular distribution of its metabolites in cultured skin fibroblasts from controls, metachromatic leukodystrophy, and globoid cell leukodystrophy.

With pulse-chase study of 1-[14C]stearic acid-labeled cerebroside sulfate (14C-CS) and subsequent subcellular fractionation by Percoll gradient, the metabolism of CS and translocation of its metabolites in human skin fibroblasts from controls, metachromatic leukodystrophy (MLD), and globoid cell leukodystrophy (GLD) were studied. In control skin fibroblasts, CS was transported to lysosome and metabolized there to galactosylceramide (GalCer) and ceramide (Cer) within 1 h. During the chase period, radioactivity was increased at plasma membrane plus Golgi as phospholipids and no accumulation of GalCer or Cer was found in lysosome. In MLD fibroblasts, 95% of 14C-CS taken up was unhydrolyzed at 24 h-chase and accumulated at not only lysosome but also plasma membrane. In GLD fibroblasts, GalCer was accumulated throughout the subcellular fractions and more accumulated mainly at plasma membrane plus Golgi with longer pulse. This translocation of lipid from lysosome seems to have considerable function, even in lipidosis, which may result in an imbalance of the sphingolipid pattern on the cell surface and these changes might be one of causes of neuronal dysfunction in sphingolipidosis.

Cloning, characterization, and chromosomal mapping of human aquaporin of collecting duct.

We recently cloned a cDNA of the collecting duct apical membrane water channel of rat kidney, which is important for the formation of concentrated urine (Fushima, K., S. Uchida, Y. Hara, Y. Hirata, F. Marumo, and S. Sasaki. 1993. Nature [Lond.]. 361:549-552). Since urine concentrating ability varies among mammalian species, we examined whether an homologous protein is present in human kidney. By screening a human kidney cDNA library, we isolated a cDNA clone, designated human aquaporin of collecting duct (hAQP-CD), that encodes a 271-amino acid protein with 91% identity to rat AQP-CD. mRNA expression of hAQP-CD was predominant in the kidney medulla compared with the cortex, immunohistochemical staining of hAQP-CD was observed only in the collecting duct cells, and the staining was dominant in the apical domain. Functional expression study in Xenopus oocytes confirmed that hAQP-CD worked as a water channel. Western blot analysis of human kidney medulla indicated that the molecular mass of hAQP-CD is 29 kD, which is the same mass expected from the amino acid sequence. Chromosomal mapping of the hAQP-CD gene assigned its location to chromosome 12q13. These results could be important for future studies of the pathophysiology of human urinary concentration mechanisms in normal and abnormal states.

Transcription-reverse transcription concerted reaction and minimal residual disease in axillary sentinel nodes of breast cancer.

AIMS: To investigate the use of transcription-reverse transcription concerted reaction (TRC) to detect axillary lymph node metastases. METHODS: Metastases in 423 lymph nodes obtained from 50 breast cancer patients were investigated by routine pathological hematoxylin and eosin (H and E) staining and quantitative analysis of carcinoembryonic antigen (CEA) mRNA by TRC. Enhanced pathological studies, serial sectioning and immunohistochemistry were conducted for cases which were negative by routine pathology, but positive by TRC. RESULTS: Pathological examination identified metastatic disease in 67 lymph nodes. TRC CEA mRNA results were concordant with 89.8% of these cases at a threshold of 100 copies. TRC identified 30 false negative nodes, which was reduced to 15 by excluding node biopsies yielding less than 40 microg total RNA. Twelve nodes were histologically negative for cancer, but positive according to TRC. Serial sectioning and immunohistochemical analysis of these nodes revealed macrometastatic lesions in three, micrometastasis in one, and isolated tumor cells in two. CONCLUSION: TRC for the detection of CEA mRNA may complement routine pathological examination by sentinel lymph node biopsy (SNB) in early breast cancer. We have started an enhanced pathological examination with serial sectioning on all excised sentinel nodes to set the best threshold for the TRC method.

Backward-masking: the effect of the duration of the second stimulus on recognition of the first stimulus.

OBJECTIVE: We recorded event-related magnetic fields following a target stimulus followed by a masking stimulus to investigate the visual backward masking effect using a helmet-type magnetoencephalography system in humans. METHODS: In the target stimulus with masking stimulus conditions, duration of the target stimulus was constant at 16 ms, and duration of the masking stimulus was altered (16, 48 and 144 ms). The target stimulus was masked by the 144-ms masking stimulus, but not by the 16-ms masking stimulus, and was obscured by the 48-ms masking stimulus. For control conditions (Single-condition), event-related magnetic fields were recorded following the sole presentation of the masking stimulus for 32, 64 or 160 ms. RESULTS: One major response was obtained at 180 ms after the onset of the stimulation in each condition. The equivalent current dipole of one major response was estimated to lie in the occipital lobe, but there was a relatively large inter-individual difference. There was no significant difference in latency between the target stimulus with masking stimulus conditions and Single-conditions. In the target stimulus with masking stimulus conditions with the 48- and 144-ms masking stimulus, the root mean square value did not differ from that in the respective Single-condition, while the root mean square value for the target stimulus with masking stimulus conditions with the 16-ms masking stimulus was significantly smaller than that in the Single-condition with the 32-ms masking stimulus, but not different from that in the Single-condition with the 16-ms masking stimulus. CONCLUSIONS: The peak latency of one major response depended on the onset of the first stimulus for both the target stimulus with masking stimulus conditions and Single-condition, but the root mean square value depended on the duration of the masking stimulus. We concluded that the temporal information for the target stimulus was preserved during the masking effect, while the figural information was interrupted by the masking stimulus. Our results suggested that temporal factors for the stimulus were processed differently from those responsible for the object's recognition during backward masking.

A longitudinal study to identify constraints to dairy cattle health and production in rural smallholder communities in Northern Vietnam.

The objective of the study was to investigate constraints to dairy cattle health and production in rural smallholder communities in northern Vietnam, one of the target areas of the Vietnam government's dairy development programme. A total of 99 dairy farms (11 per commune) were recruited from 9 of 32 communes in Ba Vi District, using random two-stage cluster sampling. After the initial questionnaire interviews were conducted, farms were visited at three monthly intervals over a period of 1 year. Information on several health and production parameters relating to the study cattle was collected. Using multiple indicator modelling, it was found that Fasciola infestation, farmers who had been involved in dairying for longer (not indicative of better management skills), larger herd size, and cattle being kept in a shed were linked to reduced reproductive performances.

Prognostic significance of polysialic acid expression in resected non-small cell lung cancer.

Polysialic acid (PSA) is a carbohydrate attached mainly to the neural cell adhesion molecule. Because PSA is composed of a linear homopolymer of alpha-2-8-linked sialic acid residues and has a large negative charge, the presence of PSA attenuates the adhesive property of neural cell adhesion molecule and increases cellular motility. In an earlier study, we demonstrated that PSA and STX, a polysialyltransferase, were associated with tumor progression in non-small cell lung cancer (NSCLC) (F. Tanaka et al., Cancer Res., 60: 3072-3080, 2000). Therefore, in the present study, to assess the prognostic significance of PSA in resected NSCLC, a total of 236 patients who underwent complete resection for pathological (p)-stage I-IIIa disease were reviewed retrospectively. PSA was expressed in 44 of 236 (18.6%) patients, and the expression was correlated with p-stage disease. For all p-stage patients, 5-year survival rates for those with PSA-positive and PSA-negative tumors were 52.1% and 71.3%, respectively, demonstrating a significantly worse prognosis for the PSA-positive patients (P = 0.012). Analysis for only p-stage I patients also demonstrated a significantly worse prognosis for the PSA-positive patients; 5-year survival rates of the PSA-positive and the PSA-negative patients were 45.1% and 83.5%, respectively, (P < 0.001). In addition, there proved to be no difference in the postoperative survival among p-stage I, II, and IIIa patients when PSA expression was positive. Multivariate analysis confirmed that PSA expression was an independent factor to predict poor prognosis in resected NSCLC. These results suggested that PSA could be an important clinical marker and that preoperative induction and/or postoperative adjuvant therapies should be performed for PSA-positive NSCLC, even if the disease is classified as p-stage I.

Inhibitory mechanism by polysialic acid for lamina-specific branch formation of thalamocortical axons.

During development, thalamocortical axons form arbors primarily in layer 4 of the neocortex. This lamina-specific branch formation was studied in cultures of rat thalamic explants grown next to chemically fixed cortical slices. After a week in vitro, thalamic axons formed branches specifically in the target layer of fixed cortical slices, regardless of the orientation of the ingrowth. This in vitro system permits a direct assessment of contributions of membrane-associated molecules to thalamic axon branch formation. To this end, the present study uses three enzymatic perturbations: chondroitinase, phosphatidylinositol phospholipase C, or the polysialic acid (PSA)-specific endoneuraminidase (endo N). With endo N pretreatment of cortex, the number of branch points was increased significantly, whereas branch tip length was decreased. In addition, the localization of branch points to the target layer was weakened considerably. These features of branch formation were not altered by the other two enzymatic treatments, except that branch tips were shortened by chondroitinase treatment to the same extent as in endo N treatment. These results suggest that membrane-bound components are involved in lamina-specific branch formation of thalamocortical axons, and in particular that PSA moieties contribute to laminar specificity by inhibiting branch emergence in inappropriate layers.

Cholesterol modulates organic cation transport activity and lipid fluidity in rat renal brush-border membranes.

The role of cholesterol in organic cation transport was studied in rat renal brush-border membranes. H+ gradient-dependent uptake of the organic cation tetraethylammonium in brush-border membrane vesicles was stimulated by cholesterol enrichment in a dose-dependent manner. The dissipation rate of the H+ gradient, a driving force for organic cation transport in brush-border membranes, was reduced by cholesterol enrichment. Tetraethylammonium uptake in the absence of H+ gradient was also stimulated by cholesterol enrichment. These findings indicate that cholesterol modulates tetraethylammonium uptake by affecting the intrinsic activity of the organic cation transporter and the H+ gradient dissipation rate. Therefore, cholesterol content should be an important determinant for organic cation transport in renal brush-border membranes.

Molecular mechanisms of organic cation transport in OCT2-expressing Xenopus oocytes.

The molecular mechanisms of organic cation transport by rat OCT2 was examined in the Xenopus oocyte expression system. When extracellular Na+ ions were replaced with K+ ions, uptake of tetraethylammonium (TEA) by OCT2-expressing oocytes was decreased, suggesting that TEA uptake by OCT2 is dependent on membrane potential. Kinetic analysis revealed that the decreased TEA uptake by ion substitution was caused at least in part by decreased substrate affinity. Acidification of extracellular buffer resulted in decreased uptake of TEA, whereas TEA efflux from OCT1- and OCT2-expressing oocytes was not stimulated by inward proton gradient, in consistent with basolateral organic cation transport in the kidney. Inhibition of TEA uptake by various organic cations revealed that apparent substrate spectrum of OCT2 was similar with that of OCT1. However, the affinity of procainamide to OCT1 was higher than that to OCT2. Uptake of 1-methyl-4-phenylpyridinium was stimulated by OCT2 as well as OCT1, but uptake of levofloxacin, a zwitterion, was not stimulated by both OCTs. These results suggest that OCT2 is a multispecific organic cation transporter with the characteristics comparable to those of the basolateral organic cation transporter in the kidney.

Role of chloride on carrier-mediated transport of p-aminohippurate in rat renal basolateral membrane vesicles.

Effect of inorganic anions on p-amino[3H]hippurate transport in renal basolateral membranes has been studied using the vesicles preloaded with unlabeled p-aminohippurate (countertransport condition). The uptake of p-amino[3H]hippurate was stimulated by the outward gradient of unlabeled p-aminohippurate and the labeled substrate was accumulated into the vesicles against its concentration gradient in the presence of Cl-. The substitution of SCN- and SO4(2-) for Cl- in both sides of the vesicles depressed the initial rate and the overshoot magnitude of p-amino[3H]hippurate uptake. These results suggest that Cl- may play an important role for the carrier-mediated transport system of organic anion in renal basolateral membranes.

Carrier-mediated transport of cephalexin via the dipeptide transport system in rat renal brush-border membrane vesicles.

Carrier-mediated transport of aminocephalosporin antibiotics by renal brush-border membrane vesicles has been studied in relation to the transport systems for dipeptides and amino acids. Dipeptides such as L-carnosine (beta-alanyl-L-histidine) and L-phenylalanylglycine competitively inhibited the uptake of cephalexin, but amino acids did not. Cephalexin uptake was stimulated by the countertransport effect of L-carnosine in the normal and papain-treated vesicles, and by the effect of L-phenylalanylglycine only in the papain-treated vesicles. In the papain-treated vesicles, the hydrolysis of dipeptides was markedly decreased, and the specific activity for cephalexin transport was increased approx. 2-fold because of the partial removal of membrane proteins. These results suggest that carrier-mediated transport of cephalexin can be transported by the system for dipeptides in renal brush-border membranes.

Enzyme activities and sodium-dependent active D-glucose transport in apical membrane vesicles isolated from kidney epithelial cell line (LLC-PK1).

Apical membrane vesicles were isolated from the confluent LLC-PK1 cells by nitrogen cavitation and Mg/EGTA precipitation methods. The specific activities of marker enzymes for apical membranes were enriched 8- to 18-fold relative to those in the homogenate. D-[3H]Glucose uptake into the vesicles was stimulated in the presence of Na+ gradient (overshoot phenomenon), and the values of apparent Km and Vmax for Na+-dependent component of D-glucose uptake were 0.3 mM and 5.8 nmol/mg protein per min, respectively.

Carrier-mediated transport systems of tetraethylammonium in rat renal brush-border and basolateral membrane vesicles.

Transport of [3H]tetraethylammonium, an organic cation, has been studied in brush-border and basolateral membrane vesicles isolated from rat kidney cortex. Some characteristics of carrier-mediated transport for tetraethylammonium were demonstrated in brush-border and basolateral membrane vesicles; the uptake was saturable, was stimulated by the countertransport effect, and showed discontinuity in an Arrhenius plot. In brush-border membrane vesicles, the presence of an H+ gradient ( [H+]i greater than [H+]o) induced a marked stimulation of tetraethylammonium uptake against its concentration gradient (overshoot phenomenon), and this concentrative uptake was completely inhibited by HgCl2. In contrast, the uptake of tetraethylammonium by basolateral membrane vesicles was unaffected by an H+ gradient. Tetraethylammonium uptake by basolateral membrane vesicles was significantly stimulated by a valinomycin-induced inside-negative membrane potential, while no effect of membrane potential was observed in brush-border membrane vesicles. These results suggest that tetraethylammonium transport across brush-border membranes is driven by an H+ gradient via an electroneutral H+-tetraethylammonium antiport system, and that tetraethylammonium is transported across basolateral membranes via a carrier-mediated system and this process is stimulated by an inside-negative membrane potential.

Transport of procainamide in a kidney epithelial cell line LLC-PK1.

Transport of procainamide, an anti-arrhythmic drug, was investigated in LLC-PK1 kidney epithelial cell line. The uptake of procainamide by LLC-PK1 monolayers cultured in plastic dishes was temperature-dependent, saturable and inhibited by organic cations such as cimetidine and N-acetylprocainamide. An aminocephalosporin antibiotic, cephalexin, also inhibited procainamide uptake, but an organic anion, p-aminohippurate, did not. The uptake of procainamide was greater at an alkaline external pH than at an acidic pH. In addition, procainamide uptake increased when intracellular pH was decreased and the uptake decreased when the intracellular pH was increased by ammonium chloride treatment, indicating the involvement of an H+/procainamide antiport system in apical membrane. The basolateral to apical flux of procainamide across LLC-PK1 monolayers cultured on permeable supports was 2.5-times larger than the apical to basolateral flux, and only the former process was inhibited by other organic cations. These findings suggest that LLC-PK1 cells can transport procainamide by the organic cation transport system and that procainamide is transported unidirectionally from basolateral to apical side across the cell monolayers.

A simple method for the isolation of basolateral plasma membrane vesicles from rat kidney cortex. Enzyme activities and some properties of glucose transport.

A procedure for preparing basolateral membrane vesicles from rat renal cortex was developed by differential centrifugation and Percoll density gradient centrifugation, and the uptake of D-[3H] glucose into these vesicles was studied by a rapid filtration technique. (Na+ + K+)-ATPase, the marker enzyme for basolateral membranes, was enriched 22-fold compared with that found in the homogenate. The rate of D-glucose uptake was almost unaffected by Na+ gradient (no overshoot).