Using serum troponins to screen for cardiac involvement and assess disease activity in the idiopathic inflammatory myopathies.

Objectives: Limitations in the methods available for identifying cardiac involvement and accurately quantifying disease activity in the idiopathic inflammatory myopathies (IIMs) may contribute to poor outcomes. We investigated the utility of different serum muscle damage markers [total creatine kinase (CK), cardiac troponin T (cTnT) and cardiac troponin I (cTnI)] to address these issues. Methods: We assessed disease activity and cardiac involvement using the International Myositis Assessment and Clinical Studies Group core set measures in 123 participants with confirmed adult-onset IIM from the UK and Denmark. Total CK, cTnT and cTnI were measured. Associations were assessed using logistic regression and Spearman's ranked correlation. Results: Cardiac involvement (n = 18) was associated with higher cTnI levels, independent of overall disease activity [adjusted odds ratio 1.03 (95% CI 1.01, 1.05); P = 0.002]. An abnormal cTnI had the highest specificity and positive predictive value for cardiac involvement (95% and 62%, respectively). In those with a normal CK but elevated cTnT or cTnI, an association with increased disease activity scores was observed. Serum cTnT correlated with the physician (ρ = 0.39) and patient-assessed (ρ = 0.28) global visual analogue scales and HAQ (ρ = 0.41) more strongly than CK or cTnI levels. cTnT was the only marker to correlate with manual muscle testing scores (ρ = -0.24). Conclusion: Serum cTnI testing may have a role in screening for cardiac involvement in IIMs. Abnormal levels of serum cTnT and cTnI are associated with increased disease activity, including in those with a normal CK.

Lifestyle factors and the metabolic syndrome in Schizophrenia: a cross-sectional study.

BACKGROUND: Cardiometabolic disease is more common in patients with schizophrenia than the general population. AIM: The purpose of the study was to assess lifestyle factors, including diet and exercise, in patients with schizophrenia and estimate the prevalence of metabolic syndrome. METHODS: This is a cross-sectional study of a representative group of outpatients with schizophrenia in Salford, UK. An interview supplemented by questionnaires was used to assess diet, physical activity, and cigarette and alcohol use. Likert scales assessed subjects' views of diet and activity. A physical examination and relevant blood tests were conducted. RESULTS: Thirty-seven people were included in the study. 92% of men had central adiposity, as did 91.7% of women (International Diabetes Federation Definition). The mean age was 46.2 years and mean illness duration was 11.6 years. 67.6% fulfilled criteria for the metabolic syndrome. The mean number of fruit and vegetable portions per day was 2.8 ± 1.8. Over a third did not eat any fruit in a typical week. 42% reported doing no vigorous activity in a typical week. 64.9% smoked and in many cigarette use was heavy. The Likert scale showed that a high proportion of patients had insight into their unhealthy lifestyles. CONCLUSIONS: Within this sample, there was a high prevalence of poor diet, smoking and inadequate exercise. Many did not follow national recommendations for dietary intake of fruit and vegetables and daily exercise. These factors probably contribute to the high prevalence of metabolic syndrome. Many had insight into their unhealthy lifestyles. Thus, there is potential for interventions to improve lifestyle factors and reduce the risk of cardiometabolic disease.

Use of a first-line urine protein-to-creatinine ratio strip test on random urines to rule out proteinuria in patients with chronic kidney disease.

BACKGROUND: Urine protein strip tests are often used in the ward or clinic as first-line measures of proteinuria. The ability of a semi-quantitative meter-read strip test for the protein:creatinine ratio, Multistix* PRO 10LS (Siemens Medical Solutions, Tarrytown, USA), was assessed as a first-line test to exclude significant proteinuria in the monitoring of patients with established chronic kidney disease. METHODS: Eighty-six patients attending a hospital renal outpatient clinic collected three random urine samples during a 24-h period. Random urine protein:creatinine ratios measured by the strip test were compared to the laboratory estimation of 24-h protein excretion on that same day. RESULTS: At significant protein excretion of 0.3 g/24 h, the strips elicited negative predictive values in the range of 91.2-94.1% and negative likelihood ratios of 0.01-0.12, using all the random urines. Receiver-operator characteristic curve analysis also demonstrated good performance with all samples. CONCLUSIONS: The strip test allows the physician to rule out significant proteinuria at the patient consultation on a random urine sample, obviating the need for specially collected samples, and with the added benefit of reducing the need for a lengthy and costly quantitative laboratory follow-up by approximately 40-48%.

Characterization of the freeze sensitivity of a hepatitis B vaccine.

Recent studies have revealed that vaccines containing aluminum adjuvant are exposed to sub-zero temperatures while in the cold chain more frequently than was previously believed. This raises concerns that these freeze-sensitive vaccines may be damaged and offer inadequate protection. This study was undertaken to characterize the immediate qualitative changes of one such vaccine, hepatitis B, caused by freeze exposure. Hepatitis B vaccine was subjected to freezing temperatures ranging from 0 degrees C to -20 degrees C for up to three episodes with durations ranging from 1 hour to 7 days. The vaccine was analyzed for freezing point, particle size distribution, tertiary structure, and in vitro and in vivo potency. Whether or not hepatitis B vaccine freezes was shown to be dependent on an array of factors including temperature, rate of temperature change, duration of exposure, supercooling effects and vibration. Vaccine exposed to "mild" freezing (-4 degrees C or warmer) temperatures did not freeze and remained qualitatively unaltered. Single or repeated freezing events at temperatures of -10 degrees C or lower were associated with aggregation of the adjuvant-antigen particles, structural damage of the antigen, and reduction of immunogenicity in mice. Damage to the vaccine increased with duration of freezing, lower temperature, and the number of freezing episodes. With vibration, vaccine froze at -6 degrees C after 1 hour and damage occurred. Freezing and freeze damage to vaccines containing aluminum salt adjuvant represent real risks to the effectiveness of immunization and should be prevented by strengthening the cold chain system or, alternatively, development of freeze-stable vaccine formulations.

Caeruloplasmin oxidase activity: measurement in serum by use of o-dianisidine dihydrochloride on a microplate reader.

Background The enzymatic method of caeruloplasmin measurement is based on copper-dependent oxidase activity. The advantage of the oxidase determination is that it has a much lower detection limit compared with immunoassay-based methods. It has found its application in both the diagnosis of Wilson's disease and also in the monitoring of patients' response to treatment. Methods The method previously described in literature was adapted for use on a 96-well plate. Caeruloplasmin oxidase activity results were derived from the equation: caeruloplasmin oxidase activity = (A15-A5) × 185 U/L. Results Repeatability (intra-batch) imprecision ranged from 6 to 15% and intermediate (inter-batch) imprecision varied from 7 to 16% for caeruloplasmin oxidative activities of 14, 29, 45 and 99 U/L. Between 3 and 92 U/L, the assay appeared linear with a regression coefficient R2 = 0.9958. The lower limit of quantification was 4 U/L. Samples were stable over a five-week period at 4℃ and for at least four freeze-thaw cycles. There was a statistically significant difference between the areas under ROC curve for copper-to-caeruloplasmin ratios between caeruloplasmin oxidative activity and immunoassay-based methods ( P < 0.0171). The reference interval for caeruloplasmin activity was determined to be 12-166 U/L. Conclusions Using the oxidative assay provides a cost-effective means of estimating caeruloplasmin concentrations. The method is easily adaptable to a 96-well plate format that facilitates high throughput of samples in a busy laboratory. The enzymatic method is more sensitive and specific for differentiating between Wilson's and non-Wilson's when compared with immunoassay-based methods.

Novel prenyl-linked benzophenone substrate analogues of mycobacterial mannosyltransferases.

PPM (polyprenol monophosphomannose) has been shown to act as a glycosyl donor in the biosynthesis of the Man (mannose)-rich mycobacterial lipoglycans LM (lipomannan) and LAM (lipoarabinomannan). The Mycobacterium tuberculosis PPM synthase (Mt-Ppm1) catalyses the transfer of Man from GDP-Man to polyprenyl phosphates. The resulting PPM then serves as a donor of Man residues leading to the formation of an alpha(1-->6)LM intermediate through a PPM-dependent alpha(1-->6)mannosyltransferase. In the present study, we prepared a series of ten novel prenyl-related photoactivatable probes based on benzophenone with lipophilic spacers replacing several internal isoprene units. These probes were excellent substrates for the recombinant PPM synthase Mt-Ppm1/D2 and, on photoactivation, several inhibited its activity in vitro. The protection of the PPM synthase activity by a 'natural' C(75) polyprenyl acceptor during phototreatment is consistent with probe-mediated photoinhibition occurring via specific covalent modification of the enzyme active site. In addition, the unique mannosylated derivatives of the photoreactive probes were all donors of Man residues, through a PPM-dependent mycobacterial alpha(1-->6)mannosyltransferase, to a synthetic Manp(1-->6)-Manp-O-C(10:1) disaccharide acceptor (where Manp stands for mannopyranose). Photoactivation of probe 7 led to striking-specific inhibition of the M. smegmatis alpha(1-->6)mannosyltransferase. The present study represents the first application of photoreactive probes to the study of mycobacterial glycosyltransferases involved in LM and LAM biosynthesis. These preliminary findings suggest that the probes will prove useful in investigating the polyprenyl-dependent steps of the complex biosynthetic pathways to the mycobacterial lipoglycans, aiding in the identification of novel glycosyltransferases.

Diagnostic accuracy of the urinary albumin: creatinine ratio determined by the CLINITEK Microalbumin and DCA 2000+ for the rule-out of albuminuria in chronic kidney disease.

An increased urinary albumin excretion (albuminuria) is an established test for the early detection of renal disease and is also recognized as a risk factor for cardiovascular disease and mortality in a number of clinical settings. There is an established body of data which shows that a random urinary albumin:creatinine ratio (ACR) based on a random urine sample correlates well with 24-hour urinary albumin excretion measurement. However, there is little data to show whether specific point-of-care testing devices can be used to rule-in or rule-out increased urinary albumin excretion in comparison to a 24-hour urinary albumin excretion measurement. This study evaluated the ability to rule-in or rule-out albuminuria in a cohort of patients attending a renal outpatient clinic, using the urinary ACR determined by the CLINITEK Microalbumin (Siemens Healthcare Diagnostics Inc., Deerfield, US) a semi-quantitative strip test, and by the DCA 2000+ (Siemens Healthcare Diagnostics Inc.) a quantitative cassette based test using 3 random urine samples collected within a 24-hour period compared to 24-hour urinary albumin measurement. The CLINITEK system was shown to be a reliable test for ruling out increased urinary albumin excretion with negative likelihood ratios less than 0.05 above the 24-hour urinary albumin excretion rate of 30 mg/24 h (threshold for microalbuminuria), and less than 0.01 above the albumin excretion rate of 100 mg/24 h. The DCA 2000+ system demonstrated similar performance as a rule-out test, with likelihood ratios of less than 0.02 at 24-hour albumin excretion rates above 30 mg/24 h. Both the CLINITEK and DCA 2000+ systems could be used to rule-out increased urinary albumin excretion at the albumin excretion cut-off rate of 30 mg/24 h in this cohort of patients.

Protein and albumin-to-creatinine ratios in random urines accurately predict 24 h protein and albumin loss in patients with kidney disease.

BACKGROUND: Random urine protein-to-creatinine (PCR) and albumin-to-creatinine (ACR) ratios have been proposed as alternatives to 24 h urine measurements to simplify sample collection and overcome errors. The aim of this study was to examine the ability of PCR and ACR to predict urinary 24 h protein and albumin loss, respectively, in patients with kidney disease, and determine the most appropriate time of collection. METHODS: Eighty-three patients were recruited from a renal outpatient clinic. In a 24 h period, each collected an early-morning urine (EMU), second and third voids, and the remaining urine passed that day. PCR and ACR were determined in random urines and compared with the 24 h loss of protein and albumin, respectively. RESULTS: For all patients, median (range) 24 h urine protein and albumin losses were 220 (30-15600) and 60 (<8-10,557) mg, respectively. Ratios derived from each of three random urines correlated well with 24 h protein or albumin loss (Spearman's r(s) > 0.87, P < 0.0001). Receiver operator characteristic (ROC) curve analysis showed PCR accurately predicted both an abnormal 24 h urine protein > or =150 mg/24 h (areas under curves [AUC] 0.90-0.92) and significant proteinuria above 300 mg/24 h (AUC between 0.97 and 1.00). ACR accurately predicted both an abnormal 24 h urine albumin > or =30 mg/24 h (AUC 0.98 to 0.99) and frank albuminuria at > or =300 mg/24 h or > or =700 mg/24 h (AUC between 0.99 and 1.00). EMU and random urines performed equally well in predicting proteinuria and albuminuria from PCR and ACR, respectively. CONCLUSIONS: By careful choice of cut-offs, both PCR and ACR can be used in patients with kidney disease to rule in or rule out abnormal 24 h losses of protein and albumin. EMU and, importantly, random samples can be used as surrogates for 24 h urine collections.

Development of a freeze-stable formulation for vaccines containing aluminum salt adjuvants.

Vaccines containing aluminum salt adjuvants are prone to inactivation following exposure to freeze-thaw stress. Many are also prone to inactivation by heat. Thus, for maximum potency, these vaccines must be maintained at temperatures between 2 degrees C and 8 degrees C which requires the use of the cold chain. Nevertheless, the cold chain is not infallible. Vaccines are subject to freezing during both transport and storage, and frozen vaccines are discarded (under the best circumstances) or inadvertently administered despite potentially reduced potency. Here we describe an approach to minimize our reliance on the proper implementation of the cold chain to protect vaccines from freeze-thaw inactivation. By including PEG 300, propylene glycol, or glycerol in a hepatitis B vaccine, particle agglomeration, changes in the fluorescence emission spectrum--indicative of antigen tertiary structural changes--and losses of in vitro and in vivo indicators of potency were prevented following multiple exposures to -20 degrees C. The effect of propylene glycol was examined in more detail and revealed that even at concentrations too low to prevent freezing at -10 degrees C, -20 degrees C, and -80 degrees C, damage to the vaccine could be prevented. A pilot study using two commercially available diphtheria, tetanus toxoid, and acellular pertussis (DTaP) vaccines suggested that the same stabilizers might protect these vaccines from freeze-thaw agglomeration as well. It remains to be determined if preventing agglomeration of DTaP vaccines preserves their antigenic activity following freeze-thaw events.

Characterization of a thermostable hepatitis B vaccine formulation.

Cold chain requirements for vaccine storage and distribution are both economic and logistical burdens for immunization programs, especially those in lower-resource settings. Inadvertent exposure of vaccines to both heat and freezing temperatures within such cold chains are frequently occurring problems in both developing and industrialized countries. Here we report on a new hepatitis B vaccine formulation that is stable against repeated freezing at -20 degrees C and is also stable for 12 months at 37 degrees C. The thermostable vaccine contains all the components of the original vaccine plus 7.5% (v/v) propylene glycol, 40mM phosphate, and 40mM histidine with a final pH of 5.2. The propylene glycol is responsible for the freeze stability while the other components are essential for the heat stability. This formulation was found to be well tolerated in rabbits without any significant local or systemic side effects. The improved stability of this hepatitis B vaccine could be a key factor in ensuring vaccine effectiveness, extending immunization coverage, simplifying immunization logistics, and reducing the costs associated with the cold chain.

Liver transplant in a four-month-old child with biliary atresia, unilateral pulmonary agenesis, and diaphragmatic hernia: first case report.

Bilateral pulmonary agenesis (PA) is a rare embryological defect incompatible with life. Unilateral PA has a wide range of clinical presentations: its prognosis depends on the presence and severity of other associated anomalies. Fetal biliary atresia has been associated with a number of congenital anomalies, but the etiology is still not understood. An unusual case of a child with right PA, right diaphragmatic hernia, and delayed diagnosed biliary atresia leading to liver failure is presented herein. At the age of 4 months the patient was referred to the Transplant Department at Children Healthcare of Atlanta at Egleston with cholestasis and failure to thrive. With a rapidly progressive liver insufficiency, this child was evaluated for liver transplantation. In the absence of any respiratory symptom, the patient received a deceased donor size-matched left lateral segment liver transplant, which covered the diaphragmatic defect, with no further repair required. Twenty-seven months post-transplant, the patient has good graft function, a normal Z-score and is thriving. In spite of the increased physiological and surgical challenges (absence of right lung tissue, hemi-diaphragm, and ectopic position of the liver in the right chest), liver transplantation was performed with positive outcome in this high-risk child. Whether PA, may have developmentally contributed to expression of biliary atresia will need further investigation.

Successful ABO-incompatible pediatric liver transplantation utilizing standard immunosuppression with selective postoperative plasmapheresis.

Transplanting blood group A, B, or O (ABO)-incompatible (ABO-I) liver grafts has resulted in lower patient and graft survival with an increased incidence of vascular and biliary complications and rejection. We report that, without modification of our standard immunosuppression protocol, crossing blood groups is an acceptable option for children requiring liver transplantation. In our study, ABO-I liver grafts -- regardless of recipient age -- have comparable long-term survival (mean follow-up of 3.25 yr) with ABO-compatible grafts without any difference in rejection, vascular or biliary complications. From January 1, 1999 to October 1, 2005, we studied 138 liver transplants in 121 children: 16 (13.2%) received an ABO incompatible liver allograft. One-year actuarial patient survival for ABO-matched grafts vs. ABO-I grafts was 93.0% and 100%, respectively, whereas graft survival was 83.4% and 92.3%. Additionally, 6 of 16 (37.5%) ABO-I transplanted children had 8 rejection episodes, whereas 47 patients (44.8%) had 121 rejection episodes in the ABO-compatible group. There were no vascular complications and 2 biliary strictures in the ABO-I group. Plasmapheresis was not used for pretransplantation desensitization and was only required in 1 posttransplantation recipient. No child was splenectomized. Six of the 16 children were older than 13 yr of age, suggesting the possibility of successfully expanding this technique to an older population. In conclusion, our outcomes may support the concept of using ABO-I grafts in a more elective setting associated with split and living donor liver transplants.

Low incidence of hepatic artery thrombosis after pediatric liver transplantation without the use of intraoperative microscope or parenteral anticoagulation.

The risk of hepatic artery thrombosis (HAT) after pediatric liver transplantation (PLT) has been reported to range from 0 to 25%. We report our experience focusing on the interrelationships between risk factors, surgical technique and the incidence of HAT after liver transplantation in the pediatric age group. From February 18, 1997 to December 31, 2003, 150 consecutive liver transplants were performed in 132 pediatric patients. There were similar numbers of whole grafts when compared with partial grafts, 80 (53.3%) vs. 70 (46.7%), p = 0.30. Four grafts (2.7%) developed HAT. Of the grafts with HAT, three were successfully revascularized within the first 24 h. Only one graft (0.66%) was lost to HAT. A single surgeon utilizing 3.5-6.0 magnification loupes performed all but one hepatic arterial anastomoses. All patients were followed postoperatively by a daily ultrasound protocol and with anticoagulation of aspirin and alprostadil only. Living and deceased donor left lateral segment grafts had an increased rate of HAT when compared with whole liver grafts. HAT with subsequent graft loss may be minimized in PLT with the use of surgical loupes only, anticoagulation utilizing aspirin, alprostadil, and daily ultrasounds.

Lipid length controls antigen entry into endosomal and nonendosomal pathways for CD1b presentation.

CD1 proteins present various glycolipid antigens to T cells, but the cellular mechanisms that control which particular glycolipids generate T cell responses are not understood. We show here that T cell recognition of glucose monomycolate antigens with long (C(80)) alkyl chains involves the delivery of CD1b proteins and antigens to late endosomes in a process that takes several hours. In contrast, analogs of the same antigen with shorter (C(32)) alkyl chains are rapidly, but inefficiently, presented by cell surface CD1b proteins. Dendritic cells (DCs) preferentially present long-chain glycolipids, which results, in part, from their rapid internalization and selective delivery of antigens to endosomal compartments. Nonprofessional antigen-presenting cells, however, preferentially present short-chain glycolipids because of their lack of prominent endosomal presentation pathways. Because long alkyl chain length distinguishes certain microbial glycolipids from common mammalian glycolipids, these findings suggest that DCs use a specialized endosomal-loading pathway to promote preferential recognition of glycolipids with a more intrinsically foreign structure.