Expansion of immature, nucleated red blood cells by transient low-dose methotrexate immune tolerance induction in mice.

Biological treatments such as enzyme-replacement therapies (ERT) can generate anti-drug antibodies (ADA), which may reduce drug efficacy and impact patient safety and consequently led to research to mitigate ADA responses. Transient low-dose methotrexate (TLD-MTX) as a prophylactic ITI regimen, when administered concurrently with ERT, induces long-lived reduction of ADA to recombinant human alglucosidase alfa (rhGAA) in mice. In current clinical practice, a prophylactic ITI protocol that includes TLD-MTX, rituximab and intravenous immunoglobulin (optional), successfully induced lasting control of ADA to rhGAA in high-risk, cross-reactive immunological material (CRIM)-negative infantile-onset Pompe disease (IOPD) patients. More recently, evaluation of TLD-MTX demonstrated benefit in CRIM-positive IOPD patients. To more clearly understand the mechanism for the effectiveness of TLD-MTX, non-targeted transcriptional and proteomic screens were conducted and revealed up-regulation of erythropoiesis signatures. Confirmatory studies showed transiently larger spleens by weight, increased spleen cellularity and that following an initial reduction of mature red blood cells (RBCs) in the bone marrow and blood, a significant expansion of Ter-119+ CD71+ immature RBCs was observed in spleen and blood of mice. Histology sections revealed increased nucleated cells, including hematopoietic precursors, in the splenic red pulp of these mice. This study demonstrated that TLD-MTX induced a transient reduction of mature RBCs in the blood and immature RBCs in the bone marrow followed by significant enrichment of immature, nucleated RBCs in the spleen and blood during the time of immune tolerance induction, which suggested modulation of erythropoiesis may be associated with the induction of immune tolerance to rhGAA.

A novel method of continuous cage-side monitoring of hyperthyroid cats treated with radio-iodine.

A continuous monitoring system (MGP DM2000X) was assessed for monitoring γ radiation emissions and determining appropriate isolation times for hyperthyroid cats treated with radioactive iodine (I(131)). Daily radiation emitted by 12 cats who had received a range of doses of I(131) (80-200 MBq) was measured and average background radiation readings deducted. The effective half-lives of the I(131) in the cats were found to have a median of 2.54 days (range 1.40-3.24 days). Cats treated with 200 MBq emitted 5 µGy/day more exposure than cats treated with lower doses throughout the study period (P=0.032). All cats were found to emit a total radiation dose exposure less than 100 µGy (range 0-43 µGy) during days 18-21 of isolation. The potential additional dose exposure to owners was calculated at various days that might be considered for the cats to be returned to their owners. Using this provisional data, maximum isolation periods at this institution could be safely reduced to 17 days as long as certain precautions are followed. This preliminary study demonstrated that this novel cage-side monitoring system can be used to calculate the effective half-life of I(131) and to measure γ radiation exposure from treated cats, which may assist other institutions in determining appropriate isolation times for individual cats.

Safety, tolerability, and pharmacokinetics of 6-month daily dosing of an oral formulation of cyclosporine (ATOPICA for cats®) in cats.

Cyclosporine was proven efficacious in the treatment of feline hypersensitivity dermatitis. This target animal study was conducted to evaluate the safety, tolerability, and pharmacokinetics of ATOPICA for Cats® (cyclosporine oral solution, USP) MODIFIED following 6-month daily dosing in cats. Forty healthy cats (four cats/sex/group) received 0, 8 (1×), 16 (2×), 24 (3×), or 40 (5×) mg/kg cyclosporine once daily for 6 months (183 days). Body weight, food consumption, ophthalmoscopic, physical examinations including neurological assessments, blood pressure, electrocardiography, clinical pathology (hematology, coagulation, clinical chemistry, urinalysis), organ weights, and macroscopic and microscopic examinations were performed and assessed. In addition, blood concentrations of cyclosporine were measured at the pretreatment trough on Days 1, 2, 7, 14, 31, 91, 154, and 182, and post-treatment on Days 1, 31, and 182. Adverse effects possibly related to treatment included prolonged APTT and one report each of bone marrow hypocellularity and lymphoma; all occurred in cats treated with doses more than 16 mg/kg. There was no significant accumulation of cyclosporine beyond the first week of treatment. Results confirm that ATOPICA for Cats is safe and well tolerated in cats without unexpected accumulation beyond the first week of treatment when administered as directed.

Fasting and postprandial volumes of the undisturbed colon: normal values and changes in diarrhea-predominant irritable bowel syndrome measured using serial MRI.

BACKGROUND: Previous assessments of colon morphology have relied on tests which were either invasive or used ionizing radiation. We aimed to measure regional volumes of the undisturbed colon in healthy volunteers (HV) and patients with diarrhea-predominant irritable bowel syndrome (IBS-D). METHODS: 3D regional (ascending, transverse, and descending) colon volumes were measured in fasting abdominal magnetic resonance (MR) images of 75 HVs and 25 IBS-D patients. Thirty-five of the HV and all 25 IBS-D subjects were fed a standard meal and postprandial MRI data obtained over 225 min. KEY RESULTS: Colonic regions were identified and 3D maps from cecum to sigmoid flexure were defined. Fasted regional volumes showed wide variation in both HVs being (mean ± SD) ascending colon (AC) 203 ± 75 mL, transverse (TC) 198 ± 79 mL, and descending (DC) 160 ± 86 mL with no difference from IBS-D subjects (AC 205 ± 69 mL, TC 232 ± 100 mL, and DC 151 ± 71 mL, respectively). The AC volume expanded by 10% after feeding (p = 0.007) in the 35 HV possibly due to increased ileo-colonic inflow. A later rise in AC volume occurred from t = 90 to t = 240 min as the meal residue entered the cecum. In contrast, IBS-D subjects showed a much reduced postprandial response of the AC (p < 0.0001) and a greater increase in TC volume after 90 min (p = 0.0244) compared to HV. CONCLUSIONS & INFERENCES: We have defined a normal range of the regional volumes of the undisturbed colon in fasted and fed states. The AC in IBS-D appeared less able to accommodate postprandial inflow which may account for faster colonic transit.

A multicentre study comparing two methods for serum free light chain analysis.

BACKGROUND: Serum free light chain analysis is now well established in the investigation of monoclonal gammopathies. In the UK there has, until recently, been a single supplier of kits for such analysis. Recently, a second method using monoclonal antisera was introduced. We have compared the performance of these two kits in four routine laboratories. METHOD: Samples submitted for routine analysis (327 samples, 258 [79%] from patients with B-cell lymphoproliferative disease) for serum free light chains were tested by both technologies (Freelite, Binding Site and N Latex FLC, Siemens), according to the manufacturers' instructions. RESULTS: Qualitative data were available by both methods on 313 samples for serum free kappa chains and 324 samples for lambda free light chains. We found poor correspondence of 81% for kappa and 74% for lambda. Five percent of samples were significantly discordant in these assays. CONCLUSIONS: These assays perform very differently in clinical practice. They cannot be used interchangeably, especially if monitoring patient responses to therapy.

Removal of mercaptans from a gas stream using continuous adsorption-regeneration.

The removal of the mercaptan, 1-methyl-1-propanethiol, from aqueous solutions using a non-porous, electrically conducting carbon-based adsorbent (Nyex 1000) was investigated. The adsorption process was found to be rapid (equilibrium capacity achieved within 5 minutes) with low adsorptive capacity (of the order of 0.4 mg g(-1)) when compared with activated carbon. Electrochemical regeneration of the Nyex 1000 in a simple divided electrochemical cell within a sequential batch treatment unit restored 100% of the adsorbent's adsorptive capacity using treatment times as low as 20 minutes by passing a current of 0.5 A. The sorptive characteristics of a Nyex-water slurry were also modelled and investigated both in a bubble column and in a continuous adsorption-regeneration treatment system. It was demonstrated that the continuous removal-destruction system could achieve a step reduction in challenge gas concentration of approximately 75% for a period of 35 minutes with a current of 5 Amps. This was attributed to mass transfer enhanced by a combination of adsorption and chemical reaction with free chlorine species generated in the electrochemical process.

Plasma adenosine deaminase isoform 2 in cancer patients undergoing chemotherapy.

Adenosine deaminase (AD), a purine salvage enzyme, exists as AD isoform 1 (AD1) and AD isoform 2 (AD2). Plasma AD has been advocated for the screening and monitoring of cancer, as AD2 activity is increased in conditions associated with tumour growth. Plasma AD2 was measured before and seven to 10 days after the first dose of chemotherapy in patients with different tumours. A 'tumour regression score' was assessed independently based on radiological changes seen in the tumour following completion of chemotherapy. Changes in plasma AD2 were then compared with the tumour regression score. Following first-dose chemotherapy, plasma AD2 decreased on average from 22.7 +/- 10.5 U/L to 15.0 +/- 4.6 U/L. The percentage decrease in plasma AD2 correlated with the tumour regression score (r=0.5, P=0.028). These data suggest plasma AD2 may have a role in determining tumour response to treatment.

Dietary and physical activity patterns in children with fatty liver.

BACKGROUND/OBJECTIVES: To examine lifestyle patterns (diet, physical activity, energy expenditure) and metabolic variables (insulin resistance, oxidative stress, inflammation) in children with fatty liver detected by sonography. SUBJECTS/METHODS: Body composition (fat-free mass, body mass index-z), waist circumference (WC), dietary intake and energy expenditure were determined in 38 patients (ages 5-19 years) with fatty liver in whom specific causative liver disorders had been excluded. Laboratory investigations included liver biochemistries, C-reactive protein, tumor necrosis factor-alpha, glutathione peroxidase, vitamin E, and erythrocyte-glutathione. RESULTS: In all, 36 of 38 children were overweight/obese; 37 had WC indicative of abdominal obesity. They displayed fasting hyperinsulinemia (n=15), hypertriglyceridemia (n=14), and hypoadiponectinemia (5.5+/-1.9 s.d. microg/ml; n=23) and insulin resistance (homeostasis model of insulin resistance (HOMA-IR)>3; n=21). Alanine aminotransferase (ALT) was elevated in 28 (43-556 U/l; median=56). Some inflammatory markers were elevated, whereas antioxidants were decreased. Diet was characterized by high saturated-, low polyunsaturated-fat, high fructose and sucrose intakes. Fructose intake was independently associated with insulin resistance and decreased serum adiponectin, regardless of serum ALT (P<0.05). Low and subnormal intakes of omega-3 fatty acids (C20:5 (n-3) and C22:6 (n-3)) were associated with abnormal serum ALT (P=0.006) and elevated HOMA-IR (P=0.01). Findings were similar in children 11 years old. Physical activity was low in both age groups. CONCLUSIONS: Children with fatty liver detected sonographically have metabolic features of non-alcoholic fatty liver disease. Their diets are high in fructose and low in polyunsaturated fatty acid. Their activity patterns are sedentary. These lifestyle features may contribute to liver damage and can be a focus for therapeutic intervention.

Safety and tolerability of 3-week and 6-month dosing of Deramaxx (deracoxib) chewable tablets in dogs.

Although non-steroidal anti-inflammatory drugs (NSAIDs) are effective in reducing pain and inflammation, these agents have adverse effects. Selective inhibitors of COX-2 are an alternative to traditional NSAIDs. Deramaxx [Novartis Animal Health US, Inc. (NAH), Greensboro, NC, USA] contains the selective COX-2 inhibitor, deracoxib, and is approved for the relief of pain and inflammation associated with orthopedic surgery and osteoarthritis in dogs. The safety of Deramaxx was evaluated in two target animal safety studies: 40 dogs (four dogs/sex/group) received 0, 4, 6, 8, or 10 mg/kg/day deracoxib once daily for 21 days; and 60 dogs (five dogs/sex/group) received 0, 2, 4, 6, 8, or 10 mg/kg/day deracoxib once daily for 6 months. There was a dose-dependent trend towards increased blood urea nitrogen (BUN) in treated dogs, however mean BUN values remained within the reference range at the labeled doses. In both trials, histopathology revealed focal renal tubular degeneration/regeneration in some dogs receiving >or=6 mg/kg/day deracoxib. Focal renal papillary necrosis was seen in one dog treated with 8 mg/kg/day and in three dogs receiving 10 mg/kg/day deracoxib on the 6-month study. No other parameters of renal function were adversely affected for either study. Results show that Deramaxx is safe and well-tolerated in dogs when administered as directed.

Physiological and functional impact of an unsupervised but supported exercise programme for claudicants.

OBJECTIVES: To evaluate an unsupervised home-based exercise programme for physiological, functional, and quality of life impact in patients with symptomatic peripheral arterial disease. DESIGN: Prospective cohort with exercise intervention. MATERIALS: Human performance laboratory with non-invasive haemodynamic assessment facilities. METHODS: Forty-seven patients with symptomatic peripheral arterial disease (mean age 67.6+/-7 years, 33 males) participated in an unsupervised home-based exercise programme. Heart rate (HR), ankle brachial blood pressure index (ABPI), leg blood flow (BF), and blood lactate were measured before and after a graded treadmill walk at baseline and after the 12-week exercise programme. Maximum walking distance (MWD) during the treadmill walk was measured at baseline and 12 weeks. Exercise compliance, functional parameters, and quality of life (VascuQoL) were assessed by questionnaire. RESULTS: MWD, leg BF, and VascuQoL scores increased significantly, while resting HR, exercise HR, and end of walk rate-pressure-product (RPP) decreased significantly after 12 weeks. Exercise compliance was significantly correlated with increase in MWD (r=0.89, p<0.001) and QOL score improvement (r=0.61, p<0.001). CONCLUSIONS: This supported but unsupervised exercise programme generated improvements in walking distance and leg blood flow without detectable increases in cardiorespiratory work. Exercise compliance is related to MWD and VascuQoL score in a dose-response manner.

Gene expression changes following acute hydrogen sulfide (H2S)-induced nasal respiratory epithelial injury.

Hydrogen sulfide (H2S) is a naturally occurring gas that is also associated with several industries. The potential for widespread human inhalation exposure to this toxic gas is a public health concern. The nasal epithelium is especially susceptible to H2S-induced pathology. Injury to and regeneration of the nasal respiratory mucosa occurred in animals with ongoing H2S exposure, suggesting that the regenerated respiratory epithelium under-goes an adaptive response and becomes resistant to further injury. To better understand this response, ten-week-old male Sprague-Dawley rats were exposed nose-only to either air or 200 ppm H2S for three hours per day for one day or five consecutive days. Nasal respiratory epithelial cells at the site of injury and regeneration were laser capture microdissected, and gene expression profiles were generated at three, six, and twenty-four hours after the initial three-hour exposure and at twenty-four hours after the fifth exposure using the Affymetrix Rat Genome 230 2.0 microarray. Gene ontology enrichment analysis showed that H2S exposure altered gene expression associated with a variety of biological processes, including cell cycle regulation, protein kinase regulation, and cytoskeletal organization and biogenesis. Surprisingly, our results did not show a significant change in cytochrome oxidase gene expression or bioenergetics.

Reduction of congenital nystagmus in a patient after smoking cannabis.

INTRODUCTION: Smoking cannabis has been described to reduce acquired pendular nystagmus in MS, but its effect on congenital nystagmus is not known. PURPOSE: To report the effect of smoking cannabis in a case of congenital nystagmus. METHODS: A 19-year-old male with congenital horizontal nystagmus presented to the clinic after smoking 10 mg of cannabis. He claimed that the main reason for smoking cannabis was to improve his vision. At the next clinic appointment, he had not smoked cannabis for 3weeks. Full ophthalmologic examination and eye movement recordings were performed at each visit. RESULTS: Visual acuity improved by 3 logMar lines in the left eye and by 2 logMar lines in the right eye after smoking cannabis. The nystagmus intensities were reduced by 30% in primary position and 44%, 11%, 10% and 40% at 20-degree eccentricity to the right, left, elevation and depression, respectively, after smoking cannabis. CONCLUSION: Cannabis may be beneficial in the treatment of congenital idiopathic nystagmus (CIN). Further research to clarify the safety and efficacy of cannabis in patients with CIN, administered for example by capsules or spray, would be important.

Mutations in WNT7A cause a range of limb malformations, including Fuhrmann syndrome and Al-Awadi/Raas-Rothschild/Schinzel phocomelia syndrome.

Fuhrmann syndrome and the Al-Awadi/Raas-Rothschild/Schinzel phocomelia syndrome are considered to be distinct limb-malformation disorders characterized by various degrees of limb aplasia/hypoplasia and joint dysplasia in humans. In families with these syndromes, we found homozygous missense mutations in the dorsoventral-patterning gene WNT7A and confirmed their functional significance in retroviral-mediated transfection of chicken mesenchyme cell cultures and developing limbs. The results suggest that a partial loss of WNT7A function causes Fuhrmann syndrome (and a phenotype similar to mouse Wnt7a knockout), whereas the more-severe limb truncation phenotypes observed in Al-Awadi/Raas-Rothschild/Schinzel phocomelia syndrome result from null mutations (and cause a phenotype similar to mouse Shh knockout). These findings illustrate the specific and conserved importance of WNT7A in multiple aspects of vertebrate limb development.

In vitro assessment of copper-induced toxicity in the human hepatoma line, Hep G2.

Copper, though essential, is highly toxic when present in excess, as in Wilson disease, a genetic disorder of hepatic copper metabolism. We hypothesized that mitochondria are a major target of copper-induced cytotoxicity in Wilson disease. We used the human hepatoma line Hep G2 to examine copper-mediated cytotoxicity and three different methods to assess organelle damage: MTT assay (mitochondria), neutral red (NR; lysosomes) and Trypan blue exclusion assay (TB; plasma membrane). For all assays, cells at approximately 60% confluence in microtitre plates were incubated with CuCl(2) (concentration range: 50-100-150-200 microM) for 24 or 48 h. Results were expressed as percent of untreated control. At 24 h, cytotoxicity as detected by NR assay was significantly higher at all concentrations of copper than for MTT or TB ( p<0.005 at all concentrations). Cytotoxicity as detected by MTT was higher than that detected by TB at all concentrations except at 200 microM (p<0.05 for 50 microM, p<0.005 for 100 microM, p = 0.001 for 150 microM). Results at 48 h were similar (NR versus others: p <0.001 MTT versus TB: NS except at 150 microM where p<0.01). We investigated reactive oxygen species (ROS) production in copper-associated hepatocytoxicity by incubating sub-confluent cells with 2('),7(')-dichlorodihydrofluorescein diacetate dye plus copper (concentration range: 0-200 microM) for 1-1.5 h. Copper, but not zinc, produced significant increases in ROS (p<0.001). In summary, Hep G2 lysosomes appeared more susceptible to Cu-mediated damage than mitochondria; the cell membrane was highly resistant to damage.

Cree encephalitis is allelic with Aicardi-Goutiéres syndrome: implications for the pathogenesis of disorders of interferon alpha metabolism.

Aicardi-Goutiéres syndrome (AGS) is an early onset, progressive encephalopathy characterised by calcification of the basal ganglia, white matter abnormalities, and a chronic cerebrospinal fluid (CSF) lymphocytosis. Cree encephalitis shows phenotypic overlap with AGS although the conditions have been considered distinct because of immunological abnormalities observed in Cree encephalitis. We report that levels of interferon alpha (IFN-alpha), a marker of AGS, are raised in Cree encephalitis. Moreover, linkage analysis indicates that the disorders are allelic and refines the AGS1 locus to a 3.47 cM critical interval. Our data show that a CSF lymphocytosis is not necessary for the diagnosis of AGS and strongly suggest that AGS and pseudo-TORCH syndrome are the same disorder. Recognition of immunological dysfunction as part of the AGS phenotype provides further evidence of a primary pathogenic role for abnormal IFN-alpha production in AGS.

Guanosine deaminase in human serum and tissue extracts--a reappraisal of the products.

Of the human salvage enzymes that deaminate ribonucleosides, two--cytidine deaminase and adenosine deaminase--have been found particularly useful for diagnostic purposes. In humans, no enzymes are present that can directly deaminate the bases of these ribonucleosides. Indeed, the only enzyme present that can directly deaminate a base is guanine deaminase, and the diagnostic usefulness of this enzyme has been well documented. The aim of this study is to identify the origin of the ammonia formed when human sera and tissue extracts are incubated with buffered guanosine, and to clarify whether the ammonia comes from the deamination of guanosine by guanosine deaminase or is produced as a result of deamination of guanine formed as a breakdown product of guanosine by purine nucleoside phosphorylase (PNP). Apparent deamination of guanosine by guanosine deaminase in human sera and tissue extracts was found to be due to two enzymes acting in tandem when the products of the reaction were examined by HPLC. The ribose was first removed from guanosine by PNP to form guanine, which was then deaminated to xanthine by guanine deaminase.

Histological improvement with lamivudine therapy for de novo hepatitis B occurring in an anti-HBs-positive child after bone marrow transplantation.

We describe a 14-year-old bone marrow transplant recipient who was anti-HBs-positive before the procedure and afterwards developed acute infection with hepatitis B virus (HBV). Liver biopsies taken while symptomatic showed portal fibrosis progressing to cirrhosis. The patient responded to lamivudine treatment with HBeAg seroconversion and significant regression of fibrosis. Although the source and timing of HBV exposure remain unclear, the potential for severe hepatitis B infection following bone marrow transplant warrants caution. This case demonstrates that a symptomatic HBV infection can occur in an immunocompromised patient who had originally been anti-HBs-positive.