Effects of EDTA on chemiluminescent immunoassay measurement of ACTH, cortisol, and thyroid hormones in dogs.

When measuring blood hormones, pre-analytical sample handling can impact the quality of the results. Previous studies have shown improved stability of canine cortisol in ethylenediaminetetraacetic acid (EDTA) plasma compared to serum and interchangeability of serum and plasma when cortisol is measured by radioimmunoassay. Additionally, cortisol samples were also interchangeable when measured by chemiluminescent immunoassay if the EDTA concentration was consistent with that of optimally filled tubes, whereas excess EDTA interfered with the measurement of cortisol and serum and EDTA plasma were not interchangeable when measuring total thyroxine (TT4). The main limitation of these studies was that they were performed by spiking pooled serum samples with EDTA or in previously collected blood samples submitted to a clinical pathology laboratory. The purpose of the present study was to evaluate the effect of EDTA on the measurement of adrenocorticotropic hormone (ACTH), cortisol, TT4, free thyroxine (FT4), and thyroid stimulating hormone (TSH) in healthy dogs using the Siemens IMMULITE 1000. Whole blood from forty dogs was aliquoted into three Monoject sample tubes: no additive, completely filled EDTA tube, and 50% filled EDTA tube. Handling and storage conditions were identical, and all samples were analyzed on the same day. Bland-Altman plots and Passing-Bablok regression were used to assess agreement and risks for error, respectively. Proportional errors were found between serum and plasma samples for ACTH, cortisol, TT4, FT4, and TSH; systematic errors were also found for FT4. There was poor agreement and clinically significant differences between the measured concentrations of all hormones in serum and plasma, proving that these sample types are not interchangeable. Incompletely filled EDTA tubes were associated with significantly lower ACTH concentrations compared to completely filled EDTA tubes. When measured by chemiluminescent immunoassays that utilize alkaline phosphatase at the reporter enzyme, serum should be used for cortisol, TT4, FT4, and TSH, while plasma from completely filled EDTA tubes should be used for ACTH.

Dimethyl fumarate modulates the Treg-Th17 cell axis in patients with psoriasis.

BACKGROUND: Dimethyl fumarate (DMF) is the active ingredient of Skilarence™ and Tecfidera™, which are used for the treatment of psoriasis and multiple sclerosis, respectively. Various immunomodulatory mechanisms of action have been identified for DMF; however, it is still unclear what effects DMF exerts in vivo in patients with psoriasis. OBJECTIVES: In this study we examined the effects of DMF, both in vivo and in vitro, on T cells, which play a key role in the pathogenesis of psoriasis. METHODS: The frequency of T-cell subsets was examined by flow cytometry in untreated patients with psoriasis or those treated with DMF. The effects of DMF in vitro on T-cell survival, activation and proliferation, and cell-surface thiols were assessed by flow cytometry. RESULTS: In patients with psoriasis treated with DMF we observed an increase in the frequency of T regulatory (Treg) cells and a decrease in T helper (Th)17 lineage cells and the associated cytokines interleukin-17, interleukin-22 and granulocyte-macrophage colony-stimulating factor. T cells cultured in vitro with DMF exhibited reduced viability, and inhibition of activation and proliferation in response to stimulation due to the oxidative effects of DMF. However, the frequency of Treg cells increased in the presence of DMF due to their heightened ability to resist DMF-induced oxidative stress. CONCLUSIONS: DMF enhanced the ratio of Treg cells to Th17 cells in patients with psoriasis, in patients with multiple sclerosis and in vitro. Furthermore, our data suggest that this is at least in part as a result of the differential effects of DMF on Treg cells compared with conventional T cells.

IL-17 in inflammatory skin diseases psoriasis and hidradenitis suppurativa.

The skin is one of the most important organs in the body, providing integrity and acting as a barrier to exclude microbes, allergens and chemicals. However, chronic skin inflammation can result when barrier function is defective and immune responses are dysregulated or misdirected against harmless or self-antigens. During the last 15 years interleukin (IL)-17 cytokines have emerged as key players in multiple inflammatory disorders, and they appear to be especially prominent in skin inflammation. IL-17 cytokines produced by T cells and other cell types potently activate keratinocytes to promote inflammation in a feed-forward loop. Given this key pathogenic role of the IL-17 pathway in autoimmune and inflammatory disease, it has been the focus of intense efforts to target therapeutically. The inflammatory effects of IL-17 can be targeted directly by blocking the cytokine or its receptor, or indirectly by blocking cytokines upstream of IL-17-producing cells. Psoriasis has been the major success story for anti-IL-17 drugs, where they have proven more effective than in other indications. Hidradenitis suppurativa (HS) is another inflammatory skin disease which, despite carrying a higher burden than psoriasis, is poorly recognized and under-diagnosed, and current treatment options are inadequate. Recently, a key role for the IL-17 pathway in the pathogenesis of HS has emerged, prompting clinical trials with a variety of IL-17 inhibitors. In this review, we discuss the roles of IL-17A, IL-17F and IL-17C in psoriasis and HS and the strategies taken to target the IL-17 pathway therapeutically.

Altered metabolic pathways regulate synovial inflammation in rheumatoid arthritis.

Rheumatoid arthritis is characterized by synovial proliferation, neovascularization and leucocyte extravasation leading to joint destruction and functional disability. The blood vessels in the inflamed synovium are highly dysregulated, resulting in poor delivery of oxygen; this, along with the increased metabolic demand of infiltrating immune cells and inflamed resident cells, results in the lack of key nutrients at the site of inflammation. In these adverse conditions synovial cells must adapt to generate sufficient energy to support their proliferation and activation status, and thus switch their cell metabolism from a resting regulatory state to a highly metabolically active state. This alters redox-sensitive signalling pathways and also results in the accumulation of metabolic intermediates which, in turn, can act as signalling molecules that further exacerbate the inflammatory response. The RA synovium is a multi-cellular tissue, and while many cell types interact to promote the inflammatory response, their metabolic requirements differ. Thus, understanding the complex interplay between hypoxia-induced signalling pathways, metabolic pathways and the inflammatory response will provide better insight into the underlying mechanisms of disease pathogenesis.

The pathogenic role of dendritic cells in non-infectious anterior uveitis.

BACKGROUND: Anterior uveitis (AU) is characterised by infiltration of immune cells into the anterior chamber of the eye. Dendritic cells (DC) are professional antigen presenting cells that initiate and promote inflammation. This study aims to characterise DC in AU and to examine the effects of aqueous humor (AqH) on DC maturation and function. METHODS: The frequency and phenotype of AU and healthy control (HC) circulating DC was examined. AU and HC AqH was immunostained and assessed by flow cytometry. The effect of AU and HC AqH on DC activation and maturation was examined and subsequent effects on CD4+ T cell proliferation assessed. RESULTS: AU peripheral blood demonstrated decreased circulating myeloid and plasmacytoid DC. Within AU AqH, three populations of CD45+ cells were significantly enriched compared to HC; DCs (CD11c+ HLA-DR+), neutrophils (CD15+ CD11c+) and T cells (CD4+ and CD8+). A significant increase in IFNγ, IL8 and IL6 was observed in the AU AqH, which was also significantly higher than that of paired serum. AU AqH induced expression of CD40 and CD80 on DC, which resulted in increased T cell proliferation and the production of GM-CSF, IFNγ and TNFα. CONCLUSION: DC are enriched at the site of inflammation in AU. Our data demonstrate an increase in inflammatory mediators in the AU inflamed microenvironment. AU AqH can activate DC, leading to subsequent proliferation and activation of effector T cells. Thus, the AU microenvironment contributes to immune cell responses and intraocular inflammation.

Pharmacodynamics and pharmacokinetics of insulin aspart assessed by use of the isoglycemic clamp method in healthy cats.

The objective of this study was to determine the pharmacodynamics (PD) and pharmacokinetics (PK) of insulin aspart in healthy cats following intramuscular (IM) and subcutaneous (SC) injection. Eight healthy, purpose-bred cats were used in a randomized, crossover study design. Each cat had 2 isoglycemic clamps performed, one after receiving 0.25 IU/kg of insulin aspart by IM injection and one after receiving the same dose by SC injection. The two isoglycemic clamps were performed on different days, at least 48 h apart. The blood glucose, plasma endogenous insulin, and plasma insulin aspart concentrations were measured and the glucose infusion rate (GIR) was recorded during the clamp. The GIR over time was used to create a time-action curve for each clamp which was used to describe the PD of insulin aspart. Data that are normally distributed are reported as mean ± SD, while data that are not normally distributed are reported as median (25-75 percentile). When compared to the PD data that have been reported for regular insulin in healthy cats, insulin aspart had a more rapid onset (IM: 10 min [10-21.25 min], SC: 12.5 min [10-18.75 min]) and shorter duration of action (IM: 182.5 ± 34.33 min, SC: 159.38 ± 41.87 min). The onset of action (P = 0.795), time to peak action (P = 0.499), duration of action (P = 0.301), and total metabolic effect (P = 0.603) did not differ with route of administration; however, SC administration did result in a higher maximum plasma insulin aspart concentration (IM: 1,265.17 pmol/L [999.69-1,433.89 pmol/L], SC: 3,278.19 pmol/L [2,485.29-4,132.01 pmol/L], P = 0.000) and larger area under the insulin aspart vs time curve (IM: 82,662 ± 30,565 pmol/L, SC: 135,060 ± 39,026 pmol/L, P = 0.010). Insulin aspart has a rapid onset of action and short duration of effect in healthy cats when administered by IM and SC injection. Although it cannot be assumed that the PD and PK of insulin aspart will be the same in cats with diabetic ketoacidosis (DKA), our data support further investigation into the use of SC insulin aspart as an alternative to regular insulin for the treatment of DKA in cats.

Validation of Ultrasonography for Assessment of Gastric Emptying Time in Healthy Cats by Radionuclide Scintigraphy.

BACKGROUND: The prevalence of gastric emptying (GE) disorders in cats is unknown due to lack of clinically applicable diagnostic tests. OBJECTIVES: The principal aim of this study was to assess correlation between scintigraphic and ultrasonographic measurements of GE time (GET) in healthy cats. Additionally, variability of ultrasonographic GET, and correlation between scintigraphy and ultrasonographic parameters of gastric motility were evaluated. ANIMALS: Eight healthy domestic shorthair cats. METHODS: Prospective study. Scintigraphic GET was determined using a solid test meal containing 4 mCi 99m Tc-mebrofenin. Each cat had 3 separate ultrasonographic assessments of GE, performed independent of scintigraphic assessment, after solid test meal consumption. The motility index (MI) of antral contractions was plotted against time and time for each fraction of the area under the MI curve determined. Ultrasonographic GET and MI were correlated to scintigraphic GET. RESULTS: Scintigraphic GET (mean ± SD) for 25, 50, and 75% GE was 103 ± 32 minutes, 196 ± 45 minutes, and 288 ± 62 minutes, whereas sonographic GET for 25, 50, and 75% GE was 106 ± 13 minutes, 203 ± 19 minutes, and 305 ± 27 minutes. There was good correlation between scintigraphic and sonographic GET (r = 0.72-0.82) at 45-90% fractional GE and between scintigraphic GET and time of corresponding MI curve fraction (r = 0.78-0.86) at 40-90% fraction of the MI curve. There was moderate intraindividual variability for sonographic GET and MI curve fraction times as well as significant variation among individuals. CONCLUSIONS AND CLINICAL IMPORTANCE: Ultrasonography is a valid alternative to scintigraphy for assessment of solid-phase GE and allows assessment of postprandial gastric motility in healthy cats.

Interruption of enzyme replacement therapy in Gaucher disease.

In Australia, 58 patients with Gaucher disease were managed by a Gaucher Disease Advisory Committee (GDAC) through a centrally adminis-tered national programme, the Life Savings Drug Program (LSDP). In June 2009, Genzyme Corporation, which manufactures imiglucerase (Cerezyme), the only enzyme replacement therapy (ERT) registered for the treatment of Gaucher disease in Australia at that time, announced that due to a viral contamination problem there would be no further shipments of Cerezyme to Australia prior to the end of 2009. The GDAC allocated available drug supplies in order to maintain treatment to those most in need on a hierarchal clinical severity basis. A cohort of 24 patients with Type 1 Gaucher disease was withdrawn from therapy, 22 of whom had no discernible clinically significant adverse effects when reviewed off therapy for up to 6 months. In this paper, we review the course of 20 of the patients who have been on imiglucerase for periods of at least 24 months after the end of their 'drug holiday'. No patient experienced a bone crisis nor clinical nor magnetic resonance imaging evidence of new avascular necrosis events during this period. Two years after recommencing ERT after a 6-month drug holiday, no patient had developed an overt irreversible complication of their Gaucher disease, with the majority returning to their previous clinical status.

Cytologic Diagnosis of Heterobilharzia americana Infection in a Liver Aspirate From a Dog.

Heterobilharzia americanais a trematode of the Schistosomatidae family that infects dogs, raccoons, and other mammals as definitive hosts. This parasite is considered endemic in the southern Atlantic and Gulf coasts; however, only a few cases are reported. A 7-year-old dog from Louisiana was referred for persistent hypercalcemia, hyperglobulinemia, and weight loss. Abdominal ultrasound revealed diffuse hyperechogenicity of the liver with several hyperechoic nodules of varying size. Cytologic examination of fine-needle aspirates of the liver revealed few ovoid to round basophilic thin-walled eggshell fragments and rare ciliated miracidia.H. americanaeggs were identified on fecal sedimentation.

Factors associated with symptom-specific psychological and functional impact among acoustic neuroma patients.

INTRODUCTION: The main purpose of this study was to investigate the psychological and functional impact attributed to acoustic neuroma symptoms. MATERIALS AND METHODS: A sample of 207 acoustic neuroma patients completed a study-specific questionnaire about the severity, frequency, and psychological and functional impact of 9 acoustic neuroma symptoms. RESULTS: The survey response rate was 56.4 per cent. All symptoms had some degree of psychological impact for the majority of participants; hearing loss was the symptom most often reported to have a severe psychological impact. The majority of respondents reported functional impact attributed to hearing loss, balance disturbance, dizziness, eye problems, headache and fatigue; balance disturbance was the symptom most often reported to have a severe functional impact. For most symptoms, psychological and functional impact were related to severity and frequency. CONCLUSION: Of the acoustic neuroma symptoms investigated, hearing loss and balance disturbance were the most likely to have a severe psychological and functional impact, respectively.

Altered temporal correlations in resting-state connectivity fluctuations in children with reading difficulties detected via MEG.

In this study we investigate systematic patterns of rapidly changing sensor-level interdependencies in resting MEG data obtained from 23 children experiencing reading difficulties (RD) and 27 non-impaired readers (NI). Three-minute MEG time series were band-passed and subjected to blind source separation (BSS) prior to estimating sensor interdependencies using the weighted phase synchronization measure (wPLI). Dynamic sensor-level network properties were then derived for two network metrics (global and local efficiency). The temporal decay of long-range temporal correlations in network metrics (LRTC) was quantified using the scaling exponent (SE) in detrended fluctuation analysis (DFA) plots. Having established the reliability of SE estimates as robust descriptors of network dynamics, we found that RD students displayed significantly reduced (a) overall sensor-level network organization across all frequency bands (global efficiency), and (b) temporal correlations between sensors covering the left temporoparietal region and the remaining sensors in the ß3 band (local efficiency). Importantly, both groups displayed scale-free global network connectivity dynamics. The direct application of DFA to MEG signals failed to reveal significant group differences. Results are discussed in relation to prior evidence for disrupted temporoparietal functional circuits for reading in developmental reading disability.

Factors associated with anxiety and depression in the management of acoustic neuroma patients.

The objectives of this study were to describe anxiety and depression levels among acoustic neuroma patients; examine differences in anxiety and depression across the acoustic neuroma management options of microsurgery, radiation and observation; and to investigate management, medical and demographic factors that might predict anxiety and depression in this patient group. A cross-sectional questionnaire was completed by 205 adults diagnosed with, or treated for, a unilateral acoustic neuroma within five years of questionnaire distribution. Median age of participants was 57.0 years, and 120 (58.5%) were female. Anxiety and depression were measured using the Hospital Anxiety and Depression Scale (HADS). Clinically significant anxiety was reported by 29.8% of participants and 10.2% were depressed. Mean anxiety and depression scores did not differ from general population norms. No significant differences in anxiety and depression were found across management options. Time since management, number of symptoms and comorbid medical conditions predicted anxiety, while depression was predicted by number of symptoms. This appears to be the first study among acoustic neuroma patients in which anxiety and depression were compared across management options. Treating physicians should be aware that as the number of acoustic neuroma symptoms increases, so may the likelihood of clinically significant anxiety and depression.

Dietary protein counting as an alternative way of maintaining metabolic control in phenylketonuria.

OBJECTIVE: To compare a gram protein exchange system (1g=50-mg Phenylalanine) with a unit exchange system (1unit=15-mg Phenylalanine) and its effect on the blood Phenylalanine (Phe) levels and acceptance in the dietary management for children and adolescents with Phenylketonuria. METHODS: In Phase One, participants were randomised to continue counting Phe unit exchanges (n=8) or changed to counting gram protein exchanges (n=10), using a new diet chart developed in-house. Foods containing less than 20mg Phe per serve were now considered "free." Interim data analysis confirmed no significant deterioration in Phe levels of the study group and the control group was changed to protein counting.In Phase Two, 18 participants were educated to use an updated version of the in-house diet chart - in this version foods containing less than 50mg Phe per serve were considered "free."In both phases, attitudes to PKU and its management were evaluated at baseline and 6months. Phenylalanine and tyrosine levels were measured from filter paper blood spots by tandem mass spectrometry. RESULTS: Phase One: Phe levels over 6months were comparable to pre-study levels (mean Phe pre 366µmol/L+/- 169, mean Phe post change=388µmol/L+/- 160).Phase Two: Four participants had a significant improvement in blood Phe levels, nine showed no significant change and one participant's levels were significantly higher. There was incomplete data on four participants. All participants preferred the freer diet chart. CONCLUSION: Protein exchanges (foods containing less than 50mg Phe/serve uncounted) are an alternative method of measuring Phe intake in the dietary management of Phenylketonuria.

Medical contribution to the comprehensive approach.

The coordination of the Military, Diplomatic and Economic/Development levers of power, the so-called "hard" and "soft" powers, to bring about the strategic aims of governments, supported by Non-Governmental Organisations and international organisations, is commonly known as the Comprehensive Approach (CA). The CA is now part of both NATO and UK military doctrine and concepts development. This article describes how medical branch HQ Allied Rapid Reaction Corp, as part of the HQs work to operationalise the CA, has sought and developed training opportunities to improve individual skill-sets to enable the branch to better contribute to the CA process.

T cells in multiple sclerosis and experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is a demyelinating inflammatory disorder of the central nervous system (CNS), which involves autoimmune responses to myelin antigens. Studies in experimental autoimmune encephalomyelitis (EAE), an animal model for MS, have provided convincing evidence that T cells specific for self-antigens mediate pathology in these diseases. Until recently, T helper type 1 (Th1) cells were thought to be the main effector T cells responsible for the autoimmune inflammation. However more recent studies have highlighted an important pathogenic role for CD4(+) T cells that secrete interleukin (IL)-17, termed Th17, but also IL-17-secreting γδ T cells in EAE as well as other autoimmune and chronic inflammatory conditions. This has prompted intensive study of the induction, function and regulation of IL-17-producing T cells in MS and EAE. In this paper, we review the contribution of Th1, Th17, γδ, CD8(+) and regulatory T cells as well as the possible development of new therapeutic approaches for MS based on manipulating these T cell subtypes.

OAS1: a multiple sclerosis susceptibility gene that influences disease severity.

BACKGROUND: Type 1 interferons upregulate oligoadenylate synthetase 1 (OAS1). A single nucleotide polymorphism (SNP) in exon 7 of OAS1 results in differential RNAseL enzyme activity, the A allele coding for a truncated form with low activity and the G conferring high activity. We hypothesized that OAS1 genotypes would influence both susceptibility to multiple sclerosis (MS) and disease activity with the AA genotype being overrepresented and the GG genotype underrepresented in relapsing-remitting MS (RRMS) with increased disease activity. METHODS: We examined OAS1 genotype distribution in 401 patients with MS, 394 healthy controls, and 178 patients with RRMS receiving interferon-beta (IFNbeta) assessed as 1) having no or minimal disease activity on IFNbeta, 2) having disease activity despite IFNbeta, and 3) 65 patients with RRMS with highly active disease. RESULTS: The OAS1 genotype distribution differed between patients with MS and controls (p = 0.000003), with lower frequency of GG homozygotes in patients with MS (6%) compared with controls (17%). In relation to disease severity, 34 (32%) patients with no or minimal disease activity on IFNbeta had the AA and 8 (8%) the GG genotype; of patients with disease activity despite IFNbeta, 27 (51%) were AA, while only 1 (2%) was GG (p = 0.03). Median time to first relapse on IFNbeta was 24 months in patients with RRMS with AA genotype and 33 months with AG or GG genotype (p = 0.04). The GG genotype was absent in 65 patients with highly active RRMS (p = 0.03). CONCLUSIONS: A functional OAS1 SNP, AA genotype, confers susceptibility to MS and the GG genotype may protect against increased disease activity.

Investigation of first year biotic and abiotic influences on the recruitment of pike Esox lucius over 48 years in Windermere, UK.

Estimated pike Esox lucius recruitment varied by a factor of 16 for females from 1944 to 1991 and by a factor of 27 for males from 1943 to 1990 in Windermere, a temperate, mesotrophic U.K. lake. No significant stock-recruitment relationships were found, but analysis with general additive models (GAMs) revealed that early autumnal water temperature, strength and direction of the North Atlantic Oscillation displacement (corresponding to different climatic conditions in winter) and zooplankton abundance but above all, late summer water temperature were important explanatory variables over the entire time series. Female recruitment was also influenced by young-of-the-year winter temperature. There was no evidence that perch Perca fluviatilis year-class strength, lake level or the summer position of the Gulf Stream influenced recruitment. The fitted models explained up to c. 65% of the overall observed variation between years.