ACU193, a Monoclonal Antibody that Selectively Binds Soluble Aß Oligomers: Development Rationale, Phase 1 Trial Design, and Clinical Development Plan.

BACKGROUND: Alzheimer's disease is a large and growing unmet medical need. Clinical trial designs need to assess disease-related outcomes earlier to accelerate the development of better treatments for Alzheimer's disease. ACU193 is a monoclonal antibody that selectively targets amyloid ß oligomers, thought to be the most toxic species of Aß that accumulates early in AD and contributes to downstream pathological effects. Nonclinical data indicate that ACU193 can reduce the toxic effects of amyloid ß oligomers. ACU193 is currently being investigated in a phase 1 clinical trial designed with the properties described in this report. This phase 1 trial is designed to provide data to enable a go/no-go decision regarding the initiation of a subsequent phase 2/3 study. OBJECTIVES: To design a phase 1 study that assesses target engagement and incorporates novel measures to support more rapid development of a potential disease-modifying treatment for Alzheimer's disease. DESIGN: The INTERCEPT-AD trial for ACU193 is an ongoing randomized, placebo-controlled phase 1a/b study that assesses safety, tolerability, pharmacokinetics, target engagement, clinical measures, and several Alzheimer's disease biomarkers, including novel digital and imaging biomarkers. SETTING: For INTERCEPT-AD, brief inpatient stays for patients in the single ascending dose portion of the study, with the remainder of the evaluations being performed as outpatients at multiple clinical trial sites in the U.S. PARTICIPANTS: Patients with early Alzheimer's disease (mild cognitive impairment or mild dementia with a positive florbetapir positron emission tomography scan). INTERVENTION: ACU193 administered intravenously at doses of 2- 60 mg/kg. MEASUREMENTS: Safety assessments including magnetic resonance imaging for the presence of amyloid-related imaging abnormalities, clinical assessments for Alzheimer's disease including the Alzheimer's Disease Rating Scale-cognition and Clinical Dementia Rating scale, pharmacokinetics, a measure of target engagement, and digital and imaging biomarkers, including a computerized cognitive test battery and a measure of cerebral blood flow using arterial spin labelling magnetic resonance imaging. RESULTS: A phase 1 study design was developed for ACU193 that allows collection of data that will enable a go/no-go decision for initiation of a subsequent adaptive phase 2/3 study. CONCLUSIONS: A phase 1a/b trial and an overall clinical development plan for an Alzheimer's disease treatment can be designed that maintains patient safety, allows informed decision-making, and achieves an accelerated timeline by using novel biomarkers and adaptive study designs.

Transport and Barrier Functions in Rainbow Trout Trunk Skin Are Regulated by Environmental Salinity.

The mechanisms underpinning ionic transport and barrier function have been relatively well characterised in amphibians and fish. In teleost fish, these processes have mostly been characterised in the gill and intestine. In contrast, these processes remain much less clear for the trunk skin of fish. In this study, we measured barrier function and active transport in the trunk skin of the rainbow trout, using the Ussing chamber technique. The effects of epithelial damage, skin region, salinity, and pharmacological inhibition were tested. Skin barrier function decreased significantly after the infliction of a superficial wound through the removal of scales. Wound healing was already underway after 3 h and, after 24 h, there was no significant difference in barrier function towards ions between the wounded and control skin. In relation to salinity, skin permeability decreased drastically following exposure to freshwater, and increased following exposure to seawater. Changes in epithelial permeability were accompanied by salinity-dependent changes in transepithelial potential and short-circuit current. The results of this study support the idea that barrier function in rainbow trout trunk skin is regulated by tight junctions that rapidly respond to changes in salinity. The changes in transepithelial permeability and short circuit current also suggest the presence of an active transport component. Immunostaining and selective inhibition suggest that one active transport component is an apical V-ATPase. However, further research is required to determine the exact role of this transporter in the context of the trunk skin.

Analysis of the Relationship of Cognitive Impairment and Functional Impairment in Mild Alzheimer's Disease in EXPEDITION 3.

BACKGROUND: Clinical progression of Alzheimer's disease is characterized by impairment in cognition and function. OBJECTIVE: To assess the relationship between cognitive and functional impairment in mild Alzheimer's disease. DESIGN: Spearman's rank correlations between cognitive and functional measures were calculated. Autoregressive cross-lagged panel analyses were used to determine the temporal relationship between cognitive and functional decline. SETTING: Post-hoc analysis of clinical trial data. PARTICIPANTS: Placebo-treated patients with mild Alzheimer's disease from the Phase 3 solanezumab study EXPEDITION 3. INTERVENTION: Placebo. MEASUREMENTS: Cognitive and functional measures were assessed at baseline and at six post-baseline time points through Week 80. RESULTS: Correlation between cognitive and functional measures was 0.41 at baseline and 0.65 at Week 80. Autoregressive cross-lagged panel analysis demonstrated that cognitive impairment preceded and predicted subsequent functional decline, but functional scores did not predict cognitive outcomes. CONCLUSIONS: This study supports the hypothesis that functional impairment predictably follows cognitive decline in mild Alzheimer's disease dementia.

Calcium mobilisation following shell damage in the Pacific oyster, Crassostrea gigas.

Shell growth of oysters requires calcium uptake from the environment and transport to the area of shell formation. A shell regeneration assay in combination with radiolabelled calcium was used to investigate uptake and distribution of calcium to different tissues and hemolymph fractions in Pacific oysters, Crassostrea gigas (Bivalvia, Ostreoida). Oysters were notched at the shell margin and subsequently sampled for hemolymph and grading of shell regeneration during a two week experimental period. Half of the oysters were additionally exposed to (45)Ca and sampled for hemolymph and tissues. Total plasma calcium concentrations increased in notched oysters compared to controls on 1, 2 and 7days after notching. A decrease in plasma calcium levels was apparent on day 4, for both total and ionic calcium. The shell regeneration assay in the notched oysters resulted in a visible deposition of CaCO3 onto the regenerate from day 7 onwards. This was coinciding with an increased uptake of total calcium on days 11 and 14 as well as free, i.e. ionic and ligand-bound calcium, on day 14. At day 1, notching also increased calcium uptake into the mantle tissues, in areas above the notch and near the hinge. During the experiment, both the total hemocyte count and the number of granulocytes increased in notched compared to control oysters. The present study suggests that induced shell damage results in a dynamic regulation of the calcium uptake from the environment and the distribution of calcium within the body, starting directly after notching. Increases in both total calcium concentrations and uptake rates coincided with the visible depositions of CaCO3 on the regenerate shell. C. gigas was found to transport calcium mainly in the ionic form in the hemolymph, with only minor parts being bound to proteins or smaller ligands. Hemolymph measurement also revealed that C. gigas is able to regulate the extracellular concentrations of calcium and potassium. The changes in plasma calcium concentrations and speciation, concomitant with increases in granulocytes indicate that multiple calcium transport processes are activated after induced shell damage.

Beliefs about medicines are strongly associated with medicine-use patterns among the general population.

AIMS: To investigate self-reported beliefs and perceived sensitivity to medicines and their effects in relation to self-reported use of medicines and herbal remedies. METHODS: A survey sent to 13,931 randomly selected Swedish adults included the Beliefs about Medicines Questionnaire-General (BMQ-General) Questionnaire and the Perceived Sensitivity to Medicines Scale (PSM). The survey also asked about individuals' use of prescribed and over-the-counter (OTC) medicines and herbal remedies in the past month. We examined all associations between scores on the BMQ-General subscales and PSM in relation to the use of medicines and herbal remedies, using analysis of covariance adjusted for potential confounders. RESULTS: Among 7099 respondents, those using herbal remedies exclusively believed strongly that prescription and OTC medicines are harmful and overprescribed. Respondents using prescription and OTC medicines reported more positive beliefs [coefficient 0.67 (95% CI 0.47-0.87) and 0.70 (95% CI 0.51-0.90)] on the benefits of medicines compared with those using herbal remedies [-0.18 (95% CI -0.57-0.20)]. Perceived sensitivity to medicines was higher among those using herbal remedies only [1.25 (95% CI 0.46-2.03)] compared with those using no medicines (reference 0) or prescription [-0.44 (95% CI -0.84 to -0.05)] or OTC [-0.27 (95% CI -0.66-0.12)] medicines alone. CONCLUSION: Respondents using prescription and/or OTC medicines reported stronger positive beliefs about the benefits of medicines in general, supporting the hypothesis that beliefs influence medicine use. Therefore, addressing beliefs and concerns about medicines during patient counselling may influence medicine use, particularly regarding unintentional non-adherence.

Role of parenting styles in adolescent substance use: results from a Swedish longitudinal cohort study.

OBJECTIVE: Adolescent substance use is an area of concern because early substance use is associated with a higher risk of adverse outcomes. Parenting style, defined as the general style of parenting, as well as substance-specific parenting practices may influence children's substance use behaviour. The present study aims to probe the impact of parenting style on adolescent substance use. METHOD: A cohort of 1268 adolescents (48% girls), aged 12-13 years at baseline, from 21 junior high schools was assessed in the first semester of junior high school, and then again in the last semester of the 9th grade, 32 months later. Parenting style, operationalised as a fourfold classification of parenting styles, including established risk factors for adolescent substance use, were measured at baseline. RESULTS: Neglectful parenting style was associated with worse substance use outcomes across all substances. After adjusting for other proximal risk factors in multivariate analyses, parenting style was found to be unrelated to substance use outcomes with one exception: authoritative parenting style was associated with less frequent drinking. Association with deviant peers, delinquent behaviour, provision of alcohol by parents, and previous use of other substances were associated with substance use outcomes at follow-up. CONCLUSIONS: The results of the present study indicate that parenting style may be less important for adolescent substance use outcomes than what has previously been assumed, and that association with deviant peers and delinquent behaviour may be more important for adolescent substance use outcomes than general parenting style.

Correlation between Cognition and Function across the Spectrum of Alzheimer's Disease.

BACKGROUND: Both cognitive and functional deterioration are characteristic of the clinical progression of Alzheimer's disease (AD). OBJECTIVES: To systematically assess correlations between widely used measures of cognition and function across the spectrum of AD. DESIGN: Spearman rank correlations were calculated for cognitive and functional measures across datasets from various AD patient populations. SETTING: Post-hoc analysis from existing databases. PARTICIPANTS: Pooled data from placebo-treated patients with mild (MMSE score ≥20 and ≤26) and moderate (MMSE score ≥16 and ≤19) AD dementia from two Phase 3 solanezumab (EXPEDITION/2) and two semagecesatat (IDENTITY/2) studies and normal, late mild cognitive impairment (LMCI) and mild AD patients from the Alzheimer's Disease Neuroimaging Initiative 2-Grand Opportunity (ADNI-2/GO). Intervention (if any): Placebo (EXPEDITION/2 and IDENTITY/2 subjects). MEASUREMENTS: Cognitive and functional abilities were measured in all datasets. Data were collected at baseline and every three months for 18 months in EXPEDITION and IDENTITY studies; and at baseline, 6, 12, and 24 months in the ADNI dataset. RESULTS: The relationship of cognition and function became stronger over time as AD patients progressed from preclinical to moderate dementia disease stages, with the magnitude of correlations dependent on disease stage and the complexity of functional task. The correlations were minimal in the normal control population, but became stronger with disease progression. CONCLUSIONS: This analysis found that measures of cognition and function become more strongly correlated with disease progression from preclinical to moderate dementia across multiple datasets. These findings improve the understanding of the relationship between cognitive and functional clinical measures during the course of AD progression and how cognition and function measures relate to each other in AD clinical trials.

Stunning fish with CO2 or electricity: contradictory results on behavioural and physiological stress responses.

Studies that address fish welfare before slaughter have concluded that many of the traditional systems used to stun fish including CO2 narcosis are unacceptable as they cause avoidable stress before death. One system recommended as a better alternative is electrical stunning, however, the welfare aspects of this method are not yet fully understood. To assess welfare in aquaculture both behavioural and physiological measurements have been used, but few studies have examined the relationship between these variables. In an on-site study aversive behaviours and several physiological stress indicators, including plasma levels of cortisol and ions as well as blood physiological variables, were compared in Arctic char (Salvelinus alpinus) stunned with CO2 or electricity. Exposure to water saturated with CO2 triggered aversive struggling and escape responses for several minutes before immobilization, whereas in fish exposed to an electric current immobilization was close to instant. On average, it took 5 min for the fish to recover from electrical stunning, whereas fish stunned with CO2 did not recover. Despite this, the electrically stunned fish had more than double the plasma levels of cortisol compared with fish stunned with CO2. This result is surprising considering that the behavioural reactions were much more pronounced following CO2 exposure. These contradictory results are discussed with regard to animal welfare and stress physiological responses. The present results emphasise the importance of using an integrative and interdisciplinary approach and to include both behavioural and physiological stress indicators in order to make accurate welfare assessments of fish in aquaculture.

Pharmacoepidemiology at Nordic School of Public Health NHV: Examples from 1999 to 2014.

BACKGROUND: Pharmacoepidemiology is a branch of public health and had a place at the Nordic School of Public Health. Courses, Master's theses and Doctorates of Public Health (DrPH) in Pharmacoepidemiology were a relatively minor, but still important part of the school's activities. METHODS: This paper gives a short background, followed by some snapshots of the activities at NHV, and then some illustrative case-studies. These case-studies list their own responsible co-authors and have separate reference lists. RESULTS: In the Nordic context, NHV was a unique provider of training and research in pharmacoepidemiology, with single courses to complete DrPH training, as well as implementation of externally-funded research projects. CONCLUSIONS: With the closure of NHV at the end of 2014, it is unclear if such a comprehensive approach towards pharmacoepidemiology will be found elsewhere in the Nordic countries.

A Combined Measure of Cognition and Function for Clinical Trials: The Integrated Alzheimer's Disease Rating Scale (iADRS).

It is generally recognized that more sensitive instruments for the earliest stages of Alzheimer's disease (AD) are needed. The integrated Alzheimer's Disease Rating Scale (iADRS) combines scores from 2 widely accepted measures, the Alzheimer's Disease Assessment Scale-Cognitive subscale (ADAS-Cog) and the Alzheimer's Disease Cooperative Study - instrumental Activities of Daily Living (ADCS-iADL). Disease progression and treatment differences as measured by the iADRS were analyzed using data from solanezumab EXPEDITION, EXPEDITION2, and EXPEDITION-EXT Studies; semagacestat IDENTITY Study; and donepezil ADCS - mild cognitive impairment (ADCS-MCI) Study. Psychometric properties of the iADRS were established through principal component analysis (PCA) and estimation of contributions of subscores and individual item scores to the iADRS total score. The iADRS performed better than most composites and scales in detecting disease progression and comparably or better than individual scales in detecting treatment differences. PCA demonstrated the iADRS can be divided into two principal components primarily representing cognitive items and instrumental ADLs. Dynamic ranges of the subscales were similar across all studies, reflecting approximately equal contributions from both subscales to the iADRS total score. In item analyses, every item contributed to the total score, with varying strength of contributions by item and across data sets. The iADRS demonstrated acceptable psychometric properties and was effective in capturing disease progression from MCI through moderate AD and treatment effects across the early disease spectrum. These findings suggest the iADRS can be used in studies of mixed populations, ensuring sensitivity to treatment effects as subjects progress during studies of putative disease-modifying agents.

Development of intestinal ion-transporting mechanisms during smoltification and seawater acclimation in Atlantic salmon Salmo salar.

This study investigated the expression of ion transporters involved in intestinal fluid absorption and presents evidence for developmental changes in abundance and tissue distribution of these transporters during smoltification and seawater (SW) acclimation of Atlantic salmon Salmo salar. Emphasis was placed on Na(+) , K(+) -ATPase (NKA) and Na(+) , K(+) , Cl(-) co-transporter (NKCC) isoforms, at both transcriptional and protein levels, together with transcription of chloride channel genes. The nka α1c was the dominant isoform at the transcript level in both proximal and distal intestines; also, it was the most abundant isoform expressed in the basolateral membrane of enterocytes in the proximal intestine. This isoform was also abundantly expressed in the distal intestine in the lower part of the mucosal folds. The protein expression of intestinal Nkaα1c increased during smoltification. Immunostaining was localized to the basal membrane of the enterocytes in freshwater (FW) fish, and re-distributed to a lateral position after SW entry. Two other Nka isoforms, α1a and α1b, were expressed in the intestine but were not regulated to the same extent during smoltification and subsequent SW transfer. Their localization in the intestinal wall indicates a house-keeping function in excitatory tissues. The absorptive form of the NKCC-like isoform (sub-apically located NKCC2 and/or Na(+) , Cl(-) co-transporter) increased during smoltification and further after SW transfer. The cellular distribution changed from a diffuse expression in the sub-apical regions during smoltification to clustering of the transporters closer to the apical membrane after entry to SW. Furthermore, transcript abundance indicates that the mechanisms necessary for exit of chloride ions across the basolateral membrane and into the lateral intercellular space are present in the form of one or more of three different chloride channels: cystic fibrosis transmembrane conductance regulator I and II and chloride channel 3.

Plasma growth hormone-binding protein levels in Atlantic salmon Salmo salar during smoltification and seawater transfer.

Specific growth hormone (GH)-binding protein (Ghbp) was purified from Atlantic salmon Salmo salar and rainbow trout Oncorhynchus mykiss plasma with immunoprecipitation and characterized in cross-linking studies using autoradiography and western blots. The size of the Ghbp was estimated to be c. 53 kDa. A radioimmunoassay was established to measure Ghbp in salmonids, using antibodies specific against the extracellular segment of the S. salar growth hormone receptor 1 (grh1; GenBank AY462105). Plasma Ghbp levels were measured in S. salar smolts in fresh water and after transfer to seawater (SW; experiments 1 and 2), and in post-smolts kept at different salinities (0, 12, 22 and 34) for 3 months (experiment 3). A transient increase in plasma Ghbp, which lasted for 1 month or less, was noted in smolts after transfer to SW. Concomitantly, plasma GH and gill Na(+) -K(+) -ATPase activity increased during smoltification (in experiment 2). No difference in plasma Ghbp was evident between post-smolts kept at different salinities, although the fish kept at salinity 34 had higher plasma GH than the group kept at salinity 22 and higher hepatic ghr1 expression than post-smolts kept at salinity 12. This suggests that plasma Ghbp regulation may respond to salinity changes in the short term. The lack of correlation between Ghbp, plasma GH and hepatic ghr1 expression in the long-term post-smolt experiment indicates that Ghbp levels may be regulated independently of other components of the endocrine GH system in salmonids.

Influence of refill adherence method when comparing level of adherence for different dosing regimens.

PURPOSE: To examine the impact of two methods when estimating refill adherence in patients using bisphosphonates with different dosing regimens. METHODS: In the Swedish Prescribed Drug Register, 18,203 new users of bisphosphonates aged 18-85 years were identified between 1 July 2006 and 30 June 2007 and followed for a maximum of 2 years. The patients were categorised based on dosing regimen: one tablet daily, one tablet weekly, switching between these regimens, and other regimens. Refill adherence was estimated with Continuous measure of Medication Acquisition (CMA, adherent if CMA ≥ 80 %) and the maximum gap method (adherent if gaps <45 days). Differences in adherence between patients in the groups were assessed with logistic regression models controlling for confounding factors. RESULTS: The proportion of patients classified as adherent was higher using CMA compared with patients classified as adherent using the maximum gap method. Patients on one tablet weekly had significantly lower adherence compared with patients on one tablet daily in the main analyses of both methods (the maximum gap method: 73 % vs. 80 %; adjusted OR=0.71; 95 % CI 0.57-0.89 and CMA: 84 % vs. 88 %, adjusted OR=0.75; 95 % CI 0.57-0.99). Patients using the other two dosing regimens had significantly lower adherence compared with patients on one tablet daily using both methods. CONCLUSION: Choice of method has an impact on the estimates of refill adherence to bisphosphonates. Patients on one tablet weekly dosing had lower adherence compared with patients on one tablet daily dosing using both methods.

Factors associated with switching and combination use of antidepressants in young Swedish adults.

AIMS: Little is known on factors associated with switching and combination use of antidepressants. Our aim was to describe such use and to analyse the association with socioeconomic factors and level of care in Swedish adults aged 20-34 years. METHODS: Individuals, aged 20-34 years, who purchased an antidepressant in January-June 2006, and who had not purchased any antidepressant in the preceding 6 months (n = 24,897) were followed from 6 up to 12 months. Among those who purchased ≥ 2 antidepressant substances, switchers were defined as those who did not fulfil the requirements for combination use. Data on purchased antidepressants and socioeconomic characteristics were obtained from the Swedish Prescribed Drug Register and Statistics Sweden. The association between (i) ≥ 2 antidepressants or (ii) switching, respectively, and socioeconomic factors as well as level of care was analysed with multiple logistic regression. RESULTS: A total of 4254 individuals (17%) purchased ≥ 2 antidepressant substances, and the remaining 20,643 (83%) purchased one antidepressant. The adjusted odds ratio (OR) for purchase of ≥ 2 antidepressants (vs. purchase of one antidepressant only) was higher among those who started on mirtazapine compared with selective serotonin re-uptake inhibitors: 2.23 (95% confidence interval: 1.93-2.57), and lower in individuals with high education: 0.64 (0.54-0.75), and shorter length of follow-up: 0.73 (0.62-0.85). Among those with ≥ 2 antidepressants, 71.6% were classified as switchers. The adjusted OR for switching (vs. combination use) were higher among divorced/widows/widowers: 1.61 (1.05-2.49), and lower among individuals with short university education: 0.58 (0.43-0.78), those starting on mirtazapine: 0.78 (0.62-0.97), and when treatment was initiated in psychiatric care: 0.75 (0.63-0.88). CONCLUSIONS: One of six new users purchased at least two antidepressants, the majority were classified as switchers. Purchase patterns were associated with socioeconomic characteristics, in particular level of education, type of first purchased antidepressant, and level of care initiating treatment.

The Nordic prescription databases as a resource for pharmacoepidemiological research--a literature review.

PURPOSE: All five Nordic countries have nationwide prescription databases covering all dispensed drugs, with potential for linkage to outcomes. The aim of this review is to present an overview of therapeutic areas studied and methods applied in pharmacoepidemiologic studies using data from these databases. METHODS: The study consists of a Medline-based structured literature review of scientific papers published during 2005-2010 using data from the prescription databases in Denmark, Finland, Iceland, Norway, and Sweden, covering 25 million inhabitants. Relevant studies were analyzed in terms of pharmacological group, study population, outcomes examined, type of study (drug utilization vs. effect of drug therapy), country of origin, and extent of cross-national collaboration. RESULTS: A total of 515 studies were identified. Of these, 262 were conducted in Denmark, 97 in Finland, 4 in Iceland, 87 in Norway, and 61 in Sweden. Four studies used data from more than one Nordic country. The most commonly studied drugs were those acting on the nervous system, followed by cardiovascular drugs and gastrointestinal/endocrine drugs. A total of 228 studies examined drug utilization and 263 focused on the effects and safety of drug therapy. Pregnant women were the most commonly studied population in safety studies, whereas prescribers' adherence to guidelines was the most frequent topic of drug utilization studies. CONCLUSIONS: The Nordic prescription databases, with their possibility of record-linkage, represent an outstanding resource for assessing the beneficial and adverse effects of drug use in large populations, under routine care conditions, and with the potential for long-term follow-up.

Glucose-lowering treatment and clinical results in 163 121 patients with type 2 diabetes: an observational study from the Swedish national diabetes register.

AIMS: To analyse clinical characteristics and treatment results in unselected type 2 diabetes mellitus (T2DM) patients, with non-pharmacological treatment as well as the most commonly used pharmacological glucose-lowering treatment regimens, in everyday clinical practice. METHODS: In this population-based cross-sectional study, information was linked from the Swedish National Diabetes Register, Prescribed Drug Register and Patient Register. T2DM patients with non-pharmacological treatment and T2DM patients continuously using the 12 most common pharmacological treatment regimens were included in the study (n = 163121). RESULTS: There were statistically significant differences in clinical characteristics between the groups. Patients with insulin-based treatment regimens had the longest duration of diabetes and more cardiovascular risk factors than the T2DM-population in general. The proportion of patients reaching HbA1c ≤ 7% varied between 70.1% (metformin) and 25.0% [premixed insulin (PMI) + SU) in patients with pharmacological treatment. 84.8% of the patients with non-pharmacological treatment reached target. Compared to patients on metformin, patients on other pharmacological treatments had a lower likelihood, with hazard ratios ranging from 0.58; 95% confidence interval (CI), 0.54-0.63 to 0.97;0.94-0.99, of having HbA1c ≤ 7% (adjusted for covariates). Patients on insulin-based treatments had the lowest likelihood, while non-pharmacological treatment was associated with an increased likelihood of having HbA1c ≤ 7%. CONCLUSION: This nation-wide study shows insufficiently reached treatment goals for haemoglobin A1c (HbA1c) in all treatment groups. Patients on insulin-based treatment regimens had the longest duration of diabetes, more cardiovascular risk factors and the highest proportions of patients not reaching HbA1c target.

Slow release cortisol implants result in impaired innate immune responses and higher infection prevalence following experimental challenge with infectious pancreatic necrosis virus in Atlantic salmon (Salmo salar) parr.

Stress can affect the immune system and increase susceptibility to various diseases but knowledge of the underlying mechanisms is scarce. There is a complex interaction between the immune system and the endocrine system of vertebrates. In fish, cortisol is a key hormone regulating stress response and recent studies have also suggested that this hormone can affect the immune system, where cortisol is mainly regarded as an immunosuppressive factor. The aim of the present study was to examine the impact of chronically elevated levels of cortisol on the immune response and susceptibility to experimental infection with infectious pancreatic necrosis virus (IPNV). Further, the effect of IPNV challenge on circulating levels of cortisol was investigated. Atlantic salmon parr were implanted intraperitoneally with sustained-release implants of bovine of cortisol (50 µg cortisol g(-1) body weight in an implant based on vegetable lipids). Vehicle implants were used as control (sham-injected). At 45 days after implantation (DAI), fish were challenged with a low virulent isolate of IPNV (by immersion). Samples of plasma, liver and head kidney was taken from fish before and 24 h, 48 h, 7 days week and 21 days post infection (DPI). Cortisol level in plasma was measured using radioimmunoassay and gene expression in liver and head kidney was analyzed with real-time PCR (RT-PCR). Infection prevalence in infected fish was assessed by virus culture and RT-PCR of head kidney samples. Cortisol implantation compared with sham-implanted fish had increased levels of plasma cortisol at 45 DAI. The relative expression of Interferon alpha-1 (IFNα-1), Myxo virus-1 Mx, Heat-shock protein 70 (HSP70), Serum amyloid A (SAA), Glucocorticoid receptor (GR) and Heat-shock protein 90 (HSP90) tends to be down-regulated by cortisol implantation. There was a higher prevalence of fish with detectable levels of IPNV, as measured by cell culture and RT-PCR, in the cortisol-implanted group challenged with IPNV (0 = 0.0305) relative to the group that received a sham implantation. Further, cortisol seems to delay the induction of the antiviral IFNα-1 pathway and Mx mRNA expression. This study shows that elevated plasma cortisol level leads to an impaired innate immune response, and higher virus (IPNV) prevalence in Atlantic salmon parr.

Disturbance of the intestinal mucosal immune system of farmed Atlantic salmon (Salmo salar), in response to long-term hypoxic conditions.

The gastrointestinal (GI) tract has many important biological functions. One is to serve as a barrier between the fish and the external environment. A decreased physical barrier function of the intestine may lead to increased inflow of luminal content and subsequent activation of the intestinal mucosal immune system. This activation is governed by the ability of various compounds to induce cytokine release and immune cell activity, leading to an immune response. In mammals, the impact of stress on the intestinal barrier is well documented and results in increased intestinal permeability and thus increased stimulation of the mucosal immune system. Fish reared in sea cages may at times be exposed to unfavourable environmental conditions leading to chronic stress and disturbed intestinal integrity. This change in permeability may increase the exposure of the mucosal immune system to activating compounds. In the present study, the effect of a prolonged stress on the intestinal mucosal immune system of fish is therefore addressed. Atlantic salmon were exposed to low levels (50%) of dissolved oxygen (DO) for 6-7 weeks in consecutive experiments performed at 8 and 16 °C. Immune parameters were assessed in terms of mRNA expression of the key cytokines, interleukin-1ß (IL-1ß), IL-8, IL-10, interferon-γ (IFNγ) and transforming growth factor-ß (TGFß) as well as the immune regulatory inhibitor of nuclear factor κB (IκB). In the experiment at 8 °C also mucosal neutrophil infiltration was monitored. Subjecting the fish to low DO levels at 8 °C resulted in an increased mucosal neutrophil infiltration together with a down-regulation of IκB. At the higher temperature, 16 °C, low DO levels created decreased expression of the pro-inflammatory cytokine IL-1ß in both intestinal regions as well as an increased expression of IL-10 in the proximal intestine. These results suggest that husbandry conditions in sea cages with DO levels as low as 50% clearly affects the intestinal mucosal immune system and results in a chronic inflammation. Moreover, the effects of low DO levels on the immune factors examined were more pronounced in the 16 °C experiment suggesting additive effects of high temperatures.

Effect of biofilm formation on antimicrobial tolerance of Flavobacterium psychrophilum.

Treatment of bacterial fish diseases can be complicated by resistant bacterial biofilms harbouring pathogenic bacteria and causing recurrent exposure of fish to infections. In this study, the effect of biofilm formation on antimicrobial tolerance was examined using three bacterial isolates of the fish pathogen Flavobacterium psychrophilum and two antimicrobial agents, oxytetracycline and flumequine, commonly used in aquaculture. Planktonic and biofilm cells were exposed to a minimum inhibitory concentration (MIC), to a 3 × MIC concentration and to an environmental concentration level of each antimicrobial in 96-well microtitre plates after which growth on agar plates was measured. The type strain NCIMB1947 of F. psychrophilum was further used to study the development of antimicrobial resistance in biofilm cells. The results suggest that at high bacterial densities (>10(7) CFU mL(-1)), biofilm cells of F. psychrophilum are less susceptible to antimicrobial agents. Furthermore, the results imply that biofilm cells of F. psychrophilum may rapidly develop resistance to both oxytetracycline and flumequine if exposed to subinhibitory concentrations of these antimicrobials.