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Background and Objectives: MicroRNAs (miRNAs) are regulators of gene expression and have been reported to be dysregulated in people with multiple sclerosis (pwMS). Autologous hematopoietic stem cell transplantation (aHSCT) is an immune-ablative treatment intervention for pwMS. Currently, it is unknown if aHSCT affects expression levels of miRNAs in CSF. We explored the ability of circulating miRNA to discriminate between pwMS and healthy controls (HCs) and investigated whether these miRNAs were affected by treatment with aHSCT.

Methods: Using quantitative reverse transcription PCR, 87 miRNAs were analyzed in CSF samples of a discovery cohort (baseline: 4 & HC: 4). The top 22 miRNAs discriminating between pwMS and HCs were then analyzed in 187 CSF samples of a validation cohort (pwMS: 50, HC: 32). Samples, failing quality control or being follow-ups to baseline samples with quality control issues, were excluded from further analyses. The remaining 133 samples (HC: 29, MS: baseline: 33, 1 year: 30, 2 years: 26, 3-5 years: 15) were analyzed for expression of the top 22 miRNAs.

Results: Twelve miRNAs were dysregulated in pwMS compared with HC (q < 0.05). Associations with clinical and analytical parameters were observed in relation to all 12 miRNAs; however, a cluster of 4 miRNAs (miR-16-5p, miR-21-5p, miR-150-5p, and miR-146a-5p) with strong correlations (r > 0.60, p < 0.001) with multiple parameters was identified. Of the 12 miRNAs, 8 were differentially expressed in pwMS with gadolinium-enhancing lesions at baseline and 4 by prior disease-modifying treatment class (p < 0.05). These 4 miRNAs correlated strongly with each other, decreased after aHSCT, and remained low throughout the follow-up period (p < 0.05). Target and pathway analysis of these revealed association with biological processes affecting cytokine production, inflammatory response, and regulation of myelin maintenance.

Discussion: miRNAs are dysregulated in CSF from pwMS and particularly in patients with less effective treatments and/or higher inflammatory disease activity. A 4-miRNA signature with elevated expression of miR-16-5p, miR-21-5p, miR-150-5p, and miR-146a-5p was recurring in multiple analyses. After intervention with aHSCT, the expression levels approached the levels of the HCs, suggesting a potent treatment effect.

Objective: Primary aldosteronism (PA) is a common cause of hypertension. It entails elevated morbidity and mortality that do not sufficiently improve with conventional antihypertensive therapy. Screening for PA by plasma aldosterone–renin ratio (ARR) enables discovery and specific treatment of affected patients. By screening primary care patients with hypertension and evaluating them further according to the Endocrine Society guidelines, we aimed to assess the prevalence of PA, the factors affecting biochemical diagnostics, and the outcome of lateralization studies and of specific treatment of the discovered PA cases.

Design, patients, and methods: Prospective evaluation of screening for PA was conducted in 1,181 patients. Screening by ARR was performed under current therapy, but without mineralocorticoid receptor antagonists (MRA), under normokalemia, and confirmed by the intravenous saline suppression test, SST#1. Those with results in a defined gray zone underwent therapy adjustment and then completed SST#2. Plasma aldosterone and ARR were compared under different stages of the diagnostic process. All patients with PA were offered adrenal venous sampling, or, in certain cases, adrenocortical-specific positron emission tomography. Lateralizing cases were offered laparoscopic adrenalectomy. Patients with bilateral disease were treated with MRA. Treatment results were assessed after a minimum of 6 months.

Results: A total of 53 discovered cases of (mostly mild) PA corresponded to its prevalence of 4.5%. Initial seated ARR was higher than recumbent ARR before SST#1. At SST#2, initial ARR and final aldosterone were higher than at SST#1. Localizing studies (accepted by 45 patients) found 14 lateralized cases. Of the 11 operated cases, 4 had aldosterone-producing adenoma, and the remainder had micro- and macronodular histopathology. A total of 31 patients had bilateral PA. Both surgical and conservative treatments were well tolerated and led to improved blood pressure and higher renin, indicating risk amelioration.

Conclusions: PA is prevalent among primary care patients with hypertension and can be screened for under current antihypertensive therapy. Aldosterone-producing adenoma was rare in this cohort. The study results support active screening of primary care patients with hypertension for PA in order to offer appropriate treatment options.

Background: Primary aldosteronism (PA) is the principal cause of secondaryhypertension; it leads to significantly elevated cardiovascular morbidity andmortality, but only a fraction of its cases ever get detected, partially due todiagnostic procedures that are difficult to perform and to interpret. Morestraightforward diagnostic methods are needed. Lateralized, or unilateral PA(uPA), is best treated by surgery. Bilateral PA (bPA) is treated medically.Aim: The aim of our study was to explore microRNA (miRNA) in peripheral bloodas markers of PA, uPA and bPA.

Methods: In groups of subjects with primary hypertension (HT, n = 11), bPA (n =12), and uPA (n = 16), peripheral serum was used for isolation of total RNA, librarypreparation, and NGS sequencing to achieve a comparative analysis of miRNAexpression. Five-fold cross-validation support vector machine learning (ML)models were employed to search for miRNA that could be used as markers ofPA and its forms.

Results: In our cohort of patients, the discovered combinations of miRNAs could,with a high level of accuracy, sensitivity, and specificity, characterize thedifference between HT and PA, as well as between a combined group of HT +bPA vs. uPA. The differentiating parameters were moderately good forcomparison of bPA vs. uPA.

Conclusion: Within our patient cohort, and using ML, the study identifieddistinctly different miRNA profiles between HT and PA, as well as between bPAand uPA. Further validation studies may lead to the emergence of a new tool forclinical diagnostics of PA.

BACKGROUND: Inflammatory bowel disease (IBD; mainly ulcerative colitis and Crohn's disease) is associated with the development of colorectal cancer (CRC) referred to as colitis-associated colorectal cancer (CAC). In inflammatory flares of IBD, the production of luminal nitric oxide (NO) increases due to the increased inducible nitric oxide synthase (iNOS) activity in inflamed tissue. It is believed that iNOS parallels pro-inflammatory interleukin-1β (IL-1β). How these biomarkers relate to CAC pathogenesis or survival is unknown.

AIM: The primary aim of this study was to investigate iNOS and IL-1β immunoreactivity in CAC tumors in comparison with CRC and normal colonic mucosa, and the secondary aim was to determine if immunoreactivity correlates with 5-year survival of CAC.

METHODS: Immunohistochemistry was performed on tissue sections as follows: CAC (n = 59); sporadic CRC (sCRC) (n = 12); colonic mucosa >2 cm outside sCRC margin (normal mucosa) (n = 22); paracancerous IBD (pIBD) (n = 12). The expression of iNOS and IL-1β was quantified separately for epithelium and stroma. Data were evaluated using the Mann-Whitney U-test and the log-rank test for 5-year Kaplan-Meier survival curves. Results were compared with online mRNA databases.

RESULTS: Immunoreactivity occurred predominantly in epithelial cells and to lesser extent in stroma. Compared with normal mucosa, immunoreactivity for iNOS (P < 0.01) and IL-1β (P < 0.005) was higher in CAC epithelium. In CAC stroma, iNOS immunoreactivity was lower than normal mucosa (P < 0.001), whereas IL-1β was higher (P < 0.05). Immunoreactivity differences of iNOS or IL-1β among CAC patients failed to correlate with 5-year survival. These findings were supported by online mRNA databases.

CONCLUSION: Consistent with high NO production in IBD, there is more iNOS in CAC epithelium, albeit not in stroma. This immunoreactivity difference exists for IL-1β in both epithelium and stroma. The intervention of arginine or iNOS activity for CAC chemotherapy is not straightforward.

Introduction; Despite its popularity in research, there is very little scientifically validated knowledge about the best practices on zebrafish (Danio rerio) husbandry, which has led to several facilities having their own husbandry protocols. This study was performed to expand knowledge on the effects of enrichment and fish density on the welfare of zebrafish, with hopes of providing a scientific basis for future recommendations and legislations.

Methods: Zebrafish were reared at three different stocking densities, (1, 3 or 6 fish/L), in tanks with or without environmental enrichment. Agonistic behavior was observed twice a week for 9 weeks directly in the housing tanks. Aspects of welfare is known to be reflected in neuroendocrine stress responses. Thus, cortisol secretion in response to lowering the water level was analyzed for each group. In addition, we assessed cortisol secretion in response to confinement and risk-taking behavior (boldness) using the novel tank diving test for individual fish. At termination of the experiment fish were subjected to stress by transfer to a novel environment and brain tissue was sampled for analysis of brain monoaminergic activity.

Results: Fish kept at the lowest density (1 fish/L) showed a significantly higher level of aggression than fish kept at 3 or 6 fish/L. Moreover, fish kept at this low density showed significantly higher cortisol secretion on a group level than fish kept at the higher stocking densities, when subjected to lowering of the water level. Keeping fish at 1 fish/L also had effects on brain monoamines, these fish showing higher brain dopamine concentrations but lower dopamine turnover than fish kept at higher densities. Neither stocking density or enrichment had any clear effects on the behavior of individual fish in the novel tank diving test. However, fish kept at high densities showed lower and more variable growth rates than fish kept at 1 fish/L.

Discussion: Taken together these results suggest that zebrafish should not be kept at a density of 1 fish/L. The optimal stocking density is likely to be in the range of 3-6 fish/L.

NDST1 (glucosaminyl N-deacetylase/N-sulfotransferase) is a key enzyme in heparan sulfate (HS) biosynthesis, where it is responsible for HS N-deacetylation and N-sulfation. In addition to the full length human enzyme of 882 amino acids, here designated NDST1A, a shorter form containing 825 amino acids (NDST1B) is synthesized after alternative splicing of the NDST1 mRNA. NDST1B is mostly expressed at a low level, but increased amounts are seen in several types of cancer where it is associated with shorter survival. In this study, we aimed at characterizing the enzymatic properties of NDST1B and its effect on HS biosynthesis. Purified recombinant NDST1B lacked both N-deacetylase and N-sulfotransferase activities. Interestingly, HEK293 cells overexpressing NDST1B synthesized HS with reduced sulfation and altered domain structure. Fluorescence resonance energy transfer-microscopy demonstrated that both NDST1A and NDST1B had the capacity to interact with the HS copolymerase subunits EXT1 and EXT2 and also to form NDST1A/NDST1B dimers. Since lysates from cells overexpressing NDST1B contained less NDST enzyme activity than control cells, we suggest that NDST1B works in a dominant negative manner, tentatively by replacing the active endogenous NDST1 in the enzyme complexes taking part in biosynthesis.

BACKGROUND: Haptoglobin (Hp), a liver derived acute phase inflammatory protein (APP), has scarcely been studied in juvenile idiopathic arthritis (JIA). Hp can occur in blood as two isoforms (Hp1 and Hp2) in precursor and mature forms. Routine clinical chemistry immunoturbidimetry does not discern these forms. It is unknown how different forms relate to disease activity in JIA. Our aims were to determine allele frequency and plasma concentrations of different Hp forms at higher versus lower JIA disease activity and compare to other APPs.

METHODS: Plasma from JIA (n = 77) and healthy (n = 42) children were analyzed for apparent Hp allelic frequency and densitometric concentrations of alpha forms by Western blot (WB). Polymerase chain reaction (PCR) (buffy coat) was performed in a subset to estimate conformity with genetics. At higher versus lower juvenile arthritis disease activity score (JADAS27) (which includes erythrocyte sedimentation rate (ESR)), total mature Hp concentration from WB was compared and correlated against immunoturbidimetry and total protein, albumin, serum amyloid A (SAA) and C-reactive protein (CRP).

RESULTS: At 300-fold dilution needed to study mature forms in Western blot, precursors were undetectable. Hp2 contributed most signal in most samples. Hp allele frequency was similar in JIA and controls. Both mature forms, taken separately or by sum, declined following treatment, but remained above concentrations of healthy controls, even in a remission subset that achieved JADAS27 < 1. Densitometry correlated with immunoturbidimetry. Hp concentrations correlated with JADAS27, albumin (negatively), CRP and SAA with immunoturbidimetric method correlating strongest to JADAS27 (Spearman R ~ 0.6, p < 0.0001).

CONCLUSION: Hp allele frequency in JIA is similar to the general population, indicating that children with JIA should have the same possibility as in healthy children to produce preHp2 (zonulin), thought to increase intestinal permeability. Circulating Hp concentrations largely parallel other APPs and ESR; none of these measures correlate very strongly to JADAS27 score but Hp can be measured from capillary sampling which is impossible with ESR.