Optimizing zebrafish rearing-Effects of fish density and environmental enrichment.

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.

High iNOS and IL-1ß immunoreactivity are features of colitis-associated colorectal cancer tumors, but fail to predict 5-year survival.

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-1ß (IL-1ß). How these biomarkers relate to CAC pathogenesis or survival is unknown. Aim: The primary aim of this study was to investigate iNOS and IL-1ß 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-1ß 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-1ß (P < 0.005) was higher in CAC epithelium. In CAC stroma, iNOS immunoreactivity was lower than normal mucosa (P < 0.001), whereas IL-1ß was higher (P < 0.05). Immunoreactivity differences of iNOS or IL-1ß 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-1ß in both epithelium and stroma. The intervention of arginine or iNOS activity for CAC chemotherapy is not straightforward.

Haptoglobin in Juvenile Idiopathic Arthritis.

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.

A dominant negative splice variant of the heparan sulfate biosynthesis enzyme NDST1 reduces heparan sulfate sulfation.

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.