Estuarine fish health assessment: evidence of wastewater impacts based on nitrogen isotopes and histopathology

Sewage effluent is a powerful agent of ecological change in estuaries. While the effects of sewage pollution on water quality are usually well documented, biological responses of exposed organisms are not. We quantified health impacts in the form of pathological tissue changes across multiple organs in estuarine fish exposed to elevated levels of treated wastewater. Structural pathologies were compared in wild populations of four fish species from two subtropical estuaries on the east coast of Australia that differ substantially in the amount of direct wastewater loadings. Uptake of sewage-derived  nitrogen by fish was traced with stable nitrogen isotopes. Pathologies were common in the liver, spleen, gill, kidney and muscle tissues, and included granulomas, melanomacrophage aggregates, and multiple deformities of the gill epithelia. Tissue deformities were more frequent in fish exposed directly to wastewater discharges. Mullet (Valamugil georgii) were most affected, with only a single specimen free of pathologies in the sewage-impacted estuary. Similarly, in those fish that had structural abnormalities, more deformities were generally found in individuals from sites receiving sewage. These spatial contrasts in impaired fish health correspond to significantly enriched δ₁₅N values in fish muscle as a consequence of fish assimilating sewage-N. Overall, the pattern of lower health and enriched δ₁₅N values in fish from sewage-impacted areas suggests that organism health is lowered by sewage inputs to estuaries. Measurements of organism health are required to understand the effects of sewage on estuarine ecosystems, and histopathology of fishes is a powerful tool to achieve this.

Heterozygous tx mice have an increased sensitivity to copper loading: implications for Wilson's Disease carriers

Wilson's disease carriers constitute 1% of the human population. It is unknown whether Wilsons disease carriers are at increased susceptibility to copper overload when exposed to chronically high levels of ingested copper. This study investigated the effect of chronic excess copper in drinking water on the heterozygous form of the Wilson’s disease mouse model – the toxic milk (tx) mouse. Mice were provided with drinking water containing 300 mg/l copper for 4–7, 8–11, 12–15 or 16–20 months. At the completion of the study liver, spleen, kidney and brain tissue were analyzed by atomic absorption spectroscopy to determine copper concentration. Plasma ceruloplasmin oxidase activity and liver histology were also assessed. Chronic copper loading resulted in significantly increased liver copper in both tx heterozygous and tx homozygous mice, while wild type mice were resistant to the effects of copper loading. Copper loading effects were greatest in tx homozygous mice, with increased extrahepatic copper deposition in spleen and kidney – an effect absent in heterozygote and wild type mice. Although liver histology in homozygous mice was markedly abnormal, no histological differences were noted between heterozygous and wild type mice with copper loading. Tx heterozygous mice have a reduced ability to excrete excess copper, indicating that half of the normal liver Atp7b copper transporter activity is insufficient to deal with large copper intakes. Our results suggest that Wilsons disease carriers in the human population may be at increased risk of copper loading if chronically exposed to elevated copper in food or drinking water.

Liver cell transplantation leads to repopulation and functional correction in a mouse model of Wilson's disease

Background and Aim: The toxic milk (tx) mouse is a non-fatal animal model for the metabolic liver disorder, Wilson's disease. The tx mouse has a mutated gene for a copper-transporting protein, causing early copper accumulation in the liver and late accumulation in other tissues. The present study investigated the efficacy of liver cell transplantation (LCT) to correct the tx mouse phenotype.

Methods: Congenic hepatocytes were isolated and intrasplenically transplanted into 3–4-month-old tx mice, which were then placed on various copper-loaded diets to examine its influence on repopulation by transplanted cells. The control animals were age-matched untransplanted tx mice. Liver repopulation was determined by comparisons of restriction fragment length polymorphism ratios (DNA and mRNA), and copper levels were measured by atomic absorption spectroscopy.

Results: Repopulation in recipient tx mice was detected in 11 of 25 animals (44%) at 4 months after LCT. Dietary copper loading (whether given before or after LCT, or both) provided no growth advantage for donor cells, with similar repopulation incidences in all copper treatment groups. Overall, liver copper levels were significantly lower in repopulated animals (538 ± 68 µg/g, n = 11) compared to non-repopulated animals (866 ± 62 µg/g, n = 14) and untreated controls (910 ± 103 µg/g, n = 6; P < 0.05). This effect was also seen in the kidney and spleen. Brain copper levels remained unchanged.

Conclusion: Transplanted liver cells can proliferate and correct a non-fatal metabolic liver disease, with some restoration of hepatic copper homeostasis after 4 months leading to reduced copper levels in the liver and extrahepatic tissues, but not in the brain.

Chronological changes in tissue copper, zinc and iron in the toxic milk mouse and effects of copper loading

The toxic milk (tx) mouse is a rodent model for Wilson disease, an inherited disorder of copper overload. Here we assessed the effect of copper accumulation in the tx mouse on zinc and iron metabolism. Copper, zinc and iron concentrations were determined in the liver, kidney, spleen and brain of control and copper-loaded animals by atomic absorption spectroscopy. Copper concentration increased dramatically in the liver, and was also significantly higher in the spleen, kidney and brain of control tx mice in the first few months of life compared with normal DL mice. Hepatic zinc was increased with age in the tx mouse, but zinc concentrations in the other organs were normal. Liver and kidney iron concentrations were significantly lower at birth in tx mice, but increased quickly to be comparable with control mice by 2 months of age. Iron concentration in the spleen was significantly higher in tx mice, but was lower in 5 day old tx pups. Copper-loading studies showed that normal DL mice ingesting 300 mg/l copper in their diet for 3 months maintained normal liver, kidney and brain copper, zinc and iron levels. Copper-loading of tx mice did not increase the already high liver copper concentrations, but spleen and brain copper concentrations were increased. Despite a significant elevation of copper in the brain of the copper-loaded tx mice no behavioural changes were observed. The livers of copper-loaded tx mice had a lower zinc concentration than control tx mice, whilst the kidney had double the concentration of iron suggesting that there was increased erythrocyte hemolysis in the copper-loaded mutants.

Intranasal vaccination with proinsulin DNA induces regulatory CD4+ T cells that prevent experimental autoimmune diabetes

Insulin, an autoantigen in type 1 diabetes, when administered mucosally to diabetes-prone NOD mice induces regulatory T cells (T_reg) that protect against diabetes. Compared with protein, Ag encoded as DNA has potential advantages as a therapeutic agent. We found that intranasal vaccination of NOD mice with plasmid DNA encoding mouse proinsulin II-induced CD4⁺ T_reg that suppressed diabetes development, both after adoptive cotransfer with "diabetogenic" spleen cells and after transfer into NOD mice given cyclophosphamide to accelerate diabetes onset. In contrast to prototypic CD4⁺CD25⁺ T_reg, CD4⁺ T_reg induced by proinsulin DNA were both CD25⁺ and CD25^– and not defined by markers such as glucocorticoid-induced TNFR-related protein (GITR), CD103, or Foxp3. Intriguingly, despite induction of T_reg and reduced islet inflammation, diabetes incidence in proinsulin DNA-treated mice was unchanged. However, diabetes was prevented when DNA vaccination was performed under the cover of CD40 ligand blockade, known to prevent priming of CTL by mucosal Ag. Thus, intranasal vaccination with proinsulin DNA has therapeutic potential to prevent diabetes, as demonstrated by induction of protective T_reg, but further modifications are required to improve its efficacy, which could be compromised by concomitant induction of pathogenic immunity.

Pharmacokinetics of recombinant human endostatin in rats

The pharmacokinetics of recombinant human endostatin (rh-Endo) has not been established in the rat, although this species of animal is commonly used in the pharmacological studies of rh-Endo. This study aimed to investigate the pharmacokinetics, tissue distribution, and excretion of rh-Endo in rats. 125I-radiolabeled rh-Endo was administered to healthy rats by intravenous (i.v) bolus injection at 1.5, 4.5 and 13.5 mg/kg. The maximum plasma concentration (Cmax) and area under the plasma concentration versus time curve (AUC) of rh-Endo increased proportionally with the increase of the dosage. There were no significant differences in total body clearance (CL) and elimination half-life (t1/2beta) of rh-Endo among the three dosages used. A 93.5% and 2.2% of the radioactivity was recovered in the urine and feces, respectively, in bile-duct intact rats; whereas only 0.1% of the total radioactivity was excreted into the bile in bile-duct cannulated rats. rh-Endo was rapidly and widely distributed in the liver, kidneys, spleen and lungs. Furthermore, a significant allometric relationship between CL, but not volume of distribution (Vd) and t1/2beta of rh-Endo, and the body weight was observed across mouse, rat and monkey, with the predicted values in humans significantly lower than those observed in cancer patients. rh-Endo exhibited a linear pharmacokinetics in rats and it is mainly excreted through the urine.

Cost-effectiveness of a post-splenectomy registry for prevention of sepsis in the asplenic

Background: Overwhelming, sometimes fatal infections represent a lifelong risk after surgical removal of the spleen, or in patients who develop hyposplenism as a consequence of illnesses. This risk may be reduced by all or a combination of vaccination, antibiotic prophylaxis and education. We aimed to determine if a registry approach to delivering these interventions would be cost effective using our own experience and published data.
Method: The decision model compared a cohort of 1,000 people covered by a registry to a cohort of 1,000 people with no registry. The impact of the registry was assessed in terms of achieved rates of vaccination, chemoprophylaxis and education, consequent outcomes of overwhelming post-splenectomy infection (OPSI) and mortality (years of life lived). The cost-effectiveness of the registry compared with no registry was estimated in terms of additional cost per case of OPSI avoided and as additional cost per life year gained.
Results: In the first two years, the additional cost of the registry was $152,611 per case of OPSI avoided or $205,931 per life year gained. After this initial registration period the costeffectiveness improves over time, such that over the cohort lifetime a post-splenectomy register is associated with an additional cost of $105,159 per case of OPSI avoided or $16,113 per life year gained.
Conclusion: A registry-based approach is likely to prove cost effective in terms of mortality and rates of OPSI avoided.

1-14C-Linoleic Acid distrubution in various tissue lipids of guinea pigs following an oral dose

A recent study on the metabolism of 1-¹⁴C-α-linolenic acid in the guinea pig revealed that the fur had the highest specific activity of all tissues examined, 48 h after dosing. The present study investigated the pattern of tissue lipid labeling following an oral dose of 1-¹⁴C-linoleic acid after the animals had been dosed for the same time as above. Guinea pigs were fed one of two diets with a constant linoleic acid content (18% total fatty acids) and a different content of α-linolenic acid (0.3 or 17.3%) from weaning for 3 wk and 1-¹⁴C-linoleic acid was given orally to each animal for 48 h prior to sacrifice. The most highly labeled tissues (dpm/mg of linoleic acid) were liver, followed by brain, lung and spleen, heart, kidney and adrenal and intestines, in both diet groups. The liver had almost a three-fold higher specific activity than skin and fur which was more extensively labeled than the adipose and carcass. Approximately two-thirds of the label in skin plus fur was found in the fur which, because of a low lipid mass, would indicate that the fur was highly labeled. All tissues derived from animals on the diet with the low α-linolenic acid level were significantly more labeled than the tissues from the animals on the high α-linolenic acid diet, by a factor of 1.5 to 3. The phospholipid fraction was the most highly labeled fraction in the liver, free fatty acids were the most labeled fraction in skin & fur, while triacyglycerols were the most labeled in the carcass and adipose tissue. In these tissues, more than 90% of the radioactivity was found in fatty acids with 2-double bonds in the tissue lipids. These data indicate that the majority of label found in guinea pig tissues 48 h after dosing was still associated with a fatty acid fraction with 2-double bonds, which suggests there was little metabolism of linoleic acid to more highly unsaturated fatty acids in this time frame. In this study, the labeling of guinea pig tissues with linoleic acid, 48 h after dosing, was quite different from the labeling with α-linolenic acid reported previously. The retention of the administered radioactivity from ¹⁴C-linoleic acid in the whole body lipids was 1.6 times higher in the group fed the low α-linolenic acid diet (diet contained a total of 1.8 g PUFA/100 g diet)compared with the group fed the high α-linolenic acid diet (diet contained 3.6 g PUFA/100 g diet). The lack of retention of ¹⁴C-labeled lipids in the whole body would be consistent with an increased rate of β-oxidation of the labeled fatty acid on the diet rich in PUFA, a result supported by other studies using direct measurement of labeled carbon dioxide.

ORP3 splice variants and their expression in human tissues and hematopoietic cells

ORP3 is a member of the newly described family of oxysterol-binding protein (OSBP)-related proteins (ORPs). We previously demonstrated that this gene is highly expressed in CD34+ hematopoietic progenitor cells, and deduced that the "full-length" ORP3 gene comprises 23 exons and encodes a predicted protein of 887 amino acids with a C-terminal OSBP domain and an N-terminal pleckstrin homology domain. To further characterize the gene, we cloned ORP3 cDNA from PCR products and identified multiple splice variants. A total of eight isoforms were demonstrated with alternative splicing of exons 9, 12, and 15. Isoforms with an extension to exon 15 truncate the OSBP domain of the predicted protein sequence. In human tissues there was specific isoform distribution, with most tissues expressing varied levels of isoforms with the complete OSBP domain; while only whole brain, kidney, spleen, thymus, and thyroid expressed high levels of the isoforms associated with the truncated OSBP domain. Interestingly, the expression in cerebellum, heart, and liver of most isoforms was negligible. These data suggest that differential mRNA splicing may have resulted in functionally distinct forms of the ORP3 gene.

Virulence determinants of infectious bursal disease virus

The very virulent (vv) pathotype of infectious bursal disease virus (IBDV) has spread rapidly throughout Europe, Asia, and the Middle East. Although Australia is currently unaffected, there remains the potential for incursion of an exotic isolate. The aim of this study was to identify putative virulence determinants of IBDV to facilitate the development of improved diagnostic assays for detection and characterisation of vvIBDV isolates. Sequencing of Indonesian vvIBDV Tasik94 revealed a unique substitution [ A�¨S222] in the hypervariable region (HVR) of viral protein (VP) VP2, which did not appear to impinge on virulence or antigenicity. Phylogenetic analyses indicated that Tasik94 was closely related to Asian and European vvIBDV strains. Extensive alignment of deduced protein sequences across the HVR of VP2 identified residuesI242 I256 and I294 as putative markers of the vv phcnotype. Comparison of the pathology induced by mildly-virulent Australian IBDV 002/73 and Indonesian vvIBDV Tasik94, revealed that histological lesions in the spleen, thymus and bone marrow were restricted to Tasik94-infected birds, suggesting the enhanced pathogenicity of vvIBDV might be attributed to replication in non-bursal lymphoid organs. The biological significance of the VP2 HVR in virulence was assessed using recombinant viruses generated by reverse genetics. Both genomic segments of Australian IBDV 002/73, and recombinant segment A constructs in which the HVR of 002/73 was replaced with the corresponding region of either tissue culture-adapted virus or vvIBDV (Tasik94), were cloned behind T7 RNA polymerase promoter sequences. In vitro transcription/translation of each construct resulted in expression of viral proteins. Co-transfection of synthetic RNA transcripts initiated replication of both tissue culture-adapted parental and recombinant viruses, however attempts to rescue non-adapted viruses in specific-pathogen-free (SPF) chickens were unsuccessful. Nucleotide sequence variation in the HVR of VP2 was exploited for the development of a new diagnostic assay to rapidly detect exotic IBDV isolates, including vvIBDV, using reverse transcription polymerase chain reaction (RT-PCR) amplification and Bmrl restriction enzyme digestion. The assay was capable of differentiating between endemic and exotic IBDV in 96% of 105 isolates sequenced to date.

Bioassay detects soluble MAdCAM-1 in body fluids

Mucosal addressin cell adhesion molecule (MAdCAM-1) is a key player in mediating the infiltration of leucocytes into chronically inflamed tissues. Five anti-MAdCAM-1 monoclonal antibodies (mAb), designated 17F5, 201F7, 314G8, 377D10 and 355G8, were generated by fusion of P3 × 63Ag8.653 myeloma cells with spleen cells from BALB/c mice immunized with recombinant human MAdCAM-1-Fc. The latter four mAb recognize the ligand-binding first Ig domain, and block T -cell adhesion to MAdCAM-1. The non-blocking mAb 17F5 recognizes the mucin domain. Extensive analysis of a large panel of paraffin-embedded human tissues revealed that the 314G8 mAb detected MAdCAM-1 on venules in the spleen and small intestine. MAdCAM-1 was strongly expressed in the synovium of osteoarthritis patients, predominantly on the endothelial lining of blood vessels, but also within the vessel lumen. An ELISA, based on mAb 377D10 and 355G8, was developed to determine whether soluble MAdCAM-1 was present in body fluids, and to measure the levels present. The assay detected soluble MAdCAM-1 in the serum and urine of healthy donors, at levels similar to those of soluble forms of the related CAM, ICAM-1 and VCAM-1. The anti-MAdCAM-1 antibodies and assay developed here may be useful therapeutically in the treatment of inflammation in humans. Similarly, they may be useful diagnostically to monitor the presence and levels of MAdCAM-1.

Consequences of immunodominant epitope deletion for minor influenza virus-specific CD8+-T-cell responses

The extent to which CD8+ T cells specific for other antigens expand to compensate for the mutational loss of the prominent DbNP366 and DbPA224 epitopes has been investigated using H1N1 and H3N2 influenza A viruses modified by reverse genetics. Significantly increased numbers of CD8+ KbPB1703+ , CD8+ KbNS2114+, and CD8+ DbPB1-F262+ T cells were found in the spleen and in the inflammatory population recovered by bronchoalveolar lavage from mice that were first given the -NP-PA H1N1 virus intraperitoneally and then challenged intranasally with the homologous H3N2 virus. The effect was less consistent when this prime-boost protocol was reversed. Also, though the quality of the response measured by cytokine staining showed some evidence of modification when these minor CD8+-T-cell populations were forced to play a more prominent part, the effects were relatively small and no consistent pattern emerged. The magnitude of the enhanced clonal expansion following secondary challenge suggested that the prime-boost with the -NP-PA viruses gave a response overall that was little different in magnitude from that following comparable exposure to the unmanipulated viruses. This was indeed shown to be the case when the total response was measured by ELISPOT analysis with virus-infected cells as stimulators. More surprisingly, the same effect was seen following primary challenge, though individual analysis of the CD8+ KbPB1703+ , CD8+ KbNS2114+, and CD8+ DbPB1-F262+ sets gave no indication of compensatory expansion. A possible explanation is that novel, as yet undetected epitopes emerge following primary exposure to the -NP-PA deletion viruses. These findings have implications for both natural infections and vaccines.

Differential tumor necrosis factor receptor 2-mediated editing of virus-specific CD8+ effector T cells

Much of the CD8+ T cell response in H2b mice with influenza pneumonia is directed at the nucleoprotein366-374 (NP366) and acid polymerase224-233 (PA224) peptides presented by the H2Db MHC class I glycoprotein. These DbNP366- and DbPA224-specific T cell populations are readily analyzed by staining with tetrameric complexes of MHC+ peptide (tetramers) or by cytokine production subsequent to in vitro stimulation with the cognate peptides. The DbPA224-specific CD8+ effector T cells make more tumor necrosis factor (TNF) α than the comparable CD8+DbNP366+ set, a difference reflected in the greater sensitivity of the CD8+DbPA224+ population to TNF receptor (TNFR) 2-mediated apoptosis under conditions of in vitro culture. Freshly isolated CD8+DbNP366+ and CD8+DbPA224+ T cells from influenza-infected TNFR2-/- mice produce higher levels of IFN-γ and TNF-α after in vitro stimulation with peptide, although the avidity of the T cell receptor-epitope interaction does not change. Increased numbers of both CD8+DbPA224+ and CD8+DbNP366+ T cells were recovered from the lungs (but not the spleens) of secondarily challenged TNFR2-/- mice, a pattern that correlates with the profiles of TNFR expression in the TNFR2+/+ controls. Thus, it seems that TNFR2-mediated editing of influenza-specific CD8+ T cells functions to limit the numbers of effectors that have localized to the site of pathology in the lung but does not modify the size of the less activated responder T cell populations in the spleen. Therefore, the massive difference in magnitude for the secondary, although not the primary, response to these DbNP366 and DbPA224 epitopes cannot be considered to reflect differential TNFR2-mediated T cell editing.

Re-framing the riot : journalism, creative non-fiction and telling the story of Australia's black history

On 19 November 2004, an Aboriginal man was arrested on Palm Island, off the coast of Townsville in northern Queensland. He was taken to the local watch house on a drunk and disorderly charge. An hour later, he lay dead on a cell floor. His liver, an autopsy showed, had been split in half and his spleen ruptured. But when that autopsy report also found that Mulrunji Doomadgee’s severe injuries were not caused by force, the Palm Island Indigenous community, enraged and grief-stricken, went looking for payback.

The Palm Island “riots” ensured that this Aboriginal death in custody made international news headlines where others barely got a mention, if at all (Hollinsworth, 2005). The ensuing Coronial Inquest and criminal prosecution of the arresting Queensland police officer, Chris Hurley, also were covered consistently by the news media. Senior Sergeant Hurley has, however, so far escaped punishment and the Queensland media’s most recent report of the case was to tell how the Qld Police Union now funds a legal bid to clear his name. Meanwhile, little is heard in the news media of the Doomadgee family, the Palm Island community, or of other deaths in custody occurring steadily through the 18 years since the Royal Commission that was supposed to implement a raft of preventative recommendations.

While the news media’s framing of these issues has most often followed historically predictable and ultimately racist lines, a work of creative non-fiction tells the story with warranted complexity and power. Chloe Hooper’s The Tall Man: Death and Life on Palm Island documents Cameron Doomadgee’s death, the riots, and the ensuing legal farce from the front row. Hooper, in the tradition of Truman Capote, arrived at Palm Island as a white writer from a big city. But by “walking the talk” – being with the Doomadgee family and their community through the hearings and after, Hooper was given extraordinary access to community, history, and significant cultural nuance barely identified by, let alone understood by, non-Indigenous readers.

By focussing on Hooper’s experience with sources and court reporting, compared with some print media coverage, this paper will consider the comparative roles of journalism and creative non-fiction in re-framing the Palm Island “riot”. It will suggest that Hooper’s work subverts some dominant (and racist) news media representations of Australian Indigenous peoples through its use of source relationships in an extended narrative structure.

Histological health indices : linking complex pollutant exposure with tissue alteration in sand flathead Platycephalus bassensis

Port Phillip Bay, Victoria, is a pollutant-impacted environment that is extensively fished both professionally and recreationally. Consumption of contaminated fish represents a potential threat to human health, and fish exposed to environmental contaminants may themselves be affected in a similar fashion. This study describes a fish health index based on histological alterations identified in multiple organs of the sand flathead Platycephalus bassensis. Alterations were evident in tissues from all individuals assessed, with common pathologies observed in the gills, skin, kidney, liver and spleen. Alterations commonly present included necrosis, melanomacrophage centres, inflammation and multiple alterations of the gill epithelium (e.g. hyperplasia and hypertrophy). Fish health, calculated using severity of histological alterations, differed significantly across Port Phillip Bay, with heavily industrialized regions of Altona and St. Helens showing greatest alteration prevalence across multiple organs. This study indicates that the health of P.bassensis from Altona, St. Helens, and Mornington to a lesser extent, are currently compromised, potentially due to complex pollutant exposures which require further investigation