Hyperuricaemia, gout and allopurinol in the CKD Queensland registry.

INTRODUCTION: There is scant data on the role of hyperuricaemia, gout and allopurinol treatment in chronic kidney disease (CKD). Therefore, our aim is to investigate the possible associations between hyperuricaemia, gout, prescription of allopurinol and renal outcomes in patients with CKD. METHODS: The retrospective cohort study involved 1123 Royal Brisbane and Women's Hospital (RBWH) patients, enrolled in the CKD.QLD registry from May 2011 to August 2017. Patients were divided into two uric acid categories, with uric acid ≤ 0.36 mmol/L and > 0.36 mmol/L. Association of delta estimated glomerular filtration rate (eGFR) with gout, allopurinol treatment and hyperuricaemia were analysed. RESULTS: Patients with an entry urate > 0.36 mmol/L were older, had higher body mass index (BMI) and worse baseline kidney function. Proportion of patients with gout, hyperuricaemia and allopurinol treatment increased with advanced CKD stages. Age-adjusted analysis revealed a significant association between serum urate level and delta eGFR, with no significant association between gout, treatment with allopurinol and delta eGFR. Furthermore, neither gout nor the prescription of allopurinol had a significant effect on the time to renal death (composite end point of kidney replacement therapy or death). CONCLUSION: Hyperuricaemia seemed to be independently associated with faster CKD progression or renal death. This was not observed with gout or prescription of allopurinol. Furthermore, allopurinol was not associated with decreased incidence of cardiovascular events. These data suggest that hyperuricaemia is likely the effect and not the cause of CKD or CKD progression.

Atypical haemolytic uraemic syndrome treated with the complement inhibitor eculizumab: the experience of the Australian compassionate access cohort.

BACKGROUND/AIM: This study aimed to report the clinical characteristics and outcomes of Australian patients treated with eculizumab for atypical haemolytic uraemic syndrome (aHUS). METHODS: A retrospective cohort study was undertaken of all patients in Australia treated with eculizumab provided in a compassionate access programme for a clinical diagnosis of aHUS using prospectively collected clinical data. RESULTS: A total of 10 patients with a median age of 23.5 years (interquartile range (IQR) 24.83 years) received compassionate access eculizumab for aHUS in Australia. Eight patients were female, and three had a family history of aHUS. Three received eculizumab for an initial acute aHUS presentation, three for relapsing and refractory acute aHUS, two for de novo aHUS post-renal transplantation, and one each for aHUS recurrence post-transplantation and facilitation of transplantation with a history of aHUS. The median duration of eculizumab therapy has been 911.5 days (IQR 569 days) with a cumulative exposure of 9184 days. At baseline all patients had renal and extra-renal aHUS involvement, with up to three non-renal organs affected. All but one patient, who died from uncontrollable gastrointestinal aHUS manifestations, have continued. The nine continuing patients achieved remission of aHUS. Two of the four patients requiring renal replacement therapy (RRT) at eculizumab commencement subsequently ceased RRT. Clinical events occurring in this cohort while on eculizumab treatment included neutropenia (two), posterior reversible encephalopathy syndrome (one), cardiomyopathy (one), pulmonary embolus (one), antibody-mediated rejection resulting in renal graft failure (one), iron deficiency (one), gastrointestinal haemorrhage (one) and death (one). CONCLUSION: Eculizumab has been an effective therapy for aHUS in this cohort, including when other therapies have failed.

Sustained remission of systemic lupus erythematosus related calciphylaxis.

Calciphylaxis continues to present a clinical challenge for patient management. As in this case, it can be associated with connective tissue disease (CTD) such as systemic lupus erythematosus (SLE). Unlike previous reported cases, long-term remission has been attained. This provides some insight into methods of therapy as well as potential pathogenic models for this disease.

Characterisation of the chemical and biological properties of molecules with QSAR/QSPR and chemical grouping, and its application to a group of alkyl ethers.

This study presents a QSAR/QSPR modelling and chemical grouping (read-across) approach to provide information on the biological properties of a group of aliphatic ethers, with accurate biological predictions restricted to those physico-chemical and (eco)toxicological properties where the performance of QSAR/QSPR has been shown to be acceptable. The mathematical methods used ranged from multivariate regression models to PLS (partial least-squares), SVM (support vector machines) and Sammon's mapping. A novel grouping approach, based on a set of key descriptors, has been proposed to give a compact picture of the structural and biological properties of the compounds, and to provide a more mechanistic basis for the interpretations of chemical groups. Besides being a straightforward case study, the paper also exemplifies the capabilities and limitations of the methods in predictive toxicology on a more general level.

Improved survival of microencapsulated islets during in vitro culture and enhanced metabolic function following transplantation.

AIMS/HYPOTHESIS: The aim of this study was to determine whether a simple alginate capsule can prolong islet survival and function during long-term tissue culture. We also wanted to observe the ability of these encapsulated islets to restore glucose responsiveness to diabetic recipients, along with the quantity of islets required to do so. METHODS: We compared the recovery and metabolic function of encapsulated canine islets with that of non-encapsulated canine islets following 1, 2 or 3 weeks of tissue culture. These culture preparations were also transplanted into diabetic nude mice and compared for their ability to reverse diabetes. Furthermore, short-term cultured encapsulated and non-encapsulated islets were transplanted in varying numbers to determine the minimum dose required to normalise blood glucose and prolong recipient survival. RESULTS: Islet recovery following 1, 2 and 3 weeks of tissue culture was significantly higher when islets were encapsulated. When these islets were recovered at 1, 2 and 3 weeks and transplanted into diabetic nude mice, survival at 100 days was 100% for all encapsulated groups, versus 66%, 33% and 33% respectively for the non-encapsulated islets. Additionally, substantially fewer short-term cultured islets were required to normalise blood glucose when the islets were encapsulated. Recipients of encapsulated islets also had significantly longer survival times than recipients of non-encapsulated preparations. CONCLUSIONS/INTERPRETATION: This study demonstrates that encapsulation of islets with purified alginate improves islet survival and function in vitro and in vivo.

Chronic pulmonary effects of respirable methylene diphenyl diisocyanate (MDI) aerosol in rats: combination of findings from two bioassays.

Two independent bioassays are available which have examined the potential carcinogenicity of monomeric and polymeric methylene diphenyl diisocyanate (MDI) following long-term inhalation exposure in rats. These studies are not directly comparable, however, due to differences in design and conduct of the in-life phase, and differences in nomenclature used for some of the histopathological findings. This paper presents a definitive overview ofthe pulmonary toxicity of MDI developed following a thorough review of both investigations. As part of this process, the test materials and the designs of the studies were compared, and an in-depth review of lung lesions was conducted by an independent reviewing pathologist. This included the re-examination of the original lung slides, supported by an analysis of the exposure regimens, the results of which were used to develop an accurate profile of the doses received by the animals in the two studies. Histopathological findings were then combined with this information to give an overall dose-response curve for both studies as a whole. The range of total inhalation exposures to MDI was calculated as 559, 1972, 2881, 6001, 17,575 and 17,728 mgh/m3. Major pulmonary effects included increased lung weights together with bronchiolo-alveolar adenomas and hyperplasia, and interstitial fibrosis which occurred consistently in both studies, indicating a very similar qualitative response of the lungs to polymeric and monomeric MDI. The quantitative response of the lung was clearly dose-related in each study, and when the studies were considered as a whole a reasonable overall dose-response relationship was apparent for major lung lesions. Lung tumours (in low incidences) only occurred at the highest dose level in both studies (17,575 and 17,728 mgh/m3). For inflammatory and other non-neoplastic pulmonary changes, the lowest dose examined (559 mgh/m3) was regarded as a no-observed-adverse-effect-level for both polymeric and monomeric MDI. It was concluded that the results of the two studies could be combined to serve as a basis for human risk assessment of MDI.

Ninety-day feeding study in Fischer-344 rats of highly refined petroleum-derived food-grade white oils and waxes.

Subchronic 90-day feeding studies were conducted in male and female Fischer-344 (F-344) rats on highly refined white mineral oils and waxes representative of those used for food applications. The goal was to help clarify the mixed results found in other toxicity studies with laboratory animals. Seven white oils and 5 waxes were fed at dietary doses of 20,000, 2,000, 200, and 20 ppm and compared with control groups on untreated diet; toxicity was assessed at 90 days and also after a reversal period of 28 days and/or 85 days. Higher molecular-sized hydrocarbons (microcrystalline waxes and the higher viscosity oils) were without biological effects. Paraffin waxes and low- to midviscosity oils produced biological effects that were inversely related to molecular weight, viscosity, and melting point; oil type and processing did not appear to be determinants. Biological effects were more pronounced in females than in males. Effects occurred mainly in the liver and mesenteric lymph nodes and included increased organ weights, microscopic inflammatory changes, and evidence for the presence of saturated mineral hydrocarbons in affected tissues. Inflammation of the cardiac mitral valve was also observed at high doses in rats treated with paraffin waxes. Further studies are required to elucidate the mechanism for the responses observed and the relevance of these inflammatory responses in the F-344 rat to other species, including humans.

The FMRFamide-like neuropeptide AF2 is present in the parasitic nematode Haemonchus contortus.

Peptides belonging to the FMRFamide family are widely distributed amongst invertebrates. We report here on the isolation of the FMRFamide neuropeptide AF2 (Lys-His-Glu-Tyr-Leu-Arg-Phe-NH2) from the parasitic nematode Haemonchus contortus. Immunocytochemical techniques showed that FMRFamide-like material was distributed in several regions of these organisms including nerve cords and cell bodies of the central nervous system. AF2 was isolated using a method that employed 6 steps of reverse-phase HPLC. The concentration of AF2 in this organism was approximately 30 pmol/g of nematode.

A novel inhibitory prostanoid receptor in piglet saphenous vein.

A range of prostanoid agonists were tested for activity on isolated ring preparations of piglet saphenous vein. The selective TxA2-mimetic (TP-receptor agonist), U-46619, contracted the preparation in a concentration-related fashion. These contractions were inhibited by the TP-receptor blocking drug, GR32191B, producing a pA2 of 7.8 (slope = 1.6). Prostanoid-induced relaxant responses were studied on preparations which had been pre-contracted using an EC60 concentration of phenylephrine (mean EC60 = 0.97 microM), in the presence of GR32191B (1 microM), to block contractile TP-receptors. Under these conditions, PGD2, PGE2, PGF2 alpha, PGI2, and U-46619, all caused concentration-related relaxation. PGE2 was the most potent agonist (EC50 = 0.23nM), whereas, all of the other agonists were at least 1,000-fold weaker, providing strong evidence for the presence of inhibitory EP-receptors. The selective synthetic EP-agonists, sulprostone (EP1/EP3) and AH13205X (EP2), were next tested for relaxant activity. While both compounds caused concentration-related relaxant activity, they were respectively 6,000 and 11,000-fold less potent than PGE2. The potent TP-receptor blocking drugs, AH22921X and AH23848B, were both weak antagonists of PGE2 but not isoproterenol-induced relaxant responses of piglet saphenous vein in a concentration-related fashion. These two compounds had pA2 values against PGE2 of 5.3 and 5.4 respectively, with regression slopes not significantly different from unity. In contrast, neither compound at a concentration of 30 microM had any antagonist activity against prostanoid-induced effects on guinea-pig fundus (EP1), rabbit ear artery (EP2) or guinea-pig vas deferens (EP3). In conclusion, the piglet saphenous vein contains TP-receptors mediating smooth muscle contraction, and a PGE2-specific (EP) receptor mediating relaxation. The inhibitory EP-receptor does not appear to be of the EP1, EP2 or EP3-subtypes, and appears therefore to be a novel subtype which we tentatively term EP4, and the potent TP-receptor blocking drugs, AH22921X and AH23848B, appear to be weak, but specific EP4-receptor blocking drugs.

Development changes to gut microflora metabolism in mice.

Developmental changes in the activities of bacterial nitrate reductase, nitroreductase and beta-glucuronidase and their response to fermentable dietary fibre, were investigated in caecal contents from suckling mice (2-week-old) and in mice aged 4-24 weeks fed either a purified fibre-free diet or that diet supplemented with 5% (w/w) pectin. There was no apparent age-related trend common to the three enzymes studied. Nitrate reductase activity in the mice fed the fibre-free diet did not markedly alter with age. Pectin administration, however, was associated with a significant increase in nitrate reductase activity, particularly in 4-week-old mice. Nitroreductase activity exhibited an overall upward trend in mice from 2 to 12 weeks and thereafter decreased. Caecal beta-glucuronidase activity in mice increased sharply between 2 weeks and 4 weeks of age, thereafter not changing significantly until the 24th week. Pectin feeding had no consistent effect on activities either of nitroreductase or beta-glucuronidase. The changes in enzyme activities with age were not related to the concentration of bacteria in the caecum, which was highest in the 2-week-old mice. We conclude that the weaning is a period in which marked changes in caecal bacterial enzyme activities can occur.

Effects of Escherichia coli lipopolysaccharide on nitrate synthesis and on nitrosation of proline in rats.

Male Ola:SD rats were fed purified diets containing 5 or 20% lactalbumin as the protein source, with or without concomitant administration of Escherichia coli lipopolysaccharide (50-250 micrograms/kg, ip), and changes in 24-hr urinary nitrate excretion, plasma urea, plasma-nitrate pool size and 24-hr urinary nitrosoproline excretion were measured. Urinary nitrate and urinary 14C-nitrosoproline excretion (after oral [14C]proline administration) were significantly greater for rats receiving the high-protein diet compared with those on the low-protein diet. The co-administration of lipopolysaccharide increased nitrate excretion in both diet groups (although the increase was greatest (relatively) in the animals fed 5% lactalbumin), but did not significantly alter urinary nitrosoproline excretion by either group. Plasma urea concentrations and plasma-nitrate pool size were increased by a high-protein diet and/or lipopolysaccharide administration. These findings suggest that treatments which alter the availability of nitrate in vivo are not necessarily associated with increased nitrosation of proline.

Continuous culture of human faecal bacteria as an in vitro model for the colonic microflora.

To investigate the role of human gut bacteria in the metabolism of potentially reactive compounds we have developed an in vitro model of the human faecal microflora using a two-stage continuous culture inoculated with human faeces. The cultured bacterial population retained many of the bacteriological and biochemical characteristics of the flora present in the faecal sample used for inoculation. Obligate anaerobes were the predominant bacterial types found in vitro and included Bacteroides ovatus and Bifidobacterium adolescentis. A comparison of in vivo (faeces) and in vitro bacterial enzyme activities that are known to be involved in the biotransformation of potentially toxic compounds found the activities of hydrolytic enzymes to be similar but reductive enzymes exhibited higher activities in the continuous culture model. When substrates of the enzymes were added to the culture vessel, the enzymes were induced to varying extents. The short-chain fatty acid profile in the culture was almost identical to that in faeces with the order of abundance being the same in two systems. These results indicate that the continuous culture of faecal bacteria can provide a suitable model for studying bacterial interactions and biotransformation of the human colonic flora.

Modified mutagen activation in hepatic fractions from rats fed dietary rutin--interaction between gut flora and host metabolism.

Male Sprague-Dawley rats were fed a purified diet or one supplemented with the glycosidic plant flavonoid (+)rutin for 14 days. Rutin treatment significantly increased caecal bacterial beta-glucosidase activity (responsible for the conversion of rutin to the flavonoid quercetin) and there was an associated increase in the capacity of hepatic fractions (S-9) to activate the food pyrolysis products IQ, MeIQ and MeIQx to bacterial mutagens in vitro. Hepatic conversion of aflatoxin B1 to a mutagen was unaltered while in vitro activation of quercetin was significantly lower in tissue fractions from the rutin-fed rats compared with those from controls. Rutin treatment was without effect, however, on a number of hepatic cytochrome P-450-dependent mixed-function oxidase activities. The results suggest that products of bacterial metabolism of rutin formed in the hindgut may influence the activity of hepatic enzymes involved in the activation of certain classes of mutagen.

Ciguatera and mannitol: experience with a new treatment regimen.

Ciguatera is a distressing, hitherto-untreatable and not rare disease which results from the eating of ciguatoxin-contaminated fish from tropical and subtropical waters. We report here the results of a pilot study to assess the efficacy of mannitol therapy in ciguatera poisoning. Twelve adult patients (six men) have been treated, five of whom--who were ill acutely--experienced a significant benefit from this therapy, in three cases, with a hitherto-unexperienced dramatic reversal of symptoms. We conclude that an intravenous infusion of 1.0 g/kg of mannitol which is given over 45 minutes, after rehydration if required, can be of significant benefit to at least some acutely intoxicated victims. We postulate either a reduction of axonal oedema, or a scavenger effect, or both, as the mechanism of the beneficial effects of mannitol. Ciguatoxin is rich in hydroxyl groups, and causes microscopic oedema of neural tissue. If our conclusion of the beneficial effects of mannitol therapy is confirmed, this will offer the first effective therapy for acute phases of this disease, and has promise of preventing much long-term morbidity.

The influence of incubation pH on the activity of rat and human gut flora enzymes.

The activities of three bacterial biotransformation enzymes (beta-glucuronidase, beta-glucosidase, nitrate reductase) were determined in suspensions of rat caecal contents or human faeces over the pH range 6-8. All three enzymes were influenced by pH, as exemplified by beta-glucosidase activity which diminished as pH increased. In other instances the rat and human flora showed distinct profiles, with nitrate reductase activity undetectable in human faeces below pH 6.6, whereas the rat caecal flora displayed optimal reduction of nitrate around neutrality. The most pronounced host-species difference was found with beta-glucuronidase, which showed maximal activity at pH 6.0 in human faecal bacteria, while the rat caecal flora expressed greatest activity at pH 8.0. All three enzyme activities were associated with that fraction of rat caecal or human faecal material sedimented by centrifugation at 5000 g for 15 min, with little or no metabolism occurring in the 11,000 g supernatant fluid. The results demonstrate that pH has a pronounced effect on the enzymic activity of bacterial preparations from rat and human sources.

Developmental changes in hepatic activation of dietary mutagens by mice.

Metabolic activation of the food mutagens 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and aflatoxin B1 by female BALB/c mice of different ages (2-24 weeks) was investigated in vivo and in vitro using Salmonella typhimurium TA98 as the indicator organism. The in vivo activation of the three mutagens was investigated in 4- and 24-week-old mice using an intrasanguineous host-mediated assay. All three compounds showed reduced levels of activation with the older hosts. Hepatic S9 fractions from female mice of varying ages between 2 and 24 weeks were used in the in vitro mutagenicity assay. To achieve optimal activation to bacterial mutagens, 5% S9 was required for aflatoxin B1 and Trp-P-2 and 10% S9 for MeIQ; age of donor generally had little effect on the profile of these protein activation curves. Under these optimal conditions MeIQ and Trp-P-2 both exhibited, as before, age-dependent decreases in activation over a wide range of mutagen concentrations, however the in vitro activation of aflatoxin showed no consistent change with age. Spectrophotometric measurements of S9 cytochrome P-450 content showed a decrease in concentration with increasing age, but this was not sufficient to account for changes observed in hepatic mutagen activation. However, changes in the activities of certain cytochrome P-450 isoenzymes and cytosolic GSH-transferases, which in turn result in changes in the activation and detoxification capacity of the liver, would appear to explain age-dependent changes in the activity of mutagens in vivo.

The role of gut micro-organisms in the metabolism of deoxynivalenol administered to rats.

1. Oral administration of deoxynivalenol (DON) to control rats resulted in the appearance of a de-epoxy metabolite in urine and faeces. 2. When DON was administered to rats treated with antibiotics to deplete their gut microflora there was very little excretion of radioactivity as the de-epoxy metabolite in faeces or urine. 3. Incubation of DON with a strictly anaerobic preparation of gut contents resulted in the progressive appearance of de-epoxy DON during a 24 h incubation period. 4. Incubation of DON with liver homogenate did not result in the appearance of the de-epoxy DON metabolite. 5. These results indicate that the presence of de-epoxy DON in rat excreta, following the oral administration of DON, is the result of metabolism by micro-organisms in the gut.

Influence of starches of low digestibility on the rat caecal microflora.

1. Male Sprague-Dawley rats were fed on either a purified, fibre-free diet or a diet in which half the maize starch was replaced with uncooked amylomaize or potato starch (equivalent to 100 or 200 g amylase-resistant starch (ARS)/kg diet respectively). Changes in short-chain fatty acids (SCFA), pH, ammonia and a number of bacterial variables in caecal contents were then assessed. 2. Both ARS supplements decreased caecal content pH by approximately 1-2 units, with an associated reduction in ammonia concentration. Potato starch significantly decreased the concentration of SCFA in the hindgut, while amylomaize supplementation increased propionic and butyric acids but decreased the occurrence of minor, branched-chain fatty acids. 3. Caecal bacterial biotransformation activities (beta-glucosidase (EC 3.2.1.21), beta-glucuronidase (EC 3.2.1.31), reduction of p-nitrobenzoic acid, apparent ammonia formation) were consistently decreased by both ARS sources. 4. The results demonstrate that amylase-resistant carbohydrate altered toxicologically important functions in the large-intestinal flora of the rat.