Transcriptional profiling of chronic clinical hepatic schistosomiasis japonica indicates reduced metabolism and immune responses

Schistosomiasis is a significant cause of human morbidity and mortality. We performed a genome-wide transcriptional survey of liver biopsies obtained from Chinese patients with chronic schistosomiasis only, or chronic schistosomiasis with a current or past history of viral hepatitis B. Both disease groups were compared with patients with no prior history or indicators of any liver disease. Analysis showed in the main, downregulation in gene expression, particularly those involved in signal transduction via EIF2 signalling and mTOR signalling, as were genes associated with cellular remodelling. Focusing on immune associated pathways, genes were generally downregulated. However, a set of three genes associated with granulocytes, MMP7, CLDN7, CXCL6 were upregulated. Differential gene profiles unique to schistosomiasis included the gene Granulin which was decreased despite being generally considered a marker for liver disease, and IGBP2 which is associated with increased liver size, and was the most upregulated gene in schistosomiasis only patients, all of which presented with hepatomegaly. The unique features of gene expression, in conjunction with previous reports in the murine model of the cellular composition of granulomas, granuloma formation and recovery, provide an increased understanding of the molecular immunopathology and general physiological processes underlying hepatic schistosomiasis.

Prevention of retinal capillary basement membrane thickening in diabetic dogs by a non-steroidal anti-inflammatory drug

AIMS/HYPOTHESIS: To investigate the effect of treatment with the non-steroidal anti-inflammatory drug Sulindac on the early vascular pathology of diabetic retinopathy in the dog, and it's effect on recognised biochemical indices of hyperglycaemia-related pathophysiology. METHODS: Experimental diabetes (streptozotocin/alloxan) was induced in 22 male beagle dogs and 12 of the animals were assigned at random to receive oral Sulindac (10 mg/kg daily). Age- and sex-matched control animals were maintained as non-diabetic controls. After 4 years, several morphological parameters were quantified in the retinal microvasculature of each animal group using an established stereological method. Also, the following diabetes-associated biochemical parameters were analysed: accumulation of advanced glycation end products (AGEs), red blood cell polyol levels and antioxidant status. RESULTS: Diabetes increased red blood cell sorbitol levels when compared to non-diabetic controls (p<or =0.05), however, there was no difference in sorbitol levels between the untreated and the treated diabetic animals. No significant differences were found in red blood cell myoinositol levels between the three groups of animals. Pentosidine and other AGEs were increased two- to three-fold in the diabetic animals (p<or =0.001) although treatment with Sulindac did not affect their accumulation in diabetic skin collagen or alter diabetes-induced rises in plasma malondialdehyde. Retinal capillary basement membrane volume was significantly increased in the untreated diabetic dogs compared to non-diabetic controls or Sulindac-treated diabetic animals (p<or =0.0001). CONCLUSION/INTERPRETATION: This study has confirmed the beneficial effect of a non-steroidal anti-inflammatory drug on the early vascular pathology of diabetic retinopathy. However the treatment benefit was not dependent on inhibition of polyol pathway activity, advanced glycation, or oxidative stress.

Acute complications and drug misuse are important causes of death for children and young adults with type 1 diabetes - Results from the Yorkshire Register of Diabetes in Children and Young Adults

OBJECTOVE - To examine mortality rates and causes of death among subjects diagnosed with type I diabetes aged

Phosphoramidates of 2'-beta-D-arabinouridine (AraU) as phosphate prodrugs; design, synthesis, in vitro activity and metabolism

2'-Beta-D-arabinouridine (AraU), the uridine analogue of the anticancer agent AraC, was synthesized and evaluated for antiviral activity and cytotoxicity. In addition, a series of AraU monophosphate prodrugs in the form of triester phosphoramidates (ProTides) were also synthesized and tested against a range of viruses, leukaemia and solid tumour cell lines. Unfortunately, neither the parent compound (AraU) nor any of its ProTides showed antiviral activity, nor potent inhibitory activity against any of the cancer cell lines. Therefore, the metabolism of AraU phosphoramidates to release AraU monophosphate was investigated. The results showed carboxypeptidase Y, hog liver esterase and crude CEM tumor cell extracts to hydrolyse the ester motif of phosphoramidates with subsequent loss of the aryl group, while molecular modelling studies suggested that the AraU l-alanine aminoacyl phosphate derivative might not be a good substrate for the phosphoramidase enzyme Hint-1. These findings are in agreement with the observed disappearance of intact prodrug and concomitant appearance of the corresponding phosphoramidate intermediate derivative in CEM cell extracts without measurable formation of araU monophosphate. These findings may explain the poor antiviral/cytostatic potential of the prodrugs.

Primed, constant infusion with [H-2(3)]serine allows in vivo kinetic measurement of serine turnover, homocysteine remethylation, and transsulfuration processes in human one-carbon metabolism

Background: One-carbon metabolism involves both mitochondrial and cytosolic forms of folate-dependent enzymes in mammalian cells, but few in vivo data exist to characterize the biochemical processes involved.

Objective: We conducted a stable-isotopic investigation to determine the fates of exogenous serine and serine-derived one carbon units in homocysteine remethylation in hepatic and whole-body metabolism.

Design: A healthy man aged 23 y was administered [2,3,3 H-2(3)]serine and [5,5,5-H-2(3)]leucine by intravenous primed, constant infusion. Serial plasma samples were analyzed to determine the isotopic enrichment of free glycine, serine, leucine, methionine, and cystathionine. VLDL apolipoprotein B-100 served as an index of liver free amino acid labeling.

Results: [H-2(1)]Methionine and [H-2(2)]methionine were labeled through homocysteine remethylation. We propose that [H-2(2)]methionine occurs by remethylation with [H-2(2)]methyl groups (as 5-methyltetrahydrofolate) formed only from cytosolic processing of [H-2(3)]serine, whereas [H-2(1)]methionine is formed with labeled one-carbon units from mitochondrial oxidation of C-3 serine to [H-2(1)]formate to yield cytosolic [H-2(1)]methyl groups. The labeling pattern of cystathionine formed from homocysteine and labeled serine suggests that cystathionine is derived mainly from a serine pool different from that used in apolipoprotein B-100 synthesis.

Conclusions: The appearance of both [H-2(1)]- and [H-2(2)]methionine forms indicates that both cytosolic and mitochondrial metabolism of exogenous serine generates carbon units in vivo for methyl group production and homocysteine remethylation. This study also showed the utility of serine infusion and indicated functional roles of cytosolic and mitochondrial compartments in one-carbon metabolism.

Vitamin B-6 deficiency in rats reduces hepatic serine hydroxymethyltransferase and cystathionine beta-synthase activities and rates of in vivo protein turnover, homocysteine remethylation and transsulfuration

Vitamin B-6 deficiency causes mild elevation in plasma homocysteine, but the mechanism has not been clearly established. Serine is a substrate in one-carbon metabolism and in the transsulfuration pathway of homocysteine catabolism, and pyridoxal phosphate (PLP) plays a key role as coenzyme for serine hydroxymethyltransferase (SHMT) and enzymes of transsulfuration. In this study we used [H-2(3)]serine as a primary tracer to examine the remethylation pathway in adequately nourished and vitamin B-6-deficient rats pi and 0.1 mg pyridoxine (PN)/kg diet]. [H-2(3)]Leucine and [1-C-13]methionine were also used to examine turnover of protein and methionine pools, respectively, All tracers were injected intraperitoneally as a bolus dose, and then rats were killed (n = 4/time point) after 30, 60 and 120 min. Rats fed the low-PN diet had significantly lower growth and plasma and liver PLP concentrations, reduced liver SHMT activity, greater plasma and liver total homocysteine concentration, and reduced liver S-adenosylmethionine concentration. Hepatic and whole body protein turnover were reduced in vitamin B-6-deficient rats as evidenced by greater isotopic enrichment of [H-2(3)]leucine. Hepatic [H-2(2)]methionine production from [H-2(3)]serine via cytosolic SHMT and the remethylation pathway was reduced by 80.6% in vitamin B-6 deficiency. The deficiency did not significantly reduce hepatic cystathionine-beta-synthase activity, and in vivo hepatic transsulfuration flux shown by production of [H-2(3)]cysteine from the [H-2(3)]serine increased over twofold. In contrast, plasma appearance of [H-2(3)]cysteine was decreased by 89% in vitamin B-6 deficiency. The rate of hepatic homocysteine production shown by the ratio of [1-C-13]homocysteine/[1-C-13]methionine areas under enrichment vs. time curves was not affected by vitamin B-6 deficiency. Overall, these results indicate that vitamin B-6 deficiency substantially affects one-carbon metabolism by impairing both methyl group production for homocysteine remethylation and flux through whole-body transsulfuration.

Comparison of 5% versus 15% sucrose intakes as part of a eucaloric diet in overweight and obese subjects:effects on insulin sensitivity, glucose metabolism, vascular compliance, body composition and lipid profile. A randomised controlled trial

AIMS: The effect of dietary sucrose on insulin resistance and the pathogenesis of diabetes and vascular disease is unclear. We assessed the effect of 5% versus 15% sucrose intakes as part of a weight maintaining, eucaloric diet in overweight/obese subjects.

METHODS: Thirteen subjects took part in a randomised controlled crossover study (M:F 9:4, median age 46 years, range 37-56 years, BMI 31.7±0.9 kg/m(2)). Subjects completed two 6 week dietary periods separated by 4 week washout. Diets were designed to have identical macronutrient profile. Insulin action was assessed using a two-step hyperinsulinaemic euglycaemic clamp; glucose tolerance, vascular compliance, body composition and lipid profiles were also assessed.

RESULTS: There was no change in weight or body composition between diets. There was no difference in peripheral glucose utilization or suppression of endogenous glucose production. Fasting glucose was significantly lower after the 5% diet. There was no demonstrated effect on lipid profiles, blood pressure or vascular compliance.

CONCLUSION: A low-sucrose diet had no beneficial effect on insulin resistance as measured by the euglycaemic glucose clamp. However, reductions in fasting glucose, one hour insulin and insulin area under the curve with the low sucrose diet on glucose tolerance testing may indicate a beneficial effect and further work is required to determine if this is the case. Clinical Trial Registration number ISRCTN50808730.

The role of microneedles for drug and vaccine delivery

INTRODUCTION: Transdermal drug delivery offers a number of advantages for the patient, not only due to its non-invasive and convenient nature, but also due to factors such as avoidance of first-pass metabolism and prevention of gastrointestinal degradation. It has been demonstrated that microneedles (MNs) can increase the number of compounds amenable to transdermal delivery by penetrating the skin's protective barrier, the stratum corneum, and creating a pathway for drug permeation to the dermal tissue below.

AREAS COVERED: MNs have been extensively investigated for drug and vaccine delivery. The different types of MN arrays and their delivery capabilities are discussed in terms of drugs, including biopharmaceutics and vaccines. Patient usage and effects on the skin are also considered.

EXPERT OPINION: MN research and development is now at the stage where commercialisation is a viable possibility. There are a number of long-term safety questions relating to patient usage which will need to be addressed moving forward. Regulatory guidance is awaited to direct the scale-up of the manufacturing process alongside provision of clearer patient instruction for safe and effective use of MN devices.

The androgen receptor fuels prostate cancer by regulating central metabolism and biosynthesis

The androgen receptor (AR) is a key regulator of prostate growth and the principal drug target for the treatment of prostate cancer. Previous studies have mapped AR targets and identified some candidates which may contribute to cancer progression, but did not characterize AR biology in an integrated manner. In this study, we took an interdisciplinary approach, integrating detailed genomic studies with metabolomic profiling and identify an anabolic transcriptional network involving AR as the core regulator. Restricting flux through anabolic pathways is an attractive approach to deprive tumours of the building blocks needed to sustain tumour growth. Therefore, we searched for targets of the AR that may contribute to these anabolic processes and could be amenable to therapeutic intervention by virtue of differential expression in prostate tumours. This highlighted calcium/calmodulin-dependent protein kinase kinase 2, which we show is overexpressed in prostate cancer and regulates cancer cell growth via its unexpected role as a hormone-dependent modulator of anabolic metabolism. In conclusion, it is possible to progress from transcriptional studies to a promising therapeutic target by taking an unbiased interdisciplinary approach.

Novel opportunities for thymidylate metabolism as a therapeutic target

For over 40 years, the fluoropyrimidine 5-fluorouracil (5-FU) has remained the central agent in therapeutic regimens employed in the treatment of colorectal cancer and is frequently combined with the DNA-damaging agents oxaliplatin and irinotecan, increasing response rates and improving overall survival. However, many patients will derive little or no benefit from treatment, highlighting the need to identify novel therapeutic targets to improve the efficacy of current 5-FU-based chemotherapeutic strategies. dUTP nucleotidohydrolase (dUTPase) catalyzes the hydrolysis of dUTP to dUMP and PPi, providing substrate for thymidylate synthase (TS) and DNA synthesis and repair. Although dUTP is a normal intermediate in DNA synthesis, its accumulation and misincorporation into DNA as uracil is lethal. Importantly, uracil misincorporation represents an important mechanism of cytotoxicity induced by the TS-targeted class of chemotherapeutic agents including 5-FU. A growing body of evidence suggests that dUTPase is an important mediator of response to TS-targeted agents. In this article, we present further evidence showing that elevated expression of dUTPase can protect breast cancer cells from the expansion of the intracellular uracil pool, translating to reduced growth inhibition following treatment with 5-FU. We therefore report the implementation of in silico drug development techniques to identify and develop small-molecule inhibitors of dUTPase. As 5-FU and the oral 5-FU prodrug capecitabine remain central agents in the treatment of a variety of malignancies, the clinical utility of a small-molecule inhibitor to dUTPase represents a viable strategy to improve the clinical efficacy of these mainstay chemotherapeutic agents.

Inhibition of O-GlcNAc transferase activity reprograms prostate cancer cell metabolism

Metabolic networks are highly connected and complex, but a single enzyme, O-GlcNAc transferase (OGT) can sense the availability of metabolites and also modify target proteins. We show that inhibition of OGT activity inhibits the proliferation of prostate cancer cells, leads to sustained loss of c-MYC and suppresses the expression of CDK1, elevated expression of which predicts prostate cancer recurrence (p=0.00179). Metabolic profiling revealed decreased glucose consumption and lactate production after OGT inhibition. This decreased glycolytic activity specifically sensitized prostate cancer cells, but not cells representing normal prostate epithelium, to inhibitors of oxidative phosphorylation (rotenone and metformin). Intra-cellular alanine was depleted upon OGT inhibitor treatment. OGT inhibitor increased the expression and activity of alanine aminotransferase (GPT2), an enzyme that can be targeted with a clinically approved drug, cycloserine. Simultaneous inhibition of OGT and GPT2 inhibited cell viability and growth rate, and additionally activated a cell death response. These combinatorial effects were predominantly seen in prostate cancer cells, but not in a cell-line derived from normal prostate epithelium. Combinatorial treatments were confirmed with two inhibitors against both OGT and GPT2. Taken together, here we report the reprogramming of energy metabolism upon inhibition of OGT activity, and identify synergistically lethal combinations that are prostate cancer cell specific.

Microneedle arrays as transdermal and intradermal drug delivery systems: Materials science, manufacture and commercial development

Transdermal drug delivery offers a number of advantages for the patient, due not only its non-invasive and convenient nature, but also factors such as avoidance of first pass metabolism and prevention of gastrointestinal degradation. It has been demonstrated that microneedle arrays can increase the number of compounds amenable to transdermal delivery by penetrating the skin's protective barrier, the stratum corneum, and creating a pathway for drug permeation to the dermal tissue below. Microneedles have been extensively investigated in recent decades for drug and vaccine delivery as well as minimally invasive patient monitoring/diagnosis. This review focuses on a range of critically important aspects of microneedle technology, namely their material composition, manufacturing techniques, methods of evaluation and commercial translation to the clinic for patient benefit and economic return. Microneedle research and development is finally now at the stage where commercialisation is a realistic possibility. However, progress is still required in the areas of scaled-up manufacture and regulatory approval.

Characterising granuloma regression and liver recovery in a murine model of schistosomiasis japonica

For hepatic schistosomiasis the egg-induced granulomatous response and the development of extensive fibrosis are the main pathologies. We used a Schistosoma japonicum-infected mouse model to characterise the multi-cellular pathways associated with the recovery from hepatic fibrosis following clearance of the infection with the anti-schistosomal drug, praziquantel. In the recovering liver splenomegaly, granuloma density and liver fibrosis were all reduced. Inflammatory cell infiltration into the liver was evident, and the numbers of neutrophils, eosinophils and macrophages were significantly decreased. Transcriptomic analysis revealed the up-regulation of fatty acid metabolism genes and the identification of Peroxisome proliferator activated receptor alpha as the upstream regulator of liver recovery. The aryl hydrocarbon receptor signalling pathway which regulates xenobiotic metabolism was also differentially up-regulated. These findings provide a better understanding of the mechanisms associated with the regression of hepatic schistosomiasis.