Development and validation of an HPLC method for the rapid and simultaneous determination of 6-mercaptopurine and four of its metabolites in plasma and red blood cells

An HPLC method has been developed and validated for the rapid determination of mercaptopurine and four of its metabolites; thioguanine, thiouric acid, thioxanthine and methylmercaptopurine in plasma and red blood cells. The method involves a simple treatment procedure based on deproteinisation by perchloric acid followed by acid hydrolysis and heating for 45 min at 100 degrees C. The developed method was linear over the concentration range studied with a correlation coefficient >0.994 for all compounds in both plasma and erythrocytes. The lower limits of quantification were 13, 14, 3, 2, 95 pmol/8 x 101 RBCs and 2, 5, 2, 3, 20 ng/ml plasma for thioguanine, thiouric acid, mercaptopurine, thioxanthine and methylmercaptopurine, respectively. The method described is selective and sensitive enough to analyse the different metabolites in a single run under isocratic conditions. Furthermore, it has been shown to be applicable for monitoring these metabolites in paediatric patients due to the low volume requirement (200 mu l of plasma or erythrocytes) and has been successfully applied for investigating population pharmacokinetics, pharmacogenetics and non-adherence to therapy in these patients. (C) 2008 Elsevier B.V. All rights reserved.

Response of red blood cell folate to intervention: implications for folate recommendations for the prevention of neural tube defects

Committees worldwide have set almost identical folate recommendations for the prevention of the first occurrence of neural tube defects (NTDs). We evaluate these recommendations by reviewing the results of intervention studies that examined the response of red blood cell folate to altered folate intake. Three options are suggested to achieve the extra 400 mu g folic acid/d being recommended by the official committees: increased intake of folate-rich foods, dietary folic acid supplementation, and folic acid fortification of food. A significant increase in foods naturally rich in folates was shown to be a relatively ineffective means of increasing red blood cell folate status in women compared with equivalent intakes of folic acid-fortified food, presumably because the synthetic form of the vitamin is more stable and more bioavailable. Although folic acid supplements are highly effective in optimizing folate status, supplementation is not an effective strategy for the primary prevention of NTDs because of poor compliance. Thus, food fortification is seen by many as the only option likely to succeed. Mandatory folic acid fortification of grain products was introduced recently in the United States at a level projected to provide an additional mean intake of 100 mu g folic acid/d, but some feel that this policy does not go far enough. A recent clinical trial predicted that the additional intake of folic acid in the United States will reduce NTDs by >20%, whereas 200 mu g/d would be highly protective and is the dose also shown to be optimal in lowering plasma homocysteine, with possible benefits in preventing cardiovascular disease. Thus, an amount lower than the current target of an extra 400 mu g/d may be sufficient to increase red blood cell folate to concentrations associated with the lowest risk of NTDs, but further investigation is warranted to establish the optimal amount.

Exposure to aflatoxin B1 in utero is associated with DNA methylation in white blood cells of infants in The Gambia

BACKGROUND: Exposure to environmental toxins during embryonic development may lead to epigenetic changes that influence disease risk in later life. Aflatoxin is a contaminant of staple foods in sub-Saharan Africa, is a known human liver carcinogen and has been associated with stunting in infants.

METHODS: We have measured aflatoxin exposure in 115 pregnant women in The Gambia and examined the DNA methylation status of white blood cells from their infants at 2-8 months old (mean 3.6 ± 0.9). Aflatoxin exposure in women was assessed using an ELISA method to measure aflatoxin albumin (AF-alb) adducts in plasma taken at 1-16 weeks of pregnancy. Genome-wide DNA methylation of infant white blood cells was measured using the Illumina Infinium HumanMethylation450beadchip.

RESULTS: AF-alb levels ranged from 3.9 to 458.4 pg/mg albumin. We found that aflatoxin exposure in the mothers was associated to DNA methylation in their infants for 71 CpG sites (false discovery rate < 0.05), with an average effect size of 1.7% change in methylation. Aflatoxin-associated differential methylation was observed in growth factor genes such as FGF12 and IGF1, and immune-related genes such as CCL28, TLR2 and TGFBI. Moreover, one aflatoxin-associated methylation region (corresponding to the miR-4520b locus) was identified.

CONCLUSIONS: This study shows that maternal exposure to aflatoxin during the early stages of pregnancy is associated with differential DNA methylation patterns of infants, including in genes related to growth and immune function. This reinforces the need for interventions to reduce aflatoxin exposure, especially during critical periods of fetal and infant development.

High frequency of donor chimerism after allogeneic transplantation of CD34+-selected peripheral blood cells

Ex vivo T cell depletion of allogeneic grafts is associated with a high (up to 80%) rate of mixed chimerism (MC) posttransplantation. The number of transplanted progenitor cells is an important factor in achieving complete donor chimerism in the T cell depletion setting. Use of granulocyte colony-stimulating factor (G-CSF) peripheral blood allografts allows the administration of large numbers of CD34+ cells. We studied the chimeric status of 13 patients who received allogeneic CD34+-selected peripheral blood progenitor cell transplants (allo-PBPCTs/CD34+) from HLA-identical sibling donors. Patients were conditioned with cyclophosphamide (120 mg/kg) and total-body irradiation (13 Gy in four fractions). Apheresis products were T cell-depleted by the immunoadsorption avidin-biotin method. The median number of CD34+ and CD3+ cells infused was 2.8x10(6)/kg (range 1.9-8.6x10(6)/kg) and 0.4x10(6)/kg (range 0.3-1x10(6)/kg), respectively. Molecular analysis of the engraftment was performed using polymerase chain reaction (PCR) amplification of highly polymorphic short tandem repeat (PCR-STR) sequences in peripheral blood samples. MC was detected in two (15%) of 13 patients. These two patients relapsed at 8 and 10 months after transplant, respectively. The remaining 11 patients showed complete donor chimerism and were in clinical remission after a maximum follow-up period of 24 months (range 6-24 months). These results were compared with those obtained in 10 patients who were treated with T cell-depleted bone marrow transplantation by means of elutriation and who received the same conditioning treatment and similar amounts of CD3+ cells (median 0.45x10(6)/kg; not significant) but a lower number of CD34+ cells (median 0.8x10(6)/kg; p = 0.001). MC was documented in six of 10 patients (60%), which was significantly higher than in the allo-PBPCT/CD34+ group (p = 0.04). We conclude that a high frequency of complete donor chimerism is achieved in patients receiving allo-PBPCT/CD34+ and that this is most likely due to the high number of progenitor cells administered.

A family with erythrocytosis establishes a role for prolyl hydroxylase domain protein 2 in oxygen homeostasis

The number of red blood cells is normally tightly regulated by a classic homeostatic mechanism based on oxygen sensing in the kidney. Decreased oxygen delivery resulting from anemia induces the production of erythropoietin, which increases red cell production and hence oxygen delivery. Investigations of erythropoietin regulation identified the transcription factor hypoxia-inducible factor (HIF). HIF is now recognized as being a key regulator of genes that function in a comprehensive range of processes besides erythropoiesis, including energy metabolism and angiogenesis. HIF itself is regulated through the -subunit, which is hydroxylated in the presence of oxygen by a family of three prolyl hydroxylase domain proteins (PHDs)/HIF prolyl hydroxylases/egg-laying-defective nine enzymes. Hydroxylation allows capture by the von Hippel–Lindau tumor suppressor gene product, ubiquitination, and destruction by the proteasome. Here we describe an inherited mutation in a mammalian PHD enzyme. We show that this mutation in PHD2 results in a marked decrease in enzyme activity and is associated with familial erythrocytosis, identifying a previously unrecognized cause of this condition. Our findings indicate that PHD2 is critical for normal regulation of HIF in humans.

Recessive congenital methaemoglobinaemia: cytochrome b(5) reductase deficiency

Some 60 years ago, Quentin Gibson reported the first hereditary disorder involving an enzyme when he deduced that familial methaemoglobinaemia was caused by an enzymatic lesion associated with the glycolysis pathway in red blood cells. This disorder, now known as recessive congenital methaemoglobinaemia (RCM), is caused by NADH-cytochrome b5 reductase (cb(5)r) deficiency. Two distinct clinical forms, types I and II, have been recognized, both characterized by cyanosis from birth. In type II, the cyanosis is accompanied by neurological impairment and reduced life expectancy. Cytochrome b(5) reductase is composed of one FAD and one NADH binding domain linked by a hinge region. It is encoded by the CYB5R3 (previously known as DIA1) gene and more than 40 mutations have been described, some of which are common to both types of RCM. Mutations associated with type II tend to cause incorrect splicing, disruption of the active site or truncation of the protein. At present the description of the sequence variants of cb(5)r in the literature is confusing, due to the use of two conventions which differ by one codon position. Herein we propose a new system for nomenclature of cb(5)r based on recommendations of the Human Genome Variation Society. The development of a heterologous expression system has allowed the impact of naturally occurring variants of cb(5)r to be assessed and has provided insight into the function of cb(5)r.

Histological and ultrastructural investigation of retinal microaneurysm development in diabetic patients

BACKGROUND: Although microaneurysms are a clinicopathological hallmark of diabetic retinopathy, there have been few ultrastructural studies of these important lesions. As a result, knowledge of the mechanisms involved in the pathogenesis of microaneurysms remains fragmentary. This study provides histological and ultrastructural evidence of various stages in microaneurysm formation within the retinal vasculature. METHODS: The eyes of three type II diabetic patients, obtained within 24 hours of death, were studied by the trypsin digest technique. Eyes from two further type II diabetics were fixed in 2.5% glutaraldehyde within 12 hours of death and processed for electron microscopy. RESULTS: In the trypsin digest preparations, small saccular and fusiform microaneurysms were observed in the peripheral retinal. In the central retina, the microaneurysms ranged in morphology from thin walled, cellular forms to dense, acellular, hyalinised forms. Ultrastructurally, four distinct groups of microaneurysm were observed. Type I showed an extensive accumulation of polymorphonuclear cells into the lumen. The endothelium remained intact, although pericytes were invariably absent. Type II microaneurysms were typified by large numbers of red blood cells (RBCs) in the lumen. Endothelial cells and pericytes were completely absent. The type III microaneurysm was also non-perfused and contained aggregates of irregularly shaped RBC profiles and RBC breakdown products. Recanalisation by new vessels into the occluded lumen was observed in one microaneurysm. Type IV microaneurysms were almost or completely sclerosed, with extensive fibrosis and lipid infiltration into the lumen and basement membrane wall. CONCLUSION: This investigation describes several distinctive stages in the formation of microaneurysms during diabetic retinopathy. With reference to the pathogenesis of retinal microaneurysms, the interaction of various cell types is discussed and the significance of vascular cell death and localised hypertensive events highlighted.

Cathelicidin-like Helminth Defence Molecules (HDMs):Absence of Cytotoxic, Anti-microbial and Anti-protozoan Activities Imply a Specific Adaptation to Immune Modulation

Host defence peptides (HDPs) are expressed throughout the animal and plant kingdoms. They have multifunctional roles in the defence against infectious agents of mammals, possessing both bactericidal and immune-modulatory activities. We have identified a novel family of molecules secreted by helminth parasites (helminth defence molecules; HDMs) that exhibit similar structural and biochemical characteristics to the HDPs. Here, we have analyzed the functional activities of four HDMs derived from Schistosoma mansoni and Fasciola hepatica and compared them to human, mouse, bovine and sheep HDPs. Unlike the mammalian HDPs the helminth-derived HDMs show no antimicrobial activity and are non-cytotoxic to mammalian cells (macrophages and red blood cells). However, both the mammalian- and helminth-derived peptides suppress the activation of macrophages by microbial stimuli and alter the response of B cells to cytokine stimulation. Therefore, we hypothesise that HDMs represent a novel family of HDPs that evolved to regulate the immune responses of their mammalian hosts by retaining potent immune modulatory properties without causing deleterious cytotoxic effects.

Two Novel Dermaseptin-Like Antimicrobial Peptides with Anticancer Activities from the Skin Secretion of Pachymedusa dacnicolor

The dermaseptin antimicrobial peptide family contains members of 27–34 amino acids in length that have been predominantly isolated from the skins/skin secretions of phyllomedusine leaf frogs. By use of a degenerate primer in Rapid amplification of cDNA ends (RACE) PCR designed to a common conserved domain within the 5′-untranslated regions of previously-characterized dermaseptin encoding cDNAs, two novel members of this peptide family, named dermaseptin-PD-1 and dermaseptin-PD-2, were identified in the skin secretion of the phyllomedusine frog, Pachymedusa dacnicolor. The primary structures of both peptides were predicted from cloned cDNAs, as well as being confirmed by mass spectral analysis of crude skin secretion fractions resulted from reversed-phase high-performance liquid chromatography. Chemically-synthesized replicates of dermaseptin-PD-1 and dermaseptin-PD-2 were investigated for antimicrobial activity using standard model microorganisms (Gram-positive bacteria, Gram-negative bacteria and a yeast) and for cytotoxicity using mammalian red blood cells. The possibility of synergistic effects between the two peptides and their anti-cancer cell proliferation activities were assessed. The peptides exhibited moderate to high inhibition against the growth of the tested microorganisms and cancer cell lines with low haemolytic activity. Synergistic interaction between the two peptides in inhibiting the proliferation of Escherichia coli and human neuronal glioblastoma cell line, U251MG was also manifested.

Erythropoietin production:evidence for multiple oxygen sensing pathways

The production of erythropoietin (Epo), the glycoprotein hormone which controls red blood cell formation, is regulated by feedback mechanisms sensing tissue oxygenation. The mechanism of the putative oxygen sensor has yet to be elucidated. There is evidence that at least two pathways participate in hypoxia signal transduction. One appears to involve a specific haem protein, and a second implicates reactive oxygen species (ROS). Iron catalyses the generation of intracellular ROS and therefore alters the cellular redox state. We have investigated the effect of modulating intracellular iron content on Epo production in Hep 3B cells. Iron chelation stimulates Epo production at 20% O2 and enhances Epo production at 1% O2, but it has no additive effect on cobalt-induced Epo production. Excess molar iron inhibited Epo production in response to hypoxia, desferrioxamine (DFO) and cobalt chloride and inhibited the DFO-enhancing effect of hypoxia-induced Epo production. We found that sulphydryl oxidising agents exert a differential inhibitory effect on hypoxia-induced versus DFO-induced Epo production, providing further evidence that multiple pathways of oxygen sensing exist.

Cytosolic phospholipase A2 gene expression in rat mesangial cells is regulated post-transcriptionally

Cytosolic phospholipase A2 (cPLA2) is thought to be the rate-limiting enzyme in the arachidonic acid/eicosanoid cascade. The ability of various agonists to increase steady-state cPLA2 mRNA levels has previously been reported. The current study delineates the contributions of transcriptional and post-transcriptional processes to the regulation of cPLA2 gene expression in response to a variety of agonists in cultured rat glomerular mesangial cells. Epidermal growth factor, platelet-derived growth factor, serum and phorbol myristate acetate all increase the half-life of cPLA2 mRNA transcripts, indicating a role for post-transcriptional modulation of gene expression. The presence of three ATTTA motifs in the 3' untranslated region (3'UTR) of the rat cPLA2 cDNA is ascertained. Heterologous expression of chimeric constructs with different 3'UTRs ligated into the 3' end of the luciferase coding region reveals that the presence of the cPLA2 3'UTR results in reduced luciferase activity compared with constructs without the cPLA2 3'UTR. Furthermore, the luciferase activity in the constructs with the cPLA2 3'UTR is increased in response to the same agonists which stabilize endogenous cPLA2 mRNA. A negligible effect of these agonists on transcriptional control of cPLA2 is evident using promoter-reporter constructs expressed in transient and stable transfectants. Taken together, these results indicate predominant post-transcriptional regulation of cPLA2 mRNA levels.

STAT3 activation in circulating immune cells is related to neovascular age-related macular degeneration

Purpose:The Signal Transducer and Activator of Transcription 3 (STAT3) pathway is known to play an important role in inflammation and angiogenesis. STAT3 can be activated by IL-6 family cytokines through the receptor IL-6R/gp130. Increased IL-6 has been detected in the plasma and vitreous in neovascular age-related macular degeneration (nAMD) patients. The aim of this study was to investigate the role of the STAT3 pathway in the pathogenesis of nAMD.

Methods:Blood cells from nAMD patients (n = 11) and age-, gender-matched healthy controls (n = 13) were stimulated with IL-6 for 20 minutes. The expression of the activated form of STAT3 (p-STAT3) was examined by flow cytometry. The mRNA levels of gp130, IL-6R and the suppressor of cytokine signalling 3 (SOCS3, a negative regulator of p-STAT3) were evaluated by real-time RT-PCR. Laser-induced choroidal neovascularisation (CNV) was performed in WT C57BL/6J mice as well as in the myeloid cell specific SOCS3 deficiency mice i.e., the LysMCre-SOCS3fl/fl mice. STAT3 activation in CNV lesions was examined by western blot. The size of CNV at different times after laser treatment was measured by confocal microscopy of RPE/choroidal flatmounts.

Results:The expression of p-STAT3 in CD11b+ monocytes was significantly increased in nAMD patients compared to healthy controls, although mRNA expression of gp130, IL-6R and SOCS3 did not differ between patients and controls. The expression of p-STAT3 in the retinal and RPE/choroidal tissues was increased at 1 and 3 days after laser treatment. The administration of a STAT3 inhibitor LLL12 significantly suppressed CNV. CD11b+ monocytes from LysMCre-SOCS3fl/fl mice expressed higher levels of p-STAT3 compared to the cells from WT mice. Laser induced CNV developed earlier and were larger in LysMCre-SOCS3fl/fl mice compared to WT C57BL/6J mice.

Conclusions:Our results suggest that STAT3 activation in circulating monocytes may contribute to the development of choroidal neovascularisation in AMD, and targeting the STAT3 pathway may have therapeutic potential in nAMD.