An integrative epi-transcriptomic approach identifies the human cartilage chitinase 3-like protein 2 (CHI3L2) as a potential mediator of B12 deficiency in adipocytes.

Vitamin B12 has multiple biochemical functions including in the one-carbon cycle generating a methyl group for DNA methylation, and metabolism of fatty acids and amino acids to generate energy via the citric acid cycle. The aim of our study was to use a combined epigenomic and transcriptomic approach to identify novel genes mediating the effect of B12 on adipogenesis.Human pre-adipocytes (CHUB-S7) were treated with a range of B12 (0-500 nM) concentrations from the day of cell seeding until harvesting in discovery and validation experiments prior to genome-wide methylation analysis using the Illumina HumanMethylation 450Beadchip. For transcriptomic analysis, RNA-seq libraries were run on the Illumina HiSeq 2500. To further investigate the expression of any genes on human adipogenesis, a second human preadipocyte strain was studied (SGBS) by real-time quantitative PCR (qRT-PCR).A combined epigenetic and transcriptomic approach in differentiated human pre-adipocyte cell line, CHUB-S7, identified that the Human cartilage chitinase 3-like protein 2 (CHI3L2) gene was hypo-methylated and had increased expression in low B12 conditions. Furthermore, there was an approximately 1000-fold increase in CHI3L2 expression in the early days of adipocyte differentiation, which paralleled an increase of lipid droplets in differentiated SGBS cells and an increased expression level of markers of mature adipocytes.In summary, we have identified a potential role of the human cartilage chitinase 3-like protein 2 (CHI3L2) in adipocyte function in the presence of low B12 levels.

Areca catechu-(Betel-nut)-induced whole transcriptome changes in a human monocyte cell line that may have relevance to diabetes and obesity; a pilot study.

BACKGROUND: Betel-nut consumption is the fourth most common addictive habit globally and there is good evidence linking the habit to obesity, type 2 diabetes (T2D) and the metabolic syndrome. The aim of our pilot study was to identify gene expression relevant to obesity, T2D and the metabolic syndrome using a genome-wide transcriptomic approach in a human monocyte cell line incubated with arecoline and its nitrosated products. RESULTS: The THP1 monocyte cell line was incubated separately with arecoline and 3-methylnitrosaminopropionaldehyde (MNPA) in triplicate for 24 h and pooled cDNA indexed paired-end libraries were sequenced (Illumina NextSeq 500). After incubation with arecoline and MNPA, 15 and 39 genes respectively had significant changes in their expression (q < 0.05, log fold change 1.5). Eighteen of those genes have reported associations with T2D and obesity in humans; of these genes there was most marked evidence for CLEC10A, MAPK8IP1, NEGR1, NQ01 and INHBE genes. CONCLUSIONS: Our preliminary studies have identified a large number of genes relevant to obesity, T2D and metabolic syndrome whose expression was changed significantly in human TPH1 cells following incubation with betel-nut derived arecoline or with MNPA. These findings require validation by further cell-based work and investigation amongst betel-chewing communities.

Is famine exposure during developmental life in rural Bangladesh associated with a metabolic and epigenetic signature in young adulthood? A historical cohort study.

OBJECTIVES: Famine exposure in utero can 'programme' an individual towards type 2 diabetes and obesity in later life. We sought to identify, (1) whether Bangladeshis exposed to famine during developmental life are programmed towards diabetes and obesity, (2) whether this programming was specific to gestational or postnatal exposure windows and (3) whether epigenetic differences were associated with famine exposure. DESIGN: A historical cohort study was performed as part of a wider cross-sectional survey. Exposure to famine was defined through birth date and historical records and participants were selected according to: (A) exposure to famine in postnatal life, (B) exposure to famine during gestation and (C) unexposed. SETTING: Matlab, a rural area in the Chittagong division of Bangladesh. PARTICIPANTS: Young adult men and women (n=190) recruited to a historical cohort study with a randomised subsample included in an epigenetic study (n=143). OUTCOME MEASURES: Primary outcome measures of weight, body mass index and oral glucose tolerance tests (0 and 120 min glucose). Secondary outcome measures included DNA methylation using genome-wide and targeted analysis of metastable epialleles sensitive to maternal nutrition. RESULTS: More young adults exposed to famine in gestation were underweight than those postnatally exposed or unexposed. In contrast, more young adults exposed to famine postnatally were overweight compared to those gestationally exposed or unexposed. Underweight adults exposed to famine in gestation in utero were hyperglycaemic following a glucose tolerance test, and those exposed postnatally had elevated fasting glucose, compared to those unexposed. Significant differences in DNA methylation at seven metastable epialleles (VTRNA2-1, PAX8, PRDM-9, near ZFP57, near BOLA, EXD3) known to vary with gestational famine exposure were identified. CONCLUSIONS: Famine exposure in developmental life programmed Bangladeshi offspring towards diabetes and obesity in adulthood but gestational and postnatal windows of exposure had variable effects on phenotype. DNA methylation differences were replicated at previously identified metastable epialleles sensitive to periconceptual famine exposure.

Cyclo-oxygenase-2 (COX-2) mRNA expression and hormone receptor status in breast cancer.

AIMS: The purpose of this study was to evaluate COX-2 mRNA expression with known clinical prognostic features of breast cancer, oestrogen/progesterone receptor status, tumour size and grade. METHODS: Total RNA was extracted from 45 frozen breast tumour (invasive) and 22 normal breast tissue samples. COX-2 mRNA transcription was quantified using a real time RT-PCR assay and expressed as copy number/microg total RNA. All specimens were assessed for tumour grade, size, nodal status and presence of vascular invasion and oestrogen and progesterone receptor status. RESULTS: COX-2 mRNA was detected in all samples with a median copy number of 1.15 x 10(7) for tumours and 6.5 x 10(6) for normal samples. Expression was significantly higher in oestrogen receptor negative tumours compared to the receptor positive group. There was no correlation between COX-2 mRNA levels and tumour size, grade, nodal status and presence of vascular invasion. CONCLUSIONS: COX-2 mRNA expression is increased in oestrogen and progesterone receptor negative breast cancers.

Expression of 25-hydroxyvitamin D-1-alpha-hydroxylase mRNA in individuals with colorectal cancer.

Vitamin D prevents proliferation, promotes differentiation, and induces apoptosis of colon cells, and reduced intake or insufficiency of the vitamin in the body are associated with increased risk of colorectal cancer. Results of previous studies have suggested that mRNA that codes for 25-hydroxyvitamin D-1-alpha-hydroxylase (1 alpha OHase), which converts 25-hydroxyvitamin D to its active metabolite, might be up regulated in human colon carcinomas. We used real-time reverse transcription PCR assays to measure absolute 1 alpha OHase mRNA concentrations in the colonic mucosa of 44 individuals without cancer, and in paired healthy colon and cancerous colon samples taken from 27 individuals with the disease, to ascertain whether or not such up regulation takes place. Our results suggest that concentrations of 1 alpha OHase mRNA in tumour samples and in healthy colon samples from individuals without cancer are similar, but that concentrations are significantly lower in the paired, phenotypically healthy mucosa of individuals with cancer.

An autosomal dominant periodic fever associated with AA amyloidosis in a north Indian family maps to distal chromosome 1q.

OBJECTIVE: To investigate genetic susceptibility in the first Indian family identified as having an autosomal dominantly inherited periodic fever syndrome. The inflammatory disease was characterized chiefly by arthralgia, skin rashes, and AA amyloidosis. METHODS: Markers from known periodic fever susceptibility loci were investigated in 7 affected and 11 healthy members of a north Indian family. These included the TNFRSF1A locus (formerly known as TNFRI), which is involved in autosomal dominant tumor necrosis factor receptor-associated periodic syndrome on chromosome 12p13, the familial Mediterranean fever locus (MEFV) on chromosome 16p13, the hyperimmunoglobulinemia D and periodic fever syndrome (HIDS) locus on chromosome 12q24, and the Muckle-Wells syndrome/familial cold urticaria (MWS/FCU) locus on distal chromosome 1q44. RESULTS: Linkage to both TNFRSF1A and MEFV was definitively excluded, and DNA sequencing of these genes revealed no mutations. Furthermore, there was no evidence of linkage to the HIDS locus. In contrast, significant logarithm of odds scores for 5 markers from the MWS/FCU region were obtained in this family, and the disease segregated with the same haplotype in all affected members. CONCLUSION: We have identified an inherited inflammatory disease in a north Indian family with clinical features overlapping some of those of MWS and FCU. The susceptibility gene maps to distal chromosome 1q44, a region already implicated in both MWS and FCU. Different mutations in the same (or a closely related) gene may be responsible for an inflammatory disease with a broad phenotype among diverse ethnic populations.

Type 1 diabetes in the offspring does not increase the risk of parental type 2 diabetes in South Indians.

OBJECTIVES: (a) To study whether there was an increased prevalence of glucose intolerance in the parents of probands with Type 1 diabetes and (b) to look for any possible link between the glucose intolerance in the parents with HLA-DQB1 alleles transmitted in excess to the Type 1 diabetes offspring. Study Design and Methods From 215 families of South Indian Type 1 diabetes probands, 336 parents (170 fathers, age 30-70 years; 166 mothers, age 23-72 years) were studied by oral glucose tolerance test (GTT). Glucose intolerance in the parents was compared with the population data available. HLA-DQB1 alleles in 170 of the families were studied by the Olerup method (based on sequence specific primers) and the transmission disequilibrium test (TDT) was used to determine the Type 1 diabetes-associated DQB1 alleles. RESULTS: Among the parents 11.2% had Type 2 diabetes which was similar to the population data of 11.6%. However there was a male predominence among the diabetic parents (chi(2)=7.0, p=0.008), while in the population there was a female predominence. Prevalence of IGT was significantly more among the parents (13.6%) compared with the population data (9.1%) (chi(2)=6.43, p=0.011). Both HLA-DQB1*0201 (p<0.0001) and DQB1*0302 (p=0.0001) were positively associated with Type 1 diabetes in the probands although 21% of the probands possessed neither DQB1*0201 or DQB1*0302. The distribution of glucose tolerance categories in the parents of the probands differed according to the presence of DQB1*0302 (p= 0.035) whilst no such differences existed for DQB1*0201. CONCLUSIONS: In summary, the presence of Type 1 diabetes in the South Indian offspring does not predict a higher occurrence of Type 2 diabetes in the parents. However, there is an increased occurrence of impaired glucose tolerance (IGT) among the parents. Family based studies demonstrate increased transmission of HLA-DQB1*0201 and HLA-DQB1*0302 with Type 1 diabetes similar to North American and European Caucasian subjects. Furthermore, HLA-DQB1*0302 may be a minor determinant of glucose tolerance in parents of offspring with Type 1 diabetes.

Germline mutations in the extracellular domains of the 55 kDa TNF receptor, TNFR1, define a family of dominantly inherited autoinflammatory syndromes.

Autosomal dominant periodic fever syndromes are characterized by unexplained episodes of fever and severe localized inflammation. In seven affected families, we found six different missense mutations of the 55 kDa tumor necrosis factor receptor (TNFR1), five of which disrupt conserved extracellular disulfide bonds. Soluble plasma TNFR1 levels in patients were approximately half normal. Leukocytes bearing a C52F mutation showed increased membrane TNFR1 and reduced receptor cleavage following stimulation. We propose that the autoinflammatory phenotype results from impaired downregulation of membrane TNFR1 and diminished shedding of potentially antagonistic soluble receptor. TNFR1-associated periodic syndromes (TRAPS) establish an important class of mutations in TNF receptors. Detailed analysis of one such mutation suggests impaired cytokine receptor clearance as a novel mechanism of disease.

Exclusion of the familial Mediterranean fever locus as a susceptibility region for autosomal dominant familial Hibernian fever.

Autosomal dominant periodic fevers constitute a range of syndromes characterised by recurrent attacks of fever and abdominal pain. Familial Hibernian fever (FHF) has been described in only one United Kingdom based family, but two other Irish families have been found with similar clinical features. FHF resembles familial Mediterranean fever (FMF) in several clinical features, but the mode of inheritance of FHF is dominant whereas FMF is recessive. We have investigated whether autosomal dominant periodic fevers, in particular FHF, map to the FMF susceptibility locus (MEFV) on chromosome 16p13.3. We have used informative microsatellite markers flanking this locus to genotype members of the three families mentioned above. Two point and multipoint lod scores definitively excluded linkage to MEFV in the two larger families. A haplotype study confirmed these findings, indicating that FHF is genotypically as well as phenotypically distinct from FMF.

Linkage of familial Hibernian fever to chromosome 12p13.

Autosomal dominant periodic fevers are characterized by intermittent febrile attacks of unknown etiology and by recurrent abdominal pains. The biochemical and molecular bases of all autosomal dominant periodic fevers are unknown, and only familial Hibernian fever (FHF) has been described as a distinct clinical entity. FHF has been reported in three families-the original Irish-Scottish family and two Irish families with similar clinical features. We have undertaken a genomewide search in these families and report significant multipoint LOD scores between the disease and markers on chromosome 12p13. Cumulative multipoint linkage analyses indicate that an FHF gene is likely to be located in an 8-cM interval between D12S77 and D12S356, with a maximum LOD score (Z max) of 3.79. The two-point Z max was 3.11, for D12S77. There was no evidence of genetic heterogeneity in these three families; it is proposed that these markers should be tested in other families, of different background, that have autosomal dominant periodic fever, as a prelude to identification of the FHF-susceptibility gene.

Allelic variation in the vitamin D receptor influences susceptibility to IDDM in Indian Asians.

Vitamin D has important immunomodulatory properties and prevents development of diabetes mellitus in an animal model of insulin-dependent diabetes (IDDM). We have studied the vitamin D receptor locus as a candidate for genetic susceptibility to IDDM in Southern Indian families. We found evidence for an association of one particular vitamin D receptor allele with IDDM susceptibility in this community. Ninety-three South Indian families consisting of available parents and an affected offspring were genotyped for three vitamin D receptor polymorphisms using the restriction enzymes TaqI, ApaI and BsmI as well as an adjacent microsatellite located to 12q14 (D12S85). Transmission disequilibrium testing analysis was used to assess preferential transmission of polymorphic markers and haplotypes with IDDM. There was significant excess transmission of vitamin D receptor alleles containing the BsmI restriction site to affected offspring in these families (p = 0.016). No association was found between D12S85 and IDDM. This study suggests that a polymorphism within or close to the vitamin D receptor gene may modify susceptibility to IDDM in this ethnic group.

A gene affecting Wallerian nerve degeneration maps distally on mouse chromosome 4.

When a nerve axon is cut or crushed, the nerve fibers in the distal part of the axon, separated from the cell body, undergo a form of spontaneous degeneration, known as Wallerian degeneration. A substrain of the mouse inbred strain C57BL, known as C57BL/Ola, carries a mutant form of a gene involved in Wallerian degeneration in the peripheral and central nervous systems, and in retrograde degeneration of retinal ganglion cells. Wallerian degeneration in this substrain is abnormally slow. Previously the defect had been shown to be due to an autosomal dominant gene. The locus has been given the name and symbol Wallerian degeneration Wld, with the mutant allele Wlds (Wallerian degeneration-slow). The Wld locus has now been mapped, by using conventional and molecular markers, to the distal end of chromosome 4, near the locus of pronatriodilatin (Pnd). The order of loci (with recombination distances in centimorgans, cM) is cen-D4Mit11-8.9 +/- 1.7 cM-Fuca-2.5 +/- 0.93 cM-Akp-2-3.2 +/- 1.1 cM-D4Mit48-3.5 +/- 1.1 cM-(Wld, Pnd, D4Mit49)-0.71 +/- 0.50 cM-(Eno-1, D4Mit33)-1.4 +/- 0.70 cM-D4Mit42-2.5 +/- 0.93 cM-D4Smh6b. The information on the position of the Wld locus should be valuable in further characterization of this gene involved in nerve degeneration and regeneration.

Isoenzyme variation within the genus Cryptosporidium.

Soluble extracts of the oocysts of Cryptosporidium parvum had demonstrable, but low, activities of malate dehydrogenase (MDH, EC. 1.1.1.37), carboxylesterase (ES, EC 3.1.1.1) and lactate dehydrogenase (LDH, EC. 1.1.1.27) following thin-layer starch-gel electrophoresis. Much higher activities of glucose phosphate isomerase (GPI, EC. 5.3.1.9) and phosphoglucomutase (PGM, EC. 2.7.5.1) were found, and zymograms of these two enzymes were used to characterise isolates of C. parvum from human, bovine, ovine and cervine sources, C. muris from the brown rat and C. baileyi from young turkeys. PGM and GPI zymograms clearly distinguished between C. parvum, C. muris and C. baileyi. The five isolates of C. parvum showed the same electrophoretic mobility for GPI, whereas the PGM mobility of the single human isolate of C. parvum examined was clearly different from that of the other isolates. This is the first report of the use of isoenzymes to distinguish between species and isolates of Cryptosporidium.

Interactions between the human monocytic leukaemia THP-1 cell line and Old and New World species of Leishmania.

The human promyelocytic THP-1 cell line has been found to support the growth of Leishmania parasites. THP-1 cells, differentiated with retinoic acid, cease replication while remaining in suspension. 72 +/- 8% of THP-1 cells became infected after inoculation with promastigotes of several Old and New World Leishmania species. The resulting amastigotes (19 +/- 5 per infected cell) were easy to harvest, capable of reinfecting cultures of normal human cells and, in the case of L. major and L. infantum, caused specific lesions in BALB/c mice. This culture system should facilitate biochemical and immunological studies on amastigotes and be of use in screening anti-parasite drugs.

Vaccination trial against canine visceral leishmaniasis. Phocean Veterinary Study Group on Visceral Leishmaniasis.

In a double-blind study 393 seronegative dogs, residing in a holoendemic area for Leishmania donovani infantum infection, were randomly assigned to an immunization with a partly purified L.d. infantum-derived preparation, or received adjuvant only. During the first year of the study period the rate of infection was significantly higher in the vaccinated group than in the control one (P less than 0.05), but this difference disappeared during the second year (P = 0.44). Since a similar immunization protocol conferred resistance against experimental murine leishmaniasis, these results stress the differences that may exist between the natural hosts of Leishmania parasites and experimental animal substitutes.

[Large-scale production of amastigotes by a human monoblastoid cell line]. / Production massive d'amastigotes par une lignée de cellules monoblastoides humanines.

In this study, a human monoblastoid cell line (TPH-1) was tested in vitro for the production of Leishmania amastigotes. The number of TPH1 cells increased with time and 6 days after promastigote infection the percentage of infected cells was around 45%. Pre-treatment of TPH1 cells with retinoic acid induced the cells to differentiate into unreplicating macrophage-like cells. Ninety per cent was parasitized 6 days after promastigote infection; the number of amastigotes quintuplied during this period of time; this result was irrespective of the Leishmania species used for experiments. Viable and infective parasites were obtained from treated and nontreated cells. TPH1 cells merit further consideration for research concerning new molecules active against Leishmania.

Inoculation of BALB/c mice against Leishmania major infection with Leishmania-derived antigens isolated by gel filtration.

Gel filtration can be used as a substitute for preparative polyacrylamide gel electrophoresis in the preparation of an immunogenic, partly purified, fraction isolated from lysates of Leishmania promastigotes. This fraction, the molecular weight of which ranged from 70,000 to 53,000, when administered to BALB/c mice in associated with muramyl dipeptide induced resistance against cutaneous leishmaniasis.