Systematics and life cycles of four avian schistosomatids from Southern Cone of South America.

Relative to the numerous studies focused on mammalian schistosomes, fewer include avian schistosomatids particularly in the southern hemisphere. This is changing and current research emerging from the Neotropics shows a remarkable diversity of endemic taxa. To contribute to this effort, nine ducks (Spatula cyanoptera, S.versicolor, Netta peposaca), 12 swans (Cygnus melancoryphus) and 1,400 Physa spp. snails from Chile and Argentina were collected for adults and larval schistosomatids, respectively. Isolated schistosomatids were preserved for morphological and molecular analyses (28S and COI genes). Four different schistosomatid taxa were retrieved from birds: Trichobilharzia sp. in N. peposaca and S. cyanoptera that formed a clade; S.cyanoptera and S. versicolor hosted Trichobilharzia querquedulae; Cygnus melancoryphus hosted the nasal schistosomatid, Nasusbilharzia melancorhypha; and one visceral, Schistosomatidae gen. sp., which formed a clade with furcocercariae from Argentina and Chile from previous work. Of the physid snails, only one from Argentina had schistosomatid furcocercariae that based on molecular analyses grouped with T. querquedulae. This study represents the first description of adult schistosomatids from Chile as well as the elucidation of the life cycles of N.melancorhypha and T. querquedulae in Chile and Neotropics, respectively. Without well-preserved adults, the putative new genus Schistosomatidae gen. sp. could not be described, but its life cycle involves Chilina spp. and C. melancoryphus. Scanning electron microscopy of T. querquedulae revealed additional, undescribed morphological traits, highlighting its diagnostic importance. Authors stress the need for additional surveys of avian schistosomatids from the Neotropics to better understand their evolutionary history.

Zika Virus Infection in the Developing Mouse Produces Dramatically Different Neuropathology Dependent on Viral Strain.

Zika virus (ZIKV) infection during pregnancy has been causally linked to a constellation of neurodevelopmental deformities in the fetus resulting in a disease termed congenital Zika syndrome (CZS). Here we detail how ZIKV infection produces extensive neuropathology in the developing mouse brain and spinal cord of both sexes. Surprisingly, neuropathology differs depending on viral strain with a French Polynesian isolate producing primarily excitotoxicity and a Brazilian isolate being almost exclusively apoptotic but occurring over a prolonged period that is more likely to produce severe hypoplasia. We also show exposure can produce a characteristic pattern of infection that mirrors neuropathology and ultimately results in gross morphological deformities strikingly similar to CZS. This research provides a valuable mouse model mirroring the clinical course of disease that can be used to test potential therapies to improve treatment and gain a better understanding of the disabilities associated with CZS.SIGNIFICANCE STATEMENT Zika virus (ZIKV) infection during pregnancy has been causally linked to a constellation of neurodevelopmental deformities in the fetus resulting in a disease termed congenital Zika syndrome. Despite its devastating effects, very little is known about how ZIKV infection produces fetal neuropathology. Here we detail the temporal progression of ZIKV infection in the mouse brain and spinal cord resulting in massive neurodegeneration of infected regions. We also report a ZIKV strain from a region of Brazil with high levels of microcephaly (abnormally small head circumference) produces particularly devastating neuropathology.

An efficient method for collecting the full-length adults, fragments, and eggs of Trichobilharzia spp. from the liver of definitive hosts.

Precise identification of avian schistosomes in the genus Trichobilharzia at the species level is difficult and requires both traditional morphological and molecular techniques. To obtain satisfactory results by traditional methods, the characteristics of the intact adults or large fragments of male and females are necessary. The present study aimed to introduce a more efficient method for collecting eggs and both fragments and intact worms for morphological identification of visceral Trichobilharzia spp. Thirty-eight domestic ducks (twenty-eight fresh and ten frozen) were studied. For fresh samples, warm saline (40-45 °C) was injected into the portal vein or liver tissue, followed by slicing of the liver to small pieces in a large Petri dish. All materials were then transferred into the laboratory sieves arranged from the largest to the smallest mesh size and while crushed with the hand, washed, and filtered using a trigger water sprayer. The collected materials were studied under a stereomicroscope for parasite eggs, fragments, and full-length worms. Out of 28 freshly killed ducks, 19 (67.9%) and of 10 frozen ducks 6 (60%) were positive for visceral Trichobilharzia spp. The full-length worms and large fragments of male worms were mostly recovered with the mesh no. 150 (diameter of 106 µm) and small fragments, especially of females, and eggs with the mesh no. 270 (diameter of 53 µm). In addition to large numbers of fragments, 15 full-length adults were obtained from fresh and 2 from frozen ducks. The number of collected full-length adults was related to the worm burden. Since morphological description of different species of the genus Trichobilharzia is primarily based on the availability of adult worms, the application of methods that provide a higher number of intact males and females will result in better characterization of the species and deposition of appropriate voucher specimens. These results show the present method as a suitable tool for the collection of quality adults of visceral Trichobilharzia spp. in ducks.

Mild hypothermia ameliorates anesthesia toxicity in the neonatal macaque brain.

Sedatives and anesthetics can injure the developing brain. They cause apoptosis of neurons and oligodendrocytes, impair synaptic plasticity, inhibit neurogenesis and trigger long-term neurocognitive deficits. The projected vulnerable period in humans extends from the third trimester of pregnancy to the third year of life. Despite all concerns, there is no ethically and medically acceptable alternative to the use of sedatives and anesthetics for surgeries and painful interventions. Development of measures that prevent injury while allowing the medications to exert their desired actions has enormous translational value. Here we investigated protective potential of hypothermia against histological toxicity of the anesthetic sevoflurane in the developing nonhuman primate brain. Neonatal rhesus monkeys underwent sevoflurane anesthesia over 5 h. Body temperature was regulated in the normothermic (>36.5 °C), mild hypothermic (35-36.5 °C) and moderately hypothermic (<35 °C) range. Animals were euthanized at 8 h and brains examined immunohistochemically (activated caspase 3) and stereologically to quantify apoptotic neuronal and oligodendroglial death. Sevoflurane anesthesia was well tolerated at all temperatures, with oxygen saturations, end tidal CO2 and blood gases remaining at optimal levels. Compared to controls, sevoflurane exposed brains displayed significant apoptosis in gray and white matter affecting neurons and oligodendrocytes. Mild hypothermia (35-36.5 °C) conferred significant protection from apoptotic brain injury, whereas moderate hypothermia (<35 °C) did not. Hypothermia ameliorates anesthesia-induced apoptosis in the neonatal primate brain within a narrow temperature window (35-36.5 °C). Protection is lost at temperatures below 35 °C. Given the mild degree of cooling needed to achieve significant brain protection, application of our findings to humans should be explored further.

Quantitative ultrasound and apoptotic death in the neonatal primate brain.

Apoptosis is triggered in the developing mammalian brain by sedative, anesthetic or antiepileptic drugs during late gestation and early life. Whether human children are vulnerable to this toxicity mechanism remains unknown, as there are no imaging techniques to capture it. Apoptosis is characterized by distinct structural features, which affect the way damaged tissue scatters ultrasound compared to healthy tissue. We evaluated whether apoptosis, triggered by the anesthetic sevoflurane in the brains of neonatal rhesus macaques, can be detected using quantitative ultrasound (QUS). Neonatal (n = 15) rhesus macaques underwent 5 h of sevoflurane anesthesia. QUS images were obtained through the sagittal suture at 0.5 and 6 h. Brains were collected at 8 h and examined immunohistochemically to analyze apoptotic neuronal and oligodendroglial death. Significant apoptosis was detected in white and gray matter throughout the brain, including the thalamus. We measured a change in the effective scatterer size (ESS), a QUS biomarker derived from ultrasound echo signals obtained with clinical scanners, after sevoflurane-anesthesia in the thalamus. Although initial inclusion of all measurements did not reveal a significant correlation, when outliers were excluded, the change in the ESS between the pre- and post-anesthesia measurements correlated strongly and proportionally with the severity of apoptotic death. We report for the first time in vivo changes in QUS parameters, which may reflect severity of apoptosis in the brains of infant nonhuman primates. These findings suggest that QUS may enable in vivo studies of apoptosis in the brains of human infants following exposure to anesthetics, antiepileptics and other brain injury mechanisms.

Veterinary Forensic Toxicology.

Veterinary pathologists working in diagnostic laboratories are sometimes presented with cases involving animal poisonings that become the object of criminal or civil litigation. Forensic veterinary toxicology cases can include cases involving animal cruelty (malicious poisoning), regulatory issues (eg, contamination of the food supply), insurance litigation, or poisoning of wildlife. An understanding of the appropriate approach to these types of cases, including proper sample collection, handling, and transport, is essential so that chain of custody rules are followed and proper samples are obtained for toxicological analysis. Consultation with veterinary toxicologists at the diagnostic laboratory that will be processing the samples before, during, and after the forensic necropsy can help to ensure that the analytical tests performed are appropriate for the circumstances and findings surrounding the individual case.

Hedgehog regulates cerebellar progenitor cell and medulloblastoma apoptosis.

The external granule layer (EGL) is a proliferative region that produces over 90% of the neurons in the cerebellum but can also malignantly transform into a cerebellar tumor called the medulloblastoma (the most common malignant brain tumor in children). Current dogma considers Hedgehog stimulation a potent proliferative signal for EGL neural progenitor cells (NPCs) and medulloblastomas. However, the Hedgehog pathway also acts as a survival signal in the neural tube where it regulates dorsoventral patterning by controlling NPC apoptosis. Here we show that Hedgehog stimulation is also a potent survival signal in the EGL and medulloblastomas that produces a massive apoptotic response within hours of signal loss in mice. This toxicity can be produced by numerous Hedgehog antagonists (vismodegib, cyclopamine, and jervine) and is Bax/Bak dependent but p53 independent. Finally, since glucocorticoids can also induce EGL and medulloblastoma apoptosis, we show that Hedgehog's effects on apoptosis can occur independent of glucocorticoid stimulation. This effect may play a major role in cerebellar development by directing where EGL proliferation occurs thereby morphologically sculpting growth. It may also be a previously unknown major therapeutic effect of Hedgehog antagonists during medulloblastoma therapy. Results are discussed in terms of their implications for both cerebellar development and medulloblastoma treatment.

Candidates for twin chiral bands in 102Rh.

Excited states in 102Rh, populated in the fusion-evaporation reaction Zr94(11B,3n)102Rh at a beam energy of 36 MeV, were studied using the Indian National Gamma Array spectrometer at Inter University Accelerator Center, New Delhi. The angular correlations and the electromagnetic character of some of the gamma-ray transitions observed were investigated in detail. A new chiral candidate sister band was found. Lifetimes of exited states in both chiral candidate bands of 102Rh were measured for the first time in the A∼100 mass region by means of the Doppler-shift attenuation technique. The derived reduced transition probabilities are compared to the predictions of the two quasiparticles plus triaxial rotor model. Both experimental results and calculations do not support the presence of static chirality in 102Rh.

Sharing schistosomes: the elephant schistosome Bivitellobilharzia nairi also infects the greater one-horned rhinoceros (Rhinoceros unicornis) in Chitwan National Park, Nepal.

Because the digenetic trematode fauna of Nepal is poorly known, we began to search for schistosomes in and around Chitwan National Park (CNP) of southern Nepal. Both domestic and wild Indian elephants (Elephus maximus) are present, and we found one of two dung samples from wild elephants and 1 of 22 (4.5%) dung samples from domestic elephants to be positive for schistosome eggs. The morphology of the eggs and both cox1 and 28S sequences derived from the eggs/miracidia were consistent with Bivitellobilharzia nairi, reported here for the first time from Nepal. Also, 7 of 14 faecal samples from the Asian or greater one-horned rhinoceros (Rhinoceros unicornis) contained viable eggs indistinguishable from those of B. nairi. This identification was confirmed by comparison with both cox1 and 28S sequences from B. nairi eggs/miracidia derived from Nepalese and Sri Lankan elephants. This represents the first sequence-verified identification of a schistosome from any species of rhinoceros, and the first verified occurrence of a representative of Bivitellobilharzia (a genus of 'elephant schistosomes') in mammals other than elephants. Our work suggests that elephants and rhinos share B. nairi in CNP, even though these two members of the 'charismatic megafauna' belong to unrelated mammalian families. Their shared life style of extensive contact with freshwater habitats likely plays a role, although the snail intermediate host and mode of definitive host infection for B. nairi have yet to be documented. This report also supports Bivitellobilharzia as a monophyletic group and its status as a distinct genus within Schistosomatidae.

Extended haplotype association study in Crohn's disease identifies a novel, Ashkenazi Jewish-specific missense mutation in the NF-κB pathway gene, HEATR3.

The Ashkenazi Jewish population has a several-fold higher prevalence of Crohn's disease (CD) compared with non-Jewish European ancestry populations and has a unique genetic history. Haplotype association is critical to CD etiology in this population, most notably at NOD2, in which three causal, uncommon and conditionally independent NOD2 variants reside on a shared background haplotype. We present an analysis of extended haplotypes that showed significantly greater association to CD in the Ashkenazi Jewish population compared with a non-Jewish population (145 haplotypes and no haplotypes with P-value <10(-3), respectively). Two haplotype regions, one each on chromosomes 16 and 21, conferred increased disease risk within established CD loci. We performed exome sequencing of 55 Ashkenazi Jewish individuals and follow-up genotyping focused on variants in these two regions. We observed Ashkenazi Jewish-specific nominal association at R755C in TRPM2 on chromosome 21. Within the chromosome 16 region, R642S of HEATR3 and rs9922362 of BRD7 showed genome-wide significance. Expression studies of HEATR3 demonstrated a positive role in NOD2-mediated NF-κB signaling. The BRD7 signal showed conditional dependence with only the downstream rare CD-causal variants in NOD2, but not with the background haplotype; this elaborates NOD2 as a key illustration of synthetic association.

Molecular phylogenetics of the elephant schistosome Bivitellobilharzia loxodontae (Trematoda: Schistosomatidae) from the Central African Republic.

One of the most poorly known of all schistosomes infecting mammals is Bivitellobilharzia loxodontae. Nearly all of our available information about this species comes from the original description of worms that were obtained from an animal park-maintained elephant in Germany, probably a forest elephant Loxodonta cyclotis, originating from the present-day Democratic Republic of Congo. We obtained schistosome eggs from faecal samples from wild forest elephants from the Central African Republic. The eggs, which were similar in size and shape to those of described B. loxodontae, were sequenced for the 28S nuclear ribosomal gene and the mitochondrial cytochrome oxidase I (cox1) gene. In a phylogenetic analysis of 28S sequences, our specimens grouped closely with B. nairi, the schistosome from the Indian elephant Elephas maximus, to the exclusion of schistosomes from other genera. However, the eggs were genetically distinct (12% distance cox1) from those of B. nairi. We conclude the specimens we recovered were of B. loxodontae and confirm this is a distinct Bivitellobilharzia species. In addition to providing the first sequence data for B. loxodontae, this report also supports Bivitellobilharzia as a monophyletic group and gives the relative phylogenetic position of the genus within the Schistosomatidae. We also provide a review of the biology of this poorly known schistosome genus.

Propylene glycol produces excessive apoptosis in the developing mouse brain, alone and in combination with phenobarbital.

INTRODUCTION: Propylene glycol (PG) is a common solvent used in medical preparations. It is generally recognized as safe at regulated concentrations; however, its apoptotic potential is unknown. RESULTS: PG triggered widespread apoptotic neurodegeneration with the greatest damage at postnatal day 7 (P7). Significant apoptosis was observed at doses as low as 2 ml/kg. These findings have implications for the safety of drug preparations used in pediatric medicine. The anticonvulsant phenobarbital (PB), which alone produces apoptosis in the immature central nervous system (CNS) is prepared in 68% PG and 10% ethanol (EtOH). We assessed whether PG contributes to the neurotoxic potential of PB. The agents (both at subtoxic doses) produce significantly more apoptosis when used in combination. DISCUSSION: In conclusion, finding an alternative non-apoptotic solvent that can be used as a substitute for PG may be beneficial to patients. METHODS: C57BL/6 mice (P4-30) were exposed to PG to examine whether PG could produce apoptosis in the developing CNS.

Glucocorticoid receptor stimulation and the regulation of neonatal cerebellar neural progenitor cell apoptosis.

Glucocorticoids are used to treat respiratory dysfunction associated with premature birth but have been shown to cause neurodevelopmental deficits when used therapeutically. Recently, we established that acute glucocorticoid exposure at clinically relevant doses produces neural progenitor cell apoptosis in the external granule layer of the developing mouse cerebellum and permanent decreases in the number of cerebellar neurons. As the cerebellum naturally matures and neurogenesis is no longer needed, the external granule layer decreases proliferation and permanently disappears during the second week of life. At this same time, corticosterone (the endogenous rodent glucocorticoid) release increases and a glucocorticoid-metabolizing enzyme that protects the external granule layer against glucocorticoid receptor stimulation (11ß-Hydroxysteroid-Dehydrogenase-Type 2; HSD2) naturally disappears. Here we show that HSD2 inhibition and raising corticosterone to adult physiological levels both can independently increase neural progenitor cell apoptosis in the neonatal mouse. Conversely, glucocorticoid receptor antagonism decreases natural physiological apoptosis in this same progenitor cell population suggesting that endogenous glucocorticoid stimulation may regulate apoptosis in the external granule layer. We also found that glucocorticoids which HSD2 can effectively metabolize generate less external granule layer apoptosis than glucocorticoids this enzyme is ineffective at breaking down. This finding may explain why glucocorticoids that this enzyme can metabolize are clinically effective at treating respiratory dysfunction yet seem to produce no neurodevelopmental deficits. Finally, we demonstrate that both acute and chronic glucocorticoid exposures produce external granule layer apoptosis but without appropriate control groups this effect becomes masked. These results are discussed in terms of their implications for glucocorticoid therapy and neurodevelopment during the perinatal period.

CTLA4 -1661A/G and 3'UTR long repeat polymorphisms are associated with ulcerative colitis and influence CTLA4 mRNA and protein expression.

Reduced cytotoxic T-lymphocyte antigen 4 (CTLA4) expression has been proposed as a risk for autoimmunity. CTLA4 polymorphisms have been associated with several autoimmune diseases, including ulcerative colitis (UC). In this study, we performed genotyping for CTLA4 -1661A/G, -1722T/C and 3' untranslated region (AT)n repeat polymorphisms in 300 Chinese UC patients and in 700 healthy controls, and evaluated the effects of polymorphisms on full-length (flCTLA4) and soluble CTLA4 (sCTLA4) expression in UC patients. The frequency of the -1661G allele was higher in UC patients than in healthy controls (16.5 vs 11.4%, P=0.003, odds ratio (OR)=1.53, 95% confidence interval (95% CI): 1.17-2.01). The prevalence of (AT)n repeats of the CTLA4 gene carrying long alleles (≥116 bp) was more common in UC patients than in healthy controls (22.0 vs 6.3%, P<0.001, OR=4.21, 95% CI: 2.79-6.33), and was associated with extensive colitis (P=0.008). Among UC patients, long-allele carriers expressed lower levels of flCTLA4 and sCTLA4 mRNA and sCTLA4 protein than did short-allele carriers (P<0.001, P<0.001, P<0.001, respectively). CTLA4 gene -1661A/G and long 3' untranslated region (AT)n repeat polymorphisms are associated with UC in Central China. This is likely from decreased expressions of sCTLA4 mRNA and sCTLA4 protein. Our study suggests that CTLA4 has an important role in susceptibility for UC in Central China.

Genetic variants in the region harbouring IL2/IL21 associated with ulcerative colitis.

OBJECTIVES: Genetic susceptibility is known to play a large part in the predisposition to the inflammatory bowel diseases (IBDs) known as Crohn's disease (CD) and ulcerative colitis (UC). The IL2/IL21 locus on 4q27 is known to be a common risk locus for inflammatory disease (shown in coeliac disease, type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosus and psoriasis), while the roles that interleukin 2 (IL2) and IL21 play in the immune response also make them attractive candidates for IBD. The objective of this study was to test for association between the IL2/IL21 locus and the IBDs. METHODS: The four single nucleotide polymorphisms (SNPs) in the IL2/IL21 locus most associated with coeliac disease were genotyped in 1590 subjects with IBD and 929 controls from The Netherlands, and then replicated in a North American cohort (2387 cases and 1266 controls) and an Italian cohort (805 cases and 421 controls), yielding a total of 4782 cases (3194 UC, 1588 CD) and 2616 controls. Allelic association testing and a pooled analysis using a Cochran-Mantel-Haenszel test were performed. RESULTS: All four SNPs were strongly associated with UC in all three cohorts and reached genome-wide significance in the pooled analysis (rs13151961 p = 1.35 x 10(-10), rs13119723 p = 8.60 x 10(-8), rs6840978 p = 3.0 7x 10(-8), rs6822844 p = 2.77 x 10(-9)). A moderate association with CD was also found in the pooled analysis (p value range 0.0016-9.86 x 10(-5)). CONCLUSIONS: A strong association for the IL2/IL21 locus with UC was found, which also confirms it as a general susceptibility locus for inflammatory disease.

A new double crystal calibration system for absolute x-ray emission measurements down to ∼1 keV energies.

Over the past few years, work has been conducted at AWE to accurately characterize x-ray diffraction crystals to allow for absolute measurements of x-ray emission for our Orion opacity campaigns. Diffraction crystals are used in spectrometers on Orion to record the dispersed spectral features emitted by the laser produced plasma to obtain a measurement of the plasma conditions. Previously, based on a Manson x-ray source, our calibration system struggled to attain a high signal at the low energies required in calibration for the use of aluminum as a tracer for higher atomic number experiments. Here, we present data from the newly commissioned CTX400 x-ray source, a twin anode water cooled system, showing it to be a bright source even for ∼1 keV energies. Rocking curve measurements for three of the most commonly used crystals, namely, pentaerythritol, cesium acid phthalate, and germanium, are presented for both convex and flat forms.

Schistosoma kisumuensis n. sp. (Digenea: Schistosomatidae) from murid rodents in the Lake Victoria Basin, Kenya and its phylogenetic position within the S. haematobium species group.

Schistosoma kisumuensis n. sp. is described based on 6 adult males and 2 adult females collected from the circulatory system of 3 murid rodent species, Pelomys isseli, Mastomys natalensis, and Dasymys incomtus. Specimens were collected from a single location, Nyabera Swamp, in Kisumu, Kenya in the Lake Victoria Basin. This new species is morphologically similar to members of the S. haematobium group, currently represented by 8 species parasitizing artiodactyls and primates, including humans. Schistosoma kisumuensis differs from these species by producing relatively small Schistosoma intercalatum-like eggs (135.2 x 52.9 microm) with a relatively small length to width ratio (2.55). Comparison of approximately 3000-base-pair sequences of nuclear rDNA (partial 28S) and mtDNA (partial cox1, nad6, 12S) strongly supports the status of S. kisumuensis as a new species and as a sister species of S. intercalatum. The cox1 genetic distance between these two species (6.3%) is comparable to other pairwise comparisons within the S. haematobium group. Separation of the Congo River and Lake Victoria drainage basins is discussed as a possible factor favoring the origin of this species.

Schistosomes in the southwest United States and their potential for causing cercarial dermatitis or 'swimmer's itch'.

Cercarial dermatitis or swimmer's itch results when cercariae of schistosomes penetrate human skin and initiate inflammatory responses. The parasites typically die in the skin but in some cases may persist and infect other organs. Cercarial dermatitis is caused by a complex and poorly known assemblage of schistosome species, and can occur in any location where people come in contact with water bodies harbouring schistosome-infected snails. In North America, most cases are reported from the upper Midwest. In south-western USA, this phenomenon has not been well studied, and it is not known which schistosome species are present, or if cercarial dermatitis occurs with any regularity. As part of our ongoing studies of schistosome diversity, using morphological traits and sequence data to differentiate species, we have thus far identified eight schistosome genetic lineages from snails from New Mexico and Colorado. We have investigated two cercarial dermatitis outbreaks, one occurring in Stubblefield Lake in northern New Mexico, and one in Prospect Lake in the heart of Colorado Springs, Colorado. The New Mexico outbreak involved either one or two different avian schistosome species, both transmitted by physid snails. The Colorado outbreak was due to Trichobilharzia brantae, a species transmitted by geese and the snail Gyraulus parvus. These outbreaks are in contrast to those in northern states where schistosomes infecting snails of the family Lymnaeidae are more often responsible for outbreaks. Our survey suggests that dermatitis-causing schistosomes are not rare in the southwest, and that there are plenty of opportunities for dermatitis outbreaks to occur in this region.

MAST3: a novel IBD risk factor that modulates TLR4 signaling.

Inflammatory bowel disease (IBD) is a chronic disorder caused by multiple factors in a genetically susceptible host. Significant advances in the study of genetic susceptibility have highlighted the importance of the innate immune system in this disease. We previously completed a genome-wide linkage study and found a significant locus (IBD6) on chromosome 19p. We were interested in identifying the causal variant in IBD6. We performed a two-stage association mapping study. In stage 1, 1530 single-nucleotide polymorphisms (SNPs) were selected from the HapMap database and genotyped in 761 patients with IBD. Among the SNPs that passed the threshold for replication, 26 were successfully genotyped in 754 additional patients (stage 2). One intronic variant, rs273506, located in the microtubule-associated serine/threonine-protein kinase gene-3 (MAST3), was found to be associated in both stages (pooled P=1.8 x 10(-4)). We identified four MAST3 coding variants, including a non-synonymous SNP rs8108738, correlated to rs273506 and associated with IBD. To test whether MAST3 was expressed in cells of interest, we performed expression assays, which showed abundant expression of MAST3 in antigen-presenting cells and in lymphocytes. The knockdown of MAST3 specifically decreased Toll-like receptor-4-dependent NF-kappaB activity. Our findings are additional proofs of the pivotal role played by modulators of NF-kappaB activity in IBD pathogenesis.

An SNP linkage scan identifies significant Crohn's disease loci on chromosomes 13q13.3 and, in Jewish families, on 1p35.2 and 3q29.

Inflammatory bowel disease (IBD) is a complex genetic disorder of two major phenotypes, Crohn's disease (CD) and ulcerative colitis (UC), with increased risk in Ashkenazi Jews. Twelve genome-wide linkage screens have identified multiple loci, but these screens have been of modest size and have used low-density microsatellite markers. We, therefore, performed a high-density single-nucleotide polymorphism (SNP) genome-wide linkage study of 993 IBD multiply affected pedigrees (25% Jewish ancestry) that contained 1709 IBD-affected relative pairs, including 919 CD-CD pairs and 312 UC-UC pairs. We identified a significant novel CD locus on chromosome 13p13.3 (peak logarithm of the odds (LOD) score=3.98) in all pedigrees, significant linkage evidence on chromosomes 1p35.1 (peak LOD score=3.5) and 3q29 (peak LOD score=3.19) in Jewish CD pedigrees, and suggestive loci for Jewish IBD on chromosome 10q22 (peak LOD score=2.57) and Jewish UC on chromosome 2q24 (peak LOD score=2.69). Nominal or greater linkage evidence was present for most previously designated IBD loci (IBD1-9), notably, IBD1 for CD families at chromosome 16q12.1 (peak LOD score=4.86) and IBD6 in non-Jewish UC families at chromosome 19p12 (peak LOD score=2.67). This study demonstrates the ability of high information content adequately powered SNP genome-wide linkage studies to identify loci not observed in multiple microsatellite-based studies in smaller cohorts.