Michael J. Chorney

CD1d-expressing Dendritic Cells but Not Thymic Epithelial Cells Can Mediate Negative Selection of NKT Cells

Natural killer T (NKT) cells are a unique immunoregulatory T cell population that is positively selected by CD1d-expressing thymocytes. Previous studies have shown that NKT cells exhibit autoreactivity, which raises the question of whether they are subject to negative selection. Here, we report that the addition of agonist glycolipid α-galactosylceramide (α-GalCer) to a fetal thymic organ culture (FTOC) induces a dose-dependent disappearance of NKT cells, suggesting that NKT cells are susceptible to negative selection. Overexpression of CD1d in transgenic (Tg) mice results in reduced numbers of NKT cells, and the residual NKT cells in CD1d-Tg mice exhibit both an altered Vβ usage and a reduced sensitivity to antigen. Furthermore, bone marrow (BM) chimeras between Tg and WT mice reveal that CD1d-expressing BM-derived dendritic cells, but not thymic epithelial cells, mediate the efficient negative selection of NKT cells. Thus, our data suggest that NKT cells developmentally undergo negative selection when engaged by high-avidity antigen or abundant self-antigen.

A Quantitative Trait Locus Associated With Cognitive Ability in Children

Quantitative trait loci (QTLs) associated with general cognitive ability (g) were investigated for several groups of children selected for very high or for average cognitive functioning. A DNA marker in the gene for insulin-like growth factor-2 receptor (IGF2R) on Chromosome 6 yielded a significantly greater frequency of a particular form of the gene (allele) in a high-g group (.303; average IQ = 136, N = 51) than in a control group (.156; average IQ = 103, N = 51). This association was replicated in an extremely-high-g group (all estimated IQs > 160, N = 52) as compared with an independent control group (average IQ = 101, N = 50), with allelic frequencies of .340 and .169, respectively. Moreover, a high-mathematics-ability group (N = 62) and a high-verbal-ability group (N = 51) yielded results that were in the same direction but only marginally significant (p = .06 and .08, respectively).

DNA markers associated with high versus low IQ: The IQ quantitative trait loci (QTL) project

General cognitive ability (intelligence, often indexed by IQ scores) is one of the most highly heritable behavioral dimensions. In an attempt to identify some of the many genes (quantitative trait loci; QTL) responsible for the substantial heritability of this quantitative trait, the IQ QTL Project uses an allelic association strategy. Allelic frequencies are compared for the high and low extremes of the IQ dimension using DNA markers in or near genes that are likely to be relevant to neural functioning. Permanent cell lines have been established for low-IQ (mean IQ=82;N=18), middle-IQ (mean IQ=105;N=21), and high-IQ (mean IQ=130;N=24) groups and for a replication sample consisting of even more extreme low-IQ (mean IQ=59;N=17) and high-IQ (mean IQ=142;N=27) groups. Subjects are Caucasian children tested from 6 to 12 years of age. This first report of the IQ QTL Project presents allelic association results for 46 two-allele markers and for 26 comparisons for 14 multiple-allele markers. Two markers yielded significant (p<.01) allelic frequency differences between the high- and the low-IQ groups in the combined sample—a new HLA marker for a gene unique to the human species and a new brain-expressed triplet repeat marker (CTGB33). The prospects for harnessing the power of molecular genetic techniques to identify QTL for quantitative dimensions of human behavior are discussed.

Is hemochromatosis a risk factor for Alzheimer's Disease?

Excess iron accumulation in the brain is a consistent observation in Alzheimers Disease. Iron affects amyloid precursor protein (AbetaPP) processing and promotes deposition of Abeta. Iron is also among the most potent biological toxins because of its ability to react with oxygen to form reactive oxygen species. Consequently, elucidation of the mechanisms associated with maintaining brain iron homeostasis is fundamentally important to understanding the underlying pathogenesis in AD. The iron overload disorder, Hemochromatosis, is the most common genetic disorder (1:200) so a significant percentage of AD patients can be expected to carry this mutation. Heterozygotes for this mutation also have an increased, but sub-clinical iron burden. Given the high percentage of the population who are at significant risk for iron overload, we propose that the hemochromatosis mutation be considered as a confounding factor when evaluating the contribution of genetic associations with AD and treatment strategies and efficacy. Two recent papers and new evidence presented here that the protein associated with hemochromatosis is expressed on blood vessels, choroid plexus and the ependymal cells in the brain are offered as support for this proposal.

Lysozymelike activity in the hemolymph of Biomphalaria glabrata challenged with bacteria

Abstract Levels of lysozyme activity have been determined in the serum and cells of untreated Biomphalaria glabrata and in snails that had been challenged with heat-killed Bacillus megaterium and water at 1, 2, and 4 hr postinjection. Lysozyme activities have also been ascertained in sham-injected snails at 1, 2, and 4 hr postchallenge. Our results indicate significant alterations in the serum lysozyme activity levels at 2 and 4 hr postchallenge with bacteria and at 1 hr postinjection of water. Also, there is a significant increase in cell lysozyme activity at 1 hr postchallenge with B. megaterium . It is concluded that lysozyme is released from phagocytes into serum as a result of challenge with B. megaterium . Although the exact role of the released enzyme is uncertain, it is hypothesized that it may serve as a humoral defense molecule.

Allelic Associations between 100 DNA Markers and High versus Low IQ.

For DNA markers in or near genes of neurological relevance, allelic frequencies were compared for groups of White children high and low in IQ in an attempt to identify specific genes responsible for the substantial heritability of IQ scores. We previously reported results for 60 DNA markers and we now describe results for 40 additional markers. One sample consisted of high- and low-IQ groups with average IQs of 130 (N = 24) and 82 (N = 18), respectively. A replication sample was more extreme, including groups with average IQs of 142 (N = 27) and 59 (N = 17). Three of the 40 markers yielded significant allelic frequency differences between the high- and low-IQ groups in the original sample. In the replication sample, two of these markers (alcohol dehydrogenase 5 and the beta polypeptide of nerve growth factor) yielded results in the same direction but were not significant. The third marker (EST00083), derived from a cDNA hippocampal library, was also significant in the replication sample. As described in another article (Skuder et al., 1995) in this issue, this marker was found to involve mitochondrial DNA (mtDNA) rather than nuclear DNA. The unexpected nature of this marker suggests caution in claiming that the replicated association for EST00083 is indeed a quantitative trait loci (QTL) for IQ until the association receives additional support. This study provides statistical power to detect associations that account for about 2% of the IQ variance in the population. We are currently obtaining samples four times larger that will provide statistical power to detect allelic associations that account for considerably less than 1% of the variance.

Duodenal HFE expression and hepcidin levels determine body iron homeostasis: Modulation by genetic diversity and dietary iron availability

HFE affects the interaction of transferrin bound iron with transferrin receptors (TfR) thereby modulating iron uptake. To study genetically determined differences in HFE expression we examined individual HFE levels in C57BL/Sv129 mice and assessed their relationship to the regulation of iron homeostasis in the duodenum and the liver, and their regulation by diet. We found an up to 14-fold variation in inter-individual expression of HFE mRNA in the duodenum. Mice with high duodenal HFE mRNA expression presented with significantly higher levels of TfR and DMT-1 mRNAs and an increased IRP-1 binding affinity as compared to mice with low HFE levels. Duodenal HFE expression was positively associated with serum iron and liver HFE levels. Dietary iron supplementation decreased HFE in the duodenum but not in the liver. This was paralleled by reduced amounts of DMT-1 and FP-1 in the duodenum while the expression of DMT-1, FP-1, and hepcidin in the liver were increased with dietary iron overload. Duodenal and liver HFE levels are regulated by divergent penetration of as yet unelucidated modifier genes and to a much lesser extent by dietary iron. These measures control duodenal iron transport and liver iron homeostasis by modulating HFE expression either directly or via stimulation of iron sensitive regulatory molecules, such as hepcidin, which then exert their effects on body iron homeostasis.

Precise localisation of 3p25 breakpoints in four patients with the 3p-syndrome.

In patients with the 3p-syndrome, hemizygous deletion of 3p25-pter is associated with profound growth failure, characteristic facial features, and mental retardation. We performed a molecular genetic analysis of 3p25 breakpoints in four patients with the 3p- syndrome, and a fifth patient with a more complex abnormality, 46,XY,der(3)t(3;?)(p25.3;?). EBV transformed lymphoblasts from each of the patients were initially characterised using fluorescent in situ hybridisation (FISH) and polymorphic microsatellite analyses. The 3p-chromosome from each patient was isolated from the normal chromosome 3 in somatic cell hybrid lines and subsequently analysed with polymorphic and monomorphic PCR amplifiable markers from 3p25. The analysis clearly shows that all five breakpoints are distinct. Furthermore, we have identified yeast artificial chromosomes that cross the 3p25 breakpoints of all four 3p-patients. Two of the patients were deleted for the von Hippel-Lindau (VHL) tumour suppressor gene, although neither has yet developed evidence of VHL disease. The patient with the most centromeric breakpoint, between D3S1585 and D3S1263, had the most severe clinical phenotype including an endocardial cushion defect that was not observed in any of the four patients who had more telomeric breakpoints. This study should provide useful insights into critical regions within 3p25 that are involved in normal human growth and development.

γδ intraepithelial lymphocytes drive tumor necrosis factor-α responsiveness to intestinal iron challenge: relevance to hemochromatosis

Summary: The dependence of intestinal epithelial cell (IEC) growth and differentiation on intraepithelial lymphocytes (IELs) expressing the gamma/delta (γδ) T‐cell receptor (TCR), suggested a potential role for γδ+ IELs in the regulation of iron absorption. We therefore examined the levels of hepatic iron and the IEL cytokine responses in C57BL/6J control and class I and TCR knockout lines (placed on a C57BL/6J genetic background) following the administration of supplemental dietary iron. The highest level of liver iron was found in the β2‐microglobulin knockout (β2m‐/‐) mice followed by the TCR‐δ knockout (TCRδ‐/‐) animals. TCR‐α knockout (TCRα‐/‐) and control animals did not differ in their iron levels. Liver iron loading correlated inversely with rhe ability of the mice to generate an IEL tumor necrosis factor (TNE)‐α response. These observations suggest a model in which IEC iron loading is communicated to IELs via the HFE class I protein. The result of this communication is the initiation of TNE‐α release by γδ+ IELs (sustained by macrophages and dendritic cells) contributing to the upregulation of ferritin expression and possibly to the normal maintenance of the IEC apoptotic pathway.

IL-4-induced Stat6 activities affect apoptosis and gene expression in breast cancer cells

IL-4-induced Stat6 signaling is active in a variety of cell types, including immune cells and cancer cells, and plays an important role in the regulation of gene expression. Using EMSA gel shift assay and an antibody to Stat6, we phenotyped two breast cancer cell lines, ZR-75-1 being active Stat6(high) phenotype and BT-20 being defective Stat6(null) phenotype, respectively. Breast cancer cells carrying Stat6(null) phenotype exhibited increased spontaneous apoptosis compared with those carrying Stat6(high) phenotype. Expression microarray analyses demonstrated that IL-4 upregulated CCL26, SOCS1, CISH, EGLN3, and SIDT1, and downregulated DUSP1, FOS, and FOSB, respectively, in these breast cancer cells. Among those genes, CCL26 and SOCS1 were known genes regulated by IL-4/Stat6 pathway, but CISH, EGLN3, SIDT1, DUSP1, FOS, and FOSB were novel genes demonstrated to be IL-4 responsive for the first time. IL-4 also upregulated 38 genes unique to Stat6(null) BT-20 cells and 23 genes unique to Stat6(high) ZR-75-1 cells, respectively. Furthermore, Stat6(high) and Stat6(null) cells showed very different profiles of constitutively expressed genes relevant to apoptosis and metastasis among others, which serve as a valuable expression database and warrant for detailed studies of IL-4/Stat6 pathway in breast cancer.