Dysregulated hematopoiesis and a progressive neurological disorder induced by expression of an activated form of the human common beta chain in transgenic mice

Previously we described activating mutations of h beta(c), the common signaling subunit of the receptors for the hematopoietic and inflammatory cytokines, GM-CSF, IL-3, and IL-5. The activated mutant, h beta(c)FI Delta, is able to confer growth factor-independent proliferation on the murine myeloid cell line FDC-P1, and on primary committed myeloid progenitors. We have used this activating mutation to study the effects of chronic cytokine receptor stimulation. Transgenic mice were produced carrying the h beta(c)FI Delta cDNA linked to the constitutive promoter derived from the phosphoglycerate kinase gene, PGK-1. Transgene expression was demonstrated in several tissues and functional activity of the mutant receptor was confirmed in hematopoietic tissues by the presence of granulocyte macrophage and macrophage colony-forming cells (CFU-GM and CFU-M) in the absence of added cytokines. All transgenic mice display a myeloproliferative disorder characterized by splenomegaly, erythrocytosis, and granulocytic and megakaryocytic hyperplasia. This disorder resembles the human disease polycythemia vera, suggesting that activating mutations in h beta(c) may play a role in the pathogenesis of this myeloproliferative disorder. In addition, these transgenic mice develop a sporadic, progressive neurological disease and display bilateral, symmetrical foci of necrosis in the white matter of brain stem associated with an accumulation of macrophages. Thus, chronic h beta(c) activation has the potential to contribute to pathological events in the central nervous system.

Differentiation of the mononuclear phagocyte system during mouse embryogenesis: The role of transcription factor PU.1

During mouse embryogenesis, macrophage-like cells arise first in the yolk sac and are produced subsequently in the liver. The onset of liver hematopoiesis is associated with the transition from primitive to definitive erythrocyte production. This report addresses the hypothesis that a similar transition in phenotype occurs in myelopoiesis. We have used whole mount in situ hybridization to detect macrophage-specific genes expressed during mouse development. The mouse c-fms mRNA, encoding the receptor for macrophage colony-stimulating factor (CSF-1), was expressed on phagocytic cells in the yolk sac and throughout the embryo before the onset of liver hematopoiesis, Similar cells were detected using the mannose receptor, the complement receptor (CR3), or the Microphthalmia transcription factor (MITF) as mRNA markers. By contrast, other markers including the F4/80 antigen, the macrophage scavenger receptor, the S-100 proteins, S100A8 and S100A9, and the secretory product lysozyme appeared later in development and appeared restricted to only a subset of c-fms-positive cells. Two-color immunolabeling on disaggregated cells confirmed that CR3 and c-fms proteins are expressed on the same cells. Among the genes appearing later in development was the macrophage-restricted transcription factor, PU.1, which has been shown to be required for normal adult myelopoiesis. Mice with null mutations in PU.1 had normal numbers of c-fms-positive phagocytes at 11.5dpc. PU.1(-/-) embryonic stem cells were able to give rise to macrophagelike cells after cultivation in vitro. The results support previous evidence that yolk sac-derived fetal phagocytes are functionally distinct from those arising in the liver and develop via a different pathway. (C) 1999 by The American Society of Hematology.

The murine cytomegalovirus chemokine homolog, m131/129, is a determinant of viral pathogenicity

Chemokines are important mediators of the early inflammatory response to infection and modify a wide range of host immune responses. Functional homologs of cellular chemokines have been identified in a number of herpesviruses, suggesting that the subversion of the host chemokine response contributes to the pathogenesis of these viruses. Transcriptional and reverse transcription-PCR analyses demonstrated that the murine cytomegalovirus (MCMV) chemokine homolog, m131, was spliced at the 3' end to the adjacent downstream open reading frame, m129, resulting in a predicted product of 31 kDa, which is significantly larger than most known chemokines. The in vivo impact of m131/129 was investigated by comparing the replication of MCMV mutants having m131/129 deleted (Delta m131/129) with that of wild-type (wt) MCMV. Our studies demonstrate that both wt and Delta m131/129 viruses replicated to equivalent levels during the first 2 to 3 days following in vivo infection. However, histological studies demonstrated that the early inflammatory response elicited by Delta m131/129 was reduced compared with that of wt MCMV. Furthermore, the Delta m131/129 mutants failed to establish a high-titer infection in the salivary glands, These results suggest that m131/129 possesses proinflammatory properties in vivo and is important for the dissemination of MCMV to or infection of the salivary gland. Notably, the Delta m131/129 mutants were cleared more rapidly from the spleen and liver during acute infection compared with wt MCMV. The accelerated clearance of the mutants was dependent on NK cells and cells of the CD4(+) CD8(+) phenotype. These data suggest that m131/129 may also contribute to virus mechanisms of immune system evasion during early infection, possibly through the interference of NK cells and T cells.

Localization of N-acetyltransferases NAT1 and NAT2 in human tissues

Human acetyl coenzyme A-dependent N-acetyltransferase (EC 2.3.1.5) (NAT) catalyzes the biotransformation of a number of arylamine and hydrazine compounds. NAT isozymes are encoded at 2 loci; one encodes NAT1, formerly known as the monomorphic form of the enzyme, while the other encodes the polymorphic NAT2, which is responsible for individual differences in the ability to acetylate certain compounds. Human epidemiological studies have suggested an association between the acetylator phenotype and particular cancers such as those of the bladder and colon. In the present study, NAT1- and NAT2-specific riboprobes were used in hybridization histochemistry studies to localize NAT1 and NAT2 mRNA sequences in formalin-fixed, paraffin-embedded human tissue sections. Expression of both NAT1 and NAT2 mRNA was observed in liver, gastrointestinal tract tissues (esophagus, stomach, small intestine, and colon), ureter, bladder, and lung. In extrahepatic tissues, NAT1 and NAT2 mRNA expression was localized to intestinal epithelial cells, urothelial cells, and the epithelial cells of the respiratory bronchioles. The observed heterogeneity of NAT1 and NAT2 mRNA expression between human tissue types may be of significance in assessing their contribution to known organ-specific toxicities of various arylamine drugs and carcinogens.

A model for assembly and activation of the GM-CSF, IL-3 and IL-5 receptors: Insights from activated mutants of the common beta subunit

Granulocyte-macrophage colony stimulating factor (GM-CSF), Interleukin-3 (IL-3) and Interleukin-5 (IL-5) have overlapping, pleiotropic effects on hematopoietic cells, including neutrophils, eosinophils, monocytes and early progenitor cells. The high-affinity receptors for human GM-CSF, IL-3, and IL-5 share a common beta-subunit (h beta(c)), which is essential for signalling and plays a major role in recruiting intracellular signalling molecules. While activation of the cytoplasmic tyrosine kinase JAK2 appears to be the initiating event for signalling, the immediate events that trigger this are still unclear. We have isolated a number of activated mutants of h beta(c), which can be grouped into classes defined by their state of receptor phosphorylation, their requirement for alpha subunit as a cofactor, and their activities in primary cells and cell lines. We discuss these findings with regard to the stoichiometry, activation, and signalling of the normal GM-CSF/IL-3/IL-5 receptor complexes. Specifically, this work has implications for the role of the ligand-specific alpha-subunits in initiating the signalling through the beta-subunit, the role of beta subunit dimerization as a receptor trigger, and the function of receptor tyrosine phosphorylation in generating growth and survival signals. Based on the properties of the activated mutants and the recent structures of erythropoietin receptor (Epo-R) complexes, we propose a model in which (1) activation of h beta(c) can occur via alternative states that differ with respect to stoichiometry and subunit assembly, but which all mediate proliferative responses, and (2) each of the different classes of activated mutants mimics one of these alternative states. (C) 2000 International Society for Experimental Hematology. Published by Elsevier Science Inc.

Vascular smooth muscle and arterial calcification

Smooth muscle cultures can calcify under certain circumstances. As a model system these cultures therefore provide information on why calcification occurs in atherosclerotic plaques. Whether all smooth muscle cells (under certain conditions), or only specific populations, can produce this mineralization has not been resolved. Demer's group has cloned calcifying vascular cells from subcultured bovine aorta and studied them in detail. They have speculated on whether the cells are smooth muscle which have altered in phenotype, or whether they are derived from a stem cell population within the artery wall. The article argues that while the normal process of smooth muscle phenotypic modulation seen in arterial repair could account for the observations, this view may be two simplistic considering the complex nature of the artery wall. Certainly there is evidence for heterogeneity of smooth muscle cells in the artery wall and recent evidence suggests that stem cells can circulate in the blood and repopulate tissues. Further studies are required to resolve the important question as to the origin of cells which produce mineralization in atheroma.

Severity of hypertension in familial hyperaldosteronism type I: Relationship to gender and degree of biochemical disturbance

In familial hyperaldosteronism type I (FH-I), inheritance of a hybrid 11 beta-hydroxylase/aldosterone synthase gene causes ACTH-regulated aldosterone overproduction. In an attempt to understand the marked variability in hypertension severity in FH-I, we compared clinical and biochemical characteristics of 9 affected individuals with mild hypertension (normotensive or onset of hypertension after 15 yr, blood pressure never >160/100 mm Hg, less than or equal to 1 medication required to control hypertension, no history of stroke, age >18 yr when studied) with those of 17 subjects with severe hypertension (onset before 15 yr, or systolic blood pressure >180 mm Hg or diastolic blood pressure >120 mm Hg at least once, or greater than or equal to 2 medications, or history of stroke). Severe hypertension was more frequent in males (11 of 13 males vs. 6 of 13 females; P

Gene replacement with the human BRCA1 locus: tissue specific expression and rescue of embryonic lethality in mice

We have generated transgenic mice that harbor a 140 kb genomic fragment of the human BRCA1 locus (TgN.BRCA1(GEN)). We find that the transgene directs appropriate expression of human BRCA1 transcripts in multiple mouse tissues, and that human BRCA1 protein is expressed and stabilized following exposure to DIVA damage, Such mice are completely normal, with no overt signs of BRCA1 toxicity commonly observed when BRCA1 is expressed from heterologous promoters. Most importantly, however, the transgene rescues the otherwise lethal phenotype associated with the targeted hypomorphic allele (Brca1(Delta exIISA)). Brca1(-/-); TgN.BRCA1(GEN) bigenic animals develop normally and can be maintained as a distinct line. These results show that a 140 kb fragment of chromosome 17 contains all elements necessary for the correct expression, localization, and function of the BRCA1 protein, Further, the model provides evidence that function and regulation of the human BRCA1 gene can be studied and manipulated in a genetically tractable mammalian system.

Does maintaining green leaf area in sorghum improve yield under drought? I. Leaf growth and senescence

Production of sorghum [Sorghum bicolor (L.) Moench], an important cereal crop in semiarid regions of the world, is often limited by drought. When water is limiting during the grain-filling period, hybrids possessing the stay-green trait maintain more photosynthetically active leaves than hybrids not possessing this trait. To improve yield under drought, knowledge of the extent of genetic variation in green leaf area retention is required. Field studies were undertaken in north-eastern Australia on a cracking and self-mulching gray clay to determine the effects of water regime and hybrid on the components of green leaf area at maturity (GLAM). Nine hybrids varying in stay-green were grown under a fully irrigated control, postflowering water deficit, and terminal (pre- and postflowering) water deficit. Water deficit reduced GLAM by 67% in the terminal drought treatment compared with the fully irrigated control. Under terminal water deficit, hybrids possessing the B35 and KS19 sources of stay-green retained more GLAM (1260 cm(2) plant(-1)) compared with intermediate (780 cm(2) plant(-1)) and senescent (670 cm(2) plant(-1)) hybrids. RQL12 hybrids (KS19 source of stay-green) displayed delayed onset and reduced rate of senescence; A35 hybrids displayed only delayed onset. Visual rating of green leaf retention was highly correlated with measured GLAM, although this procedure is constrained by an inability to distinguish among the functional mechanisms determining the phenotype. Linking functional rather than phenotypic differences to molecular markers may improve the efficiency of selecting for traits such as stay-green.

Sporadic and familial pheochromocytomas are associated with loss of at least two discrete intervals on chromosome 1p

Pheochromocytomas are tumors of the adrenal medulla originating in the chromaffin cells derived from the neural crest. Ten % of these tumors are associated with the familial cancer syndromes multiple endocrine neoplasia type 2, von Hippel-Lindau disease (VHL), and rarely, neurofibromatosis type 1, in which germ-line mutations have been identified in RET, VHL, and NF1, respectively. In both the sporadic and familial forms of pheochromocytoma, allelic loss at 1p, 3p, 17p, and 22q has been reported, yet the molecular pathogenesis of these tumors is largely unknown. Allelic loss at chromosome 1p has also been reported in other endocrine tumors, such as medullary thyroid cancer and tumors of the parathyroid gland, as well as in tumors of neural crest origin including neuroblastoma and malignant melanoma, In this study, we performed fine structure mapping of deletions at chromosome 1p in familial and sporadic pheochromocytomas to identify discrete regions likely housing tumor suppressor genes involved in the development of these tumors. Ten microsatellite markers spanning a region of similar to 70 cM (Ipter to 1p34.3) were used to screen 20 pheochromocytomas from 19 unrelated patients for loss of heterozygosity (LOH). LOH was detected at five or more loci in 8 of 13 (61%)sporadic samples and at five or more loci in four of five (80%) tumor samples from patients with multiple endocrine neoplasia type 2. No LOH at 1p was detected in pheochromocytomas from two VHL patients, Analysis of the combined sporadic and familial tumor data suggested three possible regions of common somatic loss, designated as PCI (D1S243 to D1S244), PC2 (D1S228 to D1S507), and PC3 (D1S507 toward the centromere). We propose that chromosome Ip may be the site of at least three putative tumor suppressor loci involved in the tumorigenesis of pheochromocytomas. At least one of these loci, PC2 spanning an interval of <3.8 cM, is Likely to have a broader role in the development of endocrine malignancies.

Allele and genotype frequencies of polymorphic cytochromes P4502D6, 2C19 and 2E1 in Aborigines from Western Australia

The polymorphisms of the important xenobiotic metabolizing enzymes CYP2D6, CYP2C19 and CYP2E1 have been studied extensively in a large number of populations and show significant heterogeneity in the frequency of different alleles/genotypes and in the prevalence of the extensive and poor metabolizer phenotypes, Understanding of inter-ethnic differences in genotypes is important in prediction of either beneficial or adverse effects from therapeutic agents and other xenobiotics. Since no data were available for Australian Aborigines, we investigated the frequencies of alleles and genotypes for CYP2D6, CYP2C19 and CYP2E1 in a population living in the far north of Western Australia. Because of its geographical isolation, this population can serve as a model to study the impact of evolutionary forces on the distribution of different alleles for xenobiotic metabolizing enzymes. Twelve CYP2D6 alleles were analysed, The wild-type allele *1 was the most frequent (85.8%) and the non-functional alleles (*4, *5, *16) had an overall frequency of less than 10%. Only one subject (0.4%) was a poor metabolizer for CYP2D6 because of the genotype *5/*5, For CYP2C19, the frequencies of the *1 (wild-type) and the non-functional (*2 and *3) alleles were 50.2%, 35.5% and 14.3%, respectively. The combined CYP2C19 genotypes (*2/*2, *2/*3 or *3/*3) correspond to a predicted frequency of 25.6% for the CYP2C19 poor metabolizer phenotype, For CYP2E1, only one subject had the rare c2 allele giving an overall allele frequency of 0.2%. For CYP2D6 and CYP2C19, allele frequencies and predicted phenotypes differed significantly from those for Caucasians but were similar to those for Orientals indicating a close relationship to East Asian populations. Differences between Aborigines and Orientals in allele frequencies for CYP2D6*10 and CYP2E1 c2 may have arisen through natural selection, or genetic drift, respectively, Pharmacogenetics 11:69-76 (C) 2001 Lippincott Williams & Wilkins.

Clinical and molecular genetics of myoclonic-astatic epilepsy and severe myoclonic epilepsy in infancy (Dravet syndrome)

The majority of severe epileptic encephalopathies of early childhood are symptomatic where a clear etiology is apparent. There is a small subgroup, however, where no etiology is found on imaging and metabolic studies, and genetic factors are important. Myoclonic-astatic epilepsy (MAE) and severe myoclonic epilepsy in infancy (SMEI), also known as Dravet syndrome, are epileptic encephalopathies where multiple seizure types begin in the first few years of life associated with developmental slowing. Clinical and molecular genetic studies of the families of probands with MAE and SMEI suggest a genetic basis. MAE was originally identified as part of the genetic epilepsy syndrome generalized epilepsy with febrile seizures plus (GEFS(+)). Recent clinical genetic studies suggest that SMEI forms the most severe end of the spectrum of the GEFS(+). GEF(+) has now been associated with molecular defects in three sodium channel subunit genes and a GABA subunit gene. Molecular defects of these genes have been identified in patients with MAE and SMEI. Interestingly, the molecular defects in MAE have been found in the setting of large GEFS(+) pedigrees, whereas, more severe truncation mutations arising de novo have been identified in patients with SMEI. It is likely that future molecular studies will shed light on the interaction of a number of genes, possibly related to the same or different ion channels, which result in a severe phenotype such as MAE and SMEI. (C) 2001 Elsevier Science B.V. All rights reserved.

Expression of RANTES and CCR1 in oral lichen planus and association with mast cell migration

Background: T lymphocytes and mast cells infiltrate the lamina propria in oral lichen planus (OLP). Chemokines and their receptors are involved in T cell and mast cell migration and accumulation during the inflammatory process. Methods: In the present study, we investigated the role of RANTES and its receptors in OLP using immunohistochemistry, RT-PCR and an in vitro chemotaxis assay. Results: RANTES and CCR1 were expressed on T cells and mast cells in OLP, while OLP lesional T cell supernatants stimulated CCR1 mRNA expression in a human leukemia mast cell line (HMC-1). TNF-alpha stimulated CCR1, CCR4 and CCR5 mRNA expression in the same cell line. OLP lesional T cell supernatants stimulated HMC-1 migration, which was partly inhibited by anti-RANTES antibody. Conclusions: The present study shows, for the first time, the distribution of RANTES and CCR1 in OLR It is hypothesized that RANTES and CCR1 may play important roles in mast cell trafficking and related events in OLP.

Increased generation of dendritic cells from myeloid progenitors in autoimmune-prone nonobese diabetic mice

Aberrant dendritic cell (DC) development and function may contribute to autoimmune disease susceptibility. To address this hypothesis at the level of myeloid lineage-derived DC we compared the development of DC from bone marrow progenitors in vitro and DC populations in vivo in autoimmune diabetes-prone nonbese diabetic (NOD) mice, recombinant congenic nonbese diabetes-resistant (NOR) mice, and unrelated BALB/c and C57BL/6 (BL/6) mice. In GM-CSF/IL-4-supplemented bone marrow cultures, DC developed in significantly greater numbers from NOD than from NOR, BALB/c, and BL/6 mice. Likewise, DC developed in greater numbers from sorted (lineage(-)IL-7Ralpha(-)SCA-1(-)c-kit(+)) NOD myeloid progenitors in either GM-CSF/IL-4 or GM-CSF/stem cell factor (SCF)/TNF-alpha. [H-3]TdR incorporation indicated that the increased generation of NOD DC was due to higher levels of myeloid progenitor proliferation. Generation of DC with the early-acting hematopoietic growth factor, flt3 ligand, revealed that while the increased DC-generative capacity of myeloid-committed progenitors was restricted to NOD cells, early lineage-uncommitted progenitors from both NOD and NOR had increased DC-gencrative capacity relative to BALB/c and BL/6. Consistent with these findings, NOD and NOR mice had increased numbers of DC in blood and thymus and NOD had an increased proportion of the putative myeloid DC (CD11c(+)CD11b(+)) subset within spleen. These findings demonstrate that diabetes-prone NOD mice exhibit a myeloid lineage-specific increase in DC generative capacity relative to diabetes-resistant recombinant congenic NOR mice. We propose that an imbalance favoring development of DC from myeloid-committed progenitors predisposes to autoimmune disease in NOD mice.

Immunobiology of liver dendritic cells

Dendritic cells (DC) are rare, bone marrow-derived antigen-presenting cells that play a critical role in the induction and regulation of immune reactivity. In this article, we review the identification and characterization of liver DC, their ontogenic development, in vivo mobilization and population dynamics. In addition, we discuss the functions of DC isolated from liver tissue or celiac lymph, or propagated in vitro from liver-resident haemopoietic stem/progenitor cells. Evidence concerning the role of DC in viral hepatitis. liver tumours, autoimmune liver diseases, granulomatous inflammation and the outcome of liver transplantation is also discussed.