Rel-deficient T cells exhibit defects in production of interleukin 3 and granulocyte-macrophage colony-stimulating factor.

The c-rel protooncogene encodes a subunit of the NF-kappa B-like family of transcription factors. Mice lacking Rel are defective in mitogenic activation of B and T lymphocytes and display impaired humoral immunity. In an attempt to identify changes in gene expression that accompany the T-cell stimulation defects associated with the loss of Rel, we have examined the expression of cell surface activation markers and cytokine production in mitogen-stimulated Rel-/- T cells. The expression of cell surface markers including the interleukin 2 receptor alpha (IL-2R alpha) chain (CD25), CD69 and L-selectin (CD62) is normal in mitogen-activated Rel-/- T cells, but cytokine production is impaired. In Rel-/- splenic T cell cultures stimulated with phorbol 12-myristate 13-acetate and ionomycin, the levels of IL-3, IL-5, granulocyte- macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor alpha (TNF-alpha), and gamma interferon (IFN-gamma) were only 2- to 3-fold lower compared with normal T cells. In contrast, anti-CD3 and anti-CD28 stimulated Rel-/- T cells, which fail to proliferate, make little or no detectable cytokines. Exogenous IL-2, which restitutes the proliferative response of the anti-CD3- and anti-CD28-treated Rel-/- T cells, restores production of IL-5, TNF-alpha, and IFN-gamma, but not IL-3 and GM-CSF expression to approximately normal levels. In contrast to mitogen-activated Rel-/- T cells, lipopolysaccharide-stimulated Rel-/- macrophages produce higher than normal levels of GM-CSF. These findings establish that Rel can function as an activator or repressor of gene expression and is required by T lymphocytes for production of IL-3 and GM-CSF.

Cytochrome P450 1A1 promoter as a genetic switch for the regulatable and physiological expression of a plasma protein in transgenic mice.

Transgenic and gene knockout techniques allow for in vivo study of the consequences of adding or subtracting specific genes. However, in some instances, such as the study of lethal mutations or of the physiological consequences of changing gene expression, turning on and off an introduced gene at will would be advantageous. We have used cytochrome p450 1A1 promoter to drive expression of the human apolipoprotein E (apoE) gene in transgenic mice. In six independent lines, robust expression of the transgene depended upon injection of the inducer beta-naphthoflavone, whereas the seventh line had high basal expression that was augmented further by the inducer. The low level of basal expression in an inducer-dependent line was confirmed upon breeding the transgene onto the hypercholesterolemic apoE-deficient background. In the basal state transgene expression was physiologically insignificant, as these mice were as hypercholesterolemic as their nontransgenic apoE-deficient littermates. When injected with the inducer, plasma cholesterol levels of the transgenic mice decreased dramatically as apoE expression was induced to yield greater than physiological levels in plasma. The inducer could pass transplacentally from an injected mother to her fetuses with concomitant induction of fetal transgene mRNA. Inducer could also pass via breast milk from an injected mother to her suckling neonatal pups, giving rise to the induction of human apoE in neonate plasma. These finding suggest a strategy to temporarily ameliorate genetic deficiencies that would otherwise lead to fetal or neonatal lethality.

Attenuated virulence of chitin-deficient mutants of Candida albicans.

We have analyzed the role of chitin, a cell-wall polysaccharide, in the virulence of Candida albicans. Mutants with a 5-fold reduction in chitin were obtained in two ways: (i) by selecting mutants resistant to Calcofluor, a fluorescent dye that binds to chitin and inhibits growth, and (ii) by disrupting CHS3, the C. albicans homolog of CSD2/CAL1/DIT101/KT12, a Saccharomyces cerevisiae gene required for synthesis of approximately 90% of the cell-wall chitin. Chitin-deficient mutants have no obvious alterations in growth rate, sugar assimilation, chlamydospore formation, or germ-tube formation in various media. When growing vegetatively in liquid media, the mutants tend to clump and display minor changes in morphology. Staining of cells with the fluorescent dye Calcofluor indicates that CHS3 is required for synthesis of the chitin rings found on the surface of yeast cells but not formation of septa in either yeast cells or germ tubes. Despite their relatively normal growth, the mutants are significantly less virulent than the parental strain in both immunocompetent and immunosuppressed mice; at 13 days after infection, survival was 95% in immunocompetent mice that received chs3/chs3 cells and 10% in immunocompetent mice that received an equal dose of chs3/CHS3 cells. Chitin-deficient strains can colonize the organs of infected mice, suggesting that the reduced virulence of the mutants is not due to accelerated clearing.

Transgenic mice carrying the diphtheria toxin A chain gene under the control of the granzyme A promoter: expected depletion of cytotoxic cells and unexpected depletion of CD8 T cells.

We have generated transgenic mice bearing the diphtheria toxin A chain (DTA) gene under the control of granzyme A (GrA) promoter sequences (GrA-DTA). GrA is expressed in activated cytotoxic cells but not in their immediate progenitors. These GrA-DTA mice are deficient in cytotoxic functions, indicating that most cytotoxic cells express GrA in vivo. Surprisingly, one founder strain containing a multicopy GrA-DTA insert show a marked and selective deficiency in CD8+ cells in peripheral lymphoid organs. This depletion was not observed in thymus, where the distribution of CD4+ and CD8+ cells is normal. Moreover, the emigration of T cells from thymus is normal, indicating that the depletion occurs in the periphery. GrA-DTA mice should be useful as models to dissect the role of cytotoxic cells in immune responses and as recipients of normal and neoplastic hematopoietic cells. The selective depletion of CD8+ cells in one founder strain could have implications for postthymic T-cell development.

Mice develop normally without the H1(0) linker histone.

H1 histones bind to the linker DNA between nucleosome core particles and facilitate the folding of chromatin into a 30-nm fiber. Mice contain at least seven nonallelic subtypes of H1, including the somatic variants H1a through H1e, the testis-specific variant H1t, and the replacement linker histone H1(0). H1(0) accumulates in terminally differentiating cells from many lineages, at about the time when the cells cease dividing. To investigate the role of H1(0) in development, we have disrupted the single-copy H1(0) gene by homologous recombination in mouse embryonic stem cells. Mice homozygous for the mutation and completely lacking H1(0) mRNA and protein grew and reproduced normally and exhibited no anatomic or histologic abnormalities. Examination of tissues in which H1(0) is normally present at high levels also failed to reveal any abnormality in cell division patterns. Chromatin from H1(0)-deficient animals showed no significant change in the relative proportions of the other H1 subtypes or in the stoichiometry between linker histones and nucleosomes, suggesting that the other H1 histones can compensate for the deficiency in H1(0) by occupying sites that normally contain H1(0). Our results indicate that despite the unique properties and expression pattern of H1(0), its function is dispensable for normal mouse development.

Initial hepatic removal of chylomicron remnants is unaffected but endocytosis is delayed in mice lacking the low density lipoprotein receptor.

Two endocytic receptors, the low density lipoprotein (LDL) receptor (LDLR) and the LDLR-related protein (LRP), are thought to act in concert in the hepatic uptake of partially metabolized dietary lipoproteins, the chylomicron remnants. We have evaluated the role of these two receptors in the hepatic metabolism of chylomicron remnants in normal mice and in LDLR-deficient [LDLR (-/-)] mice. The rate of chylomicron remnant removal by the liver was normal up to 30 min after intravenous injection of chylomicrons into LDLR (-/-) mice and was unaffected by receptor-associated protein (RAP), a potent inhibitor of ligand binding to LRP. In contrast, endocytosis of the remnants by the hepatocytes, measured by their accumulation in the endosomal fraction and by the rate of hydrolysis of component cholesteryl esters, was dramatically reduced in the absence of the LDLR. Coadministration of RAP prevented the continuing hepatic removal of chylomicron remnants in LDL (-/-) mice after 30 min, consistent with blockade of the slow endocytosis by a RAP-sensitive process. Taken together with previous studies, our results are consistent with a model in which the initial hepatic removal of chylomicron remnants is primarily mediated by mechanisms that do not include LDLR or LRP, possibly involving glycosaminoglycan-bound hepatic lipase and apolipoprotein E. After the remnants bind to these alternative sites on the hepatocyte surface, endocytosis is predominantly mediated by the LDLR and also by a slower and less efficient backup process that is RAP sensitive and therefore most likely involves LRP.

Disruption of the adenosine deaminase gene causes hepatocellular impairment and perinatal lethality in mice.

We have generated mice with a null mutation at the Ada locus, which encodes the purine catabolic enzyme adenosine deaminase (ADA, EC 3.5.4.4). ADA-deficient fetuses exhibited hepatocellular impairment and died perinatally. Their lymphoid tissues were not largely affected. Accumulation of ADA substrates was detectable in ADA-deficient conceptuses as early as 12.5 days postcoitum, dramatically increasing during late in utero development, and is the likely cause of liver damage and fetal death. The results presented here demonstrate that ADA is important for the homeostatic maintenance of purines in mice.

GLUT4 overexpression or deficiency in adipocytes of transgenic mice alters the composition of GLUT4 vesicles and the subcellular localization of GLUT4 and insulin-responsive aminopeptidase

The majority of GLUT4 is sequestered in unique intracellular vesicles in the absence of insulin. Upon insulin stimulation GLUT4 vesicles translocate to, and fuse with, the plasma membrane. To determine the effect of GLUT4 content on the distribution and subcellular trafficking of GLUT4 and other vesicle proteins, adipocytes of adipose-specific, GLUT4-deficient (aP2-GLUT4-/-) mice and adipose-specific, GLUT4-overexpressing (aP2GLUT4- Tg) mice were studied. GLUT4 amount was reduced by 80 - 95% in aP2-GLUT4-/- adipocytes and increased similar to10-fold in aP2-GLUT4-Tg adipocytes compared with controls. Insulin-responsive aminopeptidase ( IRAP) protein amount was decreased 35% in aP2-GLUT4-/- adipocytes and increased 45% in aP2-GLUT4-Tg adipocytes. VAMP2 protein was also decreased by 60% in aP2-GLUT4-/- adipocytes and increased 2-fold in aP2GLUT4- Tg adipocytes. IRAP and VAMP2 mRNA levels were unaffected in aP2-GLUT4-Tg, suggesting that overexpression of GLUT4 affects IRAP and VAMP2 protein stability. The amount and subcellular distribution of syntaxin4, SNAP23, Munc-18c, and GLUT1 were unchanged in either aP2-GLUT4-/- or aP2-GLUT4-Tg adipocytes, but transferrin receptor was partially redistributed to the plasma membrane in aP2-GLUT4-Tg adipocytes. Immunogold electron microscopy revealed that overexpression of GLUT4 in adipocytes increased the number of GLUT4 molecules per vesicle nearly 2-fold and the number of GLUT4 and IRAP-containing vesicles per cell 3-fold. In addition, the proportion of cellular GLUT4 and IRAP at the plasma membrane in unstimulated aP2-GLUT4-Tg adipocytes was increased 4- and 2-fold, respectively, suggesting that sequestration of GLUT4 and IRAP is saturable. Our results show that GLUT4 overexpression or deficiency affects the amount of other GLUT4-vesicle proteins including IRAP and VAMP2 and that GLUT4 sequestration is saturable.

Influence of growth hormone on the craniofacial complex of transgenic mice

Growth hormone (GH) secretion affects bone and cartilage physiology. This study investigated the effect of GH on the size of the craniofacial structures and their angular relationship. Three different models of mice with a genetically altered GH axis were used: GH excess (giant), dwarf GH antagonist (dwarf-Ant), and dwarf GH receptor knockout (dwarf-KO) mice. Each model was compared with the corresponding wild type (Wt). Five craniofacial distances were analysed: craniofacial length, upper face height, mandibular anterior height, mandibular ramus length, and mandibular corpus length. In addition, upper and lower incisor lengths and four angular relationships, nasal bone with cranial base, maxillary plane with cranial base, mandibular plane with cranial base, and the angle of the mandible, were determined. Data were analysed by one-way ANOVA. Craniofacial length, upper face height and mandibular corpus length were significantly increased in the giant mice and significantly reduced in the dwarf mice. Mandibular anterior height and mandibular ramus length were significantly affected in the dwarf-KO mice but not in the giant mice. The length of both the upper and lower incisors was significantly increased and reduced in the giant and dwarf-KO mice, respectively. In addition, the angle of the mandible was significantly increased in the giant mice and significantly reduced in the dwarf mice. It is concluded that GH plays a major role in the growth and development of the craniofacial complex by directly and indirectly modulating the size and the angular relationships of the craniofacial structures, including the incisor teeth.

Conditional deletion of hypothalamic Y2 receptors reverts gonadectomy-induced bone loss in adult mice

Reduction in levels of sex hormones at menopause in women is associated with two common, major outcomes, the accumulation of white adipose tissue, and the progressive loss of bone because of excess osteoclastic bone resorption exceeding osteoblastic bone formation. Current antiresorptive therapies can reduce osteoclastic activity but have only limited capacity to stimulate osteoblastic bone formation and restore lost skeletal mass. Likewise, the availability of effective pharmacological weight loss treatments is currently limited. Here we demonstrate that conditional deletion of hypothalamic neuropeptide Y2 receptors can prevent ongoing bone loss in sex hormone-deficient adult male and female mice. This benefit is attributable solely to activation of an anabolic osteoblastic bone formation response that counterbalances persistent elevation of bone resorption, suggesting the Y2-mediated anabolic pathway to be independent of sex hormones. Furthermore, the increase in fat mass that typically occurs after ovariectomy is prevented by germ line deletion of Y2 receptors, whereas in male mice body weight and fat mass were consistently lower than wild-type regardless of sex hormone status. Therefore, this study indicates a role for Y2 receptors in the accumulation of adipose tissue in the hypogonadal state and demonstrates that hypothalamic Y2 receptors constitutively restrain osteoblastic activity even in the absence of sex hormones. The increase in bone formation after release of this tonic inhibition suggests a promising new avenue for osteoporosis treatment.

Expression profiling of type 2 diabetes susceptibility genes in the pancreatic islets, adipose tissue and liver of obese mice

Type 2 diabetes (T2D) is characterized by impaired beta cell function and insulin resistance. T2D susceptibility genes identified by Genome-wide association studies (GWAS) are likely to have roles in both impaired insulin secretion from the beta cell as well as insulin resistance. The aim of this study was to use gene expression profiling to assess the effect of the diabetic milieu on the expression of genes involved in both insulin secretion and insulin resistance. We measured the expression of 43 T2D susceptibility genes in the islets, adipose and liver of leptin-deficient Ob/Ob mice compared with Ob/+ littermates. The same panel of genes were also profiled in cultured rodent adipocytes, hepatocytes and beta cells in response to high glucose conditions, to distinguish expression effects due to elevated glycemia from those on the causal pathway to diabetes or induced by other factors in the diabetic microenviroment. We found widespread deregulation of these genes in tissues from Ob/Ob mice, with differential regulation of 23 genes in adipose, 18 genes in liver and one gene (Tcf7l2) in islets of diabetic animals (Ob/Ob) compared to control (Ob/+) animals. However, these expression changes were in most cases not noted in glucose-treated adipocyte, hepatocyte or beta cell lines, indicating that they may not be an effect of hyperglycemia alone. This study indicates that expression changes are apparent with diabetes in both the insulin producing beta cells, but also in peripheral tissues involved in insulin resistance. This suggests that incidence or progression of diabetic phenotypes in a mouse model of diabetes is driven by both secretory and peripheral defects. © J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart New York.

Understanding the Effect of Protein Tyrosine Phosphatase Receptor Type 0 on Mammalian Sensory Development: Behavioral Studies on PTPRO Knockout Mice

Nerve development, which includes axon outgrowth and guidance, is regulated by many protein families, including receptor protein tyrosine phosphatases (RPTP's).Protein tyrosine phosphatase receptor type 0 (PTPRO) is a type III RPTP that is important for axon growth and guidance, as observed in chicks and flies. In order to examine the effects ofPTPRO on mammalian development, standard behavioral tests were used to compare mice lacking the gene for PTPRO (ROKO mice) to wild-type (WT) mice. The ROKO mice showed a significant delay in reacting to a thermal noxious stimulus, hotplate analgesia, when compared to the WT mice suggesting deficient nociceptive function. In a rotarod test for proprioceptive function the ROKO mice exhibited a significant decrease in the amount of time spent on the rotating rod than did the WT mice. Additional proprioception tests were performed including the climb, step reflex, beam, and mesh walk tests. In the climb and step (place) test, the ROKO group had a significantly lower accuracy in performing the tests than did the WT mice. Thus, mice lacking the PTPRO gene showed behavioral deficiencies that reflect impairment in sensory function, specifically for nociception and proprioception.

iNOS-producing inflammatory dendritic cells constitute the major infected cell type during the chronic Leishmania major infection phase of C57BL/6 resistant mice.

Leishmania major parasites reside and multiply in late endosomal compartments of host phagocytic cells. Immune control of Leishmania growth absolutely requires expression of inducible Nitric Oxide Synthase (iNOS/NOS2) and subsequent production of NO. Here, we show that CD11b+ CD11c+ Ly-6C+ MHC-II+ cells are the main iNOS-producing cells in the footpad lesion and in the draining lymph node of Leishmania major-infected C57BL/6 mice. These cells are phenotypically similar to iNOS-producing inflammatory DC (iNOS-DC) observed in the mouse models of Listeria monocytogenes and Brucella melitensis infection. The use of DsRed-expressing parasites demonstrated that these iNOS-producing cells are the major infected population in the lesions and the draining lymph nodes. Analysis of various genetically deficient mouse strains revealed the requirement of CCR2 expression for the recruitment of iNOS-DC in the draining lymph nodes, whereas their activation is strongly dependent on CD40, IL-12, IFN-gamma and MyD88 molecules with a partial contribution of TNF-alpha and TLR9. In contrast, STAT-6 deficiency enhanced iNOS-DC recruitment and activation in susceptible BALB/c mice, demonstrating a key role for IL-4 and IL-13 as negative regulators. Taken together, our results suggest that iNOS-DC represent a major class of Th1-regulated effector cell population and constitute the most frequent infected cell type during chronic Leishmania major infection phase of C57BL/6 resistant mice.

Double incretin receptor knock-out (DIRKO) mice present with alterations of trabecular and cortical micromorphology and bone strength.

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Antiangiogenic And Finasteride Therapies: Responses Of The Prostate Microenvironment In Elderly Mice.

The aim of this study was to evaluate the structural and molecular effects of antiangiogenic therapies and finasteride on the ventral prostate of senile mice. 90 male FVB mice were divided into: Young (18 weeks old) and senile (52 weeks old) groups; finasteride group: finasteride (20mg/kg); SU5416 group: SU5416 (6 mg/kg); TNP-470 group: TNP-470 (15 mg/kg,) and SU5416+TNP-470 group: similar to the SU5416 and TNP-470 groups. After 21 days, prostate ventral lobes were collected for morphological, immunohistochemical and Western blotting analyses. The results demonstrated atrophy, occasional proliferative lesions and inflammatory cells in the prostate during senescence, which were interrupted and/or blocked by treatment with antiangiogenic drugs and finasteride. Decreased AR and endostatin reactivities, and an increase for ER-α, ER-β and VEGF, were seen in the senile group. Decreased VEGF and ER-α reactivities and increased ER-β reactivity were verified in the finasteride, SU5416 groups and especially in SU5416+TNP-470 group. The TNP-470 group showed reduced AR and ER-β protein levels. The senescence favored the occurrence of structural and/or molecular alterations suggesting the onset of malignant lesions, due to the imbalance in the signaling between the epithelium and stroma. The SU5416+TNP-470 treatment was more effective in maintaining the structural, hormonal and angiogenic factor balance in the prostate during senescence, highlighting the signaling of antiproliferation via ER-β.