Biomedical subjects

B H Koller

Publications and source records attributed to B H Koller.

At least 55 records · Page 3Linked to original sources

PubMed1998

Coagulation defects and altered hemodynamic responses in mice lacking receptors for thromboxane A2.

Thromboxane A2 (TXA2) is a labile metabolite of arachidonic acid that has potent biological effects. Its actions are mediated by G protein-coupled thromboxane-prostanoid (TP) receptors. TP receptors have been implicated in the pathogenesis of cardiovascular diseases. To investigate the physiological functions of TP receptors, we generated TP receptor-deficient mice by gene targeting. Tp-/- animals reproduce and survive in expected numbers, and their major organ systems are normal. Thromboxane agonist binding cannot be detected in tissues from Tp-/- mice. Bleeding times are prolonged in Tp-/- mice and their platelets do not aggregate after exposure to TXA2 agonists. Aggregation responses after collagen stimulation are also delayed, although ADP-stimulated aggregation is normal. Infusion of the TP receptor agonist U-46619 causes transient increases in blood pressure followed by cardiovascular collapse in wild-type mice, but U-46619 caused no hemodynamic effect in Tp-/- mice. Tp-/- mice are also resistant to arachidonic acid-induced shock, although arachidonic acid signifi-cantly reduced blood pressure in Tp-/- mice. In summary, Tp-/- mice have a mild bleeding disorder and altered vascular responses to TXA2 and arachidonic acid. Our studies suggest that most of the recognized functions of TXA2 are mediated by the single known Tp gene locus.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed1998

BRCA1 required for transcription-coupled repair of oxidative DNA damage.

The breast and ovarian cancer susceptibility gene BRCA1 encodes a zinc finger protein of unknown function. Association of the BRCA1 protein with the DNA repair protein Rad51 and changes in the phosphorylation and cellular localization of the protein after exposure to DNA-damaging agents are consistent with a role for BRCA1 in DNA repair. Here, it is shown that mouse embryonic stem cells deficient in BRCA1 are defective in the ability to carry out transcription-coupled repair of oxidative DNA damage, and are hypersensitive to ionizing radiation and hydrogen peroxide. These results suggest that BRCA1 participates, directly or indirectly, in transcription-coupled repair of oxidative DNA damage.

Alleles↗

PubMed1998

Allergy-associated FcRbeta is a molecular amplifier of IgE- and IgG-mediated in vivo responses.

A role for the Fc receptor beta chain (FcRbeta) in the pathogenesis of allergy has been suggested by genetic studies. FcRbeta is a subunit common to the high-affinity IgE (FcepsilonRI) and low-affinity IgG (FcgammaRIII) receptors, both of which contribute to the initiation of allergic reactions. Current in vitro data suggest that FcRbeta can function as either a positive or negative regulator, leaving a mechanistic explanation for its association with the development of atopy unclear. To address this controversy, we have generated novel mouse models relevant to human Fc receptor function. Analysis of FcepsilonRI- and FcgammaRIII-dependent responses in these mice provides unequivocal genetic evidence that FcRbeta functions as an amplifier of early and late mast cell responses and, remarkably, in vivo anaphylactic responses.

Anaphylaxis↗

PubMed1998

Urinary concentrating function in mice lacking EP3 receptors for prostaglandin E2.

The actions of prostaglandin (PG) E2 are mediated by four distinct classes of PGE2 E-prostanoid (EP) receptors (EP1 through EP4). However, the in vivo functions of the individual EP receptor subtypes have not been delineated. To study the functions of one of these subtypes, the EP3 receptor, we generated EP3-deficient (-/-) mice by gene targeting. EP3 -/- animals survived in expected numbers, reproduced, and had no obvious abnormalities in their major organ systems. Because the EP3 receptor is expressed at high levels in the renal medulla and cortical collecting duct, and because previous studies have suggested that the EP3 receptor might antagonize the effects of vasopressin in the distal nephron, we examined urinary concentrating functions in EP3 -/- mice. Basal urine osmolality (UOsm) was similar in groups of EP3 -/- and wild-type (EP3 +/+) mice. However, after inhibition of endogenous PGE2 production by indomethacin, UOsm increased significantly in EP3 +/+ but not in EP3 -/- mice. Despite this insensitivity to acute inhibition of prostanoid production, EP3 -/- mice concentrated and diluted their urine normally in response to a series of physiological stimuli. This suggests that PGE2 acts through the EP3 receptor to modulate urinary concentrating mechanisms in the kidney, but these effects are not essential for normal regulation of urinary osmolality.

Animals↗

PubMed1998

The relationship of chronic mucin secretion to airway disease in normal and CFTR-deficient mice.

In the cystic fibrosis (CF) patient, lung function decreases throughout life as a result of continuous cycles of infection, particularly with Pseudomonas aeruginosa and Staphylococcus aureus. The mechanism underlying the pathophysiology of the disease in humans has not been established. However, it has been suggested that abnormal, tenacious mucus, resulting perhaps from improper hydration from loss of Cl- secretion via the cystic fibrosis transmembrane conductance regulator (CFTR) protein, impairs clearance of bacteria from the CF airway and provides an environment favorable to bacterial growth. If this hypothesis is correct, it could explain the absence of respiratory disease in CFTR-deficient mice, since mice have only a single submucosal gland and display few goblet cells in their lower airways, even when exposed to bacteria. To test this hypothesis further, we induced allergic airway disease in CFTR-deficient mice. We found that induction of allergic airway disease in mice, unlike bacterial infection, results in an inflammatory response characterized by goblet cell hyperplasia, increased mucin gene expression, and increased production of mucus. However, we also found that disease progression and resolution is identical in Cftr-/- mice and control animals. Furthermore, we show that the presence of mucus in the Cftr-/- airway does not lead to chronic airway disease, even upon direct inoculation with S. aureus and P. aeruginosa. Therefore, factors in addition to the absence of high levels of mucus secretion protect the mouse from the airway disease seen in human CF patients.

Animals↗

PubMed1998

Characterization of Brca1 deficient mice.

BRCA1 is a nuclear phosphoprotein that is expressed in a cell cycle regulated manner in virtually all normal dividing cells. Inheritance of a mutated copy of the BRCA1 gene increases a woman's risk for developing breast and ovarian cancer (1-3). Since the tumors that arise in these individuals consistently fail to express the wild-type allele, BRCA1 is believed to encode a tumor suppressor. Loss of the remaining functional BRCA1 allele, therefore, is one of the steps leading to neoplastic transformation of some types of epithelial cells. The isolation of the murine homologue of the human BRCA1 gene opened up the possibility of using a powerful genetic approach to study the role of this gene in both normal development and tumor formation. This genetic approach involves in vitro manipulation of the genome of embryonic stem (ES) cells, stable tissue culture cell lines derived from mouse blastocysts. After introducing mutations into the murine homologue of the BRCA1 gene Brca1 in these cell lines, four groups have generated mouse lines carrying the same mutations (4-7). Surprisingly, mice carrying a single mutant Brca1 allele do not display the increased risk for breast tumors seen in humans carrying similar mutations. However, while loss of BRCA1 appears to be a one of the many events involved in tumorgenesis in humans, these mouse lines demonstrate that gene expression is essential for development; as homozygosity for each of the Brca1 mutations results in postimplantation embryonic lethality. The survival of Brca1 deficient embryos is extended by one or two days in the absence of p53 and p21 (7,8).

Journal Article↗

PubMed1997

The prostaglandin receptor EP4 triggers remodelling of the cardiovascular system at birth.

Survival of newborn placental mammals depends on closure of the ductus arteriosus (DA), an arterial connection in the fetus which directs blood away from the pulmonary circulation and towards the placenta where oxygenation occurs. Here we show that morphological changes resulting in closure of the DA in mice are virtually identical to those observed in larger mammals, including humans, and that maintenance of the DA in the open, or patent, state in fetal mice is dependent on prostaglandin synthesis. This requirement is absent in mice lacking the prostaglandin E2 EP4 receptor (EP4(-/-) mice). In EP4(-/-) mice of the 129 strain, remodelling of the DA fails to occur after birth, resulting in a left-to-right shunt of blood and subsequently in death. This suggests that the neonatal drop in prostaglandin E2 that triggers ductal closure is sensed through the EP4 receptor. In contrast, 5% of EP4(-/-) mice of mixed genetic background survive, and selective breeding of these mice leads to a 21% survival rate, suggesting that alleles at other loci can provide an alternative mechanism for ductal closure.

Animals↗

PubMed1997

Embryonic stem cell lines from MRL mice allow genetic modification in a murine model of autoimmune disease.

The MRL/MpJ-Fas(lpr) (MRL-lpr/lpr) mouse spontaneously develops a generalized autoimmune disease with features similar to those of systemic lupus erythematosus. This mouse strain provides a valuable system for identifying and characterizing the multiple genetic factors that influence the pathogenesis of autoimmune diseases. One of the most powerful means of examining the role of a specific gene product in vivo is by inactivating the gene in mouse embryonic stem (ES) cells by homologous recombination and using these cells to derive mouse lines carrying the inactivated gene. The successful application of this approach, however, requires an ES cell line that will remain stable in culture during the processes of genetic manipulation and selection. To date, ES cell lines that meet this criterion have been derived from only a few mouse strains. Here we describe the production and characterization of stable ES cell lines from the MRL mouse strain. Approximately 7% of the blastocysts derived from the MRL/MpJ+ (MRL-+/+) strain gave rise to ES cell lines, and both of the male MRL-+/+ ES cell lines tested were shown to be germline competent. We show that the MRL-+/+ ES cell lines undergo gene targeting by homologous recombination at high frequency by inactivating the gene encoding the EP2 prostaglandin receptor. These Ep2-targeted MRL ES cell lines were used to generate MRL mouse lines heterozygous for the disrupted Ep2 gene, thus demonstrating the feasibility of using a genetic approach to dissect the pathobiology of the autoimmune disease in the MRL mouse.

Animals↗

PubMed1997

Altered proliferative response by T lymphocytes of Ly-6A (Sca-1) null mice.

Ly-6A is a murine antigen which is implicated in lymphocyte activation and may be involved in activation of hematopoietic stem cells. Antibody cross-linking studies and antisense experiments have suggested that Ly-6A is a lymphocyte coactivation molecule. To better understand the function of Ly-6A, we used gene targeting to produce Ly-6A null mice which are healthy and have normal numbers and percentages of hematopoietic lineages. However, T lymphocytes from Ly-6A-deficient animals proliferate at a significantly higher rate in response to antigens and mitogens than wild-type littermates. In addition, Ly-6A mutant splenocytes generate more cytotoxic T lymphocytes compared to wild-type splenocytes when cocultured with alloantigen. This enhanced proliferation is not due to alterations in kinetics of response, sensitivity to stimulant concentration, or cytokine production by the T cell population, and is manifest in both in vivo and in vitro T cell responses. Moreover, T cells from Ly-6A-deficient animals exhibit a prolonged proliferative response to antigen stimulation, thereby suggesting that Ly-6A acts to downmodulate lymphocyte responses.

Animals↗

PubMed1997

Role of the 5-lipoxygenase-activating protein (FLAP) in murine acute inflammatory responses.

Leukotrienes are potent inflammatory mediators synthesized from arachidonic acid (AA) predominately by cells of myeloid origin. The synthesis of these lipids is believed to be dependent not only on the expression of the enzyme 5-lipoxygenase (5-LO), which catalyzes the first steps in the synthesis of leukotrienes, but also on expression of a nuclear membrane protein termed the 5-LO-activating protein (FLAP). To study the relationship of these two proteins in mediating the production of leukotrienes in vivo and to determine whether the membrane protein FLAP has additional functions in various inflammatory processes, we have generated a mouse line deficient in this protein. FLAP-deficient mice develop normally and are healthy. However, an array of assays comparing inflammatory reactions in FLAP-deficient mice and in normal controls revealed that FLAP plays a role in a subset of these reactions. Although examination of DTH and IgE-mediated passive anaphylaxis showed no difference between wild-type and FLAP-deficient animals, mice without FLAP possessed a blunted inflammatory response to topical AA and had increased resistance to platelet-activating factor-induced shock compared to controls. Also, edema associated with Zymosan A-induced peritonitis was markedly reduced in animals lacking FLAP. To determine whether these differences relate solely to a deficit in leukotriene production, or whether they reflect an additional role for FLAP in inflammation, we compared the FLAP-deficient mice to 5-LO-deficient animals. Evaluation of mice lacking FLAP and 5-LO indicated that production of leukotrienes during inflammatory responses is dependent upon the availability of FLAP and did not support additional functions for FLAP beyond its role in leukotriene production.

5-Lipoxygenase-Activating Proteins↗

PubMed1997

Collagen-induced arthritis is reduced in 5-lipoxygenase-activating protein-deficient mice.

Collagen-induced arthritis in the DBA/1 mouse is an experimental model of human rheumatoid arthritis. To examine the role of leukotrienes in the pathogenesis of this disease, we have developed embryonic stem (ES) cells from this mouse strain. Here, we report that DBA/1 mice made deficient in 5-lipoxygenase-activating protein (FLAP) by gene targeting in ES cells develop and grow normally. Zymosan-stimulated leukotriene production in the peritoneal cavity of these mice is undetectable, whereas they produce substantial amounts of prostaglandins. The inflammatory response to zymosan is reduced in FLAP-deficient mice. The severity of collagen-induced arthritis in the FLAP-deficient mice was substantially reduced when compared with wild-type or heterozygous animals. This was not due to an immunosuppressive effect, because anti-collagen antibody levels were similar in wild-type and FLAP-deficient mice. These data demonstrate that leukotrienes play an essential role in both the acute and chronic inflammatory response in mice.

5-Lipoxygenase-Activating Proteins↗

PubMed1997

Phenotypic analysis of mice lacking the highly abundant Purkinje cell- and bipolar neuron-specific PCP2 protein.

The Purkinje cell protein-2 (Pcp2, also known as L7) gene is abundantly expressed only in Purkinje cells of the cerebellum and bipolar neurons of the retina. The spatio-temporal expression pattern of this gene suggests a role for PCP2 in Purkinje cell development or normal cell physiology. A PCP2-deficient mouse was created by gene targeting to test the hypothesis that it is required for Purkinje cell development or function. Although normally present in abundance, the absence of PCP2 in null animals caused no observable cerebellar abnormalities. Behavioral analysis reveals normal abilities for balance and coordination. Null cerebellum has normal Purkinje cell numbers, morphology, and ultrastructure. Retinal bipolar neurons appear similarly unaffected. Aged null animals (22 months) were also examined and no deficits were detected using the same behavioral and histologic analyses. Although the null animal does not reveal the function of PCP2, it does rule out an essential role for PCP2 in Purkinje cell development, in Purkinje cell survival, and in at least some aspects of cerebellar function.

Animals↗

PubMed1996

Anaphylaxis mediated through a humanized high affinity IgE receptor.

Mast cells and basophils, which are activated by IgE and allergens through the high affinity IgE receptor (Fc epsilon RI), play a prominent role in anaphylaxis in the mouse. Mice deficient in this receptor become resistant to passive anaphylaxis. As a first step in developing an in vivo model that more closely mimics the IgE-mediated responses in man, we used a combination of transgenic and embryonic stem cell technology to generate a mouse line in which the murine Fc epsilon RI alpha-chain has been replaced with its human homologue. We demonstrate here that these mice express a tetrameric high affinity IgE receptor, in which the human alpha-chain associates with the murine beta- and gamma-chains, and that upon triggering with relevant Ag, this receptor mediates the initiation of the expected intracellular events. In addition, we show that the human alpha-chain restores an anaphylactic response to the nonresponsive alpha-deficient parental mouse line. This "humanized" mouse represents a potentially important model system, not only for studying the role of IgE in human immune responses, but also for testing potential therapeutic reagents that can interfere with responses mediated through the human Fc epsilon RI receptor.

Anaphylaxis↗

PubMed1996

Inhibition of 5-lipoxygenase-activating protein (FLAP) reduces pulmonary vascular reactivity and pulmonary hypertension in hypoxic rats.

Chronically elevated shear stress and inflammation are important in hypertensive lung vessel remodeling. We postulate that 5-lipoxygenase (5-LO) is a molecular determinant of these processes. Immunohistology localized the 5-LO to macrophages of normal and chronically hypoxic rat lungs and also to vascular endothelial cells in chronically hypoxic lungs only. In situ hybridization of normal and chronically hypoxic lungs demonstrated that 5-LO mRNA is expressed in macrophages. Rats hypoxic for 4 wk-developed pulmonary hypertension increased translocation of the lung 5-LO from the cytosol to the membrane fraction and increased levels of lung tissue 5-lipoxygenase-activating protein (FLAP). A FLAP ligand, 3-[l-(4-chlorobenzyl)-3-t-butyl-thio-t-isopropylindol-2-yl]-2,2- dimethylpropanoic acid (MK-886), inhibited the acute angiotensin II and hypoxia-induced pulmonary vasoconstriction in vitro and the development of chronic hypoxic pulmonary hypertension in rats in vivo. Mice bred with the deletion of the 5-LO enzyme (5-LO knockout) developed less right heart hypertrophy than age-matched 5-LO competent mice. Our results support the hypothesis that the 5-LO is involved in lung vascular tone regulation and in the development of chronic pulmonary hypertension in hypoxic rodent models.

5-Lipoxygenase-Activating Proteins↗

PubMed1996

Brca1 deficiency results in early embryonic lethality characterized by neuroepithelial abnormalities.

The breast and ovarian cancer susceptibility gene, BRCA1, has been cloned and shown to encode a zinc-finger protein of unknown function. Mutations in BRCA1 account for at least 80% of families with both breast and ovarian cancer, as well as some non-familial sporadic ovarian cancers. The loss of wild-type BRCA1 in tumours of individuals carrying one nonfunctional BRCA1 allele suggests that BRCA1 encodes a tumour suppressor that may inhibit the proliferation of mammary epithelial cells. To examine the role of BRCA1 in normal tissue growth and differentiation, and to generate a potential model for the cancer susceptibility associated with loss of BRCA1 function, we have created a mouse line carrying a mutation in one Brca1 allele. Analysis of mice homozygous for the mutant allele indicate that Brca1 is critical for normal development, as these mice died in utero between 10 and 13 days of gestation (E10-E13). Abnormalities in Brca1-deficient embryos were most evident in the neural tube, with 40% of the embryos presenting with varying degrees of spina bifida and anencephaly. In addition, the neuroepithelium in Brca1-deficient embryos appeared disorganized, with signs of both rapid proliferation and excessive cell death.

Anencephaly↗

PubMed1995

A new embryonic stem cell line from DBA/1lacJ mice allows genetic modification in a murine model of human inflammation.

The development of embryonic stem (ES) cells and their capacity to generate mice with mutations at specific loci has provided a powerful resource for functional analysis of genes in pathological processes. However, the ability to combine this technology with the large number of existing murine models of human genetic disease has been complicated by the inability to routinely generate ES cell lines from strains other than 129. Here, we report the production of a novel ES cell line derived from an inbred mouse, DBA/1lacJ. This new ES cell line undergoes homologous recombination and efficient colonization of the germline of male chimeric offspring with ES cell microinjection into C57B1/6 embryos. The DBA/1lacJ mouse is a murine model of human inflammation, therefore genetic modifications in the DBA ES cells will allow evaluation of the target gene's role in the inflammatory process.

5-Lipoxygenase-Activating Proteins↗

PubMed1995

A murine model of cystic fibrosis.

We have generated a mouse line in which the cystic fibrosis transmembrane conductance regulator (CFTR) gene has been mutated by gene targeting. Like human cystic fibrosis (CF) patients, mice lacking a functional CFTR gene, referred to as CFTR(-/-) mice, show increased numbers of goblet cells and obstruction of glands with inspissated eosinophilic secretions. The obstruction of glands often results in the destruction of gland-containing tissues in these animals. However, unlike the case in human CF patients, the most severe pathological changes in these mice were found, on preliminary analysis, to be confined to the intestinal tract and gallbladder. Although respiratory failure is the primary cause of death among humans with CF, we found only minor pathological alterations in the lungs and upper airways of our CFTR(-/-) animals. Possible explanations for the apparent lack of respiratory disease are the young age at which the animals were examined and the pathogen-free environment in which they were housed. In this manuscript, we examine the respiratory and other organ systems of CFTR(-/-) mice that have survived to adulthood. We also report on initial experiments in which CFTR(-/-) mice have been exposed to bacterial pathogens, and we present data on a single animal that displayed severe respiratory disease.

Animals↗

PubMed1994

Altered inflammatory responses in leukotriene-deficient mice.

Leukotrienes have been implicated in the regulation of immune responses, including inflammation and immediate hypersensitivity reactions. Here, we describe the phenotypic analysis of leukotriene-deficient mice generated by inactivation of the 5-lipoxygenase (5LO) gene. These 5LO(-/-) mice were unable to synthesize detectable levels of leukotrienes and were more resistant to lethal anaphylaxis induced by platelet-activating factor. The intensity of an acute inflammatory response induced by arachidonic acid was similar in 5LO(-/-) mice and controls. However, the response in 5LO(-/-) mice, but not in controls, could be virtually eliminated by a cyclooxygenase inhibitor. These data suggest that inflammatory responses are modulated by arachidonic acid metabolites through a variety of interconnected mechanisms. This has important implications for understanding the early events of an inflammatory response and for designing drugs for use in therapeutic intervention.

Anaphylaxis↗