The nuclear receptor PXR is a lithocholic acid sensor that protects against liver toxicity.

The pregnane X receptor (PXR) is the molecular target for catatoxic steroids such as pregnenolone 16alpha-carbonitrile (PCN), which induce cytochrome P450 3A (CYP3A) expression and protect the body from harmful chemicals. In this study, we demonstrate that PXR is activated by the toxic bile acid lithocholic acid (LCA) and its 3-keto metabolite. Furthermore, we show that PXR regulates the expression of genes involved in the biosynthesis, transport, and metabolism of bile acids including cholesterol 7alpha-hydroxylase (Cyp7a1) and the Na(+)-independent organic anion transporter 2 (Oatp2). Finally, we demonstrate that activation of PXR protects against severe liver damage induced by LCA. Based on these data, we propose that PXR serves as a physiological sensor of LCA, and coordinately regulates gene expression to reduce the concentrations of this toxic bile acid. These findings suggest that PXR agonists may prove useful in the treatment of human cholestatic liver disease.

Disruption of the A(3) adenosine receptor gene in mice and its effect on stimulated inflammatory cells.

The A(3) adenosine receptor (A3AR) is one of four receptor subtypes for adenosine and is expressed in a broad spectrum of tissues. In order to study the function of A3AR, a mouse line carrying a mutant A(3) allele was generated. Mice homozygous for targeted disruption of the A3AR gene, A3AR(-/-), are fertile and visually and histologically indistinguishable from wild type mice. The lack of a functional receptor in the A3AR(-/-) mice was confirmed by molecular and pharmacological analyses. The absence of A3AR protein expression in the A3AR(-/-) mice was demonstrated by lack of N(6)-(4-amino-3-[(125)I]iodobenzyl)adenosine binding to bone marrow-derived mast cell membranes that were found to express high levels of A3AR in wild type mice. In A3AR(-/-) mice, the density of A(1) and A(2A) adenosine receptor subtypes was the same as in A3AR(+/+) mice as determined by radioligand binding to brain membranes. Additionally, A(2B) receptor transcript expression was not affected by ablation of the A3AR gene. A3AR(-/-) mice have basal heart rates and arterial blood pressures indistinguishable from A3AR(+/+) mice. Functionally, in contrast to wild type mice, adenosine and the A3AR-specific agonist, 2-chloro-N(6)-(3-iodobenzyl)-adenosine-5'-N-methyl-carboxamide (2-Cl-IB-MECA), elicit no potentiation of antigen-dependent degranulation of bone marrow-derived mast cells from A3AR(-/-) mice as measured by hexosaminidase release. Also, the ability of 2Cl-IB-MECA to inhibit lipopolysaccharide-induced tumor necrosis factor-alpha production in vivo was decreased in A3AR(-/-) mice in comparison to A3AR(+/+) mice. The A(2A) adenosine receptor agonist, 2-p-(2-carboxyethyl)phenylamino)-5'-N-ethylcarboxamidoadenosine, produced inhibition of lipopolysaccharide-stimulated tumor necrosis factor-alpha production in both A3AR(-/-) and A3AR(+/+) mice. These results show that the inhibition in vivo can be mediated by multiple subtypes, specifically the A(3) and A(2A) adenosine receptors, and A3AR activation plays an important role in both pro- and anti-inflammatory responses.

Decreased platelet aggregation, increased bleeding time and resistance to thromboembolism in P2Y1-deficient mice.

Platelet activation is characterized by shape change, induction of fibrinogen receptor expression and release of granular contents, leading to aggregation and plug formation. While this response is essential for hemostasis, it is also important in the pathogenesis of a broad spectrum of diseases, including myocardial infarction, stroke and unstable angina. Adenosine 5'-diphosphate (ADP) induces platelet aggregation, but the mechanism for this has not been established, and the relative contribution of ADP in hemostasis and the development of arterial thrombosis is poorly understood. We show here that the purinoceptor P2Y1 is required for platelet shape change in response to ADP and is also a principal receptor mediating ADP-induced platelet aggregation. Activation of P2Y1 resulted in increased intracellular calcium but no alteration in cyclic adenosine monophosphate (cAMP) levels. P2Y1-deficient platelets partially aggregated at higher ADP concentrations, and the lack of P2Y1 did not alter the ability of ADP to inhibit cAMP, indicating that platelets express at least one additional ADP receptor. In vivo, the lack of P2Y1 expression increased bleeding time and protected from collagen- and ADP-induced thromboembolism. These findings support the hypothesis that the ATP receptor P2Y1 is a principal receptor mediating both physiologic and pathological ADP-induced processes in platelets.

BRCA1 deficient embryonic stem cells display a decreased homologous recombination frequency and an increased frequency of non-homologous recombination that is corrected by expression of a brca1 transgene.

BRCA1 is a nuclear phosphoprotein that has been classified as a tumor suppressor based on the fact that women carrying a mutated copy of the BRCA1 gene are at increased risk of developing breast and ovarian cancer. The association of BRCA1 with RAD51 has led to the hypothesis that BRCA1 is involved in DNA repair. We describe here the generation and analysis of murine embryonic stem (ES) cell lines in which both copies of the murine homologue of the human BRCA1 gene have been disrupted by gene targeting. We show that exogenous DNA introduced into these BRCA1 deficient cells by electroporation is randomly integrated into the genome at a significantly higher rate than in wild type ES cells. In contrast, integration of exogenous DNA by homologous recombination occurs in BRCA1 deficient cells at a significantly lower rate than in wild type controls. When BRCA1 expression is re-established at 5-10% of normal levels by introduction of a Brca1 transgene into BRCA1 deficient ES cells, the frequency of random integration is reduced to wild type levels, although the frequency of homologous recombination is not significantly improved. These results suggest that BRCA1 plays a role in determining the response of cells to double stranded DNA breaks.

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.

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.

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.

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.

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.

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.

An animal model for cystic fibrosis made by gene targeting.

Cystic fibrosis results from defects in the gene encoding a cyclic adenosine monophosphate-dependent chloride ion channel known as the cystic fibrosis transmembrane conductance regulator (CFTR). To create an animal model for cystic fibrosis, mice were generated from embryonic stem cells in which the CFTR gene was disrupted by gene targeting. Mice homozygous for the disrupted gene display many features common to young human cystic fibrosis patients, including failure to thrive, meconium ileus, alteration of mucous and serous glands, and obstruction of glandlike structures with inspissated eosinophilic material. Death resulting from intestinal obstruction usually occurs before 40 days of age.

Susceptibility of beta 2-microglobulin-deficient mice to Trypanosoma cruzi infection.

The beta 2-microglobulin (beta 2m) protein associates with the products of the class I major histocompatibility (MHC) loci; this combination functions in the thymic development of and antigen presentation to CD8+ T cells. Mice in which the beta 2m gene has been disrupted by homologous recombination fail to express class I MHC gene products, and therefore lack CD8+ T cells and measurable cytotoxic T-cell responses. However, beta 2m- mice appear to have normal development of both CD4+ alpha/beta T-cell receptor (TCR+) and gamma/delta TCR+ T cells and are not overtly more susceptible than beta 2m+ mice to potential environmental agents of infection or to experimental viral infection. Here we show that beta 2m- mice suffer high parasitaemias and early death when infected with the obligate cytoplasmic protozoan parasite Trypanosoma cruzi. Despite this increased susceptibility, the beta 2m- mice are more responsive than their beta 2m+ littermates in terms of lymphokine production, making higher levels of both interleukin-2 and interferon-gamma in response to mitogen stimulation. In addition, the beta 2m- mice show essentially no inflammatory response in parasite-infected tissues. These results confirm previous experiments on mice depleted of CD8+ cells using antibody treatment in demonstrating the importance of CD8+ T cells in immune protection in T. cruzi infection. They also implicate CD8+ T cells and/or class I MHC molecules in regulation of lymphokine production and recruitment of inflammatory cells.

Toward an animal model of cystic fibrosis: targeted interruption of exon 10 of the cystic fibrosis transmembrane regulator gene in embryonic stem cells.

A gene-targeting construct was made containing 7.8 kilobases of DNA spanning exon 10 of the mouse cystic fibrosis transmembrane regulator (CFTR) gene in which part of the exon has been replaced by two neomycin-resistance (Neo) genes driven by different promoters. (This replacement introduces a chain-termination codon at amino acid position 489 in the CFTR sequence). A herpes simplex thymidine kinase gene was on each end of the construct, which was electroporated into embryonic stem (ES) cells. Colonies resistant to G418, or to G418 plus ganciclovir, were selected and screened by Southern blotting or by PCR amplification. Five pools of G418-resistant cells gave PCR products diagnostic of targeting. Four independent clones of ES cells with a disrupted CFTR gene have been isolated from these pools. The frequency of targeting was 1/2500 G418-resistant colonies. This low frequency is not the consequence of marginal expression of the Neo genes in the targeted cells. The CFTR targeting events were clustered among our experiments in a manner suggesting that some unidentified factor(s), possibly passage number, influences the recovery of CFTR-targeted cells.

Recognition of beta 2-microglobulin-negative (beta 2m-) T-cell blasts by natural killer cells from normal but not from beta 2m- mice: nonresponsiveness controlled by beta 2m- bone marrow in chimeric mice.

The role of major histocompatibility complex (MHC) class I expression in control of the sensitivity of normal cells to natural killer (NK) cells was studied by the use of mutant mice made deficient for expression of beta 2-microglobulin (beta 2m) through homologous recombination in embryonal stem cells. T-cell blasts from beta 2m-deficient (beta 2m -/-) mice were killed by NK cells from normal mice in vitro, while beta 2m +/- blasts were resistant. The beta 2m defect also affected the NK effector cell repertoire: NK cells from beta 2m -/- mice failed to kill beta 2m -/- blasts, while they retained the ability to kill the prototype NK cell target lymphoma YAC-1, although at reduced levels. The inability to recognize beta 2m -/- blasts could be transferred with beta 2m -/- bone marrow to irradiated beta 2m-expressing mice. In contrast, the development of CD8+ T cells (deficient in beta 2m -/- mice) was restored in such chimera. These results indicate that loss of MHC class I/beta 2m expression is sufficient to render normal cells sensitive to NK cells, and that the same defect in the hemopoietic system of a mouse renders its NK cells tolerant to beta 2m-deficient but otherwise normal cells. In the beta 2m -/- mice, NK cells may be selected or educated by other bone marrow cells to tolerate the MHC class I deficiency. Alternatively, the specificity may be controlled directly by the class I molecules on the NK cells themselves.

Postpartum uterine infection with Clostridium perfringens.

Clostridium perfringens is commonly present in the female genital tract. Uterine infection with this organism is a potentially fatal disease infrequently seen in obstetric practice. The manifestations of C. perfringens uterine infection are variable, ranging from endometritis to gas gangrene with fulminant septicemia. The usual precipitating event has been septic abortion, but such infections can also occur spontaneously in uterine tumors and after complicated deliveries requiring mechanical intervention. Diagnosis may be aided by radiologic techniques, and treatment involves high-dose penicillin and possibly surgery. We report two cases and review the clinical presentation and the diagnostic and therapeutic aspects of this disease.

Mycobacterium szulgai: an unusual pathogen.

Mycobacterium szulgai is an unusual pathogen that accounts for less than 1% of all cases of non-tuberculosis mycobacterial infection. Infections with this organism usually involve the lung but may involve soft tissues. Although similar to tuberculosis in its clinical presentation, infection due to M. szulgai requires different management, and it is therefore important to distinguish disease caused by M. szulgai from that caused by M. tuberculosis. Isolation of M. szulgai implies the presence of clinical disease, and when the organism is identified, treatment based on sensitivity testing should be initiated. Although no standard recommendations for treatment exist, most infections due to M. szulgai have been treated with combined high doses of isoniazid, ethambutol, and rifampin for 18-24 months. M. szulgai has been isolated worldwide; the first case of infection reported from Canada is described, and the clinical presentation, microbiologic diagnosis, and therapeutic management of M. szulgai infections are reviewed.

Measurement of gastric emptying rate.

Gastric emptying rate was measured chronically using an electromagnetic probe inserted into the proximal duodenum via a duodenal cannula. Each duodenal gush was identified and its value calculated on the basis of pre-established threshold and timing criteria which eliminate shifts in the baseline and artefacts due to the presence of particles. The net flow of digestive contents and the aboral cumulative flow were displayed online. This system has been used in sheep for continuous measurement of duodenal flow over periods of 3-5 weeks.

Computer-aided analysis of gastrointestinal myoelectric activity.

This method of analysing gastrointestinal motility involves data collection for 12 h periods and calculations from electromyographic signals at 15 s intervals; results are printed for successive 20 min epochs. In addition, phases of regular spike activity are recognized during the interdigestive periods. The system has been used in a series of comparative measurements of duodenal activity before and after feeding, and in studies involving drug-induced regular spike activity.