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PubMed2006

Vitamin D receptor is required to control gastrointestinal immunity in IL-10 knockout mice.

The vitamin D receptor (VDR) is a nuclear receptor expressed in a number of different cells of the immune system. This study was performed to determine the effect of VDR deficiency on immune function and inflammation of the gastrointestinal tract in a model of inflammatory bowel disease, namely interleukin-10 (IL-10) knockout mice. IL-10 knockout mice were generated which either could or could not respond to vitamin D (double IL-10/VDR knockout; DKO). The distribution and function of lymphocytes in both the primary and secondary lymphoid organs were compared and determined as a function of the severity of intestinal inflammation. DKO mice had normal thymic development and peripheral T-cell numbers at 3 weeks of age, but a week after intestinal disease was detected the thymus was dysplastic with a reduction in cellularity. The atrophy was coupled with increased apoptosis. The spleen weight of DKO mice increased as a result of the accumulation of red blood cells; however, there was a 50% reduction in the numbers of T and B cells. Conversely, the mesenteric lymph nodes were enlarged and contained increased numbers of lymphocytes. The T cells from DKO mice were of a memory phenotype and were hyporesponsive to T-cell receptor stimulation. Colitis in the DKO mice was associated with local and high expression of IL-2, interferon-gamma, IL-1beta, tumour necrosis factor-alpha and IL-12. The primary and secondary lymphoid organs in DKO mice are profoundly altered as a consequence of the fulminating inflammation in the gastrointestinal tract. VDR expression is required for the T cells and other immune cells to control inflammation in the IL-10 KO mice.

Animals↗

PubMed1998

The use of knockout mice to understand the role of cytokines in fever.

1. In most instances, data obtained using knockout mice to dissect the role of cytokines in fever are similar to data obtained by other, more traditional experimental techniques. 2. Interleukin (IL)-1beta appears to be critically involved in fever caused by some routes of infection/inflammation (e.g. localized inflammation with turpentine). This cytokine has only a small role in fevers caused by i.p. injection of lipopolysaccharide (LPS). These IL-1beta-induced fevers in knockout mice appear to be via the induction of IL-6, similar to LPS-induced fevers in rats. Interleukin-6 also appears to be critically involved in turpentine-induced fever. 3. The precise role of tumour necrosis factor (TNF) in fever is controversial. Data obtained from knockout mice lacking both TNF receptors do not support a pyrogenic role for TNF in fever either to i.p. injection of LPS, s.c. injection of turpentine or following caecal ligation and puncture. 4. The roles of these cytokines in fevers induced by injection of LPS, IL-1beta, turpentine and caecal ligation and puncture are summarized. The data show the complexity of the febrile response. Depending on the types of inflammatory/infectious stimuli, different cytokines play important roles. Because other cytokines are thought to be involved in fever (e.g. macrophage inflammatory protein, interferons), considerable work is still needed to dissect the precise roles of cytokines in fever.

Animals↗

PubMed2004

Stressor-related impairment of synaptic transmission in hippocampal slices from alpha-synuclein knockout mice.

The role of alpha-synuclein (alpha-Syn) has recently received considerable attention because it seems to play a role in Parkinson's disease (PD). Missense mutations in the alpha-Syn gene were found in autosomal dominant PD and alpha-Syn was shown to be a major constituent of protein aggregates in sporadic PD and other synucleinopathies. Under normal conditions, alpha-Syn protein is found exclusively in synaptic terminals. However, the potential participation of alpha-synuclein in maintaining and regulating synaptic efficacy is unknown. We have investigated the excitatory synaptic modulation of alpha-synuclein in CA1 pyramidal neurons, using the in vitro hippocampal slice technique. The 4-aminopyridine-induced increase of both spontaneous excitatory postsynaptic current (EPSC) frequency and amplitude was significantly higher in alpha-Syn wild-type than knockout mice, whereas basal spontaneous EPSC frequency and amplitude was similar in both animals. As the spontaneous synaptic activity was abolished by tetrodotoxin, which indicates that it was a result of action potential-mediated transmitter release from presynaptic terminals, spontaneous EPSC changes observed in alpha-Syn knockout mice suggest that these animals present a modification of synaptic transmission with a presynaptic origin. Presynaptic depression of evoked EPSCs by hypoxia or adenosine was significantly larger in alpha-Syn knockout than in wild-type mice, further supporting the hypothesis of regulation of synaptic transmission by alpha-Syn. Together, these observations indicate that the loss of alpha-Syn reduces synaptic efficacy when the probability of transmitter release is modified. We conclude that alpha-Syn might have important actions on the maintenance of the functional integrity of synaptic transmission and its regulation in hippocampus.

4-Aminopyridine↗

PubMed2006

Neuropathic pain is enhanced in delta-opioid receptor knockout mice.

We have evaluated the possible involvement of delta-opioid receptor (DOR) in the development and expression of neuropathic pain. For this purpose, partial ligation of the sciatic nerve was performed in DOR knockout mice and wild-type littermates. The development of mechanical and thermal allodynia, as well as thermal hyperalgesia was evaluated by using the von Frey filament model, the cold-plate test and the plantar test, respectively. In wild-type and DOR knockout mice, sciatic nerve injury led to a neuropathic pain syndrome revealed in these nociceptive behavioural tests. However, the development of mechanical and thermal allodynia, and thermal hyperalgesia was significantly enhanced in DOR knockout mice. These results reveal the involvement of DOR in the control of neuropathic pain and suggest a new potential therapeutic use of DOR agonists.

Analysis of Variance↗

PubMed2006

Differential actions of NPY on seizure modulation via Y1 and Y2 receptors: evidence from receptor knockout mice.

PURPOSE: Neuropeptide Y (NPY) has been shown to modulate seizure activities. To provide further understanding of the involvement of two of the most abundantly expressed NPY receptors, Y1 and Y2, we assessed the effect of Y1 and Y2 gene deletion on systemic kainic acid-induced seizures. We also examined the effect of rAAV-mediated hippocampal NPY overexpression on seizure susceptibility in these receptor knockout mice. METHODS: Recombinant adeno-associated viral vector overexpressing NPY (rAAV-NPY) or an empty vector control (rAAV-Empty) was injected into the hippocampus of adult C57BL/6-129/SvJ wild-type male mice and mice deficient of Y1 or Y2 receptors on the same background. Four weeks after vector injection, mice were subjected to systemic kainic acid-induced seizures, and the seizure behaviors were scored. RESULTS: The rAAV-mediated hippocampal overexpression of NPY led to a twofold reduction in seizures induced by systemic kainic acid in wild-type mice and Y1 receptor knockout mice but not in mice deficient of Y2 receptors. A differential action by the receptors was observed in the seizure-induced mortality rate, with increased fatality in Y2-/- mice. In addition, although NPY overexpression did not significantly affect the mortality rate in Y2-/- and wild-type mice, it abolished KA-induced mortality in Y1-/-mice. CONCLUSIONS: This study shows for the first time an altered susceptibility to chemically induced seizures in Y1 and Y2 knockout mice and demonstrates a differential seizure modulation mediated by these receptors via a genetic approach.

Animals↗

PubMed1998

Natural killer cell differentiation: insights from knockout and transgenic mouse models and in vitro systems.

In the last few years, the routine development of knockout and transgenic mice and the ease with which rare progenitor populations can be isolated from hematopoietic organs and cultured in vitro has facilitated significant advances in understanding the lineage and development of natural killer (NK) cells. Fluorescence-activated cell sorter analyses have identified a common lymphoid progenitor capable of giving rise to NK, T, and B cells, confirming the lymphoid origin of NK cells. Knockout and transgenic mouse models have pointed to an absolutely critical role for signals sent through the interleukin (IL)-2/15 receptor beta (CD122) chain and common gamma (gamma c) chain for NK development. Such signals are likely relayed inside the cell by the tyrosine kinase Jak3, which associates with gamma c. Recently developed IL-15 and IL-15 receptor alpha knockout mice have pinpointed IL-15 as the mediator of this signal. Other mouse models have indicated an unexpected role for flt3 ligand in early NK-cell development as well as minor roles for stem cell factor and IL-7 in expanding NK-cell progenitor numbers. Finally, in vitro culture systems have proven useful in identifying the point in NK development at which each of these signals is critical.

Animals↗

PubMed1999

Lipid antigen presentation in the immune system: lessons learned from CD1d knockout mice.

CD1 molecules represent a distinct lineage of antigen-presenting molecules that are evolutionarily related to the classical major histocompatibility complex (MHC) class I and class II molecules. Unlike the classical MHC products that bind peptides, CD1 molecules have evolved to bind lipids and glycolipids. Murine and human CD1d molecules can present glycolipid antigens such as alpha-galactosylceramide (alpha-GalCer) to CD1d-restricted natural killer (NK) T cells. Using CD1d knockout mice we demonstrated that CD1d expression is required for the development of NK T cells. These animals were also deficient in the rapid production of interleukin-4 and interferon-gamma in response to stimulation by anti-CD3 antibodies. Despite these defects, CD1d knockout animals were able to generate strong T-helper type 1 (TH1) and TH2 responses. Spleen cells from these animals neither proliferated nor produced cytokines in response to stimulation by alpha-GalCer. Repeated injection of alpha-GalCer into wild-type but not CD1d mutant mice was able to clear metastatic tumors. We further showed that alpha-GalCer can inhibit disease in diabetes-prone non-obese diabetic mice. Collectively, these findings with CD1d knockout animals indicate a critical role for CD1d-dependent T cells in various disease conditions, and suggest that alpha-GalCer may be useful for therapeutic intervention in these diseases.

Animals↗

PubMed2004

Oxytocin and estrogen receptor alpha and beta knockout mice provide discriminably different odor cues in behavioral assays.

Social behavior involves both the recognition and pro-duction of social cues. Mice with selective deletion(knockout) of either the gene for oxytocin (OT) or genes for the estrogen receptor (ER) -c or -B display impaired social recognition. In this study we demonstrate that these gene knockout mice also provide discriminably different social stimuli in behavioral assays. In an odor choice test, which is a measure of social interest and discrimination, outbred female Swiss-Webster mice discriminated the urine odors of male knock-outs IKO: OTKO, alphaERKO, betaERKO) from the odors of their wildtype littermates (WT: OTWT, alphaERWT, betaERWT). Females showed marked initial choices of the urine odors of OTWT and betaERWT males over those of OTKOand PERKO males, and alphaERKO males over alphaERWT males. The odors of OTKO and betaERKO males also induced aversive, analgesic responses, with the odors of WTs having no significant effects. Odors of both the alphaERWT andalphaERKO males induced aversive, analgesic responses,with the odors of the WT inducing significantly greater analgesia. The odors of restraint stressed WT and KO males also elicited analgesia with, again, females dis-playing significantly greater responses to the odors of stressed OTKO and betaERKO males than their WTs, and significantly lower analgesia to the odors of stressedalphaERKO than alphaERWT males. These findings show that the KO mice are discriminated from their WTs on the basis of odor and that the various KOs differ in the relative attractiveness/aversiveness of their odors. Therefore, in behavioral assays one causal route by which gene inactivation alters the social behavior of knockout mice may be mediated through the partners'modified responses to their odors.

Animals↗

PubMed2006

Vasopressin 1a receptor knockout mice have a subtle olfactory deficit but normal aggression.

Two receptors for vasopressin (Avp) are expressed in the brain, the Avp 1a receptor (Avpr1a) and the Avp 1b receptor (Avpr1b). To investigate the role of Avpr1a in behaviors in mice more extensively, we generated a line of mice lacking a functional Avpr1a (knockout, Avpr1a(-/-)). We first performed a baseline phenotypic screen of the Avpr1a knockouts followed by a more detailed analysis of their circadian rhythms and olfactory function. When free-running in constant darkness, the Avpr1a(-/-) mice have a longer circadian tau than the wild types. There are also subtle olfactory deficits in Avpr1a(-/-) mice as measured in an olfactory habituation/dishabituation test and in the discrimination of female urine from male urine using an operant testing paradigm. An extensive body of research has shown that manipulation of the Avpr1a alters behavior, including aggression and social recognition. Therefore, we expected profound behavioral deficits in mice lacking the Avpr1a gene. Contrary to our expectations, social aggression, anxiety-like behavior and social recognition are unaffected in this line of Avpr1a knockout mice. These data suggest either that the Avpr1a is not as critical as we thought for social behavior in mice or, more likely, that the neural circuitry underlying aggression and other social behaviors compensates for the life-long loss of the Avpr1a. However, the olfactory deficits observed in the Avpr1a(-/-) mice suggest that Avp and Avpr1a drugs may affect behavior, in part, by modulation of chemosensory systems.

Aggression↗

PubMed1997

Insights into the neurobiology of impulsive behavior from serotonin receptor knockout mice.

Although the concept of impulsivity has proved useful in human and animal studies of addiction, violent aggression, and violent suicide, and has been recognized as an important component of human behavior, little research has been done to understand the underlying psychobiological mechanisms. We explore the concept of impulsivity and its relation with the neurotransmitter serotonin in the context of aggressive behavior and behavior associated with positive reinforcement using a knockout mouse that lacks one of the serotonin receptors, the 5-HT1B receptor. This knockout mouse shows more impulsive aggression, acquires cocaine self-administration faster, and drinks more alcohol than the corresponding wild-type control. We show that the impulsive characteristics of these mice are not due to change in cognitive functions: in a cognitive task involving a choice between a small immediate one and a larger more delayed reward, knockout mice showed intact choice and timing capabilities and good discrimination of reward amounts. Thus, this mouse may prove an animal model of addiction and motor impulsivity.

Animals↗

PubMed2003

Knockout studies defining different roles for melanocortin receptors in energy homeostasis.

Proopiomelanocortin (POMC) is expressed in the arcuate nucleus of the hypothalamus (ARC) and the commissural nucleus of the solitary tract (cNTS). Post-translational processing of POMC produces two melanocortin receptor ligands, alpha- and gamma-melanocyte-stimulating hormone (MSH). Two melanocortin receptors (MC3R, MC4R) are expressed in brain regions receiving projections of POMC fibers, most of which also receive projections from a population of ARC neurons that co-express neuropeptide Y (NPY) and the MC3R/MC4R antagonist agouti-related peptide (AgRP). MC4R haploinsufficient humans and MC4R knockout (MC4RKO) mice exhibit increased adiposity and linear growth. MC4RKO mice exhibit hyperleptinemia and hyperinsulinemia and sometimes, but not always, develop type 2 diabetes (T2D). Individually housed MC4RKO mice fed low-fat diets are not hyperphagic when food intake is corrected for lean mass, whereas hyperphagia is observed after the introduction of diets with increased fat content. POMC knockout (POMCKO) mice are similar in that the severity of hyperphagia increases with the introduction of high-fat diets. By contrast, targeted deletion of the MC3R in the mouse results in increased adiposity despite the absence of hyperphagia. MC3RKO mice also exhibit reduced linear growth and lean mass; while MC3RKO mice are hyperleptinemic and hyperinsulinemic, the development of T2D has not been reported. The MC4R, but not the MC3R, is required for the stimulation of energy expenditure in response to melanocortin agonists and voluntary hyperphagia. Evidence for altered physical activity has also been reported for both knockout models. Analysis of MC4RKO mice indicates that this receptor is involved in rapidly coordinating energy consumption with energy expenditure through diet-induced thermogenesis and activity.

Animals↗

PubMed2002

Molecular and pharmacological characterization of GABA(A) receptor alpha1 subunit knockout mice.

GABA(A) receptors mediate fast inhibitory neurotransmission in the central nervous system (CNS), and approximately half of these receptors contain alpha1 subunits. GABA(A) receptor alpha1 subunits are important for receptor assembly and specific pharmacological responses to benzodiazepines. Plasticity in GABA(A) receptor alpha1 subunit expression is associated with changes in CNS excitability observed during normal brain development, in animal models of epilepsy, and upon withdrawal from alcohol and benzodiazepines. To examine the role of alpha1 subunit-containing GABA(A) receptors in vivo, we characterized receptor subunit expression and pharmacological properties in cerebral cortex of knockout mice with a targeted deletion of the alpha1 subunit. The mice are viable but exhibit an intention tremor. Western blot analysis confirms the complete loss of alpha1 subunit peptide expression. Stable adaptations in the expression of several GABA(A) receptor subunits are observed in the fifth to seventh generations, including decreased expression of beta2/3 and gamma2 subunits and increased expression of alpha2 and alpha3 subunits. There was no change in alpha4, alpha5, or delta subunit peptide levels in cerebral cortex. Knockout mice exhibit loss of over half of GABA(A) receptors measured by [(3)H]muscimol, [(3)H]2-(3-carboxyl)-3-amino-6-(4-methoxyphenyl)-pyridazinium bromide ([(3)H]SR-95531), and t-butylbicyclophosphoro[(35)S]thionate ([(35)S]TBPS) binding. [(3)H]Ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate ([(3)H]Ro15-4513) binding is reduced by variable amounts in different regions across brain. GABA(A) receptor alpha1(-/-) mice lose all high-affinity [(3)H]zolpidem binding and about half of [(3)H]flunitrazepam binding in the cerebral cortex. The potency and maximal efficacy of muscimol-stimulated (36)Cl(-) uptake in cerebral cortical synaptoneurosomes are reduced in alpha1(-/-) mice. Furthermore, knockout mice exhibit increased bicuculline-induced seizure susceptibility compared with wild-type mice. These data emphasize the significance of alpha1 subunit expression and its involvement in the regulation of CNS excitability.

Animals↗

PubMed2006

Carcinogen and anticancer drug transport by Mrp2 in vivo: studies using Mrp2 (Abcc2) knockout mice.

The ATP-binding-cassette (ABC) transporter multidrug resistance protein (MRP) 2 (ABCC2) forms a natural barrier and efflux system for various (conjugates of) drugs, other xenotoxins, and endogenous compounds. To obtain insight in the pharmacological and physiological functions of Mrp2, we generated Mrp2 knockout mice, which were viable and fertile but suffered from mild hyperbilirubinemia due to impaired excretion of bilirubin monoglucuronides into bile. The mice also had an 80-fold decreased biliary glutathione excretion and a 63% reduced bile flow. Levels of Mrp3 (Abcc3) in liver and Mrp4 (Abcc4) in kidney of Mrp2-/- mice were approximately 2-fold increased. After oral administration of the food-derived carcinogens [(14)C]PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) and [14C]IQ (2-amino-3-methylimidazo[4,5-f]quinoline) plasma values were 1.9- and 1.7-fold higher in Mrp2-/- mice versus wild-type mice, respectively, demonstrating the role of Mrp2 in restricting exposure to these compounds. At a high dose of 50 mg/kg of the drug [3H]methotrexate, the plasma area under the curve for i.v. administration was 1.8-fold higher in Mrp2-/- mice (1345+/-207 versus 734+/-81 min.microg/ml). No clear plasma concentration difference arose at low dose (1 mg/kg). Subsequently, Mdr1a/b/Mrp2 knockout mice were generated. Their biliary excretion of doxorubicin after i.v. administration (5 mg/kg) was 54-fold decreased (0.32+/-0.13 versus 17.30+/-6.59 nmol/g liver in wild type), and a role for both Mdr1a/b and Mrp2 in this process was revealed. Our results demonstrate that the Mrp2-/- mouse provides a valuable tool for studies of the impact of Mrp2 on behavior of drugs and other toxins, especially when combined with other ABC transporter knockout mice.

Animals↗

PubMed2005

Oral benzo[a]pyrene in Cyp1 knockout mouse lines: CYP1A1 important in detoxication, CYP1B1 metabolism required for immune damage independent of total-body burden and clearance rate.

CYP1A1 and CYP1B1 metabolically activate many polycyclic aromatic hydrocarbons (PAHs), including benzo[a]pyrene, to reactive intermediates associated with toxicity, mutagenesis, and carcinogenesis. Paradoxically, however, Cyp1a1-/- knockout mice are more sensitive to oral benzo[a]pyrene exposure, compared with wild-type Cyp1a1+/+ mice (Mol Pharmacol 65:1225, 2004). To further investigate the mechanism for this enhanced sensitivity, Cyp1a1-/-, Cyp1a2-/-, and Cyp1b1-/- single-knockout, Cyp1a1/1b1-/- and Cyp1a2/1b1-/- double-knockout, and Cyp1+/+ wild-type mice were analyzed. After administration of oral benzo[a]pyrene (125 mg/kg/day) for 18 days, Cyp1a1-/- mice showed marked wasting, immunosuppression, and bone marrow hypocellularity, whereas the other five genotypes did not. After 5 days of feeding, steady-state blood levels of benzo[a]pyrene were approximately 25 and approximately 75 times higher in Cyp1a1-/- and Cyp1a1/1b1-/- mice, respectively, than in wild-type mice. Benzo[a]pyrene-DNA adduct levels were highest in liver, spleen, and marrow of Cyp1a1-/- and Cyp1a1/1b1-/- mice. Many lines of convergent data obtained with oral benzo[a]pyrene dosing suggest that: 1) inducible CYP1A1, probably in both intestine and liver, is most important in detoxication; 2) CYP1B1 in spleen and marrow is responsible for metabolic activation of benzo[a]pyrene, which results in immune damage in the absence of CYP1A1; 3) both thymus atrophy and hepatocyte hypertrophy are independent of CYP1B1 metabolism but rather may reflect long-term activation of the aryl hydrocarbon receptor; and 4) the magnitude of immune damage in Cyp1a1-/- and Cyp1a1/1b1-/- mice is independent of plasma benzo[a]pyrene and total-body burden and clearance. Thus, a balance between tissue-specific expression of the CYP1A1 and CYP1B1 enzymes governs sensitivity of benzo[a]pyrene toxicity and, possibly, carcinogenicity.

Administration, Oral↗

PubMed1997

5-Hydroxytryptamine1B receptors modulate the effect of cocaine on c-fos expression: converging evidence using 5-hydroxytryptamine1B knockout mice and the 5-hydroxytryptamine1B/1D antagonist GR127935.

Serotonergic transmission has been suggested to modulate the effects of cocaine. However, the specific receptors and brain structures underlying this phenomenon have not been identified. To test the possible contribution of the 5-hydroxytryptamine1B (5-HT1B) receptor, we studied the induction of the immediate-early gene c-fos elicited by cocaine in knockout mice lacking this receptor. 5-HT1B knockout mice display a markedly reduced effect of cocaine on c-fos induction in different brain structures, most notably in the striatum. In addition, the administration to wild-type mice of the 5-HT1B receptor agonist RU24969 results in a striatal induction of c-fos expression very similar to that induced by cocaine in its time course, cellular and anatomical distribution, and pharmacology. Here, we also report the ability of a 5-HT1D receptor antagonist, GR127935, to antagonize 5-HT1B receptors in vivo. Finally, when administered to wild-type mice, GR127935 reduces the increase in striatal c-fos expression elicited by cocaine. These converging lines of evidence obtained with the knockout mice and 5-HT(1B/1D) antagonist indicate that cocaine acts as an indirect agonist of 5-HT1B receptors in vivo and demonstrate that activation of 5-HT1B receptors contributes to the cellular responses elicited by cocaine.

Animals↗

PubMed2026

Construction and characterization of novel Mycobacterium tuberculosis-derived triple and quadruple knockout vaccines against tuberculosis.

Tuberculosis (TB) is a deadly disease that claims the lives of over a million people each year worldwide. The Bacille Calmette-Guérin vaccine has long been used to protect against TB, but it produces variable effects across different populations and fails to protect against adult pulmonary TB. Therefore, there is an urgent need for alternative vaccines that can offer better protection. We have developed a strategy for the rational deletion of virulence-related genes in Mycobacterium tuberculosis (Mtb) to create hyperattenuation that also enhances immunogenicity. Previously, we generated both single (∆fbpA) and double knockout (DKO) (∆fbpA-∆sapM) mutants of Mtb and assessed their immunogenicity and efficacy using mice. Herein, we have created triple knockout (TKO) and quadruple knockout (QKO) strains to enhance the immunogenicity and safety of the DKO strain by deleting the zmp1 and dosR genes. The resulting TKO strains, TKO-Z (∆fbpA-∆sapM-∆zmp1) and TKO-D (∆fbpA-∆sapM-∆dosR), and the QKO strain (∆fbpA-∆sapM-∆zmp1-∆dosR), were evaluated for their immunogenicity and safety in mice. Whereas TKO-Z and QKO strains exhibited superior immunogenicity compared to the DKO strain, their protective efficacy in mice was comparable. However, survival studies involving SCID mice indicated that the QKO strain was highly attenuated. Therefore, rational deletion of genes in Mtb seems to be an innovative approach for developing safer and more efficacious vaccines against TB.

Animals↗

PubMed1998

NF-kappaB-mediated inhibition of apoptosis is required for encephalomyocarditis virus virulence: a mechanism of resistance in p50 knockout mice.

Apoptosis is a central host defense mechanism to eliminate virus-infected cells. Activation of NF-kappaB suppresses apoptosis following some types of stimulation in vitro. To test the physiological importance of this pathway in vivo, we studied murine encephalomyocarditis virus (EMCV) infection in mice and cell lines defective in NF-kappaB1 (p50) signaling. As previously reported, we find that all p50 knockout (p50 -/-) mice survive an EMCV infection that readily kills normal mice. By introducing the p50 mutation into interferon (IFN) type I receptor knockout (IFNRI -/-) mice, we find that this resistance is not mediated by IFN-beta as previously thought. While no IFNRI -/- mice survive, the double-knockout mice survive 60% of the time. The survival is tightly linked to the animals' ability to clear the virus from the heart in vivo. Using murine embryonic fibroblasts (MEF) derived from wild-type, p50 -/-, and p65 -/- embryos, we found that NF-kappaB is not required for the replication cycle of EMCV. However, during these experiments we observed that p50 -/- and p65 -/- MEF infected with EMCV undergo enhanced, premature cytotoxicity. Upon examination of this cell death, we found that EMCV infection induced both plasma membrane and nuclear changes typical of apoptosis in all cell lines. These apoptotic processes occurred in an accelerated and pronounced way in the NF-kappaB-defective cells, as soon as 6 h after infection, when virus is beginning to be released. Previously, only the RelA (p65) subunit of NF-kappaB has been shown to play a role in suppressing apoptosis. In our studies, we find that p50 is equally important in suppressing apoptosis during EMCV infection. Additionally, we show that suppression of apoptosis by NF-kappaB1 is required for EMCV virulence in vivo. The attenuation in p50 -/- mice can be explained by rapid apoptosis of infected cells which allows host phagocytes to clear infected cells before the viral burst leading to a reduction of the viral burden and survival of the mice.

Animals↗

PubMed2003

Synergistic effects of Y2 and Y4 receptors on adiposity and bone mass revealed in double knockout mice.

Neuropeptide Y regulates numerous physiological processes via at least five different Y receptors, but the specific roles of each receptor are still unclear. We previously demonstrated that Y2 receptor knockout results in a lean phenotype, increased cancellous bone volume, and an increase in plasma pancreatic polypeptide (PP), a ligand for Y4 receptors. PP-overexpressing mice are also known to have a lean phenotype. Deletion of the Y4 receptor also produced a lean phenotype and increased plasma PP levels. We therefore hypothesized that part of the Y2 phenotype results from increased PP action on Y4 receptors and tested this in PP transgenic Y4(-/-) and Y2(-/-) Y4(-/-) double knockout mice. Bone mass was not altered in Y4 knockout mice. Surprisingly, despite significant hyperphagia, Y2(-/-) Y4(-/-) mice retained a markedly lean phenotype, with reduced body weight, white adipose tissue mass, leptinemia, and insulinemia. Furthermore, bone volume was also increased threefold in Y2(-/-) Y4(-/-) mice, and this was associated with enhanced osteoblastic activity. These changes were more pronounced than those observed in Y2(-/-) mice, suggesting synergy between Y2 and Y4 receptor pathways. The lack of bone changes in PP transgenic mice suggests that PP alone is not responsible for the bone mass increases but might play a major role in the lean phenotype. However, a synergistic interaction between Y2 and Y4 pathways seems to regulate bone volume and adiposity and could have important implications for possible interventions in obesity and for anabolic treatment of osteoporotic bone loss.

Animals↗