Genetic Background Of Mice Research Articles

Protection from Experimental Cerebral Malaria with a Single Dose of Radiation-Attenuated, Blood-Stage Plasmodium berghei Parasites

BackgroundWhole malaria parasites are highly effective in inducing immunity against malaria. Due to the limited success of subunit based vaccines in clinical studies, there has been a renewed interest in whole parasite-based malaria vaccines. Apart from attenuated sporozoites, there have also been efforts to use live asexual stage parasites as vaccine immunogens.Methodology and ResultsWe used radiation exposure to attenuate the highly virulent asexual blood stages of the murine malaria parasite P. berghei to a non-replicable, avirulent form. We tested the ability of the attenuated blood stage parasites to induce immunity to parasitemia and the symptoms of severe malaria disease. Depending on the mouse genetic background, a single high dose immunization without adjuvant protected mice from parasitemia and severe disease (CD1 mice) or from experimental cerebral malaria (ECM) (C57BL/6 mice). A low dose immunization did not protect against parasitemia or severe disease in either model after one or two immunizations. The protection from ECM was associated with a parasite specific antibody response and also with a lower level of splenic parasite-specific IFN-γ production, which is a mediator of ECM pathology in C57BL/6 mice. Surprisingly, there was no difference in the sequestration of CD8+ T cells and CD45+ CD11b+ macrophages in the brains of immunized, ECM-protected mice.ConclusionsThis report further demonstrates the effectiveness of a whole parasite blood-stage vaccine in inducing immunity to malaria and explicitly demonstrates its effectiveness against ECM, the most pathogenic consequence of malaria infection. This experimental model will be important to explore the formulation of whole parasite blood-stage vaccines against malaria and to investigate the immune mechanisms that mediate protection against parasitemia and cerebral malaria.

Issues and solutions of conditional gene targeting

Conditional gene targeting, based on the site-specific recombination system such as Cre-loxP, plays a vital role in the study of gene functions and the generation of disease mouse models. It was always under consideration that there were problems in the Cre-loxP recombination system, such as illegal expression pattern of Cre transgene, variation of Cre recombination efficiency and toxicity of Cre recombinase, as well as the potential influences of genetic background, breeding strategy, experimental control and gene compensation. Oversights of these issues may have a profound influence on the accuracy of gene functional dissection and conditional gene targeting mice phenotypic interpretation. Accordingly, solutions should be adopted including delicate regulative control of temporal-spatial specific Cre expression, detailed detection of Cre recombination efficiency, reduction of Cre toxicity, simplification of mouse genetic background, optimization of breeding, setting up of proper control and combined conditional gene targeting.

Minor Abnormalities of Testis Development in Mice Lacking the Gene Encoding the MAPK Signalling Component, MAP3K1

In mammals, the Y chromosome is a dominant male determinant, causing the bipotential gonad to develop as a testis. Recently, cases of familial and spontaneous 46,XY disorders of sex development (DSD) have been attributed to mutations in the human gene encoding mitogen-activated protein kinase kinase kinase 1, MAP3K1, a component of the mitogen-activated protein kinase (MAPK) signal transduction pathway. In individuals harbouring heterozygous mutations in MAP3K1, dysregulation of MAPK signalling was observed in lymphoblastoid cell lines, suggesting a causal role for these mutations in disrupting XY sexual development. Mice lacking the cognate gene, Map3k1, are viable and exhibit the eyes open at birth (EOB) phenotype on a mixed genetic background, but on the C57BL/6J genetic background most mice die at around 14.5 dpc due to a failure of erythropoiesis in the fetal liver. However, no systematic examination of sexual development in Map3k1-deficient mice has been described, an omission that is especially relevant in the case of C57BL/6J, a genetic background that is sensitized to disruptions to testis determination. Here, we report that on a mixed genetic background mice lacking Map3k1 are fertile and exhibit no overt abnormalities of testis development. On C57BL/6J, significant non-viability is observed with very few animals surviving to adulthood. However, an examination of development in Map3k1-deficient XY embryos on this genetic background revealed no significant defects in testis determination, although minor abnormalities were observed, including an increase in gonadal length. Based on these observations, we conclude that MAP3K1 is not required for mouse testis determination. We discuss the significance of these data for the functional interpretation of sex-reversing MAP3K1 mutations in humans.

Differential levels of resistance to disease induction and development of relapsing experimental autoimmune encelphalomyelitis in two H-2b-restricted mouse strains

Besides the major histocompatibility complex (MHC) genes, background genes are believed to influence the encephalitogenicity of SJL(H-2s) and B10.S (H-2s) mice responding to myelin basic protein (MBP). A new mouse strain was constructed to study the effects of the SJL genetic background in mice responding to H-2b-restricted neuroantigens. Although the SJL.B (H-2b) mouse remained resistant to MBP in active EAE induction, the disease severity was uniformly higher in MOG-induced active EAE and in MBP-induced adoptive EAE when compared to those of B6 (H-2b) mice. Treatment of mice with anti-CD25 antibodies prior to immunization caused 60% of SJL.B mice to become susceptible to MBP-induced EAE while only 14% of B6 mice were converted. In addition, MOG-induced EAE in SJL.B mice followed a remitting–relapsing disease course while B6 mice only exhibited monophasic or chronic episodes. The new SJL.B mouse strain provides a valuable tool for studying EAE resistance and remitting–relapsing disease in H-2b mice.

T Cell Receptor Clonotype Influences Epitope Hierarchy in the CD8+ T Cell Response to Respiratory Syncytial Virus Infection

CD8+ T cell responses are important for recognizing and resolving viral infections. To better understand the selection and hierarchy of virus-specific T cell responses, we compared the T cell receptor (TCR) clonotype in parent and hybrid strains of respiratory syncytial virus-infected mice. K(d)M2(82-90) (SYIGSINNI) in BALB/c and D(b)M(187-195) (NAITNAKII) in C57Bl/6 are both dominant epitopes in parent strains but assume a distinct hierarchy, with K(d)M2(82-90) dominant to D(b)M(187-195) in hybrid CB6F1/J mice. The dominant K(d)M2(82-90) response is relatively public and is restricted primarily to the highly prevalent Vβ13.2 in BALB/c and hybrid mice, whereas D(b)M(187-195) responses in C57BL/6 mice are relatively private and involve multiple Vβ subtypes, some of which are lost in hybrids. A significant frequency of TCR CDR3 sequences in the D(b)M(187-195) response have a distinct "(D/E)WG" motif formed by a limited number of recombination strategies. Modeling of the dominant epitope suggested a flat, featureless structure, but D(b)M(187-195) showed a distinctive structure formed by Lys(7). The data suggest that common recombination events in prevalent Vβ genes may provide a numerical advantage in the T cell response and that distinct epitope structures may impose more limited options for successful TCR selection. Defining how epitope structure is interpreted to inform T cell function will improve the design of future gene-based vaccines.

Difference in Resistance toStreptococcus pneumoniaeInfection in Mice

Streptococcus pneumoniae is a major pathogen that causes various diseases, including pneumonia and sepsis, as millions of people suffer from S. pneumoniae infection worldwide. To better understand the immune and inflammatory responses to S. pneumoniae, we produced murine models. To investigate the differences between intranasal and intratracheal infection, BALB/c mice were infected with S. pneumoniae D39 intranasally or intratracheally. Mice showed no significant differences in survival rates, body weight changes, and bacterial loads. To investigate resistance and susceptibility among mouse strains, BALB/c, C57BL/6J, tumor necrosis factor-α (TNF-α) knockout, and interleukin-10 (IL-10) knockout mice were infected with S. pneumoniae D39 via intranasal or intravenous routes. In this study, BALB/c and C57BL/6J mice were resistant, IL-10 knockout mice were intermediate, and TNF-α knokout mice were susceptible to S. pneumoniae infection. These data show that intranasal and intratracheal infection induced similar results after S. pneumoniae infection, and the genetic background of mice must be considered when studying S. pneumoniae infection in vivo.

Multiple Apoptotic Defects In Hematopoietic Cellsfrom Mice Lacking Lipocalin 24p3.

AbstractAbstract 1560The lipocalin mouse 24p3 has been implicated in diverse physiological processes including apoptosis, iron trafficking, development and innate immunity. Studies from our lab as well as others demonstrated the pro-apoptotic activity of 24p3 in a variety of cultured models. However, a general role for the lipocalin 24p3 in the homeostasis of the hematopoietic system has not been tested in vivo. To study the role of 24p3 in apoptosis, we derived 24p3 null mice on 129 SVE and C57BL/6 genetic backgrounds, which enabled us to test, for the first time, the in vivo role of 24p3 without any confounders. We find that homozygous 24p3-/- mice have apoptotic defects in several hematopoietic lineages, including neutrophils, cytokine-dependent mast cells, thymocytes and erythroid cells. The progressive accumulation of lymphoid, myeloid and erythroid cells in 24p3-/- mice demonstrate the importance of 24p3 in the regulation of homeostasis of hematopoietic cells in adults. Thymocytes isolated from 24p3 null mice displayed resistance to apoptosis-induced by Dexamethasone. In addition, Dexamethasone-induced apoptosis is also reduced in 24p3-/- mice. Collectively, these results indicate that 24p3 is an important target gene involved in GC-mediated apoptosis of lymphocytes and other hematopoietic cell types. Finally, our results also indicate that the genetic background of mice can modulate the effects of 24p3 deficiency. In summary, the results presented here, in conjunction with those of previous studies, reveal 24p3 as a regulator of the hematopoietic compartment with important roles in normal physiology and disease progression. Disclosures:No relevant conflicts of interest to declare.

Cytomegalovirus microRNAs Facilitate Persistent Virus Infection in Salivary Glands

Micro (mi)RNAs are small non-coding RNAs that regulate the expression of their targets' messenger RNAs through both translational inhibition and regulation of target RNA stability. Recently, a number of viruses, particularly of the herpesvirus family, have been shown to express their own miRNAs to control both viral and cellular transcripts. Although some targets of viral miRNAs are known, their function in a physiologically relevant infection remains to be elucidated. As such, no in vivo phenotype of a viral miRNA knock-out mutant has been described so far. Here, we report on the first functional phenotype of a miRNA knock-out virus in vivo. During subacute infection of a mutant mouse cytomegalovirus lacking two viral miRNAs, virus production is selectively reduced in salivary glands, an organ essential for virus persistence and horizontal transmission. This phenotype depends on several parameters including viral load and mouse genetic background, and is abolished by combined but not single depletion of natural killer (NK) and CD4+ T cells. Together, our results point towards a miRNA-based immunoevasion mechanism important for long-term virus persistence.

Distinct role of T helper Type 17 immune response for Graves' hyperthyroidism in mice with different genetic backgrounds

T helper type 17 (Th17) cells, a newly identified effector T-cell subset, have recently been shown to play a role in numerous autoimmune diseases, including iodine-induced autoimmune thyroiditis in non-obese diabetic (NOD)-H2h4 mice, which had previously been thought Th1-dominant. We here studied the role of Th17 in Graves' hyperthyroidism, another thyroid-specific autoimmune disease, in a mouse model. Two genetically distinct BALB/c and NOD-H2h4 strains with intact or disrupted IL-17 genes (IL-17+/+ or IL-17− / − ) were immunized with adenovirus (Ad) expressing the thyrotropin receptor (TSHR) A-subunit (Ad-TSHR289). Both IL-17+/+ and IL-17− / − mice developed anti-TSHR antibodies and hyperthyroidism at equally high frequencies on the BALB/c genetic background. In contrast, some IL-17+/+, but none of IL-17− / − , mice became hyperthyroid on the NOD-H2h4 genetic background, indicating the crucial role of IL-17 for development of Graves' hyperthyroidism in non-susceptible NOD-H2h4, but not in susceptible BALB/c mice. In the T-cell recall assay, splenocytes and lymphocytes from the draining lymph nodes from either mouse strains, irrespective of IL-17 gene status, produced IFN-γ and IL-10 but not other cytokines including IL-17 in response to TSHR antigen. Thus, the functional significance of Th17 may not necessarily be predictable from cytokine expression patterns in splenocytes or inflammatory lesions. In conclusion, this is, to our knowledge, the first report showing that the role of Th17 cells for the pathogenesis of a certain autoimmune disease depends on the mouse genetic backgrounds.

Emphysema is associated with increased inflammation in lungs of atherosclerosis-prone mice by cigarette smoke: implications in comorbidities of COPD

BackgroundChronic obstructive pulmonary disease is associated with numerous vascular effects including endothelial dysfunction, arterial stiffness and atherogenesis. It is also known that a decline in lung function is associated with increased cardiovascular comorbidity in smokers. The mechanism of this cardiopulmonary dual risk by cigarette smoke (CS) is not known. We studied the molecular mechanisms involved in development of emphysema in atherosclerosis-prone apolipoprotein E-deficient (ApoE-/-) mice in response to CS exposure.MethodsAdult male and female wild-type (WT) mice of genetic background C57BL/6J and ApoE-/- mice were exposed to CS, and lung inflammatory responses, oxidative stress (lipid peroxidation products), mechanical properties as well as airspace enlargement were assessed.Results and DiscussionThe lungs of ApoE-/- mice showed augmented inflammatory response and increased oxidative stress with development of distal airspace enlargement which was accompanied with decline in lung function. Interestingly, the levels and activities of matrix metalloproteinases (MMP-9 and MMP-12) were increased, whereas the level of eNOS was decreased in lungs of CS-exposed ApoE-/- mice as compared to air-exposed ApoE-/- mice or CS-exposed WT mice.ConclusionThese findings suggest that CS causes premature emphysema and a decline of lung function in mice susceptible to cardiovascular abnormalities via abnormal lung inflammation, increased oxidative stress and alterations in levels of MMPs and eNOS.

Abstract 949: Different lineages of carcinogenesis in prostate lobes in the transgenic adenocarcinoma of mouse prostate (TRAMP) model in C57BL/6 background and their responses to chemopreventive selenium

Abstract The transgenic adenocarcinoma of the mouse prostate (TRAMP) model is based on probasin promoter-driven expression of SV40 T-antigen (Tag) to inactivate p53 and Rb tumor suppressor to propel proliferative lesions and aggressive cancers in the prostate. The genetic background of mice (FVB vs. C57BL/6) has been known to profoundly affect the pathogenesis profiles and disease severity. A recent study suggested prostate lobe specificity of neuro-endocrine (NE) carcinomas vs. glandular epithelial lesions, challenging the classical notion of single-lineage disease progression from early epithelial lesions to poorly differentiated carcinomas. Because of the increasing use of this model for chemoprevention studies, it is critical to further characterize the lobe-specificity of lesion lineages in the prostate and determine their responses to chemopreventive agents. To this end, we carefully dissected the dorsolateral (DLP) and ventral prostate (VP) and macroscopic tumors from 30 male C57BL/6 TRAMP mice at 24-weeks of age and fixed tissues in 10% formalin for pathology diagnosis. Our results show that macroscopic tumors were Tag+, synaptophysin (Syp)+, androgen-receptor (AR)− and E-cadherin− NE carcinomas (poorly differentiated carcinomas) and were dissected exclusively from VP, not DLP. Lesions identified in lymph node, liver or lung of such NE-carcinoma bearing mice showed concordant patterns of biomarkers with the primary prostate NE-carcinomas and represent their metastases. In the DLP, we found lesions ranging from mild (formerly as low grade PIN), moderate to atypical hyperplasia of Tag (formerly as moderately differentiated adenocarcinoma) with Tag+, AR+ and E-cadherin+ expression and no NE-carcinoma. For assessing the impact of selenium on the long term survival from different lineages of prostate lesions, we analyzed archival tissues from a survival study published recently (Wang et al, Cancer Prev Res 2009): TRAMP mice received a daily oral dose of 3 mg Se/kg body weight as methylseleninic acid (MSeA) beginning at 10 weeks (n=31) or 16 weeks of age (n=30), or water (n=31) until euthanasia or 50 weeks of age. MSeA supplementation significantly delayed death from NE-carcinomas (30% life-time incidence). MSeA also prolonged survival of mice with enlarged seminal vesicle containing epithelial-stromal tumors that were Tag+, Syp−, E-cadherin+ and AR+/low in the older TRAMP mice (>30 weeks). Interestingly, mice with enlarged seminal vesicle often had normal sized prostate. In summary, the C57BL/6 TRAMP mouse model represents two lineages of prostate carcinogenesis with an involvement of seminal vesicle tumorigenesis in older mice. Future chemoprevention studies should incorporate this new knowledge for efficacy assessment and for molecular target validations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 949.

Epithelial Stress and Apoptosis Underlie Hermansky-Pudlak Syndrome–associated Interstitial Pneumonia

The molecular mechanisms underlying Hermansky-Pudlak syndrome-associated interstitial pneumonia (HPSIP) are poorly understood but, as in idiopathic pulmonary fibrosis, may be linked to chronic alveolar epithelial type II cell (AECII) injury. We studied the development of fibrosis and the role of AECII injury in various murine models of HPS. HPS1, HPS2, and HPS6 monomutant mice, and HPS1/2 and HPS1/6 double-mutant and genetic background mice, were killed at 3 and 9 months of age. Quantitative morphometry was undertaken in lung sections stained with hemalaun-eosin. The extent of lung fibrosis was assessed by trichrome staining and hydroxyproline measurement. Surfactant lipids were analyzed by electrospray ionization mass spectrometry. Surfactant proteins, apoptosis, and lysosomal and endoplasmic reticulum stress markers were studied by Western blotting and immunohistochemistry. Cell proliferation was measured by water-soluble tetrazolium salt-1 and bromodeoxyuridine assays. Spontaneous and slowly progressive HPSIP was observed in HPS1/2 double mutants, but not in other HPS mutants, with subpleural onset at 3 months and full-blown fibrosis at 9 months. In these mice, extensive surfactant abnormalities were encountered in AECII and were paralleled by early lysosomal stress (cathepsin D induction), late endoplasmic reticulum stress (activating transcription factor-4 [ATF4], C/EBP homologous protein [CHOP] induction), and marked apoptosis. These findings were fully corroborated in human HPSIP. In addition, cathepsin D overexpression resulted in apoptosis of MLE-12 cells and increased proliferation of NIH 3T3 fibroblasts incubated with conditioned medium of the transfected cells. Extensively impaired surfactant trafficking and secretion underlie lysosomal and endoplasmic reticulum stress with apoptosis of AECII in HPSIP, thereby causing the development of HPSIP.

Adaptation of bone and calcium metabolism to low dietary calcium (Ca) stress is affected by genetic background in mice

We tested the hypothesis that Gene × Diet interactions influence adaptation of Ca and bone metabolism to low Ca intake using inbred mice. Male C57BL/6J (B6), DBA/2J (DBA), and PWK/PhJ (PWK) mice were fed diets containing adequate (0.5%) or low (0.25%) Ca levels from weaning until 3 mo (n=6–12 per group) when 45Ca absorption (Ca Abs) was assessed after an overnight fast (n=6 per group), serum was harvested for vitamin D metabolite levels, and femura were fixed for DEXA analysis of bone mineral density (BMD). Significant differences between the lines on the 0.5% Ca diet were observed for body weight (B6>DBA>PWK), BMD (DBA>B6>PWK), serum 25 hydroxyvitamin D (B6>DBA>PWK), serum 1,25 dihydroxyvitamin D (1,25 D; DBA>B6=PWK), and Ca Abs (B6>DBA=PWK). Ca restriction reduced BMD only in DBA mice (10.6% lower) and this was partly explained by their inability to increase serum 1,25 D and Ca Abs; low Ca diet increased serum 1,25 D and Ca Abs by 70% and 60%, respectively in B6 mice and by 97% and 11%, respectively in PWK mice. These data suggest there are genetic factors influencing basal levels of vitamin D and calcium metabolism in inbred mouse lines as well as the adaptation of Ca metabolism to dietary Ca restriction in DBA mice. Future forward genetic approaches will be necessary to identify the genetic variation responsible for these physiological differences. Supported by NIH Award DK054111 and funds from Purdue University

Role of KLF2 in endocardial cushion development in mice

Krüppel‐like factor 2 (KLF2) belongs to a family of transcription factors that have 3 C2/H2 zinc finger domains. KLF2 is expressed in endothelial cells and is induced by shear stress. KLF2−/− embryos in FVB/N genetic background die by embryonic day 11.5 (E11.5) (chi‐square p = 0.0044), whereas in mixed genetic background, they die by E14.5. Our studies show that unlike WT hearts, in E9.5 KLF2 KO hearts (n = 3) the cells lining the atrioventricular (AV) cushion, the area that will become the AV valves, form multiple, disorganized layers and the number of cells is 2‐fold higher. Electron microscopy revealed that compared to WT, the E9.5 KLF2−/− AV canal is narrower in some locations but patent and lined by endothelial cells having numerous cytoplasmic processes extending toward the lumen. In E10.5 KLF2−/− embryos (n = 3), the AV and outflow tract cushions are hypoplastic and the cells lining the AV canal form multiple disorganized layers. Alcian blue staining of E10.5 KLF2−/− hearts showed an absence of glycosaminoglycans in the cardiac jelly. These findings suggest that KLF2, directly or indirectly, regulates endothelial to mesenchymal transformation in AV cushion development. The AV phenotype is observed in FVB/N KLF2−/− embryos but not in embryos of mixed background (n = 2). Therefore, KLF2 is required for normal endocardial cushion development in FVB/N genetic background mice. (Supported by NIH R01DK074694)

Production of mouse chimeras by injection of embryonic stem cells into the perivitelline space of one-cell stage embryos

Generation of mouse chimeras is useful for the elucidation of gene function. In the present report, we describe a new technique for the production of chimeras by injection of R1 embryonic stem (ES) cells into the perivitelline space of one-cell stage mouse embryos. One-cell embryos are injected with 2-6 ES cells into the perivitelline space under the zona pellucida without laser-assistance. Our embryo culture experiments reveal that ES cells injected at the one-cell stage embryo start to be incorporated into the blastomeres beginning at the 8-cell stage and form a chimeric blastocyst after 4days. We have used this approach to successfully produce a high rate of mouse chimeras in two different mouse genetic backgrounds permitting the establishment of germ line transmitters. This method allows for the earlier introduction of ES cells into mouse embryos, and should free up the possibility of using frozen one-cell embryos for this purpose.

PKCε Overexpression, Irrespective of Genetic Background, Sensitizes Skin to UVR-Induced Development of Squamous-Cell Carcinomas

Chronic exposure to UVR is the major etiologic factor in the development of human skin cancers including squamous-cell carcinoma (SCC). We have previously shown that protein Kinase C epsilon (PKCepsilon) transgenic mice on FVB/N background, which overexpress PKCepsilon protein approximately eightfold over endogenous levels in epidermis, exhibit about threefold more sensitivity than wild-type littermates to UVR-induced development of SCC. To determine whether it is PKCepsilon and not the mouse genetic background that determines susceptibility to UVR carcinogenesis, we cross-bred PKCepsilon FVB/N transgenic mice with SKH-1 hairless mice to generate PKCepsilon-overexpressing SKH-1 hairless mice. To evaluate the susceptibility of PKCepsilon SKH-1 hairless transgenic mice to UVR carcinogenesis, the mice were exposed to UVR (1-2 KJ m(-2)) three times weekly from a bank of six kodacel-filtered FS40 sunlamps. As compared with the wild-type hairless mice, PKCepsilon overexpression in SKH-1 hairless mice decreased the latency (12 weeks), whereas it increased the incidence (twofold) and multiplicity (fourfold) of SCC. The SKH hairless transgenic mice were observed to be as sensitive as FVB/N transgenic mice to UVR-induced development of SCC and expression of proliferative markers (proliferating cell nuclear antigen, signal transducers and activators of transcription 3, and extracellular signal-regulated kinase 1/2). The results indicate that PKCepsilon level dictates susceptibility, irrespective of genetic background, to UVR carcinogenesis.

Matrix Metalloproteinase-8 and the Regulation of Blood Pressure, Vascular Inflammation, and Atherosclerotic Lesion Growth

See related articles, pages 921–929 The description in the early 1990s of intriguing associations between the expression/activity of specific matrix metalloproteinases (MMPs) and signs of plaque inflammation and matrix degradation1–3 spurred an impressive effort of research to precisely define the roles of MMPs in both the development and complications of atherosclerosis. After two decades of research in this area, mechanistic studies using genetically manipulated mice with deletion or overexpression of specific MMPs, complemented with genetic epidemiological studies, have revealed more complex and, sometimes divergent, roles of MMPs in the modulation of lesion development, progression and complications.4 There are many potential explanations for these findings. Ambivalent effects of specific MMPs have been reported in different models of atherosclerosis,5 emphasizing the urgent need for clinically relevant animal models of disease complications. For example, even the use of the brachiocephalic artery of apolipoprotein (Apo)E knockout mice as a model of site-specific plaque rupture did not allow to differentiate between the roles of MMPs in plaque development versus plaque complications. A close look at this model reveals that the number of buried fibrous caps is highly proportional to lesion size, independently of mouse genetic background and MMP expression.6 The complex role of MMPs in atherosclerosis could be attributed to the diversity of biological processes relevant to this disease, which are subject to tight control by MMPs and the modulation of which could have divergent consequences for lesion development and fibrous cap stability. The MMP-controlled processes include those classically related to matrix remodeling, vascular …

A murine model of urinary tract infection

Urinary tract infections (UTIs) inflict extreme pain and discomfort to those affected and have profound medical and socioeconomic impact. Although acute UTIs are often treatable with antibiotics, a large proportion of patients suffer from multiple recurrent infections. Here, we describe and provide a protocol for a robust murine UTI model that allows for the study of uropathogens in an ideal setting. The infections in the urinary tract can be monitored quantitatively by determining the bacterial loads at different times post-infection. In addition, the simple bladder architecture allows observation of disease progression and the uropathogenic virulence cascade using a variety of microscopic techniques. This mouse UTI model is extremely flexible, allowing the study of different bacterial strains and species of uropathogens in a broad range of mouse genetic backgrounds. We have used this protocol to identify important aspects of the host-pathogen interaction that determine the outcome of infection. The time required to complete the entire procedure will depend on the number of bacterial strains and mice included in the study. Nevertheless, one should expect 4 h of hands-on time, including inoculum preparation on the day of infection, transurethral inoculation, tissue harvest and post-harvest processing for a small group of mice (e.g., 5 mice).

GPR56-Regulated Granule Cell Adhesion Is Essential for Rostral Cerebellar Development

Mutations in GPR56, an orphan G-protein-coupled receptor (GPCR), cause bilateral frontoparietal polymicrogyria (BFPP), a disorder characterized by mental retardation, seizures, motor developmental delay, and ataxia. BFPP patients have structural abnormalities of the cerebral cortex, cerebellum, and pons. To shed light on the function of GPR56 and the anatomical and behavioral defects underlying BFPP, we analyzed the cerebellum of mice lacking this GPCR. Gpr56(-/-) mice display a severe malformation of the rostral cerebellum that develops perinatally. Defects involve fusion of adjacent lobules, disrupted layering of neurons and glia, and fragmentation of the pial basement membrane. At the age of defect onset, GPR56 expression is restricted specifically to developing granule cells in the rostral cerebellum, suggesting that GPR56 regulates properties of these cells. Indeed, granule cells from the rostral region of perinatal Gpr56(-/-) cerebella show loss of adhesion to extracellular matrix molecules of the pial basement membrane. Interference RNA-mediated knockdown of GPR56 recapitulates the loss of adhesion seen in knock-outs, and reexpression of GPR56 rescues the adhesion defect in knock-out granule cells. Loss of GPR56 does not affect cell proliferation, migration, or neurite outgrowth. These studies establish a novel role for GPR56 in the adhesion of developing neurons to basal lamina molecules and suggest that this adhesion is critical for maintenance of the pia and proper cerebellar morphogenesis.

Selective enhanced constriction and attenuated dilation in leptin deficient mice.

We tested responses to endothelin‐1 (ET), interleukin‐6 (IL6) and IL8 in leptin deficient diabetic mice (db/db or ob/ob), in comparison to C57 genetic background mice in the absence or presence of inhibitors. The cremaster muscle of anesthetized (50 mg/kg Nembutal) mice (n=63) was prepared for intravital microscopy. Arteriole(A)/venule(V) pairs were chosen for study; baseline diameters A 26±8 ?m, V 50±13 ?m were not different between strains. Micropipette applied adenosine (10‐4M) was attenuated in A and V from db/db or nitroarginine (LNNA) pre‐treated C57 vs. control C57. Phenylephrine (10‐4M) constriction was unchanged in A, but attenuated in V from db/db, ob/ob or LNNA vs. C57. ET1 dose dependent constriction (C57 logM EC50 ‐10±5 A; ‐5±4 V) was significantly attenuated in either db/db or ob/ob mice, yet enhanced with LNNA pretreatment in C57. Responses to IL6 or IL8 were biphasic and dose dependent in A from C57, while V showed only constriction. Responses to IL6 were blocked by PD142893. IL6 caused only constriction in A and V of ob/ob or db/db. IL8 caused biphasic responses in some diabetics. Leptin deficient diabetes thus shows attenuated constriction to ET1, selective absence of dilation to IL6 and inconsistent absence of dilation to IL8. NIH DK68401