Survival analysis and microarray profiling identify Cd40 as a candidate for the Salmonella susceptibility locus, Ity5.

The outcome of infection with Salmonella Typhimurium in mouse models of human typhoid fever is dependent upon a coordinated complex immune response. A panel of recombinant congenic strains (RCS) derived from reciprocal backcross of A/J and C57BL/6J mice was screened for their susceptibility to Salmonella infection and two susceptibility loci, Ity4 (Immunity to Typhimurium locus 4) and Ity5, were identified. We validated Ity5 in a genetic environment free of the impact of Ity4 using a cross between A/J and 129S6. Using a time-series analysis of genome-wide transcription during infection, comparing A/J with AcB60 mice having a C57BL/6J-derived Ity5 interval, we have identified the differential expression of the positional candidate gene Cd40, Cd40-associated signaling pathways, and the differential expression of numerous genes expressed in neutrophils. CD40 is known to coordinate T cell-dependent B-cell responses and myeloid cell activation. In fact, CD40 signaling is altered in A/J mice as seen by impaired IgM upregulation during infection, decreased Ig class switching, neutropenia, reduced granulocyte recruitment in response to infection and inflammation, and decreased ERK1/2 activity. These results suggest that altered CD40 signaling and granulocyte recruitment in response to infection are responsible for the Ity5-associated Salmonella susceptibility of A/J mice.

Mapping of a quantitative trait locus controlling susceptibility to Coxsackievirus B3-induced viral hepatitis.

The pathogenesis of coxsackieviral infection is a multifactorial process involving host genetics, viral genetics and the environment in which they interact. We have used a mouse model of Coxsackievirus B3 infection to characterize the contribution of host genetics to infection survival and to viral hepatitis. Twenty-five AcB/BcA recombinant congenic mouse strains were screened. One, BcA86, was found to be particularly susceptible to early mortality; 100% of BcA86 mice died by day 6 compared with 0% of B6 mice (P=0.0012). This increased mortality was accompanied by an increased hepatic necrosis as measured by serum alanine aminotransferase (ALT) levels (19547±10556 vs 769±109, P=0.0055). This occurred despite a predominantly resistant (C57BL/6) genetic background. Linkage analysis in a cohort (n=210) of (BcA86x C56Bl/10)F2 animals revealed a new locus on chromosome 13 (peak linkage 101.2 Mbp, lod 4.50 and P=0.003). This locus controlled serum ALT levels as early as 48 h following the infection, and led to an elevated expression of type I interferon. Another locus on chromosome 17 (peak linkage 57.2 Mbp) was significantly linked to heart viral titer (lod 3.4 and P=0.046). These results provide new evidence for the presence of genetic loci contributing to the susceptibility of mice to viral hepatitis.

Reduced MCMV Δm157 viral clearance in the absence of TSAd.

The T cell specific adapter protein (TSAd) is expressed in activated T cells and NK cells. While TSAd is beginning to emerge as a critical regulator of Lck and Itk activity in T cells, its role in NK cells has not yet been explored. Here we have examined susceptibility to virus infections in a murine model using various viral infection models. We report that TSAd-deficient mice display reduced clearance of murine cytomegalovirus (MCMV) that lack the viral MHC class I homologue m157, which is critical for Ly49H-mediated NK cell recognition of infected cells. In this infection model, NK cells contribute in the early stages of the disease, whereas CD8+ T cells are critical for viral clearance. We found that mice infected with MCMV Δm157 displayed reduced viral clearance in the spleen as well as reduced proliferation in spleen NK cells and CD8+ T cells in the absence of TSAd. Though no other immunophenotype was detected in the infection models tested, these data suggests that in the absence of the Ly49H ligand activation, NK cell and CD8+ T cell responses may be compromised in TSAd-deficient mice.

An ENU-induced splicing mutation reveals a role for Unc93b1 in early immune cell activation following influenza A H1N1 infection.

Genetic and immunological analysis of host-pathogen interactions can reveal fundamental mechanisms of susceptibility and resistance to infection. Modeling human infectious diseases among inbred mouse strains is a proven approach but is limited by naturally occurring genetic diversity. Using N-ethyl-N-nitrosourea mutagenesis, we created a recessive loss-of-function point mutation in Unc93b1 (unc-93 homolog B1 (C. elegans)), a chaperone for endosomal Toll-like receptors (TLR)3, TLR7 and TLR9, which we termed Letr for 'loss of endosomal TLR response'. We used Unc93b1(Letr/Letr) mice to study the role of Unc93b1 in the immune response to influenza A/PR/8/34 (H1N1), an important global respiratory pathogen. During the early phase of infection, Unc93b1(Letr/Letr) mice had fewer activated exudate macrophages and decreased expression of CXCL10, interferon (IFN)-γ and type I IFN. Mutation of Unc93b1 also led to reduced expression of the CD69 activation marker and a concomitant increase in the CD62L naive marker on CD4(+) and CD8(+) T cells in infected lungs. Finally, loss of endosomal TLR signaling resulted in delayed viral clearance that coincided with increased tissue pathology during infection. Taken together, these findings establish a role for Unc93b1 and endosomal TLRs in the activation of both myeloid and lymphoid cells during the innate immune response to influenza.

Susceptibility to lethal cerebral malaria is regulated by epistatic interaction between chromosome 4 (Berr6) and chromosome 1 (Berr7) loci in mice.

In humans, cerebral malaria is a rare but often lethal complication of infection with Plasmodium parasites, the occurrence of which is influenced by complex genetic factors of the host. We used a mouse model of experimental cerebral malaria (ECM) with Plasmodium berghei ANKA to study genetic factors regulating appearance of neurological symptoms and associated lethality. In a genome-wide screen of N-ethyl-N-nitrosourea-mutagenized mice derived from C57BL/6J (B6) and 129S1/SvImJ (129) mouse strains, we detected a strong interaction between the genetic backgrounds of these strains, which modulates ECM resistance. We have mapped a major gene locus to central chromosome 4 (log of the odds (LOD) 6.7; 79.6-97.3 Mb), which we designate Berr8. [corrected]. B6 alleles at Berr6 are associated with resistance, and are inherited in a co-dominant fashion. In mice heterozygous for Berr6 B6/129 alleles, resistance to ECM is strongly modulated by a second locus, Berr7, that maps to the proximal portion of chromosome 1 (LOD 4.03; 41.4 Mb). 129 alleles at Berr7 are associated with ECM resistance in a dosage-dependent fashion. Results are discussed in view of the possible role of this two-locus system in susceptibility to unrelated inflammatory conditions in mice and humans.

Veterinary education in the area of food safety (including animal health, food pathogens and surveillance of foodborne diseases).

The animal foodstuffs industry has changed in recent decades as a result of factors such as: human population growth and longer life expectancy, increasing urbanisation and migration, emerging zoonotic infectious diseases and foodborne diseases (FBDs), food security problems, technological advances in animal production systems, globalisation of trade and environmental changes. The Millennium Development Goals and the 'One Health' paradigm provide global guidelines on efficiently addressing the issues of consumer product safety, food security and risks associated with zoonoses. Professionals involved in the supply chain must therefore play an active role, based on knowledge and skills that meet current market requirements. Accordingly, it is necessary for the veterinary medicine curriculum, both undergraduate and postgraduate, to incorporate these skills. This article analyses the approach that veterinary education should adopt in relation to food safety, with an emphasis on animal health, food pathogens and FBD surveillance.

Methylenetetrahydrofolate reductase (MTHFR) deficiency enhances resistance against cytomegalovirus infection.

Folates provide one-carbon units for nucleotide synthesis and methylation reactions. A common polymorphism in the MTHFR gene (677C --> T) results in reduced enzymatic activity, and is associated with an increased risk for neural tube defects and cardiovascular disease. The high prevalence of this polymorphism suggests that it may have experienced a selective advantage under environmental pressure, possibly an infectious agent. To test the hypothesis that methylenetetrahydrofolate reductase (MTHFR) genotype influences the outcome of infectious disease, we examined the response of Mthfr-deficient mice against mouse cytomegalovirus (MCMV) infection. Acute MCMV infection of Mthfr(-/-) mice resulted in early control of cytokine secretion, decreased viral titer and preservation of spleen immune cells, in contrast to Mthfr wild-type littermates. The phenotype was abolished in MTHFR transgenic mice carrying an extra copy of the gene. Infection of primary fibroblasts with MCMV showed a decrease in viral replication and in the number of productively infected cells in Mthfr(+/-) fibroblasts compared with wild-type cells. These results indicate that Mthfr deficiency protects against MCMV infection in vivo and in vitro, suggesting that human genetic variants may provide an advantage in the host response against certain pathogens.

Forward genetic dissection of immunity to infection in the mouse.

Forward genetics is an experimental approach in which gene mapping and positional cloning are used to elucidate the molecular mechanisms underlying phenotypic differences between two individuals for a given trait. This strategy has been highly successful for the study of inbred mouse strains that show differences in innate susceptibility to bacterial, parasitic, fungal, and viral infections. Over the past 20 years, these studies have led to the identification of a number of cell populations and critical biochemical pathways and proteins that are essential for the early detection of and response to invading pathogens. Strikingly, the macrophage is the point of convergence for many of these genetic studies. This has led to the identification of diverse pathways involved in extracellular and intracellular pathogen recognition, modification of the properties and content of phagosomes, transcriptional response, and signal transduction for activation of adaptive immune mechanisms. In models of viral infections, elegant genetic studies highlighted the pivotal role of natural killer cells in the detection and destruction of infected cells.

Complex genetic control of host susceptibility to coxsackievirus B3-induced myocarditis.

The pathogenesis of viral myocarditis is a multifactorial process involving host genetics, viral genetics and the environment in which they interact. We have used a model of infection with coxsackievirus B3 (CVB3) to characterize the contribution of host genetics to viral myocarditis in mice of different genetic backgrounds but with a common H2 haplotype: A/J and B10.A-H2(a). Here we have used Evans blue dye as a quantitative biomarker for susceptibility to CVB3-induced myocarditis in addition to histopathological semiquantitative measures. We have found evidence of linkage between susceptibility to viral myocarditis and three loci. A locus on chromosome 1 centered on D1Mit200 was linked to sarcolemmal disruption in males (P=0.00005), a second locus on chromosome 4 centered on D4Mit81 was also linked to sarcolemmal disruption in males (P=0.0022). A third locus on distal chromosome 3 centered on D3Mit19 was linked to myocardial infiltration, with a logarithm of odds (LOD) score of 4.7 (P=0.0045), as well as sarcolemmal disruption in females (P=0.0015). These results provide strong evidence for the presence of loci contributing to the susceptibility of mice to viral myocarditis.

NK cell recognition of mouse cytomegalovirus-infected cells.

Abstract Natural killer (NK) cells and cytomegalovirus have been locked in an evolutionary arms race for millions of years in an attempt to overwhelm each other. Cytomegaloviruses deploy cunning disguises to avoid detection by NK cells. Studies of the mouse model of infection have shown that NK cells deploy multiple mechanisms to deal with mouse cytomegalovirus (MCMV) infection, which involve receptors of the C-lectin type superfamily. Remarkably, these receptors have two additional common features: They map to the same genetic region, known as the NK cell gene complex; and they recognize MHC class I-related structures. While reviewing these attack-counterattack measures, this chapter points to the central role that recognition of the MCMV-infected cells by NK cells plays in host resistance to infection.

Genetic control of innate immune responses against cytomegalovirus: MCMV meets its match.

Cytomegalovirus (CMV) is a widespread pathogen that is responsible for severe disease in immunocompromised individuals and probably, associated with vascular disease in the general population. There is increasing evidence that cells of the innate immune system play a key role in controlling this important pathogen. This is particularly evident in the experimental murine CMV (MCMV) model of infection which has revealed an important role for natural killer (NK) cells in controlling early viral replication after infection with MCMV. In this model, different strains of inbred mice exhibit striking differences in their level of susceptibility to MCMV infection. Genetic studies, performed almost 10 years ago, revealed that this pattern of susceptibility/resistance can be attributed to a single genetic locus termed Cmv1 and recently several groups that have been working on the mapping and identification of Cmv1 have met with success. Interestingly, Cmv1 is allelic to a member of the Ly49 gene family, which encode activating or inhibitory transmembrane receptors present on the surface of NK cells. All Ly49 receptors characterized to date interact with MHC class I molecules on potential target cells, resulting in the accumulation of signals to the NK to either 'kill' or 'ignore' the cell based upon the repertoire of MHC class I molecules expressed. The identification of Cmv1 as Ly49H, a stimulatory member of the Ly49 family, adds an interesting twist to the Ly49 story. Although the ligand of Ly49H is not yet known, there is already compelling evidence that the ligand is upregulated on virally infected cells, resulting in specific activation of Ly49H-expressing NK cells. This review provides an historical perspective of the MCMV infection model from its inception to the discovery of the gene responsible for the phenotype and provides a basis for further experiments aimed at understanding the role of NK cells, in general, and Ly49H, in particular, in mediating resistance to cytomegalovirus.

Pathological and genetic analysis of the degenerating muscle (dmu) mouse: a new allele of Scn8a.

Here, we describe a novel spontaneous autosomal recessive mutation in the mouse that is characterized by skeletal and cardiac muscle degeneration. We have named this mutant degenerating muscle (dmu). At birth, mutant mice are indistinguishable from their normal littermates. Thereafter, the disease progresses rapidly and a phenotype is first observed at approximately 11 days after birth; the dmu mice are weak and have great difficulty in moving. The principal cause of the lack of mobility is muscle atrophy and wasting in the hindquarters. Affected mice die at or around the time of weaning of unknown causes. Histopathological observations and ultrastructural analysis revealed muscle degeneration in both skeletal and cardiac muscle, but no abnormalities in sciatic nerves. Using linkage analysis, we have mapped the dmu locus to the distal portion of mouse chromosome 15 in a region syntenic to human chromosome 12q13. Interestingly, scapuloperoneal muscular dystrophy (SPMD) in humans has been linked to this region. SPMD patients with associated cardiomyopathy have also been described in the past. Initial analysis of candidate genes on mouse chromosome 15 reveal that although intact transcripts for Scn8a, the gene encoding the sodium channel 8a subunit, are present in dmu mice, their levels are dramatically reduced. Furthermore, genetic complementation crosses between dmu and med (mutation in Scn8a) mice revealed that they are allelic. Our results suggest that at least a portion of the dmu phenotype is caused by a down-regulation of Scn8a, making dmu a new allele of Scn8a.

Susceptibility to mouse cytomegalovirus is associated with deletion of an activating natural killer cell receptor of the C-type lectin superfamily.

Cytomegalovirus is the leading cause of congenital viral disease and the most important opportunistic infection in immunocompromised patients. We have used a mouse experimental infection model (MCMV) to study the genetic parameters of host/virus interaction. Susceptibility to infection with MCMV is controlled by Cmv1, a chromosome 6 locus that regulates natural killer (NK) cell activity against virally infected targets. Here, we use a positional cloning strategy to isolate the gene mutated at the Cmv1 locus. Cmv1 maps within a 0.35-cM interval defined by markers D6Ott8 and D6Ott115, which corresponds to a physical distance of 1.6 Mb (refs. 6-8). A transcript map of the region identified 19 genes, including members of the killer cell lectin-like receptor family a (Klra, formerly Ly49; refs. 9-12), which encode inhibitory or activating NK cell receptors that interact with MHC class I molecules. Klra genes have different copy numbers and genomic organization, and are highly polymorphic among inbred strains, making it difficult to distinguish between normal allelic variants and distinct Klra genes, or possible mutations associated with Cmv1. The recombinant inbred strain BXD-8/Ty (BXD-8; ref. 18), derived from Cmv1r C57BL/6 (B6, resistant) and Cmv1s DBA/2 (susceptible), is of particular interest because it is highly susceptible to MCMV infection despite having a B6 haplotype at Cmv1. We determined that MCMV susceptibility in BXD-8 is associated with the deletion of Klra8 (formerly Ly49h).

Cloning, expression and chromosomal location of NKX6B TO 10Q26, a region frequently deleted in brain tumors.

Nkx6-2 (former Gtx) is a murine-homeobox-containing gene localized distally on Chromosome (Chr) 7. Analysis of the expression pattern, together with DNA binding assays, suggests that this gene product might be important for differentiated oligodendrocyte function and in the regulation of myelin gene expression. We now report on the cloning and characterization of the human homolog (NKX6B). DNA sequence analysis of an 11-kb genomic fragment revealed that the complete human gene spans 1.2 kb and is composed of three exons. NKX6B is predicted to encode a polypeptide of 277 amino acids with 97% identity to mouse Nkx6-2. Northern blot experiments showed that NKX6B expression is tightly controlled in a tissue-specific fashion with the highest site of expression being the brain. Finally, using STS content mapping and RH analyis, we demonstrated that NKX6B maps to the 10q26, a region where frequent loss of heterozygosity has been observed in various malignant brain tumors. These results may implicate NKX6B as a candidate tumor suppressor gene for brain tumors, particularly for oligodendrogliomas.

Sequence-ready BAC contig, physical, and transcriptional map of a 2-Mb region overlapping the mouse chromosome 6 host-resistance locus Cmv1.

The host-resistance locus Cmv1 controls viral replication of mouse cytomegalovirus (MCMV) in the spleen of infected mice. Cmv1 maps on distal chromosome 6, very tightly linked to the Ly49 gene family within a 0.35-cM interval defined proximally by Cd94/Nkg2d and distally by D6Mit13/D6Mit111/D6Mit219/Prp/Kap. To facilitate the cloning of the gene, we have created a high-resolution physical map of the Cmv1 genetic interval that is based on long-range restriction mapping by pulsed-field gel electrophoresis, fluorescence in situ hybridization analysis of interphase nuclei, and the assembly of a cloned contig. A contig of BAC and YAC clones was assembled using probes derived from the minimal genetic interval. Individual clones from the region were validated by (1) restriction digest fingerprinting, (2) STS content mapping, (3) Southern hybridizations, and (4) sequencing and mapping of clone ends. This contig contains 25 YACs anchored by 71 STSs and 73 BACs anchored by 40 STSs. We also report the cloning of 31 new STSs and 18 new polymorphic markers. A minimum tiling path was defined that consists of either 4 YACs or 13 BACs covering 1.82 Mb between D6Ott8, the closest proximal marker, and D6Ott115, the closest distal marker. Gene distribution in the region includes 14 Ly49 genes as well as 3 new additional transcripts. This high-resolution, sequence-ready BAC contig provides a backbone for the identification of Cmv1 and its relationship with genes involved in innate immunity.

Assessment of Cmv1 candidates by genetic mapping and in vivo antibody depletion of NK cell subsets.

The mouse chromosome 6 locus Cmv1 controls resistance to infection with murine cytomegalovirus (MCMV). We have previously shown that Cmv1 is tightly linked to members of the NK gene complex (NKC) including the Ly49 gene family. To assess the candidacy of individual NKC members for the resistance locus, first we followed the co-segregation of Cd94, Nkg2d, and the well-characterized Ly49a, Ly49c and Ly49g genes with respect to Cmv1 in pre-existing panels of intraspecific backcross mice. Gene order and intergene distances (in cM) were: centromere-Cd94/Nkg2d-(0.05)-Ly49a/Ly49c/Ly49 g/Cmv1-(0. 3)-Prp/Kap/D6Mit13/111/219. This result excludes Cd94 and Nkg2d as candidates whereas it localizes the Ly49 genes within the minimal genetic interval for Cmv1. Second, we monitored the cell surface expression of individual Ly49 receptors in MCMV-infected mice over 2 weeks. The proportion of Ly49C(+) and Ly49C/I(+) cells decreased, the proportion of Ly49A(+) and Ly49G2(+) remained constant, and the cell surface density of Ly49G2 increased during infection, suggesting that NK cell subsets might have different roles in the regulation of MCMV infection. Third, we performed in vivo antibody depletion of specific NK cell subsets. Depletion with single antibodies did not affect the resistant phenotype suggesting that Ly49A(+), Ly49C(+), Ly49G2(+) and Ly49C/I(+) populations are not substantial players in MCMV resistance, and arguing for exclusion of the respective genes as candidates for Cmv1. In contrast, mice depleted with combined antibodies showed an intermediate phenotype. Whether residual NK cells, post-depletion, belong to a particular subset expressing another Ly49 receptor, or a molecule encoded by a yet to be identified gene of the NKC, is discussed.

High-resolution linkage map in the proximity of the host resistance locus Cmv1.

The mouse chromosome 6 locus Cmv1 controls replication of mouse Cytomegalovirus (MCMV) in the spleen of the infected host. In our effort to clone Cmv1, we have constructed a high-resolution genetic linkage map in the proximity of the gene. For this, a total of 45 DNA markers corresponding to either cloned genes or microsatellites were mapped within a 7.9-cM interval overlapping the Cmv1 region. We have followed the cosegregation of these markers with respect to Cmv1 in a total of 2248 backcross mice from a preexisting interspecific backcross panel of 281 (Mus spretus x C57BL/6J)F1 x C57BL/6J and 2 novel panels of 989 (A/ J x C57BL6)F1 x A/J and 978 (BALB/c x C57BL/6J)F1 x BALB/c segregating Cmv1. Combined pedigree analysis allowed us to determine the following gene order and intergene distances (in cM) on the distal region of mouse chromosome 6: D6Mit216-(1.9)-D6Mit336-(2.2)- D6Mit218-(1.0)-D6Mit52-(0.5)-D6Mit194-(0.2)-Nkrp 1/ D6Mit61/135/257/289/338-(0.4)-Cmv1/Ly49A/D6Mit370++ +- (0.3)-Prp/Kap/D6Mit13/111/219-(0.3)-Tel/D6Mit374/290/ 220/196/195/110-(1.1)-D6Mit25. Therefore, the minimal genetic interval for Cmv1 of 0.7 cM is defined by 13 tightly linked markers including 2 markers, Ly49A and D6Mit370, that did not show recombination with Cmv1 in 1967 meioses analyzed; the proximal limit of the Cmv1 domain was defined by 8 crossovers between Nkrp1/ D6Mit61/135/257/289/338 and Cmv1/Ly49A/D6Mit370, and the distal limit was defined by 5 crossovers between Cmv1/Ly49A/D6Mit370 and Prp/Kap/D6Mit13/111/219. This work demonstrates tight linkage between Cmv1 and genes from the natural killer complex (NKC), such as Nkrp1 and Ly49A, suggesting that Cmv1 may represent an NK cell recognition structure encoded in the NKC region.

Tissue distribution, genomic structure, and chromosome mapping of mouse and human eukaryotic initiation factor 4E-binding proteins 1 and 2.

Two related eukaryotic initiation factor-4E binding proteins (4E-BP1 and 4E-BP2) were recently characterized for their capacity to bind specifically to eIF4E and inhibit its function. Here, we determined the cDNA sequence, tissue distribution, genomic structure, and chromosome localization of murine and human 4E-BP1 and 4E-BP2. Mouse 4E-BP1 and 4E-BP2 consist of 117 and 120 amino acids and exhibit 91. 5 and 95.0% identity, respectively, to their human homologues. 4E-BP1 mRNA is expressed in most tissues, but is most abundant in adipose tissue, pancreas, and skeletal muscle, while 4E-BP2 mRNA is ubiquitously expressed. The structures of the mouse 4E-BP1 and 4E-BP2 were determined. The 4E-BP1 gene consists of three exons and spans approximately 16 kb. In addition, two 4E-BP1 pseudogenes exist in the mouse genome. The 4E-BP2 gene spans approximately 20 kb and exhibits an identical genomic organization to that of 4E-BP1, with the protein coding portion of the gene divided into three exons. There are no pseudogenes for 4E-BP2. The chromosomal locations of 4E-BP1 and 4E-BP2 were determined in both mice and humans by fluorescence in situ hybridization analysis. Mouse 4E-BP1 and 4E-BP2 map to chromosomes 8 (A4-B1) and 10 (B4-B5), respectively, and human 4E-BP1 and 4E-BP2 localize to chromosomes 8p12 and 10q21-q22, respectively.