Effects of Leishmania major infection on the gut microbiome of resistant and susceptible mice.

Cutaneous leishmaniasis, a parasitic disease caused by Leishmania major, is a widely frequent form in humans. To explore the importance of the host gut microbiota and to investigate its changes during L. major infection, two different groups of mouse models were assessed. The microbiome of two parts of the host gut-ileum and colon-from infected and non-infected mice were characterised by sequencing of 16S rDNA using an Ion Torrent PGM platform. Microbiome analysis was performed to reveal changes related to the susceptibility and the genetics of mice strains in two different gut compartments and to compare the results between infected and non-infected mice. The results showed that Leishmania infection affects mainly the ileum microbiota, whereas the colon bacterial community was more stable. Different biomarkers were determined in the gut microbiota of infected resistant mice and infected susceptible mice using LEfSe analysis. Lactobacillaceae was associated with resistance in the colon microbiota of all resistant mice strains infected with L. major. Genes related to xenobiotic biodegradation and metabolism and amino acid metabolism were primarily enriched in the small intestine microbiome of resistant strains, while genes associated with carbohydrate metabolism and glycan biosynthesis and metabolism were most abundant in the gut microbiome of the infected susceptible mice. These results should improve our understanding of host-parasite interaction and provide important insights into the effect of leishmaniasis on the gut microbiota. Also, this study highlights the role of host genetic variation in shaping the diversity and composition of the gut microbiome. KEY POINTS: • Leishmaniasis may affect mainly the ileum microbiota while colon microbiota was more stable. • Biomarkers related with resistance or susceptibility were determined in the gut microbiota of mice. • Several pathways were predicted to be upregulated in the gut microbiota of resistant or susceptible mice.

Mice with different susceptibility to tick-borne encephalitis virus infection show selective neutralizing antibody response and inflammatory reaction in the central nervous system.

BACKGROUND: The clinical course of tick-borne encephalitis (TBE), a disease caused by TBE virus, ranges from asymptomatic or mild influenza-like infection to severe debilitating encephalitis or encephalomyelitis. Despite the medical importance of this disease, some crucial steps in the development of encephalitis remain poorly understood. In particular, the basis of the disease severity is largely unknown. METHODS: TBE virus growth, neutralizing antibody response, key cytokine and chemokine mRNA production and changes in mRNA levels of cell surface markers of immunocompetent cells in brain were measured in mice with different susceptibilities to TBE virus infection. RESULTS: An animal model of TBE based on BALB/c-c-STS/A (CcS/Dem) recombinant congenic mouse strains showing different severities of the infection in relation to the host genetic background was developed. After subcutaneous inoculation of TBE virus, BALB/c mice showed medium susceptibility to the infection, STS mice were resistant, and CcS-11 mice were highly susceptible. The resistant STS mice showed lower and delayed viremia, lower virus production in the brain and low cytokine/chemokine mRNA production, but had a strong neutralizing antibody response. The most sensitive strain (CcS-11) failed in production of neutralizing antibodies, but exhibited strong cytokine/chemokine mRNA production in the brain. After intracerebral inoculation, all mouse strains were sensitive to the infection and had similar virus production in the brain, but STS mice survived significantly longer than CcS-11 mice. These two strains also differed in the expression of key cytokines/chemokines, particularly interferon gamma-induced protein 10 (IP-10/CXCL10) and monocyte chemotactic protein-1 (MCP-1/CCL2) in the brain. CONCLUSIONS: Our data indicate that the genetic control is an important factor influencing the clinical course of TBE. High neutralizing antibody response might be crucial for preventing host fatality, but high expression of various cytokines/chemokines during TBE can mediate immunopathology and be associated with more severe course of the infection and increased fatality.

Epistasis in iron metabolism: complex interactions between Cp, Mon1a, and Slc40a1 loci and tissue iron in mice.

Disorders of iron metabolism are among the most common acquired and constitutive diseases. Hemochromatosis has a solid genetic basis and in Northern European populations it is usually associated with homozygosity for the C282Y mutation in the HFE protein. However, the penetrance of this mutation is incomplete and the clinical presentation is highly variable. The rare and common variants identified so far as genetic modifiers of HFE-related hemochromatosis are unable to account for the phenotypic heterogeneity of this disorder. There are wide variations in the basal iron status of common inbred mouse strains, and this diversity may reflect the genetic background of the phenotypic diversity under pathological conditions. We therefore examined the genetic basis of iron homeostasis using quantitative trait loci mapping applied to the HcB-15 recombinant congenic strains for tissue and serum iron indices. Two highly significant QTL containing either the N374S Mon1a mutation or the Ferroportin locus were found to be major determinants in spleen and liver iron loading. Interestingly, when considering possible epistatic interactions, the effects of Mon1a on macrophage iron export are conditioned by the genotype at the Slc40a1 locus. Only mice that are C57BL/10ScSnA homozygous at both loci display a lower spleen iron burden. Furthermore, the liver-iron lowering effect of the N374S Mon1a mutation is observed only in mice that display a nonsense mutation in the Ceruloplasmin (Cp) gene. This study highlights the existence of genetic interactions between Cp, Mon1a, and the Slc40a1 locus in iron metabolism, suggesting that epistasis may be a crucial determinant of the variable biological and clinical presentations in iron disorders.

Positional cloning of the combined hyperlipidemia gene Hyplip1.

Familial combined hyperlipidemia (FCHL, MIM-144250) is a common, multifactorial and heterogeneous dyslipidemia predisposing to premature coronary artery disease and characterized by elevated plasma triglycerides, cholesterol, or both. We identified a mutant mouse strain, HcB-19/Dem (HcB-19), that shares features with FCHL, including hypertriglyceridemia, hypercholesterolemia, elevated plasma apolipoprotein B and increased secretion of triglyceride-rich lipoproteins. The hyperlipidemia results from spontaneous mutation at a locus, Hyplip1, on distal mouse chromosome 3 in a region syntenic with a 1q21-q23 FCHL locus identified in Finnish, German, Chinese and US families. We fine-mapped Hyplip1 to roughly 160 kb, constructed a BAC contig and sequenced overlapping BACs to identify 13 candidate genes. We found substantially decreased mRNA expression for thioredoxin interacting protein (Txnip). Sequencing of the critical region revealed a Txnip nonsense mutation in HcB-19 that is absent in its normolipidemic parental strains. Txnip encodes a cytoplasmic protein that binds and inhibits thioredoxin, a major regulator of cellular redox state. The mutant mice have decreased CO2 production but increased ketone body synthesis, suggesting that altered redox status down-regulates the citric-acid cycle, sparing fatty acids for triglyceride and ketone body production. These results reveal a new pathway of potential clinical significance that contributes to plasma lipid metabolism.

Ptprj is a candidate for the mouse colon-cancer susceptibility locus Scc1 and is frequently deleted in human cancers.

Only a small proportion of cancers result from familial cancer syndromes with Mendelian inheritance. Nonfamilial, 'sporadic' cancers, which represent most cancer cases, also have a significant hereditary component, but the genes involved have low penetrance and are extremely difficult to detect. Therefore, mapping and cloning of quantitative trait loci (QTLs) for cancer susceptibility in animals could help identify homologous genes in humans. Several cancer-susceptibility QTLs have been mapped in mice and rats, but none have been cloned so far. Here we report the positional cloning of the mouse gene Scc1 (Susceptibility to colon cancer 1) and the identification of Ptprj, encoding a receptor-type protein tyrosine phosphatase, as the underlying gene. In human colon, lung and breast cancers, we show frequent deletion of PTPRJ, allelic imbalance in loss of heterozygosity (LOH) and missense mutations. Our data suggest that PTPRJ is relevant to the development of several different human cancers.

Susceptibility loci affecting ERBB2/neu-induced mammary tumorigenesis in mice.

Twenty percent of breast cancers exhibit amplification or overexpression of ERBB2/neu and a poor prognosis. As the susceptibility genes controlling ERBB2 tumorgenesis are unknown, in a genetic mapping project we crossed transgenic mice expressing the neu oncogene under control of MMTV promoter with recombinant congenic (RC) strains, which provided a high mapping power. RC strains differed considerably in tumor latency (P = 0.0002), suggesting a strong genetic control of tumor development. Linkage analysis in neu-transgene carrying F2 hybrids between the most susceptible and most resistant RC strain revealed three mammary tumor susceptibility (Mts) loci with main effects, Mts1 (chr. 4), Mts2 (chr. 10), Mts3 (chr. 19), and two interacting loci Mts4 (chr.6) and Mts5 (chr. 8), significantly affecting mammary tumor latency. Suggestive significance levels indicated control of tumor numbers by Mts1 alone and in interaction with Mts5, and by two additional interacting loci on chromosomes 1 and 8. These loci combined explain to a large extent the tumor latency and number in individual F2 mouse. We also identified a suggestive locus on chromosome 17 controls metastasis to the lung. The loci Mts1, Mts1b, and Mts3 are located in the Naad4-4,5 and Naad19-2 LOH-regions of neu-induced mammary tumors, corresponding to the frequent human breast cancer LOH-regions 1p34/1p36, and 10q25, respectively. These results expand the knowledge of ERBB2 tumorigenesis and point to a combined control of specific tumor phenotypes by germ-line polymorphisms and somatic alterations.

Clustering of colon, lung, and other cancer susceptibility genes with protein tyrosine phosphatases and protein kinases in multiple short genomic regions.

Interactions of large gene families are poorly understood. We found that human, mouse, and rat colon and lung cancer susceptibility genes, presently considered as separate gene families, were frequently pairwise linked. The orthologous mouse map positions of 142 of 159 early discovered colon and lung cancer susceptibility genes formed 41 genomic clusters conserved >70 million years. These linked gene pairs concordantly affected both tumors and their majority was linked with two other gene families - protein tyrosine phosphatases and cancer driver protein kinases. 25% of both protein tyrosine phosphatases and protein kinases mapped <1 cM from a colon or lung cancer susceptibility gene, and 50% in <3 cM. Similar linkage was detected with most other human susceptibility genes that controlled 29 different cancer types. This concentration of tumor susceptibility genes with protein tyrosine phosphatases and driver protein kinases in multiple relatively short genomic regions suggests their possible functional diversity.

Functionally distinct regions of the locus Leishmania major response 15 control IgE or IFNγ level in addition to skin lesions.

Leishmaniasis, a disease caused by parasites of Leishmania spp., endangers more than 1 billion people living in endemic countries and has three clinical forms: cutaneous, mucocutaneous, and visceral. Understanding of individual differences in susceptibility to infection and heterogeneity of its pathology is largely lacking. Different mouse strains show a broad and heterogeneous range of disease manifestations such as skin lesions, splenomegaly, hepatomegaly, and increased serum levels of immunoglobulin E and several cytokines. Genome-wide mapping of these strain differences detected more than 30 quantitative trait loci (QTLs) that control the response to Leishmania major. Some control different combinations of disease manifestations, but the nature of this heterogeneity is not yet clear. In this study, we analyzed the L. major response locus Lmr15 originally mapped in the strain CcS-9 which carries 12.5% of the genome of the resistant strain STS on the genetic background of the susceptible strain BALB/c. For this analysis, we used the advanced intercross line K3FV between the strains BALB/c and STS. We confirmed the previously detected loci Lmr15, Lmr18, Lmr24, and Lmr27 and performed genetic dissection of the effects of Lmr15 on chromosome 11. We prepared the interval-specific recombinant strains 6232HS1 and 6229FUD, carrying two STS-derived segments comprising the peak linkage of Lmr15 whose lengths were 6.32 and 17.4 Mbp, respectively, and analyzed their response to L. major infection. These experiments revealed at least two linked but functionally distinct chromosomal regions controlling IFNγ response and IgE response, respectively, in addition to the control of skin lesions. Bioinformatics and expression analysis identified the potential candidate gene Top3a. This finding further clarifies the genetic organization of factors relevant to understanding the differences in the individual risk of disease.

Comprehensive skeletal phenotyping and linkage mapping in an intercross of recombinant congenic mouse strains HcB-8 and HcB-23.

Bone biomechanical performance is a complex trait or, more properly, an ensemble of complex traits. Biomechanical performance incorporates flexibility under loading, yield and failure load, and energy to failure; all are important measures of bone function. To date, the vast majority of work has focused on yield and failure load and its surrogate, bone mineral density. We performed a reciprocal intercross of the mouse strains HcB-8 and HcB-23 to map and ultimately identify genes that contribute to differences in biomechanical performance. Mechanical testing was performed by 3-point bending of the femora. We measured femoral diaphysis cross-sectional anatomy from photographs of the fracture surfaces. We used beam equations to calculate material level mechanical properties. We performed a principal component (PC) analysis of normalized whole bone phenotypes (17 input traits). We measured distances separating mandibular landmarks from calibrated digital photographs and performed linkage analysis. Experiment-wide α = 0.05 significance thresholds were established by permutation testing. Three quantitative trait loci (QTLs) identified in these studies illustrate the advantages of the comprehensive phenotyping approach. A pleiotropic QTL on chromosome 4 affected multiple whole bone phenotypes with LOD scores as large as 17.5, encompassing size, cross-sectional ellipticity, stiffness, yield and failure load, and bone mineral density. This locus was linked to 3 of the PCs but unlinked to any of the tissue level phenotypes. From this pattern, we infer that the QTL operates by modulating the proliferative response to mechanical loading. On this basis, we successfully predicted that this locus also affects the length of a specific region of the mandible. A pleiotropic locus on chromosome 10 with LOD scores displays opposite effects on failure load and toughness with LOD scores of 4.5 and 5.5, respectively, so that the allele that increases failure load decreases toughness. A chromosome 19 QTL for PC2 with an LOD score of 4.8 was not detected with either the whole bone or tissue level phenotypes. We conclude that first, comprehensive, system-oriented phenotyping provides much information that could not be obtained by focusing on bone mineral density alone. Second, mechanical performance includes inherent trade-offs between strength and brittleness. Third, considering the aggregate phenotypic data allows prediction of novel QTLs.

Gene-Specific Sex Effects on Susceptibility to Infectious Diseases.

Inflammation is an integral part of defense against most infectious diseases. These pathogen-induced immune responses are in very many instances strongly influenced by host's sex. As a consequence, sexual dimorphisms were observed in susceptibility to many infectious diseases. They are pathogen dose-dependent, and their outcomes depend on pathogen and even on its species or subspecies. Sex may differentially affect pathology of various organs and its influence is modified by interaction of host's hormonal status and genotype: sex chromosomes X and Y, as well as autosomal genes. In this Mini Review we summarize the major influences of sex in human infections and subsequently focus on 22 autosomal genes/loci that modify in a sex-dependent way the response to infectious diseases in mouse models. These genes have been observed to influence susceptibility to viruses, bacteria, parasites, fungi and worms. Some sex-dependent genes/loci affect susceptibility only in females or only in males, affect both sexes, but have stronger effect in one sex; still other genes were shown to affect the disease in both sexes, but with opposite direction of effect in females and males. The understanding of mechanisms of sex-dependent differences in the course of infectious diseases may be relevant for their personalized management.

Genetic Influence on Frequencies of Myeloid-Derived Cell Subpopulations in Mouse.

Differences in frequencies of blood cell subpopulations were reported to influence the course of infections, atopic and autoimmune diseases, and cancer. We have discovered a unique mouse strain B10.O20 containing extremely high frequency of myeloid-derived cells (MDC) in spleen. B10.O20 carries 3.6% of genes of the strain O20 on the C57BL/10 genetic background. It contains much higher frequency of CD11b+Gr1+ cells in spleen than both its parents. B10.O20 carries O20-derived segments on chromosomes 1, 15, 17, and 18. Their linkage with frequencies of blood cell subpopulations in spleen was tested in F2 hybrids between B10.O20 and C57BL/10. We found 3 novel loci controlling MDC frequencies: Mydc1, 2, and 3 on chromosomes 1, 15, and 17, respectively, and a locus controlling relative spleen weight (Rsw1) that co-localizes with Mydc3 and also influences proportion of white and red pulp in spleen. Mydc1 controls numbers of CD11b+Gr1+ cells. Interaction of Mydc2 and Mydc3 regulates frequency of CD11b+Gr1+ cells and neutrophils (Gr1+Siglec-F- cells from CD11b+ cells). Interestingly, Mydc3/Rsw1 is orthologous with human segment 6q21 that was shown previously to determine counts of white blood cells. Bioinformatics analysis of genomic sequence of the chromosomal segments bearing these loci revealed polymorphisms between O20 and C57BL/10 that change RNA stability and genes' functions, and we examined expression of relevant genes. This identified potential candidate genes Smap1, Vps52, Tnxb, and Rab44. Definition of genetic control of MDC can help to personalize therapy of diseases influenced by these cells.

A locus on chromosome 8 controlling tumor regionality-a new type of tumor diversity in the mouse lung.

Regional specificity of lung tumor formation has rarely been studied in mouse or human. By using crosses of strains semi-congenic for lung cancer susceptibility locus Sluc20, we have analyzed the genetic influences of Sluc20 and 5 other loci on tumor regionality in the mouse lung. We have mapped Sluc20 to a 27.92-MB proximal region of chromosome 8 and found that it controls the number and load of only those tumors that surround or are directly adjacent to the bronchi or bronchioli (peribronchial tumors). These tumors lie outside the bronchial basement membrane and tend to reach a larger size than the tumors at other locations in the lung. Similar to tumors of alveolar lineage at other locations, peribronchial tumors stain with SP-C but not CC10 antibody. The effects of Sluc20 alleles are additive because the number of peribronchial tumors in heterozygotes is intermediate. These findings show that tumor regionality in the mouse lung, which represents a novel level of lung tumor heterogeneity, is under specific genetic control. The identification of genes controlling lung tumor regionality will provide novel insights into the biology of lung tumors and potentially improve the possibilities of individualized prognosis and treatment in human lung cancer.

Loci controlling lymphocyte production of interferon c after alloantigen stimulation in vitro and their co-localization with genes controlling lymphocyte infiltration of tumors and tumor susceptibility.

Low infiltration of lymphocytes into cancers is associated with poor prognosis, but the reasons why some patients exhibit a low and others a high infiltration of tumors are unknown. Previously we mapped four loci (Lynf1­Lynf4) controlling lymphocyte infiltration of mouse lung tumors. These loci do not encode any of the molecules that are involved in traffic of lymphocytes. Here we report a genetic relationship between these loci and the control of production of IFNγ in allogeneic mixed lymphocyte cultures (MLC). We found that IFNγ production by lymphocytes of O20/A mice is lower than by lymphocytes of OcB-9/Dem mice (both H2pz) stimulated in MLC by irradiated splenocytes of C57BL/10SnPh (H2b) or BALB/ cHeA (H2d) mice, or by ConA. IFNγ production in MLCs of individual (O20 9 OcB-9)F2mice stimulated by irradiated C57BL/10 splenocytes and genotyped for microsatellite markers revealed four IFNγ-controlling loci (Cypr4-Cypr7), each of which is closely linked with one of the four Lynf loci and with a cluster of susceptibility genes for different tumors. This suggests that inherited differences in certain lymphocyte responses may modify their propensity to infiltrate tumors and their capacity to affect tumor growth.

Quantitative trait loci for biomechanical performance and femoral geometry in an intercross of recombinant congenic mice: restriction of the Bmd7 candidate interval.

Despite steady progress in identifying quantitative trait loci (QTLs) for bone phenotypes, relatively little progress has been made in moving from QTLs to identifying the relevant gene. We exploited the genetic structure of recombinant congenic mouse strains by performing a reciprocal intercross of the strains HcB-8 and HcB-23, phenotyped for body size, femoral biomechanical performance, and femoral diaphyseal geometry and mapped with R/qtl and QTL Cartographer. Significant QTLs are present on chromosomes 1, 2, 3, 4, 6, and 10. We found significant sex x QTL and cross-direction x QTL interactions. The chromosome 4 QTL affects multiple femoral anatomic features and biomechanical properties. The known segregating segment of chromosome 4 contains only 18 genes, among which Ece1, encoding endothelin-converting enzyme 1, stands out as a candidate. Endothelin signaling has been shown to promote the growth of osteoblastic metastases and to potentiate signaling via the Wnt pathway. The colocalizing chromosome 4 QTL Bmd7 (for bone mineral density 7) increases responsiveness to mechanical loading. By exploiting the short informative segment of chromosome 4 and the known biology, we propose that Ece1 is the gene responsible for Bmd7 and that it acts by increasing responsiveness to mechanical loading through modulation of Wnt signaling.

A New Polygenic Model for Nonfamilial Colorectal Cancer Inheritance Based on the Genetic Architecture of the Azoxymethane-Induced Mouse Model.

The azoxymethane model of colorectal cancer (CRC) was used to gain insights into the genetic heterogeneity of nonfamilial CRC. We observed significant differences in susceptibility parameters across 40 mouse inbred strains, with 6 new and 18 of 24 previously identified mouse CRC modifier alleles detected using genome-wide association analysis. Tumor incidence varied in F1 as well as intercrosses and backcrosses between resistant and susceptible strains. Analysis of inheritance patterns indicates that resistance to CRC development is inherited as a dominant characteristic genome-wide, and that susceptibility appears to occur in individuals lacking a large-effect, or sufficient numbers of small-effect, polygenic resistance alleles. Our results suggest a new polygenic model for inheritance of nonfamilial CRC, and that genetic studies in humans aimed at identifying individuals with elevated susceptibility should be pursued through the lens of absence of dominant resistance alleles rather than for the presence of susceptibility alleles.

Distinct genetic control of parasite elimination, dissemination, and disease after Leishmania major infection.

Elimination of pathogens is the basis of host resistance to infections; however, relationship between persisting pathogens and disease has not been clarified. Leishmania major infection in mice is an important model of host-pathogen relationship. Infected BALB/c mice exhibit high parasite numbers in lymph nodes and spleens, and a chronic disease with skin lesions, splenomegaly, and hepatomegaly, increased serum IgE levels and cytokine imbalance. Although numerous gene loci affecting these disease symptoms have been reported, genes controlling parasites' elimination or dissemination have never been mapped. We therefore compared genetics of the clinical and immunologic symptomatology with parasite load in (BALB/c x CcS-11) F2 hybrids and mapped five loci, two of which control parasite elimination or dissemination. Lmr5 influences parasite loads in spleens (and skin lesions, splenomegaly, and serum IgE, IL-4, and IFNgamma levels), and Lmr20 determines parasite numbers in draining lymph nodes (and serum levels of IgE and IFNgamma), but no skin or visceral pathology. Three additional loci do not affect parasite numbers but influence significantly the disease phenotype-Lmr21: skin lesions and IFNgamma levels, Lmr22: IL-4 levels, Lmr23: IFNgamma levels, indicating that development of L. major-caused disease includes critical regulations additional to control of parasite spread.

Novel Loci Controlling Parasite Load in Organs of Mice Infected With Leishmania major, Their Interactions and Sex Influence.

Leishmaniasis is a serious health problem in many countries, and continues expanding to new geographic areas including Europe and USA. This disease, caused by parasites of Leishmania spp. and transmitted by phlebotomine sand flies, causes up to 1.3 million new cases each year and despite efforts toward its functional dissection and treatment it causes 20-50 thousands deaths annually. Dependence of susceptibility to leishmaniasis on sex and host's genes was observed in humans and in mouse models. Several laboratories defined in mice a number of Lmr (Leishmania major response) genetic loci that control functional and pathological components of the response to and outcome of L. major infection. However, the development of its most aggressive form, visceral leishmaniasis, which is lethal if untreated, is not yet understood. Visceral leishmaniasis is caused by infection and inflammation of internal organs. Therefore, we analyzed the genetics of parasite load, spread to internal organs, and ensuing visceral pathology. Using a new PCR-based method of quantification of parasites in tissues we describe a network-like set of interacting genetic loci that control parasite load in different organs. Quantification of Leishmania parasites in lymph nodes, spleen and liver from infected F2 hybrids between BALB/c and recombinant congenic strains CcS-9 and CcS-16 allowed us to map two novel parasite load controlling Leishmania major response loci, Lmr24 and Lmr27. We also detected parasite-controlling role of the previously described loci Lmr4, Lmr11, Lmr13, Lmr14, Lmr15, and Lmr25, and describe 8 genetic interactions between them. Lmr14, Lmr15, Lmr25, and Lmr27 controlled parasite load in liver and lymph nodes. In addition, Leishmania burden in lymph nodes but not liver was influenced by Lmr4 and Lmr24. In spleen, parasite load was controlled by Lmr11 and Lmr13. We detected a strong effect of sex on some of these genes. We also mapped additional genes controlling splenomegaly and hepatomegaly. This resulted in a systematized insight into genetic control of spread and load of Leishmania parasites and visceral pathology in the mammalian organism.

Persistence of Gamma-H2AX Foci in Bronchial Cells Correlates with Susceptibility to Radiation Associated Lung Cancer in Mice.

The risk of developing radiation-induced lung cancer differs between different strains of mice, but the underlying cause of the strain differences is unknown. Strains of mice also differ in how quickly they repair radiation-induced DNA double-strand breaks (DSBs). We assayed mouse strains from the CcS/Dem recombinant congenic strain set for their efficacy in repairing DNA DSBs during protracted irradiation. We measured unrepaired γ-H2AX radiation-induced foci (RIF), which persisted after chronic 24-h gamma irradiation, as a surrogate marker for repair efficiency in bronchial epithelial cells for 17 of the CcS/Dem strains and the BALB/c founder strain. We observed a very strong correlation (R2 = 79.18%, P < 0.001) between the level of unrepaired RIF and radiogenic lung cancer incidence measured in the same strains. Interestingly, spontaneous levels of foci in nonirradiated mice also showed good correlation with lung cancer incidence when incidence data from male and female mice were combined. These results suggest that genetic differences in DNA repair capacity largely account for differing susceptibilities to radiation-induced lung cancer among CcS/Dem mouse strains, and that high levels of spontaneous DNA damage are also a relatively good marker of cancer predisposition. In a smaller pilot study, we found that the repair capacity measured in peripheral blood leucocytes also correlated well with radiogenic lung cancer susceptibility, raising the possibility that the assay could be used to detect radiogenic lung cancer susceptibility in humans.

Genetic Regulation of Guanylate-Binding Proteins 2b and 5 during Leishmaniasis in Mice.

Interferon-induced GTPases [guanylate-binding proteins (GBPs)] play an important role in inflammasome activation and mediate innate resistance to many intracellular pathogens, but little is known about their role in leishmaniasis. We therefore studied expression of Gbp2b/Gbp1 and Gbp5 mRNA in skin, inguinal lymph nodes, spleen, and liver after Leishmania major infection and in uninfected controls. We used two different groups of related mouse strains: BALB/c, STS, and CcS-5, CcS-16, and CcS-20 that carry different combinations of BALB/c and STS genomes, and strains O20, C57BL/10 (B10) and B10.O20, OcB-9, and OcB-43 carrying different combinations of O20 and B10 genomes. The strains were classified on the basis of size and number of infection-induced skin lesions as highly susceptible (BALB/c, CcS-16), susceptible (B10.O20), intermediate (CcS-20), and resistant (STS, O20, B10, OcB-9, OcB-43). Some uninfected strains differed in expression of Gbp2b/Gbp1 and Gbp5, especially of Gbp2b/Gbp1 in skin. Uninfected BALB/c and STS did not differ in their expression, but in CcS-5, CcS-16, and CcS-20, which all carry BALB/c-derived Gbp gene-cluster, expression of Gbp2b/Gbp1 exceeds that of both parents. These data indicate trans-regulation of Gbps. Infection resulted in approximately 10× upregulation of Gbp2b/Gbp1 and Gbp5 mRNAs in organs of both susceptible and resistant strains, which was most pronounced in skin. CcS-20 expressed higher level of Gbp2b/Gbp1 than both parental strains in skin, whereas CcS-16 expressed higher level of Gbp2b/Gbp1 than both parental strains in skin and liver. This indicates a trans-regulation present in infected mice CcS-16 and CcS-20. Immunostaining of skin of five strains revealed in resistant and intermediate strains STS, CcS-5, O20, and CcS-20 tight co-localization of Gbp2b/Gbp1 protein with most L. major parasites, whereas in the highly susceptible strain, BALB/c most parasites did not associate with Gbp2b/Gbp1. In conclusion, expression of Gbp2b/Gbp1 and Gbp5 was increased even in organs of clinically asymptomatic resistant mice. It suggests a hidden inflammation, which might contribute to control of persisting parasites. This is supported by the co-localization of Gbpb2/Gbp1 protein and L. major parasites in skin of resistant and intermediate but not highly susceptible mice.