Quantitative trait analysis of nickel-induced acute lung injury in mice.

The genetic determinants underlying susceptibility to acute lung injury have not been identified. Recently, we found that the strain distribution pattern for mean survival time (MST) to three irritants-ozone, ultrafine Teflon, and nickel sulfate- was shared between inbred mouse strains. For ozone-induced acute lung injury, survival was found to be a complex trait controlled by at least three quantitative trait loci (QTLs), designated Aliq1, Aliq2, and Aliq3. To explore whether similar genes might be involved in survival to acute lung injury induced by nickel sulfate, we took advantage of the 2-fold difference in MSTs between the sensitive A/J and resistant C57BL/6J mice. QTL analysis of 307 backcross mice generated from these strains identified significant linkage to chromosome 6 (proposed as Aliq4) and suggestive linkage on chromosomes 1 and 12. Loci on chromosomes 9 and 16 had lod scores (log of the odds ratio, which equals the log of the "likelihood of linkage divided by the likelihood of no linkage") below significance, but contributed to the overall response. Comparing MSTs of backcross mice with similar haplotypes identified an allelic combination of four QTLs that could account for the survival time difference between the parental strains. Similar QTL intervals on chromosomes 6 and 12 were previously identified with ozone, suggesting that the interplay between different combinations of relatively few genes might be important for irritant-induced acute lung injury survival.     

Resistance to Paracoccidioides brasiliensis in mice is controlled by a single dominant autosomal gene.

In a previous report it was shown that there are resistant, susceptible, and intermediate strains of mice to intraperitoneal Paracoccidioides brasiliensis infection. In the present work, we investigated the type of inheritance and the number of genes that determine resistance to paracoccidioidomycosis. Parental and hybrid mice were inoculated intraperitoneally with 5 X 10(6) P. brasiliensis yeast cells, and mortality was scored daily. Analysis of susceptible and resistant parental strains and of F1, F2, and backcross mice showed that the resistance to P. brasiliensis seems to be controlled genetically by a single dominant gene, which we designated the Pbr locus. The mean survival times of susceptible F2 and backcross hybrids were very similar to that of the susceptible parent. Examination of the pathological changes observed in parental and F1 mice, 6 months after infection, showed that F1 offspring presented a similar number and distribution of lesions to those of the resistant strains. The Pbr gene is not linked to H-2, Hc, and albino genes. Furthermore, resistance to paracoccidioidomycosis is controlled by an autosomal gene.     

Canavan disease: a monogenic trait with complex genomic interaction

Canavan disease (CD) is an inherited leukodystrophy, caused by aspartoacylase (ASPA) deficiency, and accumulation of N-acetylaspartic acid (NAA) in the brain. The gene for ASPA has been cloned and more than 40 mutations have been described, with two founder mutations among Ashkenazi Jewish patients. Screening of Ashkenazi Jews for these two common mutations revealed a high carrier frequency, approximately 1/40, so that programs for carrier testing are currently in practice. The enzyme deficiency in CD interferes with the normal hydrolysis of NAA, which results in disruption of myelin and spongy degeneration of the white matter of the brain. The clinical features of the disease are macrocephaly, head lag, progressive severe mental retardation, and hypotonia in early life, which later changes to spasticity. A knockout mouse for CD has been generated, and used to study the pathophysiological basis for CD. Findings from the knockout mouse indicate that this monogenic trait leads to a series of genomic interaction in the brain. Changes include low levels of glutamate and GABA. Microarray expression analysis showed low level of expression of GABA-A receptor (GABRA6) and glutamate transporter (EAAT4). The gene Spi2, a gene involved in apoptosis and cell death, showed high level of expression. Such complexity of gene interaction results in the phenotype, the proteome, with spongy degeneration of the brain and neurological impairment of the mouse, similar to the human counterpart. Aspartoacylase gene transfer trial in the mouse brain using adenoassociated virus (AAV) as a vector are encouraging showing improved myelination and decrease in spongy degeneration in the area of the injection and also beyond that site.     

Immmune complex glomerular disease in argyric mice.

A mesangiopathic glomerulonephritis with prominent mesangial immunoglobulin deposits was induced in mice by single or multiple intraperitoneal injections of bovine serum albumin. The silver-labelled basement membrane in mice which had ingested 6mM silver nitrate in drinking water proved a useful marker indetermining that the immune deposits were on the intracapillary aspect of the basement membrane.     

Genomewide search for epistasis in a complex trait: pentobarbital withdrawal convulsions in mice

The well-documented difference in pentobarbital withdrawal severity between DBA/2J and C57BL/6J mice offers the opportunity to study how differences between allelic variants influence pentobarbital withdrawal via their additive and/or dominance effects and to identify modifier loci that also influence the trait via gene-gene interactions (a form of epistasis). Previous work in our laboratory identified seven provisional quantitative trait loci (QTLs) for pentobarbital withdrawal using BXD recombinant inbred strains. To date, only one of these QTLs has been confirmed, Pbwl. We hypothesized that other loci that act epistatically may also influence genetic variance in pentobarbital withdrawal severity. Using Epistat, a program developed to carry out full-genome searches for epistasis, we identified six provisional epistatic interactions (p < .002) between the provisional QTLs and modifier loci elsewhere in the genome. Verification testing of these interactions using 404 B6D2F₂ mice provided supporting evidence that a QTL on chromosome 11 contributes to genetic variance in pentobarbital withdrawal, but only in the presence of a modifier allele on distal chromosome 1 (p = .0004). This modifier is in the same genomic vicinity as loci detected for a variety of withdrawal and seizure phenotypes.     

Neural infection in mice after cutaneous inoculation with HSV-1 is under complex host genetic control

The ability to restrict neural infection with herpes simplex virus is of potential importance because the nervous system is the main reservoir of virus between recrudescences. Genetic control of innate resistance to herpes simplex after intraperitoneal challenge is well established, but this route of infection does not mimic the progress of virus from skin to the sensory nervous system which occurs during natural infection. We show here, by Mendelian analysis, that the control of neural infection after cutaneous inoculation is complex, involving several (perhaps four) genetic loci, and is not accurately represented by genes which determine survival. The results are discussed in relation to possible underlying resistance mechanisms, particularly the control of early interferon production.     

Endogenous interleukin-12 is involved in resistance of mice to Mycobacterium avium complex infection.

Acquired cellular resistance against Mycobacterium avium complex (MAC) infections involves the induction of Th1 type gamma interferon (IFN-gamma)-producing T cells. Interleukin-12 (IL-12) is a cytokine involved in the control of IFN-gamma production by T cells and NK cells. The role of IL-12 in the response to MAC infection was investigated. Depletion of endogenous IL-12 by injection of monoclonal antibody prior to and during intranasal infection with MAC resulted in an 150- to 550-fold increase in bacterial load in lung, spleen, and liver homogenates by 10 weeks postinfection. Depletion of IL-12 abrogated the ability of spleen cell cultures to produce IFN-gamma in response to stimulus with live MAC. IL-12-depleted mice showed a 75% decrease in the number of inflammatory cells entering the lungs following intranasal infection with MAC, with significant reductions in cytotoxic activity and nitric oxide production by lung cells. This work suggests that IL-12 plays a major role in the activation of IFN-gamma-producing cells during MAC infection.     

Altered lung motion is a sensitive indicator of regional lung disease

Since lung diseases adversely affect airflow during breathing, they must also alter normal lung motion, which can be exploited to detect these diseases. However, standard imaging techniques such as CT and MRI imaging during breath-holds provide little or no information on lung motion and cannot detect diseases that cause subtle changes in lung structure. Phase-contrast X-ray imaging provides images of high contrast and spatial resolution with temporal resolutions that allow multiple images to be acquired throughout the respiratory cycle. Using X-ray phase-contrast imaging, coupled with velocimetry, we have measured lung tissue movement and determined velocity fields that define speed and direction of regional lung motion throughout a breath in normal Balb/c nude male mice and mice exposed to bleomycin. Regional maps of lung tissue motion reveal both the heterogeneity of normal lung motion, as well as abnormal motion induced by bleomycin treatment. Analysed histologically, bleomycin treatment caused pathological changes in lung structure that were heterogenous, occupying less than 12% of the lung at 6 days after treatment. Moreover, plethysmography failed to detect significant changes in compliance at either 36 h or 6 days after treatment. Detailed analysis of the vector fields demonstrated major differences (p < 0.001) in regional lung motion between control and bleomycin-treated mice at both 36 h and 6 days after treatment. The results of this study demonstrate that X-ray phase-contrast imaging, coupled with velocimetry, can detect early stage, subtle and non-uniform lung disease.     

Resistance to Plasmodium chabaudi in B10 mice: influence of the H-2 complex and testosterone.

Resistance to Plasmodium chabaudi has been examined in different inbred mouse strains bearing identical H-2 haplotypes on different genetic backgrounds as well as in H-2-congenic mouse strains on B10 background. Resistance is expressed in terms of percent survival after a challenge with 10(6) P. chabaudi-infected erythrocytes. We can show that murine resistance to P. chabaudi is under complex polygenic control involving a non-H-2 gene(s) as well as genes in both I-A and I-E subregions of the H-2 complex. Our data indicate in particular that malaria protective antigens can be presented in context with I-Ab molecules but not in context with I-Ak molecules. Resistance controlled by I-Ab does not become apparent when I-Ek molecules are coincidentally expressed. Moreover, testosterone abrogates I-Ab-controlled resistance to P. chabaudi.     

A possible genetic contribution to the alexithymia trait

Alexithymic characteristics were studied in 15 monozygotic and 18 like-sex dizygotic healthy young twin pairs in a semistructured interview. Variance analysis showed larger within-pair variances in the dizygotic than in the monozygotic twins. With some reservations, these results point to a genetic influence on the etiology of the alexithymic phenomenon.     

Oestrogen is a potent disease accelerator in SLE-prone MRL lpr/lpr mice.

The influence of oestrogen on the lupus disease in MRL/l mice has been investigated. Adult, castrated male and female MRL/l mice were administered with s.c. injections of 3.2 micrograms of 17 beta-oestradiol twice a week. The results clearly demonstrate that a relatively small dose of oestrogen is a potent accelerator of the lupus disease in this mouse strain. Thus, administration of oestrogen accelerates glomerulonephritis, lymphoproliferation and mortality. Our results also indicate that oestrogen exerts a dual effect on the immune system of MRL/l mice by depression of antigen-specific and mitogen-induced T cell responses as well as enhancement of polyclonal B cell activation and autoantibody formation. In addition, even short-term administration of oestrogen in the preclinical phase of the disease resulted in long-lasting effects as evaluated by reduced longevity and aggravation of renal disease.     

Immunological tolerance to a non-cytopathic virus is not inheritable in mice.

Male lymphocytic choriomeningitis (LCM) virus-carrier mice were mated to uninfected females and their offsprings tested for (i) LCMV virus and (ii) their potential to generate LCM virus-specific cytotoxic T cells. Among about 500 offspring tested no example of antigen-independent inheritance of tolerance to LCM virus was detected. Therefore inheritance of acquired tolerance cannot be observed in this murine infectious disease model.     

Susceptibility to malaria as a complex trait: big pressure from a tiny creature

Malaria, which is a major infectious disease worldwide, is caused by the Plasmodium parasite, one of the longest-known parasites infecting humans. The malaria situation is complicated by the emergence of drug resistance and the lack of an effective vaccine. Genetic factors play a key role in disease susceptibility, progression and outcome. Interestingly, an increasing large number of polymorphisms associated with resistance and susceptibility in humans have been found in proteins from erythrocytes, the site of Plasmodium replication. Some of these deleterious alleles have been selected by direct genetic pressure from the parasite in endemic areas of malaria. A number of additional gene effects have been mapped both in humans and in mice using population studies and experimental models of malaria, respectively. These recent studies have started to reveal additional aspects of the complex host-parasite interactions.     

Functional characterization of genetic variants in NPC1L1 supports the sequencing extremes strategy to identify complex trait genes

Resequencing genes in individuals at extremes of the population distribution constitutes a powerful and efficient strategy to identify sequence variants associated with complex traits. An excess of sequence variants at one extreme relative to the other that is not due to chance or to population stratification constitutes evidence for genetic association and implies the presence of functionally significant sequence variants. Recently, we reported that non-synonymous sequence variants in Niemann-Pick type C1-like 1 (NPC1L1), an intestinal cholesterol transporter, were significantly more common among individuals with low cholesterol absorption than in those with high cholesterol absorption. To determine whether sequence variations identified in individuals with low cholesterol absorption affect protein function, we performed studies in cultured cells and in families. Expression of the mutant proteins in Chinese hamster ovarian-K1 cells revealed that a majority (14 of 20) of the variants identified in low absorbers were associated with very low levels of NPC1L1 protein. In two extended families, mean cholesterol absorption levels, as measured using stable isotopes, were significantly lower in family members with the sequence variants than in those without the variant. These data indicate that the excess of sequence variations in individuals with extreme phenotypes reflects an enrichment of functionally significant variants. These findings are consistent with in silico predictions that some sequence variations found in healthy individuals are as deleterious to protein function as mutations that, in other genes, cause monogenic diseases. Such sequence variations may explain a significant fraction of quantitative phenotypic variation in humans.     

Chronic immune complex disease in mice: the role of antibody affinity.

Two lines of mice selectively bred for producing high and low affinity antibody to protein antigens were repeatedly injected with human serum albumin and the severity and pattern of immune complex disease induced in this way was studied in the two lines. In low affinity mice, there was a greater intensity of deposits in the glomeruli shown by immunofluorescence, and more antibody was eluted from kidney homogenates compared to high affinity line mice. In the low affinity mice, complexes were mainly on the basement membrane whereas in high affinity mice, the localization of immune complexes was predominantly mesangial. However, no significant difference in glomerular filtration rates between the two lines was obtained. The immunopathological significance of antibody affinity is discussed in the light of these results.     

Chagas' disease is attenuated in mice lacking gamma delta T cells.

The role of gamma delta T cells in the immunopathology of Chagas' disease is evaluated by monitoring the course of Trypanosoma cruzi infection in mice lacking gamma delta T cells after disruption of the T-cell receptor C delta locus. Levels of parasitemia, states of lymphocyte activation, and levels of lymphokine production as well as tissue pathology are compared in delta knockout mice and their littermates in acute and chronic phases of infection. Although the levels of circulating parasites do not significantly differ in the two groups, mortality scores and numbers of inflammatory lesions of skeletal and cardiac muscles are lower in gamma delta T cell-deficient m ice than in littermate controls. Furthermore, polyclonal lymphocyte activation, as measured by proliferative activities and numbers of B- and T-cell blasts in the spleen, are reduced in deficient mice in the acute and chronic phases of infection. Levels of gamma interferon mRNA obtained from total spleen cells, known to be a critical lymphokine in resistance to T. cruzi infection, are significantly higher in uninfected gamma delta T cell-deficient mice than in control animals and slightly above levels for littermates in the course of acute infection. Interestingly, however, in chronic phases, the levels of this lymphokine are not statistically different between the two groups of mice. These results indicate that gamma delta T cells do not play a crucial role in parasite clearance during the acute phase of the disease but contribute to the mechanisms leading to tissue damage and pathology.