BALB Mice Research Articles

Mechanistic Basis for Inflammation and Tumor Promotion in Lungs of 2,6-Di-tert-butyl-4-methylphenol-Treated Mice: Electrophilic Metabolites Alkylate and Inactivate Antioxidant Enzymes

An established model for mechanistic analysis of lung carcinogenesis involves administration of 3-methylcholanthrene to mice followed by several weekly injections of the tumor promoter 2,6-di-tert-butyl-4-methylphenol (BHT). BHT is metabolized to quinone methides (QMs) responsible for promoting tumor formation. QMs are strongly electrophilic and readily form adducts with proteins. The goal of the present study was to identify adducted proteins in the lungs of mice injected with BHT and to assess the potential impact of these modifications on tumorigenesis. Cytosolic proteins from treated mouse lungs were separated by two-dimensional electrophoresis, adducts detected by immunoblotting, and proteins identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Eight adducts were detected in the lungs of most, or all, of six experimental groups of BALB mice. Of these adducts, several were structural proteins, but others, namely, peroxiredoxin 6 (Prx6), Cu,Zn-superoxide dismutase (SOD1), carbonyl reductase, and selenium-binding protein 1, have direct or indirect antioxidant functions. When the 9000g supernatant fraction of mouse lung was treated with BHT-QM (2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone), substantial lipid peroxidation and increases in hydrogen peroxide and superoxide formation were observed. Studies with human Prx6 and bovine SOD1 demonstrated inhibition of enzyme activity concomitant with adduct formation. LC-MS/MS analysis of digests of adducted Prx6 demonstrated adduction of both Cys 91 and Cys 47; the latter residue is essential for peroxidatic activity. Analysis of QM-treated bovine SOD1 by matrix-assisted laser desorption/ionization time-of-flight MS demonstrated the predominance of a monoadduct at His 78. This study provides evidence that indicates Prx6, SOD1, and possibly other antioxidant enzymes in mouse lung are inhibited by BHT-derived QMs leading to enhanced levels of reactive oxygen species and inflammation and providing a mechanistic basis for the effects of BHT on lung tumorigenesis.

Safingol Toxicology After Oral Administration to TRAMP Mice: Demonstration of Safingol Uptake and Metabolism by N-Acylation and N-Methylation

Safingol [(2S,3S)-2-amino-1,3-octadecanediol] is an unnatural l-threo-stereoisomer of sphinganine that is cytotoxic for cancer cells in culture and is being tested in phase 1 human clinical trials. To determine if safingol can be absorbed orally and if it affects prostate cancer in a mouse strain used in prostate cancer studies, safingol was fed to TRAMP (transgenic adenocarcinoma of mouse prostate) mice for 2 weeks at 0.0125% to 0.1% w/w of the diet. Analysis of safingol and safingol metabolites in blood and tissues by liquid chromatography electrospray ionization tandem mass spectrometry revealed uptake in tissue and extensive conversion of safingol to N-acyl species (comparable to natural “ceramides”) and mono-, di-, and tri-N-methyl metabolites that have not been observed previously. Safingol caused significant hepatotoxicity at all dosages, as reflected in elevated liver alanine aminotransferase, and at the highest dose (0.1 %) caused changes in liver histology (appearance of autophagosomal vacuoles) and renal toxicity (based on elevation of blood urea nitrogen) and decreases in packed blood cell volume and body weight. Safingol did not inhibit the prostate pre-neoplastic lesion (prostate intraepithelial neoplasia) in TRAMP mice; however, additional studies at lower dosages for longer time were not pursued due to host toxicity. Safingol and its N-methyl metabolites were cytotoxic to both a human prostate cell line (DU145) and mouse BALB 3T3 cells; therefore, the host and potential antitumor toxicity may be due to multiple molecular species of safingol.

Inhibition of T Cell Alloreactivity by Gr-1+Mac-1+ Myeloid Suppressor Cells.

Graft-versus-host-disease (GVHD) remains the major complication of allogeneic stem cell transplantation (SCT). While pharmacological approaches have long been the mainstay of GVHD prevention, recent strategies have focused on cellular approaches, specifically the use of regulatory cell populations to prevent or mitigate the severity of GVHD. Gr-1+Mac-1+ myeloid-derived cells, termed myeloid suppressor cells (MSCs), are a regulatory cell population that has been shown to play an important role in the suppression of tumor and anti-infectious immunity. In these studies, we examined whether these cells were detectable in GVHD recipients and played any role in the suppression of alloreactive T cell responses in vitro and in vivo. Lethally irradiated Balb/c (H-2d) mice were transplanted with MHC-incompatible C57BL/6 (H-2b) bone marrow and spleen cells to induce GVHD. Immature myeloid cells with a Gr-1+Mac-1+ phenotype were found to constitute 60% of all splenocytes at day 10 post-SCT thereafter declining to 30% by day 21. MSCs isolated from the spleens of mice with GVHD expressed high levels of class I and II, but low levels of the costimulatory molecules CD80, CD86, CD40 and were negative for CD11c. Highly purified Gr-1+ Mac-1+ cells obtained from the spleens of GVHD mice 10 or 21 days post-SCT potently suppressed naive T cell proliferation in a standard MLC by 70% at a responder to suppressor ratio of 1:1. Notably, MSCs could also be generated in vitro from normal B6 BM after 6 days in culture with G-CSF and were equally potent at suppressing T cell alloreactivity. Suppression was partially mediated by nitric oxide (NO) as addition of the NO inhibitor L-NMA reversed 50% of the inhibitory effect. To determine whether MSCs exerted a suppressive effect in vivo, lethally irradiated Balb mice were transplanted with B6 BM plus naïve spleen cells with or without MSCs obtained from the spleens of GVHD animals 10 or 21 days post-BMT. The adoptive transfer of MSCs failed to protect mice from GVHD when assessed by overall survival, serial weight measurements or histological analysis. As an alternative approach to augment GVHD protection, G-CSF was administered for 14 days beginning at the time of BMT to enhance the in vivo survival of MSCs. G-CSF administration in both irradiated and non-irradiated MHC-incompatible GVHD mouse models, however, also had no protective effect. We reasoned that a possible explanation for the lack of an effect in vivo was that MSCs did not appropriately localize to nodal sites where GVHD is initiated. To examine this question, B6-green fluorescent protein (GFP) mice were used as donor animals to permit the detection and trafficking of MSCs in vivo. Adoptive transfer of B6 GFP-MSCs from the spleens of GVHD mice 10 days after SCT demonstrated that these cells could be detected in the secondary lymphoid organs of recipient mice 20 hours after BMT, but were completely absent by day 3, supporting the premise that the lack of protection was attributable, at least in part, to the inability of these cells to persist at nodal sites where T cell priming against host alloantigens was occurring. These results indicate that MSCs, which are present in the spleen of GVHD recipients, show potent T cell suppressive capacity in vitro, but fail to mediate a similar effect in vivo. Lack of protection appears to be due to the inability of these cells to localize to all critical secondary lymphoid sites underscoring the importance that the capability of regulatory cells to migrate to and persist at appropriate tissue sites plays in the design of cellular strategies to prevent GVHD.

In vitro cytotoxicity of dental composites based on new and traditional polymerization chemistries

The biological response to dental restorative polymer composites is mediated by the release of unpolymerized residual monomers. Several new composite formulations claim to reduce unpolymerized residual mass. The current study assessed the cytotoxic responses to several of these new formations and compared them with more traditional formulations. Our hypothesis predicted that if these new polymerization chemistries reduce unpolymerized residual mass, the cytotoxicity of these materials also should be reduced relative to traditional formulations. Materials (HerculiteXRV, Premise, Filtek Supreme, CeramxDuo, Hermes, and Quixfil) were tested in vitro in direct contact with Balb mouse fibroblasts, initially, then after aging in artificial saliva for 0, 1, 3, 5, or 8 weeks. The toxicity was determined by using the MTT assay to the estimate SDH activity. Knoop hardness of the materials also was measured at 0 and 8 weeks to determine whether surface breakdown of the materials in artificial saliva contributed to cytotoxic responses. Materials with traditional methacrylate chemistries (Herculite, Premise, Filtek Supreme) were severely (>50%) cytotoxic throughout the 8-week interval, but materials with newer chemistries or filling strategies (Hermes, CeramXDuo, and Quixfil) improved over time of aging in artificial saliva. Hermes showed the least cytotoxicity at 8 weeks, and was statistically equivalent to Teflon negative controls. Hardness of the materials was unaffected by exposure to artificial saliva. Newer polymerization and filling strategies for dental composites show promise for reducing the release of unpolymerized components and cytotoxicity.

Effects of radiofrequency on immune function of mice with hepatocellular carcinoma

目的 观察肝癌小鼠模型射频治疗后外周血肿瘤坏死因子-α(TNF-α)和白细胞介素-2(IL-2)的变化及癌组织CD54的表达情况,探讨肝癌射频治疗对机体免疫功能的影响.方法Balb's小鼠60只,随机分为2组,对照组(C组)和射频治疗组(R组),每组30只.成功制作小鼠肝细胞癌模型后,R组给予射频治疗,并于其后0 h、2 h、2周、3周、4周处死并眼球取血法取血,采用双抗体夹心ELISA法测定TNF-α和IL-2光密度值(OD),同时切除肿瘤进行免疫组化CD54表达分析.结果R组肿瘤明显缩小,其TNF-α和IL-2浓度呈递增趋势,与对照组比较差异有显著性(P＜0.05);经射频处理后的R组肝癌细胞CD54呈过度表达现象,并与各时间段TNF-α、IL-2浓度呈明显正相关(r=0.520,P＜0.01;r=0.501,P＜0.01).结论肝癌射频治疗诱使TNF-α、IL-2浓度增高,并上调细胞间粘附分子CD54的表达,从而诱导肿瘤免疫应答而发挥抗肿瘤作用。

Strain-dependent pulmonary gene expression profiles of a cystic fibrosis mouse model

Cystic fibrosis (CF) lung disease severity is influenced by unknown genetic factors apart from the disease causative gene, cystic fibrosis transmembrane conductance regulator (CFTR). Previous studies have shown the C57BL/6J congenic Cftr(-/-) (B6 CF) mouse to develop a fibrotic lung disease compared with both CF mice of the BALB/c background and wild-type animals. In this report, gene expression profiling with microarrays was used to identify genes differentially expressed in the lungs of B6 and BALB CF mice compared with non-CF littermates. Seven hundred two genes or expressed sequence tags (ESTs) were identified to be differentially expressed between the B6 CF and non-CF control lungs (P < 0.05), and, by Gene Ontology classification, the B6 CF response included the cell proliferation categories of DNA metabolism and mitosis. In the response of BALB mice to nonfunctional Cftr, 943 genes/ESTs were differentially expressed compared with controls. The biological processes of apoptosis and T and B cell proliferation were prominent in the gene list of the BALB CF strain. In support of this strain difference, increased T lymphocyte infiltration was evident in the lungs of BALB CF mice, through immunohistochemical staining, compared with the lungs from both B6 CF and non-CF control mice. Four hundred forty-four genes/ESTs were differentially expressed between B6 CF and BALB CF mice (P < 0.05, fold > 2), including 56 that map to previously identified linkage intervals. These results suggest that the variable severity of CF lung disease in this mouse model is controlled by multiple genetic factors, including those of an immune response.

Heritable renal hypoplasia and frontonasal dysplasia is associated with misexpression of six2

Renal hypoplasia and frontonasal dysplasia are malformations associated with tissues derived from mesoderm. A radiation induced mouse mutant, called Brachyrrhine (Br) exhibits both malformations. Initial microsatellite analysis placed the mutation on distal chromsome 17 in Br. The purpose of this study was to further resolve the Br locus and determine tissue expression of candidate genes. An interspecific backcross breeding strategy was designed using 3H1 Br/+ mice crossed to Castaneus and Balb mice. Segregation analysis showed that Br was inherited as a semidominant lethal mutation when outcrossed to both inbred strains. DNA was extracted from F2 progeny and scored for recombinations. Results showed that Br localized to a region just distal to D17Mit76 that included six2. Using this microsatellite as a genotypic marker, C3H1 x Balb Br/+ F1 mice were crossed and embryos were obtained at embryonic days 10.5–11.5. The embryos were subjected to whole-mount in situ hybridization (WMISH) and quantitative RT-PCR. Results showed that homozygous Br/Br animals have significantly reduced six2 expression. Quantitative RT-PCR reflected a pattern of six2 haploinsufficiency with heterozygous mutants showing about a 50% decrease in gene dosage and homozygous mutants showing about a 10% expression. It is concluded that a mutation affecting six2 expression is responsible for mesodermal differentiation pathways in the face and kidney. Supported by R01-DK064752, Meiji Yasuda Endowment, and HS-BRIN P20 PP16467

The Role of the MHC on Resistance to Group A Streptococci in Mice

The severity of infection with Streptococcus pyogenes is strongly influenced by the host's genetics. This observation extends to the murine model of streptococcal infection, where the background of the mouse strain determines the infection outcome (BALB/c are resistant, whereas C3H/HeN are susceptible). To determine the extent to which the MHC complex (H2) contributed to diseases susceptibility, the response to S. pyogenes of congenic BALB mice from a resistant background (BALB/c), but carrying the H2(k) region of susceptible C3H/HeN mice (BALB/k), was examined. BALB/k were as susceptible as the H2 donor strain (C3H/HeN). Linkage analysis performed in F(2) backcross ([BALB/c x C3H/HeN] x BALB/c) mice confirmed the presence of a susceptibility locus within the H2 region on proximal chromosome 17. The possibility that modulation of T cell responses to streptococcal superantigens (GAS-SAgs) by different H2 haplotypes may influence disease severity was examined. BALB/k exhibited a significantly stronger response at the level of cell proliferation and cytokine production to GAS-SAgs than did BALB/c mice. However, the fact that T cell-deficient SCID-C3H/HeN mice also exhibited a susceptible phenotype suggests a more important contribution of innate effector cells to disease susceptibility. Lower transcriptional levels of certain inflammation-related regulatory genes located on chromosome 17 were detected in macrophages from susceptible than in those from resistant mice in response to infection. These results suggest that susceptibility to S. pyogenes may be associated with an altered transcription of specific genes that may compromise the endogenous regulatory processes controlling the inflammatory cascade and favor the progression to sepsis.

４系統のマウスにおけるプレパルス・インヒビション―プレパルスの種類とリードタイムの効果―

The effects of the type of prepulse (PP: visual 30 ms and auditory 30 ms) and lead time (50, 100, 200 ms) on the acoustic startle inhibition (prepulse inhibition: PPI) were tested and compared among four strains of inbred mice (BALB/cA, C3H/HeJ, C57BL/6J, and DBA/2N: all n=11). Following 40 trials of habituation using single pulse (white noise: P; 115 dB, 50 ms), the prepulse session made of 36 trials was conducted. In the habituation, the startle amplitude and peak latency were measured. DBA mice showed weaker startle amplitude than the other 3 strains. The peak latency of the BALB mice was faster than the other strains. The PPI by auditory PP was largest in C57BL, followed by C3H, and the least in BALB mice. DBA mice showed no PPI effect. The PPI under visual prepulse condition was observed only in BALB strain. Contrary, DBA mice showed prepulse facilitation in the 50 ms lead time condition.

Krypton laser-induced photothrombotic distal middle cerebral artery occlusion without craniectomy in mice

Recent advances in genetical engineering of the mouse have highlighted the importance of reproducible and less invasive models of cerebral ischemia in mice. In this paper, we developed minimally invasive and reproducible model of distal middle cerebral artery (MCA) occlusion in mice using krypton (Kr) laser-induced photothrombosis. C57BL/6 or BALB mice ( n=8 each) were anesthetized with halothane. The skin was cut, the temporal muscle was retracted, and the right distal MCA was observed through the skull. A Kr laser beam of wavelength 568 nm was focused onto the MCA over the intact skull. Upon laser irradiation, intravenous administration of a rose bengal solution was begun. After 4 min of irradiation, the laser beam was refocused on the MCA just proximal to the first spot, and another 4-min irradiation was performed. Then, the right common carotid artery (CCA) was ligated. Three days later, the brain was removed, and infarct volume was determined. Infarction confined almost solely to the cortical area was produced in each mouse. Mean infarct volume in C57BL/6 mice was 25.2±13.7 mm 3. The BALB mice group showed significantly larger and more reproducible infarction (44.1±5.2 mm 3; the coefficient of variation was 12%) than did C57BL/6 mice ( P<0.005). Our photothrombosis model of stroke in mice can be performed without craniectomy, and its reproducibility is satisfactory when using BALB mice.

The Organ Blood Flow under Conditions of Motor Rest in Rodents with Different Body Mass

The volume velocity of organ blood flow (VVBF) was measured using 86RbCl in anesthetized wild rodents (birch mouse, narrow-skulled vole, and common hamster) as well as in wakeful and anesthetized Wistar rats and Balb mice. It has been shown that with increase of the rodent body mass (from 8 g to 760 g) and decrease of the specific minute volume of blood circulation (VVBF/100 g organ), VVBF is unchanged in kidney, decreases, although to a lesser extent than cardiac output (CO), in digestive tract, liver, spleen, and heart, whereas in supporting-motor and covering structures it decreases to a greater extent than CO.

Genetically based influences on the site-specific regulation of trabecular and cortical bone morphology.

The degree of site-specificity by which genes influence bone quantity and architecture was investigated in the femur of three strains of mice. Morphological indices were highly dependent on both genetic makeup as well as anatomical location showing that the assessment of bone structure from a single site cannot be extrapolated to other sites even within a single bone. The identification of genes responsible for establishing peak BMD will yield critical information on the regulation of bone quantity and quality. Whereas such knowledge may eventually uncover novel molecular drug targets or enable the identification of individuals at risk of osteoporosis, the site-specificity by which putative genotypes cause low or high bone mass (and effective bone morphology) is essentially unknown. microCT was used to determine morphological and microarchitectural features of the femora harvested from three genetically distinct strains of 4-month-old female mice, each with distinct skeletal mass (low: C57BL/6J [B6], medium: BALB/cByJ [BALB], high: C3H/HeJ [C3H]). Two trabecular regions (distal epiphysis and metaphysis) were considered in addition to four cortical regions within the metaphysis and diaphysis. Comparing morphological properties of the different trabecular and cortical femoral regions between the three strains of mice, it was apparent that high or low values of specific parameters of bone morphology could not be consistently attributed to the same genetic strain. Trabecular metaphyseal bone volume, for instance, was 385% larger in C3H mice than in B6 mice, yet the two strains displayed similar bone volume fractions in the epiphysis. Similarly, BALB mice had 48% more trabecular bone than C3H mice in the epiphysis, but there were no strain-specific differences in cortical bone area at the diaphysis. These data suggest that the genetic control of bone mass and morphology, even within a given bone, is highly site-specific and that a comprehensive search for genes that are indicative of bone quantity and quality may also have to occur on a very site-specific basis.

Behaviour in the elevated plus-maze predicts coping after subchronic mild stress in mice

This study was aimed at investigating the coping style of mice subjected to a subchronic unpredictable mild stress procedure and its relationship to initial emotional reactivity. Two inbred strains of mice, the BALB/c ByJ and the C57BL/6 J, known to exhibit distinct emotionality, have been used. They were first observed in the elevated plus-maze and the free exploratory paradigm, each provides a separation of the population in high and low emotional mice. Half of the mice of each strain were then confronted to a 2-week subchronic unpredictable mild stress and tested for their responses in different behavioural situations (consumption of a palatable food, physical state, grooming behaviours and reactivity to a conflict situation). Mice were also tested in the light/dark procedure to assess the effect of the subchronic stress on emotional reactivity. First, a relationship between initial emotional reactivity in the elevated plus-maze and behavioural coping style in response to stress was found, high emotional mice (i.e., BALB mice) displaying inhibited behaviours and less emotional mice (i.e., BL/6 mice) exhibiting few behavioural changes. Furthermore, emotional reactivity was increased in stressed mice compared with nonstressed ones.

Habituation of the Acoustic and the Tactile Startle Responses in Mice: Two Independent Sensory Processes.

To test whether habituation is specific to the stimulus modality, the authors analyzed cross-habituation between the tactile startle response' (TSR) and the acoustic startle response (ASR). The acoustic artifacts of airpuffs used to elicit the TSR were reduced by using a silencer and were effectively masked by background noise of 90-100 dB sound-pressure level. ASR was elicited by 14-kHz tones. TSR and ASR habituated in DBA and BALB mice: both the TSR and ASR habituated to a greater extent in DBA mice than in BALB mice. In both strains, habituation of the TSR did not generalize to the ASR, and vice versa. From this, the authors concluded that habituation of startle is located in the sensory afferent branches of the pathway.

Prospect of preimplantation genetic diagnosis for heritable mitochondrial DNA diseases.

To perform preimplantation genetic diagnosis for women carrying heteroplasmic mitochondrial DNA (mtDNA) mutations, it is necessary to ensure that the proportion of mutant mtDNA diagnosed in the biopsied cell gives an accurate indication of the mutant load in the remaining embryo. A heteroplasmic mouse model, carrying NZB and BALB mtDNA genotypes, was used to study the relative proportions of each mtDNA genotype in the ooplasm and first polar body of mature oocytes, and between blastomeres of early cleavage stage embryos. The levels of heteroplasmy varied widely in the gametes compared with the maternal genotype. However, the distribution of the two mtDNA genotypes was virtually identical between the ooplasm and polar body of a mature oocyte, and also between the blastomeres of each 2-, 4- and 6-8-cell embryo. Therefore, the level of heteroplasmy diagnosed from the polar body of an unfertilized oocyte or from a single blastomere of an embryo is representative of the level in the embryo as a whole. Reliable results were obtained from both polar bodies and blastomeres, but the efficiency of diagnosis was greater with blastomeres. We conclude that preimplantation genetic diagnosis is feasible for mtDNA diseases, although it should be approached with caution, as it is possible that transmission of some pathogenic mutations could behave in a different manner.

Molecular evolution of V(H)9 germline genes isolated from DBA, BALB, 129 and C57BL mouse strains and sublines.

We have used the polymerase chain reaction (PCR) in an attempt to clone and sequence the exons and hitherto unavailable contiguous flanks of all members of the small V(H) 9 germline gene family from inbred mouse strains and sublines that have had a common ancestry within the last century, and to analyze the molecular evolution of these sequences. Fifteen genuine germline genes were isolated (designated V(H) 9.1 through V(H) 9.15) from strains and sublines of DBA, BALB, 129 and C57BL inbred mice. Of the 15 genuine isolates, nine are novel: seven sequences from DBA strains and sublines ( V(H) 9.3 to V(H) 9.9) and two sequences from C57BL strains ( V(H) 9.13 and V(H) 9.14). We have identified sequencing errors and PCR recombinant artefacts in previously published sequences. We detected no sequence divergence of individual genes shared by the strains and sublines studied. However, we isolated two genes from DBA strains and sublines, V(H) 9.1 and V(H) 9.3, that differ only by five nucleotides encoding three amino acid changes that are concentrated within a 33 nucleotide (11 codon) region. Of these 11 codons, eight encode a putative antigen binding site. There were no differences in the remaining 733 nucleotides sequenced (including both 5' and 3' flanking regions). Potential explanations for the generation of V(H) 9.1 and V(H) 9.3 are discussed.

The use of the murine model of infection with Leishmania major to reveal the antagonistic effects that IL-4 can exert on T helper cell development and demonstrate that these opposite effects depend upon the nature of the cells targeted for IL-4 signaling

In contrast to mice from the majority of inbred strains, BALB mice develop aberrant Th2 responses and suffer progressive disease after infection with Leishmania major. These outcomes depend on the production of Interleukin 4, during the first 2 d of infection, by CD4 + T cells that express the Vβ4-Vα8 T cell receptors specific for a dominant I-A d restricted epitope of the LACK antigen from L. major. In contrast to this well established role of IL-4 in Th2 cell maturation, we have recently shown that, when limited to the initial period of activation of dendritic cells by L. major preceding T cell priming, IL-4 directs DCs to produce IL-12, promotes Th1 cell maturation and resistance to L. major in otherwise susceptible BALB/c mice. Thus, the antagonistic effects that IL-4 can have on Th cell development depend upon the nature of the cells (DCs or primed T cells) targeted for IL-4 signaling.

Relationship between long-term resistance to Trypanosoma cruzi and latent infection, examined by antibody production and polymerase chain reaction in mice.

Trypanosoma cruzi infections persist for the lifetime of humans and laboratory animals as either latent or pathogenic parasitism. Mice inoculated with a nonpathogenic, attenuated strain (TCC) display resistance against virulent challenge, with a strong control of parasitemia and protection against tissue lesions for more than 12 mo. Three main approaches were used to test whether protection by TCC inocula is based on a latent infection or on a "sterile" immunological memory: curative Benznidazole (Bzl) treatment, serological reactions, and detection of infection by polymerase chain reaction (PCR). If resistance is maintained in the absence of infection, it should not be reduced by Bzl treatment and TCC-inoculated animals should not maintain long-term serological or PCR reactivity. The Bzl treatment after TCC inoculations did not reduce, after periods of up to 420 days, TCC-induced resistance to challenge. But TCC inocula given during Bzl treatment conferred short-term, but not long-term. protection. Maintenance of high antibody levels and protection were better in the virulent Tulahuen (TUL) strain than in the attenuated TCC strain infections, and trypomastigote inocula of either strain were better inducers of antibodies and resistance than epimastigotes. PCR detection of T. cruzi DNA was positive in almost all TUL strain-inoculated animals and negative in immunocompetent animals inoculated with TCC epimastigotes, although high numbers of TCC trypomastigotes produced persistent PCR signals of infection in newborn BALB mice. Thus, 2 polar models were developed, where latent infection by TCC was either demonstrated or excluded. In both, resistance to virulent challenge was maintained during long periods. But late declination of antibody titers (>200 days) and resistance to challenge (>350 days) was observed in animals displaying clearance of all signals of infection.

Induction of a large deletion in mitochondrial genome of mouse cells by X-ray irradiation

Large deletions and point mutations of mitochondrial DNA (mtDNA) is causally associated with mitochondrial diseases, and accumulates with age in human tissues. In order to find out the role of oxidative stress in the generation of large mtDNA deletions, using normal Balb and severe combined immunodeficiency (SCID) mouse cells, we assessed whether X-ray irradiation induces large mtDNA deletions. Cultured cells were irradiated with X-rays and assayed for a large mtDNA deletion using a PCR technique with a specific pair of primers. X-ray doses as low as 1 Gy were effective for the induction of the large mtDNA deletion (5823 bp long), which corresponds to the human 4977 bp common deletion. The breakpoints were flanked by the 5-bp long tandem repeats, 5′-TACCC-3′. A dose-dependent induction of the large mtDNA deletion was observed, the yield being higher in normal cells than in SCID cells. The fraction of large mtDNA deletion increased in the normal cells but decreased in the SCID cells within 7 days after X-ray irradiation. As the SCID cells carry a mutation in the gene encoding DNA–PKcs, the key enzyme in DNA double-strand break repair, it can be concluded that repair of DNA strand breaks may be involved in the formation of X-ray induced large mtDNA deletions.

A locus affecting immunoglobulin isotype selection (Igis1) maps to the MHC region in C57BL, BALB/c and NOD mice.

We have previously shown that H2b mice with B10 or BALB genetic backgrounds have higher basal levels of IgG2a than H2k and H2d congenic strains and, hence, have low IgG1/IgG2a ratios, which is consistent with a T1 cytokine milieu. The phenotypic marker of the high IgG2a levels, denoted immunoglobulin isotype-1 (Igis1) was provisionally mapped telomeric of IEbeta using MHC recombinant mice. In addition, data from B10.A(2R), B10.A(1R) and B10.A(18R) mice indicated that Igis1 may lie in a 27 kb region between G7b (Sm-X5) and G7c. In the present study we confirm that Igis1 is in the H2 region using BALB and B10 congenic F2 mice. H2bb F2 mice had higher IgG2a levels than the H2dd parental strains. H2bd F1 and F2 mice on the B10 background produced IgG2a levels comparable with the H2bb parental strain, indicating that the b allele was dominant. In contrast, the H2bd F1 and F2 mice on the BALB background produced IgG2a levels between those of the H2bb and H2dd parental strains, indicating codominance of the b and d alleles. This suggests that a background gene influences regulation of IgG2a levels by Igis1. Non-obese diabetic (NOD) mice (KdIAg7IEnu11Db), which can develop type 1 diabetes, had higher levels of IgG2a than NOD-H2d congenic mice. Thus, Igis1 affects isotype selection in the presence of non-MHC diabetes genes. As type 1 diabetes is associated with T1 responses, Igis1 may affect susceptibility to this condition.