SJL/J Mice Are Highly Susceptible to Infection by Mouse Adenovirus Type 1

Mouse adenovirus type 1 (MAV-1) targets endothelial and monocyte/macrophage cells throughout the mouse. Depending on the strain of mouse and dose or strain of virus, infected mice may survive, become persistently infected, or die. We surveyed inbred mouse strains and found that for the majority tested the 50% lethal doses (LD(50)s) were >10(4.4) PFU. However, SJL/J mice were highly susceptible to MAV-1, with a mean LD(50) of 10(-0.32) PFU. Infected C3H/HeJ (resistant) and SJL/J (susceptible) mice showed only modest differences in histopathology. Susceptible mice had significantly higher viral loads in the brain and spleen at 8 days postinfection than resistant mice. Infection of primary macrophages or mouse embryo fibroblasts from SJL/J and C3H/HeJ mice gave equivalent yields of virus, suggesting that a receptor difference between strains is not responsible for the susceptibility difference. When C3H/HeJ mice were subjected to sublethal doses of gamma irradiation, they became susceptible to MAV-1, with an LD(50) like that of SJL/J mice. Antiviral immunoglobulin G (IgG) levels were measured in susceptible and resistant mice infected by an early region 1A null mutant virus that is less virulent that wild-type virus. The antiviral IgG levels were high and similar in the two strains of mice. Taken together, these results suggest that immune response differences may in part account for differences in susceptibility to MAV-1 infection.     

Transgenic Mice With Elevated Level of CuZnSOD Are Highly Susceptible to Malaria Infection

Copper/zinc superoxide dismutase (CuZnSOD) catalyses the conversion of O₂^•− into H₂O₂. Constitutive overexpression of CuZnSOD in cells and animals creates an indigenous oxidative stress that predisposes them to added insults. In this study, we used transgenic CuZnSOD (Tg-CuZnSOD) mice with elevated levels of CuZnSOD to determine whether overexpression of CuZnSOD affected the susceptibility of these mice to plasmodium infection. Acute malaria is associated with oxidative stress, mediated by redox-active iron released from the infected RBC. Two independently derived Tg-CuZnSOD lines showed higher sensitivity than control mice to infection by Plasmodium berghei (P. berghei), reflected by an earlier onset and increased rate of mortality. Nevertheless, while Tg-CuZnSOD mice were more vulnerable than control mice, the levels of parasitemia were comparable in both strains. Moreover, treatment of infected red blood cells (RBC) with oxidative stress inducers, such as ascorbate or paraquat, reduced the viability of parasites equally in both transgenic and control RBC. This further confirms that increased CuZnSOD does not support plasmodia development. The data are consistent with the possibility that the combination of increased redox-active iron and elevated H₂O₂ in the plasmodium-infected Tg-CuZnSOD mice, led to an enhanced Fenton’s reaction-mediated HO^• production, and the resulting oxidative injury renders the transgenic mice more vulnerable to parasite infection.     

Identification of Wild-Derived Inbred Mouse Strains Highly Susceptible to Monkeypox Virus Infection for Use as Small Animal Models

Infection with monkeypox virus (MPXV) causes disease manifestations in humans that are similar, although usually less severe, than those of smallpox. Since routine vaccination for smallpox ceased more than 30 years ago, there is concern that MPXV could be used for bioterrorism. Thus, there is a need to develop animal models to study MPXV infection. Accordingly, we screened 38 inbred mouse strains for susceptibility to MPXV. Three highly susceptible wild-derived inbred strains were identified, of which CAST/EiJ was further developed as a model. Using an intranasal route of infection with an isolate of the Congo Basin clade of MPXV, CAST/EiJ mice exhibited weight loss, morbidity, and death in a dose-dependent manner with a calculated 50% lethal dose (LD₅₀) of 680 PFU, whereas there were no deaths of BALB/c mice at a 10,000-fold higher dose. CAST/EiJ mice exhibited greater MPXV sensitivity when infected via the intraperitoneal route, with an LD₅₀ of 14 PFU. Both routes resulted in MPXV replication in the lung, spleen, and liver. Intranasal infection with an isolate of the less-pathogenic West African clade yielded an LD₅₀ of 7,600 PFU. The immune competence of CAST/EiJ mice was established by immunization with vaccinia virus, which induced antigen-specific T- and B-lymphocyte responses and fully protected mice from lethal doses of MPXV. The new mouse model has the following advantages for studying pathogenesis of MPXV, as well as for evaluation of potential vaccines and therapeutics: relative sensitivity to MPXV through multiple routes, genetic homogeneity, available immunological reagents, and commercial production.Monkeypox virus (MPXV), a member of the orthopoxvirus genus of the Chordopoxvirinae subfamily of the Poxviridae, was isolated in 1958 from lesions in a cynomolgous monkey that had been imported from Africa (27). The first human infections with MPXV were reported in 1972, and since then more than two thousand cases have been recorded, most in the Democratic Republic of the Congo and lesser numbers in West African countries (reviewed by Parker et al. [18]). The mortality from human monkeypox in the Congo is estimated to be 10% of infected individuals with clinical symptoms that mimic smallpox, which is caused by another member of the orthopoxvirus genus: variola virus. However, whereas the host range of variola virus is restricted to humans, serological studies indicate that MPXV naturally infects a large number of animal species, particularly squirrels and nonhuman primates. The sporadic occurrence of human monkeypox is thought to arise from close proximity and handling of infected animals. In this respect, a self-limited outbreak in the United States was traced to a shipment of West African rodents (19). Although monkeypox is a minor public health problem when compared historically to smallpox, the potential for expansion of the MPXV host range and adaptations to enhance human transmission make it prudent to continue careful surveillance. Moreover, the potential use of MPXV for bioterrorism has led to its inclusion as a select agent in the United States (http://www.selectagents.gov).Animal models are crucial for studying virus pathogenesis, and MXPV is no exception. Ground squirrels (22, 26), black-tailed prairie dogs (9, 11, 13, 30), and African dormice (23) are highly susceptible to MPXV. However, as experimental systems, each has limitations with regard to unavailability of commercial breeding, genetic heterogeneity and absence of immunological and other reagents. Laboratory mice, including BALB/c, C57BL/6, and several other mouse strains tested, were found to be resistant to MPXV disease unless impaired in innate or acquired immunity (10, 17, 24). In the present study, we tested a large group of distinct inbred strains of mice chosen for genetic diversity, inclusion of classical and wild-derived strains, and commercial availability. Of 38 inbred mouse strains tested, three wild-derived strains were highly susceptible to MPXV. One of these, CAST/EiJ, was further characterized with regard to MPXV strain sensitivity, route of inoculation, virus dissemination, immune response, and protection by vaccination and drug treatment.     

Generation of Live Offspring from Vitrified Mouse Oocytes of C57BL/6J Strain

In mammals, unfertilized oocytes are one of the most available stages for cryopreservation because the cryopreserved oocytes can be used for assisted reproductive technologies, including in vitro fertilization (IVF) and intracytoplasmic sperm injection. However, it has generally been reported that the fertility and developmental ability of the oocytes are reduced by cryopreservation. C57BL/6J mice, an inbred strain, are used extensively for the production of transgenic and knockout mice. If the oocytes from C57BL/6J mice can be successfully cryopreserved, the cryopreservation protocol used will contribute to the high-speed production of not only gene-modified mice but also hybrid mice. Very recently, we succeeded in the vitrification of mouse oocytes derived from ICR (outbred) mice. However, our protocol can be applied to the vitrification of oocytes from an inbred strain. The aim of the present study was to establish the vitrification of oocytes from C57BL/6J mice. First, the effect of cumulus cells on the ability of C57BL/6J mouse oocytes to fertilize and develop in vitro was examined. The fertility and developmental ability of oocyte-removed cumulus cells (i.e., denuded oocytes, or DOs) after IVF were reduced compared to cumulus oocyte complexes (COCs) in both fresh and cryopreserved groups. Vitrified COCs showed significantly (P<0.05) higher fertility and ability to develop into the 2-cell and blastocyst stages compared to the vitrified DOs with cumulus cells and vitrified DOs alone. The vitrified COCs developed to term at a high success rate, equivalent to the rate obtained with IVF using fresh COCs. Taken together, our results demonstrate that we succeeded for the first time in the vitrification of mouse oocytes from C57BL/6J mice. Our findings will also contribute to the improvement of oocyte vitrification not only in animals but also in clinical applications for human infertility.     

Generating Embryonic Stem Cells from the Inbred Mouse Strain DBA/2J,a Model of Glaucoma and Other Complex Diseases

Mouse embryonic stem (ES) cells are derived from the inner cell mass of blastocyst stage embryos and are used primarily for the creation of genetically engineered strains through gene targeting. While some inbred strains of mice are permissive to the derivation of embryonic stem cell lines and are therefore easily engineered, others are nonpermissive or recalcitrant. Genetic engineering of recalcitrant strain backgrounds requires gene targeting in a permissive background followed by extensive backcrossing of the engineered allele into the desired strain background. The inbred mouse strain DBA/2J is a recalcitrant strain that is used as a model of many human diseases, including glaucoma, deafness and schizophrenia. Here, we describe the generation of germ-line competent ES cell lines derived from DBA/2J mice. We also demonstrate the utility of DBA/2J ES cells with the creation of conditional knockout allele for Endothelin-2 (Edn2) directly on the DBA/2J strain background.     

Kinase Suppressor of Ras 1 Is Not Required for the Generation of Regulatory and Memory T Cells

The mammalian target of rapamycin (mTOR) kinase is a critical regulator of the differentiation of helper and regulatory CD4+ T cells, as well as memory CD8+ T cells. In this study, we investigated the role of the ERK signaling pathway in regulating mTOR activation in T cells. We showed that activation of ERK following TCR engagement is required for sustained mTOR complex 1 (mTORC1) activation. Absence of kinase suppressor of Ras 1 (KSR1), a scaffold protein of the ERK signaling pathway, or inhibition of ERK resulted in decreased mTORC1 activity following T cell activation. However, KSR1-deficient mice displayed normal regulatory CD4+ T cell development, as well as normal memory CD8+ T cell responses to LCMV and Listeria monocytogenes infection. These data indicate that despite its role in mTORC1 activation, KSR1 is not required in vivo for mTOR-dependent T cell differentiation.     

Computational and Empirical Studies Predict Mycobacterium tuberculosis-Specific T Cells as a Biomarker for Infection Outcome

Identifying biomarkers for tuberculosis (TB) is an ongoing challenge in developing immunological correlates of infection outcome and protection. Biomarker discovery is also necessary for aiding design and testing of new treatments and vaccines. To effectively predict biomarkers for infection progression in any disease, including TB, large amounts of experimental data are required to reach statistical power and make accurate predictions. We took a two-pronged approach using both experimental and computational modeling to address this problem. We first collected 200 blood samples over a 2- year period from 28 non-human primates (NHP) infected with a low dose of Mycobacterium tuberculosis. We identified T cells and the cytokines that they were producing (single and multiple) from each sample along with monkey status and infection progression data. Machine learning techniques were used to interrogate the experimental NHP datasets without identifying any potential TB biomarker. In parallel, we used our extensive novel NHP datasets to build and calibrate a multi-organ computational model that combines what is occurring at the site of infection (e.g., lung) at a single granuloma scale with blood level readouts that can be tracked in monkeys and humans. We then generated a large in silico repository of in silico granulomas coupled to lymph node and blood dynamics and developed an in silico tool to scale granuloma level results to a full host scale to identify what best predicts Mycobacterium tuberculosis (Mtb) infection outcomes. The analysis of in silico blood measures identifies Mtb-specific frequencies of effector T cell phenotypes at various time points post infection as promising indicators of infection outcome. We emphasize that pairing wetlab and computational approaches holds great promise to accelerate TB biomarker discovery.     

Transgenic Mouse Bioassay: Evidence That Rabbits Are Susceptible to a Variety of Prion Isolates

Interspecies transmission of prions is a well-established phenomenon, both experimentally and under field conditions. Upon passage through new hosts, prion strains have proven their capacity to change their properties and this is a source of strain diversity which needs to be considered when assessing the potential risks associated with consumption of prion contaminated protein sources. Rabbits were considered for decades to be a prion resistant species until proven otherwise recently. To determine the extent of rabbit susceptibility to prions and to assess the effects of passage of different prion strains through this species a transgenic mouse model overexpressing rabbit PrP^C was developed (TgRab). Intracerebral challenges with prion strains originating from a variety of species including field isolates (ovine SSBP/1 scrapie, Nor98- scrapie; cattle BSE, BSE-L and cervid CWD), experimental murine strains (ME7 and RML) and experimentally obtained ruminant (sheepBSE) and rabbit (de novo NZW) strains were performed. On first passage TgRab were susceptible to the majority of prions (Cattle BSE, SheepBSE, BSE-L, de novo NZW, ME7 and RML) tested with the exception of SSBP/1 scrapie, CWD and Nor98 scrapie. Furthermore, TgRab were capable of propagating strain-specific features such as differences in incubation periods, histological brain lesions, abnormal prion (PrP^d) deposition profiles and proteinase-K (PK) resistant western blotting band patterns. Our results confirm previous studies proving that rabbits are not resistant to prion infection and show for the first time that rabbits are susceptible to PrP^d originating in a number of other species. This should be taken into account when choosing protein sources to feed rabbits.     

Characterization of the Cytolytic T-Lymphocyte Response to a Candidate Vaccine Strain of Equine Herpesvirus 1 in CBA Mice

The cytolytic T-lymphocyte (CTL) response to respiratory infection with equine herpesvirus 1 (EHV-1) in CBA (H-2^k) mice was investigated. Intranasal (i.n.) inoculation of mice with the attenuated EHV-1 strain KyA resulted in the generation of a primary virus-specific CTL response in the draining mediastinal lymph nodes 5 days following infection. EHV-1-specific CTL could be restimulated from the spleen up to 26 weeks after the resolution of infection, indicating that a long-lived memory CTL population was generated. Depletion of CD8⁺ T cells by treatment with antibody and complement prior to assay eliminated CTL activity from both primary and memory populations, indicating that cytolytic activity in this model was mediated by class I major histocompatibility complex-restricted, CD8⁺ T cells. A single i.n. inoculation with KyA induced protective immunity against infection with the pathogenic EHV-1 strain, RacL11. The adoptive transfer of splenocytes from KyA-immune donors into sublethally irradiated recipients resulted in a greater than 250-fold reduction in RacL11 in the lung. The elimination of both CD4⁺ and CD8⁺ T cells from the transferred cells abrogated clearance of RacL11, while the selective depletion of either subpopulation alone had little effect. These results suggested that both lymphocyte subpopulations contribute to viral clearance, with either subpopulation alone being sufficient.Equine herpesvirus 1 (EHV-1) is a prevalent respiratory pathogen of horses worldwide (8, 17, 38). Infection of horses with EHV-1 results in fever, respiratory distress, abortagenic disease, and severe neurological sequelae (3, 13, 27, 35, 36). The highly contagious respiratory transmission of EHV-1 has resulted in disastrous outbreaks of disease in domestic horse populations and has had a significant economic impact on the equine industry. EHV-1 infection of the horse results in the generation of a short-lived humoral response but does not confer long-term protection, as disease often occurs following natural infection (10, 22). Although both live and inactivated vaccines are currently available for EHV-1, only relatively short-lived protection has been observed (11, 12, 24). Furthermore, it is not clear which immune functions are responsible for conferring the short-lived protection following vaccination. In addition to specific antibody responses, peripheral blood leukocytes from vaccinated horses produce gamma interferon in culture (19). EHV-1-specific, CD8⁺ class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL) have been identified in peripheral blood mononuclear cells, reaching maximal levels 2 to 3 weeks postinfection (p.i.) (4, 20). However, the effectiveness of the current vaccines in stimulating EHV-1-specific CTL and their role in protective immunity in vivo are currently not known.Horses inoculated with the attenuated EHV-1 strain Kentucky A (KyA) exhibited a reduction in clinical signs following challenge with a pathogenic EHV-1 strain (33, 34). Although the attenuated strain induced a protective response, in terms of a reduced duration of viral shedding and viremia, the ability of this strain to induce an EHV-1-specific antibody response was weaker than that of the virulent strain. This finding suggested that immune functions other than the generation of specific antibodies might be critical in the resolution of infection. To generate a more effective EHV-1 vaccine, a better understanding of the precise immune functions associated with protection and resolution from EHV-1 infection is essential.A murine model of respiratory EHV-1 infection which closely mimicked EHV-1 infection in the natural host was established in various strains of mice (5). Common features included replication in the respiratory mucosae, the development of pneumonitis, cell-associated viremia, and abortion (5, 6). Specific immune responses are important for modulating infection. In the mouse, the passive transfer of hyperimmune polyclonal rabbit EHV-1-specific antibodies into infected mice significantly reduced the viremia following challenge with live EHV-1 (5). Subsequent studies demonstrated that various EHV-1 glycoproteins, including gB, gC, gD and gH, were capable of inducing the generation of neutralizing antibodies (9, 23, 40, 49, 52, 56). Furthermore, inoculation of mice with gB, gC, and/or gD elicited a protective response against subsequent challenge with pathogenic EHV-1 (39, 49, 50, 52, 56). However, each of these EHV-1 gene products is capable of eliciting both B- and T-cell responses; thus, the role of distinct immune functions conferring protection is not clearly defined.In the most extensively studied BALB/c mouse model of EHV-1 infection, adoptive transfer experiments demonstrated that immune spleen cells isolated from mice primed with live, but not heat-killed, EHV-1 reduced viral levels in both the lungs and nasal turbinates of infected recipient mice (5). Although the specific cell population responsible for protection was not determined, the data suggested that cell-mediated immune functions may be critical in the resolution of EHV-1 infection. The adoptive transfer of defined T-cell subpopulations demonstrated that both CD4⁺ and CD8⁺ T cells play a role in controlling EHV-1 respiratory infection. The CD8⁺ T-cell subpopulation appeared to play a more dominant role, although the functions by which protection was mediated were not defined (7). If the immune response was elicited by immunization with recombinant baculovirus-derived EHV-1 glycoproteins, the response was altered so that CD4⁺ T cells were predominantly associated with protection (52). Thus, either T-cell subpopulation is likely to play an important role in the optimal response to infection.While adoptive transfer studies have implicated an important role for CD8⁺ T cells in the control of EHV-1 infection in the lungs of BALB/c mice (7), there has been no direct assessment of CD8⁺ T-cell effector functions in this model. This is due predominantly to the lack of suitable class I MHC-compatible, H-2^d-expressing murine cells that are susceptible to infection with EHV-1. However, the attenuated KyA strain of EHV-1 has been propagated in our laboratory in suspension cultures of murine L-M fibroblasts that express the H-2^k haplotype. Therefore, an alternative mouse model, using mice expressing the H-2^k haplotype, was adopted principally to assess the activation of EHV-1-specific CTL responses. Initial studies by Awan et al. (5) demonstrated that CBA (H-2^k) mice were susceptible to infection with EHV-1 strain Ab4. In the present study, CBA mice were susceptible to respiratory tract infection by both the nonpathogenic KyA and the pathogenic RacL11 strains of EHV-1. Furthermore, infection of CBA mice with the attenuated KyA strain generated a vigorous CD8⁺, class I MHC-restricted, EHV-1-specific primary CTL response in the draining mediastinal lymph nodes (MLN) and a long-term memory CTL response in the spleen. These studies provide the basis for a model system to analyze the potential importance of class I MHC-restricted CTL activity in controlling EHV-1 infection in vivo.     

DBA/2J Mice Are Susceptible to Diabetic Nephropathy and Diabetic Exacerbation of IOP Elevation

Some pathological manifestations of diabetes in the eye include retinopathy, cataracts and elevated intraocular pressure (IOP). Loss of retinal ganglion cells (RGCs) in non-proliferative stages of diabetic retinopathy and small increases in IOP in diabetic patients has raised the possibility that diabetes affects the development and progression of ocular hypertension and glaucoma. The Ins2^Akita mutation is known to cause diabetes and retinopathy on a C57BL/6J (B6) background by as early as 3 months of age. Here, the impact of the Akita mutation on glaucoma was assessed using DBA/2J (D2) mice, a widely used mouse model of ocular hypertension induced glaucoma. In D2.Ins2^Akita/+ mice, the contribution of diabetes to vascular permeability, IOP elevation, RGC loss, and glaucoma development was assessed. D2.Ins2^Akita/+ mice developed a severe diabetic nephropathy and early mortality between 6–8 months of age. This agrees with previous reports showing that the D2 background is more susceptible to diabetes than the B6 background. In addition, D2.Ins2^Akita/+ mice had vascular leakage, astrocyte reactivity and a significant increase in IOP. However no RGC loss and no anterograde axonal transport dysfunction were found at 8.5 months of age. Therefore, our data show that despite severe diabetes and an increased IOP compared to controls, RGCs do not lose axon transport or degenerate. This may be due to a DBA/2J-specific genetic modifier(s) that could provide novel and important avenues for developing new therapies for diabetic retinopathy and possibly glaucoma.     

Pathogenicity of Helicobacter ganmani in Mice Susceptible and Resistant to Infection with H. hepaticus

Helicobacter spp. are some of the most prevalent bacterial contaminants of laboratory mice. Although abundant data regarding the diseases associated with H. hepaticus infection are available, little is known about the pathogenicity of H. ganmani, which was first isolated in 2001 from the intestines of laboratory mice. The objective of this study was to evaluate the host response to H. ganmani colonization in H. hepaticus disease-resistant C57BL/6 and disease-susceptible A/J and IL10-deficient mice. Mice were inoculated with H. ganmani, H. hepaticus, or Brucella broth. Cecal lesion scores, cecal gene expression, and Helicobacter load were measured at 4 and 90 d after inoculation. At both time points, mice inoculated with H. ganmani had similar or significantly more copies of cecum-associated Helicobacter DNA than did mice inoculated with H. hepaticus. When compared with those of sham-inoculated control mice, cecal lesion scores at 4 and 90 d after inoculation were not significantly greater in H. ganmani-inoculated A/J, C57BL/6, or IL10-deficient mice. Analysis of cecal gene expression demonstrated that H. ganmani infection failed to cause significant elevations of IFNγ in A/J, C57BL/6, or IL10-deficient mice. However, in IL10-deficient mice, H. ganmani infection was associated with a significant increase in the expression of the proinflammatory cytokine IL12/23p40. Although H. ganmani infection in this study failed to induce the typhlitis that is the hallmark of H. hepaticus infection, infection with H. ganmani was associated with alterations in inflammatory cytokines in IL10-deficient mice.Abbreviations: B6, C57BL/6NCr; HPRT, hypoxanthine guanine phosphoribosyl transferase; IL10 KO, B6129P2-IL10^tm1Cgn/JSince the discovery of the link between Helicobacter pylori and chronic gastritis in 1982,¹⁷ Helicobacter spp. in humans and animals have become a field of extensive study. Due to improved detection methods, there has been a rapid expansion in our understanding and ability to detect native Helicobacter spp. in mouse models. Several reports investigating their prevalence in mice housed in research institutions have found Helicobacter spp. to be some of the most common bacterial contaminants of laboratory rodents.^2,3,12,16,23 Helicobacter hepaticus is perhaps the most notorious of the murine helicobacters, by virtue of the early realization of its pathogenicity in adult mice.^8,24 The hallmarks of infection by H. hepaticus are typhlitis, colitis, and hepatitis.¹⁰ In addition, H. hepaticus is commonly used as a microbial trigger in susceptible mouse strains used as models of inflammatory bowel disease.^5,9,19,21,28 In 2001, less than 10 y after H. hepaticus was discovered, H. ganmani was isolated from the intestines of laboratory mice.²⁶ During its initial characterization, 16S rDNA sequence analysis placed H. ganmani phylogenetically closest to H. rodentium, a urease-negative helicobacter that had been previously isolated from mouse intestines.²⁶Despite the reported endemic presence of H. ganmani in many research colonies,^2,3,12 only a few reports to date have attempted to address H. ganmani’s potential pathogenicity.^22,30 One report describes an outbreak of inflammatory bowel-like disease associated with H. ganmani infection in an otherwise Helicobacter-free conventional colony of IL10-deficient mice.²² The findings from another report describe the effect of natural colonization of IL10-deficient mice with H. ganmani, H. hepaticus, or both.³⁰ In that study, 8- to 20-wk-old mice monoinfected with H. ganmani had significantly lower lesion scores than did mice monoinfected with H. hepaticus, suggesting that infection with H. ganmani alone was not sufficient to cause severe typhlocolitis.³⁰ However, by 34 wk of age, clinical typhlocolitis (diarrhea) and grossly enlarged ceca were observed at necropsy in 2 of the 6 mice monoinfected with H. ganmani.³⁰Although these reports of naturally occurring infections have provided a glimpse into H. ganmani’s potential to produce intestinal disease in immunodeficient mice, a controlled study in immunocompetent and immunodeficient mice had not been conducted previously. The objectives of the current study were to evaluate the effect of H. ganmani infection on intestinal disease and to characterize alterations of inflammatory gene expression associated with infection. To this end, we selected A/J and IL10-deficient mice for this study because of their known susceptibility to H. hepaticus-induced typhlocolitis.^{9,13,14,19,21,28} In contrast, although C57BL/6 mice show an initial spike in inflammatory cytokines after H. hepaticus infection, they do not typically develop chronic disease.¹⁹ We did not expect C57BL/6 mice to develop H. hepaticus-induced disease, but we deemed it prudent to characterize the possible effects—through unknown mechanisms—of H. ganmani on this common strain.Previous studies characterizing cecal gene expression during H. hepaticus induced typhlocolitis demonstrated that IFNγ and IL12/23p40 (IL12/23) are key proinflammatory cytokines that drive typhlitis.¹⁹ Expression of these cytokines was increased in H. hepaticus-inoculated A/J mice but not in H. hepaticus-inoculated C57BL/6 mice.¹⁹ In addition, treatment with neutralizing monoclonal antibodies against these cytokines significantly decreased cecal lesion severity, implicating the roles of IFNγ and IL12/23 in modulating the pathogenesis of typhlitis.¹⁹ We hypothesized that characterizing the effect of H. ganmani infection on expression of IFNγ and IL12/23 would uncover aspects of the host response that are not readily apparent by histologic evaluation of cecal tissue alone.To date, our understanding of the potential for H. ganmani to cause intestinal disease has been limited to reports that focused on the evaluation of histologic disease in naturally infected IL10-deficient mice. Despite the reported endemic presence of H. ganmani in many research colonies,^2,3,12 there are no published reports of disease associated with H. ganmani infection in immunocompetent mice. In addition, H. ganmani shares close sequence homology with H. rodentium, which has been found to be nonpathogenic in monoinfected immunodeficient and immunocompetent mice.²⁰ Therefore, we hypothesized that experimental infection with H. ganmani would not produce disease in H. hepaticus-susceptible or -resistant mice.     

重组人apoE基因表达活性和转基因小鼠研究

人apoE基因组DNA,去除其自身启动子,代之以小鼠金属硫蛋白启动子,重组质粒经脂质体介导转入小鼠NIH／3T3细胞后,以人apoE基因组DNA／EcoRⅠ片段为探针检测mRNA表达,可见apoEmRNA杂交信号很强,经重金属诱导后杂交信号更强,表明MT启动子功能良好,pME表达正常．将人apoE基因组DNA用显微注射法导入小鼠受精卵雄性原核,再将胚胎移植入假孕母鼠输卵管内,仔鼠分娩四周后,自鼠尾提取DNA,鉴定人apoEDNA在小鼠染色体上的整合,最终得到有人apoE基因整合的转基因首建鼠．     

Relation of Infection to Tissue Temperature in Mice Infected with Mycobacterium marinum and Mycobacterium leprae

Intravenous and footpad infections with Mycobacterium marinum and footpad infections with M. leprae were compared in the following mouse strains: A/He, BALB/C, CBA, C3H, C57BL, C57L, DBA, 101, and CFW. The results varied a great deal according to mouse strain used. Intravenous injection of high doses of M. marinum resulted in deaths after 28 days of 100% of strain A/He, and none of strain 101; 27 days after injection, the feet and noses of all strain CBA mice, but few of the C57BL, 101, or CFW mice, were involved. Injection of a small dose of M. marinum into the footpad produced visible disease in 5 days in all of the C57BL and 101 mice, but in not more than 60% of the A/He, DBA, and CFW mice; the average amount of swelling at 17 days varied from 4.40 mm in strain C57L to 0.92 in strain 101. After footpad injection of M. leprae, the average plateau harvests varied from 1.3 x 10(7) acid-fast bacteria in strain CBA to 6.5 x 10(5) in strain C57L. The infections in CBA mice extended from the site of inoculation throughout the foot. The temperature was measured rectally, in the footpad, and in the tail. Analysis of all the results revealed little correlation among the three types of infection. There was a strong negative correlation between the tail temperature and the death rate after intravenous injection of M. marinum, and a strong positive correlation between footpad temperature and plateau harvest of M. leprae.     

In Situ Characterization of Splenic Brucella melitensis Reservoir Cells during the Chronic Phase of Infection in Susceptible Mice

Brucella are facultative intracellular Gram-negative coccobacilli that chronically infect humans as well as domestic and wild-type mammals, and cause brucellosis. Alternatively activated macrophages (M2a) induced by IL-4/IL-13 via STAT6 signaling pathways have been frequently described as a favorable niche for long-term persistence of intracellular pathogens. Based on the observation that M2a-like macrophages are induced in the spleen during the chronic phase of B. abortus infection in mice and are strongly infected in vitro, it has been suggested that M2a macrophages could be a potential in vivo niche for Brucella. In order to test this hypothesis, we used a model in which infected cells can be observed directly in situ and where the differentiation of M2a macrophages is favored by the absence of an IL-12-dependent Th1 response. We performed an in situ analysis by fluorescent microscopy of the phenotype of B. melitensis infected spleen cells from intranasally infected IL-12p40^-/- BALB/c mice and the impact of STAT6 deficiency on this phenotype. Most of the infected spleen cells contained high levels of lipids and expressed CD11c and CD205 dendritic cell markers and Arginase1, but were negative for the M2a markers Fizz1 or CD301. Furthermore, STAT6 deficiency had no effect on bacterial growth or the reservoir cell phenotype in vivo, leading us to conclude that, in our model, the infected cells were not Th2-induced M2a macrophages. This characterization of B. melitensis reservoir cells could provide a better understanding of Brucella persistence in the host and lead to the design of more efficient therapeutic strategies.     

Intestinal Resistance in the Experimental Enteric Infection of Mice with a Mouse Adenovirus

Investigation was made on the process of enteric infection with mouse adenovirus strain K87 in inbred DK1 mice and the intestinal resistance acquired through infection. The cells containing viral antigens were enumerated in most parts of the infected intestinal tract by a fluorescent antibody technique, and the infectivity titer of the virus in each part was examined in mouse kidney tissue culture. The virus was observed to grow in 3~14 days (sometimos 3~21 days) after oral challenge, and infectivity titers reached their peak after 7~14 days, when a number of viral antigen-containing cells and cells with nuclear inclusions were detected. In the mice rechallenged 28 days after the initial challenge, the virus did not grow, and no viral antigen-containing cells were found. From these results it was concluded that the main sites where the virus grows in mice are the cells which are scattered in the epithelial layer of the mucous membrane of the small intestine, and which seem to be the usual epithelial cells and not Paneth's or goblet cells. As for intestinal resistance, experiments with inactivated vaccine and with passive transfer of serum-antibodies were performed in order to find out whether neutralizing antibodies in the serum had any influence on the growth of virus in the intestinal wall, and no influences were indicated. Eighteen days or more after challenge, K87 virus-neutralizing substances were detected in the intestinal wall and in the intestinal contents of the infected mice, but not in the serum-transferred mice, though both groups of mice had equal levels of serum antibodies. The substance continued to be found until 15 weeks after challenge in the intestinal contents, and until later than 34 weeks in the intestinal walls. The nature and the possible role of the substance is discussed, but actual data will be reported in subsequent papers.     

Intestinal Resistance in the Experimental Enteric Infection of Mice with a Mouse Adenovirus

In order to find out whether the mouse adenovirus-neutralizing substance, which appeared in the intestinal tract of mice orally infected with mouse adenovirus, was an immunoglobulin, examinations were carried out for the status of 3 classes of immunoglobulin, IgA, IgG, and IgM, in the intestinal tract as well as in the serum of the mouse. In infected mice, as in uninfected mice, the serum contained much IgG, a moderate amount of IgA, and a small amount of IgM, whereas the intestinal wall showed a moderate amount of IgA, a small amount of IgG and no IgM, and the intestinal contents contained a moderate amount of IgA. Secondly, DEAE-cellulose chromatography or Sephadex G-200 gel filtration was done in order to know whether the virus-neutralizing activity was recoverable in the fractions containing some class of immunoglobulin. The result indicated that a large part of the activity in the serum was recovered in the fractions of IgG and a small part in those of IgA. In the case of the intestinal wall, a large part of the activity was found in the fractions of IgA, and only a small part in the fractions containing both IgG and IgA. In the intestinal contents, the activity was detected solely in the fractions containing IgA. Finally, when the substance from the intestinal wall was purified by DEAE and Sephadex, a parallel increase of both IgA and the virus-neutralizing activity per protein content was observed. Thus, it became clear that the mouse adenovirus-neutralizing substance in the intestinal tract was an antibody against the virus, and that it mostly belongs to IgA.