Pattern of polyomavirus replication from infection until tumor formation in the organs of athymic nu/nu mice.

Inbred athymic nu/nu BALB/c mice were injected subcutaneously with the highly oncogenic polyomavirus A2 strain, and the sites of viral DNA replication were determined by whole mouse section hybridization (T. W. Dubensky, E. A. Murphy, and L. P. Villareal, J. Virol. 50:779-783, 1984) and Southern blot analysis. We show that infection is persistent in some epithelial tissues (skin, mammary, and salivary glands), in lymphoid organs (spleen and nodes), and in mesenchymal bone tissue. Only mammary glands and bones were targets for tumor formation. Although the same pattern of infection was observed in males and females, mammary adenocarcinomas were induced exclusively in females, while the frequency of osteosarcomas was similar in both sexes. No viral DNA or lytic lesion was detected in kidney, liver, or lung tissue. The restricted targeting of polyomavirus oncogenicity in nude mice, compared with newborn immunocompetent animals, inoculated via the same route with the same virus strain, therefore does not reflect selective tissue targeting of virus replication. These results further document the influence of the age, immunological status, and genetic background of the host on the pattern of viral infection and tumor formation.     

Studies on defense mechanisms against Candida albicans infection in congenitally athymicnude (nu/nu) mice

The defense mechanisms against Candida albicans infection were studied by using a mouse thigh lesion model in congenitally athymic nude (nu/nu) mice and their normal littermates (nu/+). Nu/nu mice were more resistant to C. albicans infection than nu/+ mice judging from the course of the thigh lesion, the results of CFUs (colony-forming units) of C. albicans in the lesion, and histopathological observations. Histopathological and serological studies revealed that granulocytic cellular infiltration was predominant, and there were few indications of development of cell-mediated immunity to protect Candida infection in Candida-infected nu/nu and nu/+ mice. These results confirmed that lower susceptibility of nu/nu mice to C. albicans infection as compared with nu/ + mice was due to accelerated non-specific defense mechanisms in nu/nu mice, and that cell-mediated or humoral immunity played a minor role in the defense against Candida infection in this experimental model.Furthermore, treatment with high titer of rabbit anti-C. albicans serum was effective to control the number of Candida cells in thigh lesions of BALB/c mice.Above experimental results seem to clearly indicate the great variability of defense manifestation according to the experimental model exployed.     

L-arginine-dependent macrophage effector functions inhibit metabolic activity of Mycobacterium leprae

Recently, L-arginine has been shown to be a necessary substrate for murine-activated macrophage-mediated tumor cytostasis and microbiostasis of certain fungi, bacteria, and intracellular protozoa. We report here the effects of the L-arginine-dependent pathway of activated mouse macrophages (MO) on the obligate intracellular prokaryote, Mycobacterium leprae. Due to the inability to culture M. leprae in vitro, a simple, quantitative assay was employed to measure the metabolism/viability of M. leprae released from MO: the metabolic capacity of M. leprae to oxidize 14C-palmitic acid to 14CO2. Murine normal MO or MO activated in vitro with IFN-gamma or in vivo by injection with Corynebacterium parvum were infected with viable M. leprae freshly harvested from the footpads of nu/nu mice. Activated MO strikingly inhibited the metabolism of M. leprae; however, in L-arginine-free medium or in medium containing L-arginase, the inhibitory effects of activated MO on M. leprae metabolism were abolished. The competitive inhibitor of L-arginine, NG-monomethyl-L-arginine, also blocked the inhibitory effects of activated MO for M. leprae, but the addition of supplemental L-arginine overcame the NG-monomethyl-L-arginine-induced block. Furthermore, in the culture supernatants, the levels of NO2-, an end product of L-arginine degradation, were directly proportional to the ability of the activated MO to inhibit M. leprae metabolism. These data present five lines of evidence that suggest that activated MO utilize the L-arginine-dependent pathway to cope with M. leprae.     

Thymus differentiation and T-cell specificity in nu/nu +/+ mouse aggregation chimaeras

Thymus development and T cell differentiation were studied in mouse chimaeras produced by aggregating pre-implantation embryos of thymus-deficient nude BALB/c (nu/nu) and wild-type C57BL/6 (+/+) mice and vice versa. Chimaeras showed mosaic distribution of skin and coat pigmentation, of hair follicles, of glucosephosphate isomerase within all tested organs and of lymphocytes expressing the different major transplantation antigens (H-2). When tested for their capacity to generate vaccinia virus-specific and self-H-2 specific cytotoxic T cells, all chimaeras of BALB/c (nu/nu) H-2d in equilibrium C57BL/6 (+/+) H-2b type generated T cells of one or both parental origins that were specific for virus and for self-H-2 of the +/+ (H-2b) type only. In contrast, some BALB/c (+/+) H-2d in equilibrium C57BL/6 (nu/nu) H-2b chimaeras generated vaccinia virus-specific cytotoxic T cells specific for either H-2d (+/+) type or for H-2b (nu/nu) type. These asymmetrical results can be interpreted to indicate the following: (i) The +/+ thymus part alone is functional, but because of asymmetrical cross-reactivities of anti-self-H-2 specificities, the observed T cell restriction phenotypes differ. (ii) Both nu/nu and +/+ thymus parts are functional but immune response defects may be exaggerated in such chimaeras producing unexpected non-responsiveness to vaccinia virus linked to H-2d in H-2b (+/+) in equilibrium H-2d (nu/nu).     

A role for IL-12 receptor expression and signal transduction in host defense in leprosy.

The generation of cell-mediated immunity against intracellular infection involves the production of IL-12, a critical cytokine required for the development of Th1 responses. The biologic activities of IL-12 are mediated through a specific, high affinity IL-12R composed of an IL-12Rbeta1/IL-12Rbeta2 heterodimer, with the IL-12Rbeta2 chain involved in signaling via Stat4. We investigated IL-12R expression and function in human infectious disease, using the clinical/immunologic spectrum of leprosy as a model. T cells from tuberculoid patients, the resistant form of leprosy, are responsive to IL-12; however, T cells from lepromatous patients, the susceptible form of leprosy, do not respond to IL-12. We found that the IL-12Rbeta2 was more highly expressed in tuberculoid lesions compared with lepromatous lesions. In contrast, IL-12Rbeta1 expression was similar in both tuberculoid and lepromatous lesions. The expression of IL-12Rbeta2 on T cells was up-regulated by Mycobacterium leprae in tuberculoid but not in lepromatous patients. Furthermore, IL-12 induced Stat4 phosphorylation and DNA binding in M. leprae-activated T cells from tuberculoid but not from lepromatous patients. Interestingly, IL-12Rbeta2 in lepromatous patients could be up-regulated by stimulation with M. tuberculosis. These data suggest that Th response to M. leprae determines IL-12Rbeta2 expression and function in host defense in leprosy.     

Development of T-cell function in relation to T-cell set diversification in nu/nu mice

The differentiation pattern of splenic T-cell populations in germ- and pathogen-free nu/nu mice, as compared to nu/+ littermates, is characterized by two abnormal features: the expression of TL determinants on peripheral T cells and the delayed onset of their differentiation from the predominant Lyt-123:TL+ set into TL- cells of Lyt-1+ and Lyt-123+ phenotype, which, in these mice, does not occur until 10 weeks of age. We report here that the delayed onset of mitogen- or alloantigen-induced interleukin-2 synthesis and T-cell proliferation as well as the development of cytotoxic T-lymphocyte activity of enriched T-cell populations is strictly correlated with the time point of T-cell subset diversification in nu/nu mice and depends in particular on the presence of the Lyt-1 (TL-:Lyt-2-) T-cell set which is lacking in splenic T-cell populations of germ-free young nu/nu mice.     

Regional variations in the distributions of small intestinal intraepithelial lymphocytes (IELs) in BALB/c +/+, nu/+, and nu/nu mice

Regional variations in intraepithelial lymphocytes (IELs) in the small intestine were examined in BALB/c +/+, nu/+, and nu/nu mice. The small intestine was obtained from 11- to 12-week-old mice and divided equally into three (proximal, middle, and distal) parts. The IELs were isolated from each part of the intestine, and the total numbers of IELs in nu/+ and nu/nu mice were about a fifth of those in +/+ mice. Regional variations in the distribution of the IEL alphabeta, but not the gammadelta T-cell subset were found by use of flow cytometry in +/+ and nu/+ mice. On the other hand, such differences were not found in nu/nu mice, suggesting that thymus-independent development of T cells is not different among regions. Different local expansion of thymus-dependent alphabeta T cells may cause the regional variations seen in the distribution of alphabeta T cell IELs in +/+ and nu/+ mice.     

Electroacupuncture alters the expression of genes associated with lipid metabolism and immune reaction in liver of hypercholesterolemia mice

Acupuncture or electroacupuncture (EA) is effective in treating various metabolism disorders. Previously we found that EA at the acupoint, Fenglong (ST40), had the cholesterol-lowering effect and regulated genes expression in liver of hypercholesterolemia mice (M Li and YZ Zhang, Int J Mol Med 2007, 19: 617–629). To explain gene expression associated with EA, suppression subtractive hybridization (SSH), combined with targeted display (TD), was used and 26 up-regulated and 24 down-regulated genes with known functions were identified in hypercholesterolemia mice liver, some of which are involved in key reactions of lipid metabolism and immune reaction. Promoting lipid metabolism and suppressing inflammation via modulating mRNA expression may be the mechanism of EA inducing modulation of cholesterol concentrations.     

Demyelination and wasting associated with polyomavirus infection in nude (nu/nu) mice

Nude (nu/nu) mice bearing human tumour heterografts were affected with posterior paralysis and wasting. There was demyelination and infection of the oligodendrocytes of the spinal cord with a papovavirus. Similar virus particles and inclusion bodies were found in the bronchial epithelium, which showed histopathological changes. Similar changes were shown by the epithelia of the renal pelvis, ureter and choroid plexus. The virus was found in a transplantable human tumour, and evidence of spread by contact was also obtained. Intracerebral injection of spinal cord suspension from infected mice resulted in virus infected cutaneous carcinomata, demyelination with virus particles in the oligodendrocytes and posterior paralysis with wasting in adult nude mice. The suspension injected intraperitoneally into newborn Syrian hamsters produced tumours similar to those produced by murine polyoma. No evidence of infection was found in mice from the colony of origin. The virus was identified as murine polyoma Wild Type A2.     

Signaling lymphocytic activation molecule expression and regulation in human intracellular infection correlate with Th1 cytokine patterns.

Induction of Th1 cytokines, those associated with cell-mediated immunity, is critical for host defense against infection by intracellular pathogens, including mycobacteria. Signaling lymphocytic activation molecule (SLAM, CD150) is a transmembrane protein expressed on lymphocytes that promotes T cell proliferation and IFN-gamma production. The expression and role of SLAM in human infectious disease were investigated using leprosy as a model. We found that SLAM mRNA and protein were more strongly expressed in skin lesions of tuberculoid patients, those with measurable CMI to the pathogen, Mycobacterium leprae, compared with lepromatous patients, who have weak CMI against M. leprae. Peripheral blood T cells from tuberculoid patients showed a striking increase in the level of SLAM expression after stimulation with M. leprae, whereas the expression of SLAM on T cells from lepromatous patients show little change by M. leprae stimulation. Engagement of SLAM by an agonistic mAb up-regulated IFN-gamma production from tuberculoid patients and slightly increased the levels of IFN-gamma in lepromatous patients. In addition, IFN-gamma augmented SLAM expression on M. leprae-stimulated peripheral blood T cells from leprosy patients. Signaling through SLAM after IFN-gamma treatment of Ag-stimulated cells enhanced IFN-gamma production in lepromatous patients to the levels of tuberculoid patients. Our data suggest that the local release of IFN-gamma by M. leprae-activated T cells in tuberculoid leprosy lesions leads to up-regulation of SLAM expression. Ligation of SLAM augments IFN-gamma production in the local microenvironment, creating a positive feedback loop. Failure of T cells from lepromatous leprosy patients to produce IFN-gamma in response to M. leprae contributes to reduced expression of SLAM. Therefore, the activation of SLAM may promote the cell-mediated immune response to intracellular bacterial pathogens.     

Susceptibility of congenitally immunodeficient mice to a nonencapsulated strain of Cryptococcus neoformans.

The susceptibility of congenitally immunodeficient mice to a nonencapsulated strain of Cryptococcus neoformans (strain M7) was evaluated. Gnotobiotic mice with defined congenital defects in innate immunity (beige) or cell-mediated immunity (athymic) or with combined defects in innate and cellular immunity (beige athymic) were i.v. challenged with C. neoformans M7. The nonencapsulated strain of C. neoformans produced a persistent low-grade infection in the brains of all immunodeficient and immunocompetent mice used in this study. Immunocompetent mice (nu/+;bg/+) and immunodeficient bg/bg mice readily cleared nonencapsulated cryptococci from their kidneys, liver, lungs, and spleen. In contrast to nu/+ mice, nu/nu mice had a reduced capacity to clear nonencapsulated cryptococci from their kidneys and liver after i.v. challenge. Both bg/bg-nu/nu and bg/bg-nu/+ mice developed a low-grade infection in their kidneys, liver, lungs, and spleen, which was maintained throughout the 21-day study. Persistent infections were not due to reversion to an encapsulated state. These data indicate that a capsule may not always be necessary for C. neoformans to survive, in vivo, in tissues of immunodeficient and immunocompetent mice.     

镉诱导小鼠睾丸细胞凋亡与Caspase-3、Bcl-2和Bax mRNA的表达

一个月大雄性小鼠24只,随机分为6组,用30 μmol/kg CdCl2作用小鼠睾丸不同的时间(3 h、6 h、12 h、18 h、24 h)后,利用DNA电泳、免疫组化和半定量RT-PCR技术,分析生殖细胞凋亡过程中三种关键物质Caspase-3、Bcl-2、Bax蛋白和mRNA的表达量变化.结果显示:1) DNA各组 (除对照组外)均出现不同程度断裂.2)Caspase-3蛋白表达量一直上升,与对照组相比差异极显著;Bax蛋白在12 h前一直上升,与对照组相比差异显著,12 h后又开始下降,且与对照组相比无显著差异;Bcl-2蛋白在下降,与对照组相比差异显著.3)RT-PCR结果显示Caspase-3基因表达量减少;Bax基因表达量逐渐上升;Bcl-2基因表达量波动很大.综上所述,Caspase-3、Bcl-2和Bax三个基因可能参与了镉应激状态下小鼠睾丸组织细胞的凋亡过程.     

Interleukin 2 production by lymphoid cells from congenitally athymic (nu/nu) mice

The ability of lymphoid cells from congenitally athymic (nu/nu) mice to produce interleukin 2 (IL 2) was investigated. Spleen or lymph node cells (superficial or mesenteric) from nude mice on an N:NIH(S)II or BALB/c genetic background were stimulated with concanavalin A (Con A) or with irradiated allogeneic (DBA/2) spleen cells that had been depleted of T cells by treatment with monoclonal anti-Thy-1.2 antibody plus complement. After 24 hr, supernatants were harvested and assayed for their ability to support the proliferation of a cloned IL 2-dependent cytolytic T cell line. With this quantitative microassay, IL 2 production was not detectable in spleen and lymph nodes of 6-wk-old N:NIH(S)II nude mice; however, by 12 mo of age, IL 2 production increased more than 100-fold to reach levels comparable to control (nu/+) animals. Con A was more potent than alloantigen in the induction of IL 2 in either nude or control (nu/+) animals. Furthermore, differences in the genetic background of nude mice resulted in corresponding differences in both numbers of T cells (defined by monoclonal anti-Thy-1 antibody) and IL 2 production. By using negative selection with monoclonal antibodies plus complement, IL 2 production in aged nude mice was shown to depend upon a subpopulation of cells that expressed Thy-1 but not Lyt-2. These data thus demonstrate that a subpopulation of IL 2-producing cells with a Thy-1+ Lyt-2- surface phenotype can develop in the apparent absence of thymic influence.     

An in vitro model for the lepromatous leprosy granuloma: fate of Mycobacterium leprae from target macrophages after interaction with normal and activated effector macrophages

The lepromatous leprosy granuloma is a dynamic entity requiring a steady influx of macrophages (Mphi) for its maintenance. We have developed an in vitro model to study the fate of Mycobacterium leprae in a LL lesion, with and without immunotherapeutic intervention. Target cells, consisting of granuloma Mphi harvested from the footpads of M. leprae-infected athymic nu/nu mice, were cocultured with normal or IFN-gamma-activated (ACT) effector Mphi. The bacilli were recovered and assessed for viability by radiorespirometry. M. leprae recovered from target Mphi possessed high metabolic activity, indicating a viable state in this uncultivable organism. M. leprae recovered from target Mphi incubated with normal effector Mphi exhibited significantly higher metabolism. In contrast, bacilli recovered from target Mphi cocultured with ACT effector Mphi displayed a markedly decreased metabolic activity. Inhibition by ACT Mphi required an E:T ratio of at least 5:1, a coculture incubation period of 3-5 days, and the production of reactive nitrogen intermediates, but not reactive oxygen intermediates. Neither IFN-gamma nor TNF-alpha were required during the cocultivation period. However, cell-to-cell contact between the target and effector Mphi was necessary for augmentation of M. leprae metabolism by normal effector Mphi as well as for inhibition of M. leprae by ACT effector Mphi. Conventional fluorescence microscopy and confocal fluorescence microscopy revealed that the bacilli from the target Mphi were acquired by the effector Mphi. Thus, the state of Mphi infiltrating the granuloma may markedly affect the viability of M. leprae residing in Mphi in the lepromatous lesion.     

Apoptosis governs the elimination of Schistosoma japonicum from the non-permissive host Microtus fortis

The reed vole, Microtus fortis, is the only known mammalian host in which schistosomes of Schistosoma japonicum are unable to mature and cause significant pathogenesis. However, little is known about how Schistosoma japonicum maturation (and, therefore, the development of schistosomiasis) is prevented in M. fortis. In the present study, the ultrastructure of 10 days post infection schistosomula from BALB/c mice and M. fortis were first compared using scanning electron microscopy and transmission electron microscopy. Electron microscopic investigations showed growth retardation and ultrastructural differences in the tegument and sub-tegumental tissues as well as in the parenchymal cells of schistosomula from M. fortis compared with those in BALB/c mice. Then, microarray analysis revealed significant differential expression between the schistosomula from the two rodents, with 3,293 down-regulated (by ≥ 2-fold) and 71 up-regulated (≥ 2 fold) genes in schistosomula from the former. The up-regulated genes included a proliferation-related gene encoding granulin (Grn) and tropomyosin. Genes that were down-regulated in schistosomula from M. fortis included apoptosis-inhibited genes encoding a baculoviral IAP repeat-containing protein (SjIAP) and cytokine-induced apoptosis inhibitor (SjCIAP), genes encoding molecules involved in insulin metabolism, long-chain fatty acid metabolism, signal transduction, the transforming growth factor (TGF) pathway, the Wnt pathway and in development. TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) and PI/Annexin V-FITC assays, caspase 3/7 activity analysis, and flow cytometry revealed that the percentages of early apoptotic and late apoptotic and/or necrotic cells, as well as the level of caspase activity, in schistosomula from M. fortis were all significantly higher than in those from BALB/c mice.     

An alternative route for infecting armadillos with Mycobacterium leprae

A nine-banded armadillo was inoculated with Mycobacterium leprae in both hind footpads. The animals were usually inoculated intravenously, or intradermally in the abdominal skin. Profuse multiplication of the bacilli occurred at the injection sites after more than two years. Eventually bacteraemia developed, and large numbers of the organisms were found in skin biopsies and in lymph nodes. There was limited dissemination of the bacteria into the spleen and the liver, and peripheral nerve invasion by the bacilli was also detected. M. leprae remained viable in the liver tissue, kept frozen at -80 degrees C for three years. This experimental system would be useful in testing the effects of certain immunological and chemotherapeutic agents against M. leprae by injecting them directly at the infection site.     

Time course of gene expression profiling in the liver of experimental mice infected with Echinococcus multilocularis

Background

Alveolar echinococcosis (AE) is a severe chronic parasitic disease which behaves like a slow-growing liver cancer. Clinical observations suggest that the parasite, Echinococcus multilocularis (E. multilocularis) influences liver homeostasis and hepatic cell metabolism. However, this has never been analyzed during the time course of infection in the common model of secondary echinococcosis in experimental mice.

Methodology/Principal Findings

Gene expression profiles were assessed using DNA microarray analysis, 1, 2, 3 and 6 months after injection of E. multilocularis metacestode in the liver of susceptible mice. Data were collected at different time points to monitor the dynamic behavior of gene expression. 557 differentially expressed genes were identified at one or more time points, including 351 up-regulated and 228 down-regulated genes. Time-course analysis indicated, at the initial stage of E. multilocularis infection (month 1–2), that most of up-regulated pathways were related to immune processes and cell trafficking such as chemokine-, mitogen-activated protein kinase (MAPK) signaling, and down-regulated pathways were related to xenobiotic metabolism; at the middle stage (month 3), MAPK signaling pathway was maintained and peroxisome proliferator-activated receptor (PPAR) signaling pathway emerged; at the late stage (month 6), most of up-regulated pathways were related to PPAR signaling pathway, complement and coagulation cascades, while down-regulated pathways were related to metabolism of xenobiotics by cytochrome P450. Quantitative RT-PCR analysis of a random selection of 19 genes confirmed the reliability of the microarray data. Immunohistochemistry analysis showed that proliferating cell nuclear antigen (PCNA) was increased in the liver of E. multilocularis infected mice from 2 months to 6 months.

Conclusions

E. multilocularis metacestode definitely exerts a deep influence on liver homeostasis, by modifying a number of gene expression and metabolic pathways. It especially promotes hepatic cell proliferation, as evidenced by the increased PCNA constantly found in all the experimental time-points we studied and by an increased gene expression of key metabolic pathways.     

Infection and elimination of Mycobacterium leprae in SCID C.B.-17 mice (severe combined immunodeficiency)]

Previous studies documented that T-cell deficient nude mice failed to control M. leprae infection. In the present investigation we monitored the growth of M. leprae for up to 15 months in the SCID C.B.-17 mouse, a host deficient in both T and B lymphocytes. At 8 months post-infection 10(8) organisms/foot-pad were recovered from SCID mice vs 5 x 10(6) in normal BALB/c mice. Thereafter the number of bacilli decreased rapidly in mice infected with high-dose inoculum (10(7)); however, at all doses SCID mice eventually cleared M. leprae. During infection both T and B cells as well as serum Ig remained as low as in uninfected mice; however, in the spleen MAC-1+ cells which include macrophages and NK cells were substantially increased. These results suggest that MAC-1+ cells are involved in the anti-mycobacteria-1 defence mechanisms adopted by SCID mice to compensate their deficiency in T and B cells.