Vaccination against murine gamma-herpesvirus infection

The gamma-herpesviruses establish life-long latency in the host and are important human pathogens. T cells play a major role in controlling the initial acute infection and subsequently maintaining the virus in a quiescent state. However, the nature of the T-cell response to gamma-herpesvirus infection and the requirements for effective vaccination are poorly understood. The recent development of a murine gamma-herpesvirus (murine herpesvirus-68 [MHV-68]) has made it possible to analyze T-cell responses and test vaccination strategies in a small animal model. Intranasal infection with MHV-68 induces an acute infection in the lung and the subsequent establishment of long-term latency, which is associated with splenomegaly and an infectious mononucleosis-like syndrome. Here we review the T-cell response to different phases of the infection and the impact of vaccination against either lytic-cycle, or latency-associated T-cell epitopes.     

Changing patterns of dominance in the CD8+ T cell response during acute and persistent murine gamma-herpesvirus infection

The murine gamma-herpesvirus MHV-68 causes an acute, transient pneumonitis, followed by an infectious mononucleosis (IM)-like illness with splenomegaly, widespread latent infection of B lymphocytes and an expansion of Vbeta4+ CD8+ T cells. CD8+ T cells specific for an H-2Db-restricted epitope were prominent during the acute respiratory infection, but their prevalence declined rapidly during the mononucleosis. In contrast, CD8+ T cells specific for an H-2Kb-restricted epitope, apparently expressed by virus-infected B lymphocytes, were most numerous during the mononucleosis illness and were maintained at relatively high frequencies thereafter. The prevalence of all peptide-specific CD8+ T cells decreased during the expansion of the Vbeta4+ CD8+ population, which did not recognize any peptide epitopes identified and was apparent also in an MHC class I-deficient environment. The CD8+ T cell population recognizing productively infected epithelial cells thus differed substantially from that responding during the IM illness.     

Factors controlling levels of CD8+ T-cell lymphocytosis associated with murine gamma-herpesvirus infection.

Intranasal infection of mice with murine gamma-herpesvirus 68 (MHV-68) elicits a striking CD8+ T-cell lymphocytosis following the establishment of latency, which includes a marked increased frequency of Vbeta4+ CD8+ T cells. The Vbeta4+ CD8+ T cells do not recognize a conventional viral peptide, but are stimulated by an uncharacterized ligand expressed on latently infected, activated B cells. The selective expansion of Vbeta4+ CD8+ T cells after MHV-68 infection is observed in all mouse strains examined, although the fold-increase varies widely, ranging from less than twofold to greater than 10-fold. The factors controlling the variation are currently undefined. In the current study, CD8+ T cell activation and Vbeta4+ CD8+ T-cell frequencies were analyzed in 18 inbred strains of mice. The data show that the magnitude of the Vbeta4+ CD8+ T-cell response correlates with the degree of CD8+ T cell-activation, and that both major histocompatibility complex (MHC) and non-MHC genes contribute to the magnitude of the activation. Furthermore, the magnitude of the response does not reflect major differences in susceptibility to viral infection and/or corresponding differences in the acute response. Rather the degree of Vbeta4+ CD8+ T cell activation may be determined by differences in levels of expression of the stimulatory ligand at the peak of latency.     

Laccase expression in murine pulmonary Cryptococcus neoformans infection

Cryptococcus neoformans laccase expression during murine infection was investigated in lung tissue by immunohistochemistry and immunogold electron microscopy. Laccase was detected in the fungal cell cytoplasm, cell wall, and capsule in vivo. The amount of laccase found in different sites varied as a function of the time of infection.     

Evidence for superantigenic activity during murine malaria infection

TCR V beta usage was examined in C57BL/6 mice infected with Plasmodium yoelii. In addition to a polyclonal T cell activation, already described, a superantigenic-like activity was observed during the acute infection. This superantigenic activity induces a preferential deletion without prior expansion of CD4+ and CD8+ T cells bearing the TCR V beta 9 segment. The superantigen could be released by the parasite at different stages of its development since the deletion of V beta 9+ T cells was observed in blood and lymph nodes of mice infected either with sporozoites or with erythrocytic stages. Injection of sporozoite or parasitized erythrocytes to newborn mice led to a deletion and anergy of peripheral V beta 9+ T cells, without affecting thymic T cell populations. These observations suggest that the superantigen is released at very low concentrations during parasite development. The role of such parasite superantigenic activity in infectivity can be underlined by the observation that congenic BALB.D2 Mis1a mice lacking V beta 9 T cells are more susceptible to infection by P. yoelii.      

利用小鼠感染模型筛选与鉴定幽门螺杆菌外膜蛋白抗原

目的:利用小鼠感染模型筛选与鉴定幽门螺杆菌SS1株的外膜蛋白抗原。方法:提取SS1株的外膜蛋白进行双向电泳,用幽门螺杆菌感染的小鼠血清作免疫印迹实验,将阳性反应蛋白点进行质谱鉴定分析,将肽质量指纹谱数据输入互联网上的蛋白质数据库进行检索。结果:获得32种抗原相关蛋白。通过与已有报道的幽门螺杆菌感染人抗原比较分析,发现大部分典型的保护性抗原在本实验中都可以检测到。结论:幽门螺杆菌感染的小鼠模型适用于人用保护性幽门螺杆菌抗原的筛选;而且此研究中得到的相关抗原蛋白对于寻找与鉴定幽门螺杆菌未知保护性抗原也有参考价值。     

Antivirus antibody-dependent cell-mediated cytotoxicity during murine cytomegalovirus infection.

BALB/c mice infected with murine cytomegalovirus were studied to determine whether antibody-dependent cell-mediated cytotoxicity contributes to the immune control of this infection. Antibody-dependent killer cells from uninfected mice were used as effector cells to assay for antibody in sera of infected mice. Secondary immune sera were found to contain both cytomegalovirus-specific and autoreactive antibodies. After primary infection only cytomegalovirus-specific antibodies were found. These were detected by antibody-dependent cell-mediated cytotoxicity within 8 to 10 days after onset of infection, but usually not until day 21, by a neutralizing antibody assay. Antibody titers were about 10-fold higher by antibody-dependent cell-mediated cytotoxicity than by neutralization. The results indicate that cellular immunity to cytomegalovirus infection includes an antibody-dependent cell-mediated cytotoxicity response which is likely to be highly efficient and may contribute significantly to control of both acute and later stages of infection.     

Bcl-2 protein expression during murine development.

Bcl-2 functions as a death repressor molecule in an evolutionarily conserved cell death pathway. To further explore the role of Bcl-2 in development, we assessed its pattern of expression during murine embryogenesis. Immunohistochemical analysis demonstrates that Bcl-2 is widely expressed early in mouse fetal development in tissues derived from all three germ layers and that this expression becomes restricted with maturation. Within epithelium, the E12.5 lung bud demonstrates a proximal to distal gradient of Bcl-2 expression which is enhanced by E18.5. Bcl-2 is expressed throughout the intestinal epithelium through E14.5, but by E18.5 only cells in the crypts and lower villi express Bcl-2. In the mesoderm-derived kidney, Bcl-2 is expressed in both the ureteric bud and metanephric cap tissue at E12.5. Tubular structures also express Bcl-2, although overall levels drop as the kidney matures. Retinal neuroepithelial cells uniformly express Bcl-2 until cells begin to differentiate and then display the topographic distribution maintained into adulthood. The developing limb provides a clear example where Bcl-2 is restricted to zones of cell survival; Bcl-2 is expressed in the digital zones but not in the interdigital zones of cell death. The wide distribution of Bcl-2 in the developing mouse suggests that many immature cells require a death repressor molecule or that Bcl-2 may have roles beyond regulating developmental cell death.     

Multidrug resistance gene expression during the murine ontogeny

Multidrug resistance (MDR) was described initially for tumor cells which become resistant not only to the specific drug to which they are submitted, but also to a large range of unrelated drugs. The expression of mdr genes, responsible for the phenotype, and their product P glycoprotein (Pgp), is currently under intensive study due to their ample distribution in different organisms and their possible physiological roles which include protection against xenobiotics. In mice, three mdr isoforms expressed in some normal tissues are known. In this work, we analyzed by RT-PCR the expression of mdr1, mdr2 and mdr3 in several organs of BALB/c and C57BL/6 mice during ontogeny. A considerable variation in mdr expression among individuals of the same strain, as well as among different organs in individuals of the same age group and among different age groups, was detected. We also observed a strong tendency for the expression of a greater number of active isoforms in old mice. The large expression range of the mdr isoforms point to an important role as a natural defense system.     

Antigen presentation in the murine oral epithelium

We have previously reported that the buccal mucosa can support delayed type hypersensitivity (DTH) reactions to contact sensitizers. In the present study, we show that cells isolated from the buccal epithelium are able to present soluble exogenous antigens to specific T cells. Single cell suspensions obtained by enzymatic dispersion of buccal epithelial sheets could present the native protein antigen hen-egg lysozyme (HEL) to the I-A^k-restricted CD4⁺ T-cell hybridoma specific for a.a 46–61 on HEL. T-cell activation resulted in interleukin-2 (IL-2) production which could be inhibited by anti-major histocompatibility complex (MHC) class-II antibodies of pertinent specificity. Immunohistochemical staining of whole buccal epithelial sheets revealed that all MHC II positive cells had a dendritic morphology and expressed ATPase activity, indicating that these cells represent a major antigen-presenting cell (APC) population in this tissue. Furthermore, single cell suspensions isolated from buccal epithelium (BEC) after local in vivo administration of either a native soluble protein, a synthetic dodecapeptide, or a contact sensitizer were able to activate antigen-specific T cells ex vivo. Kinetic analyses indicated that maximal APC activity in the oral epithelium occurred within 1 hr after local antigen administration, and had essentially vanished after 24 hr. Conversely, APC activity was undetectable in draining cervico-mandibular lymph node cell suspensions recovered 1 hr after local antigen injection but became manifest after 3–24 hr. These observations suggest that dendritic cells can acquire antigens in the buccal epithelium and migrate to draining lymph nodes where they present processed antigen to MHC class II-restricted T cells. This APC population may thus be a critical element in the initiation of Th1-driven DTH responses in the oral mucosa.     

Lung function and inflammation during murine Pseudomonas aeruginosa airway infection

Background

Following any acute irritation lung function declines rapidly. Reasons for pulmonary deterioration in humans had been attributed to the action of either interleukin-6 or interleukin-8 in the lungs.

Objectives

The present study investigates the association between immune response and decline in lung function in a murine bacterial lung infection model.

Methods

Upon intratracheal inoculation of C57BL/6J mice with a sublethal dose of Pseudomonas aeruginosa lung function, cytokine, chemokine and cytometry in bronchoalveolar lavage fluid, bacterial counts and lung histology was assessed at 2, 4, 6, 8, 10, 12, 18, 24, 48, 72, 96 and 120 h post inoculation.

Results

Lung function measured by non-invasive head-out spirometry decreased most strongly between 6 and 10 h post inoculation and required up to 72 h to recover for selected parameters. CFU counts in the lungs peaked at 4 h post inoculation with subsequent decline until at 24-48 h post inoculation background levels were reached. Cytokine and chemokine responses could be separated into an early pro-inflammatory phase (2-8 h post inoculation; mainly tumor-necrosis factor α (TNFα) and interleukin-1α driven) and a late anti-inflammatory resolution phase (starting at 24 h post inoculation; mainly interleukin-10 and interleukin-4 driven). Interleukin-6 levels correlated with the deterioration of lung function. Lung histology showed maximal changes in terms of inflammation and edema between 24 and 48 h post inoculation.

Conclusions

In summary, elevated interleukin-6, high local neutrophil counts and lung edema were found to be the most characteristic signs of the transient period of deterioration of lung function.     

Myocardial expression of endothelin-1 in murine Trypanosoma cruzi infection.

Chagas' disease, caused by Trypanosoma cruzi, is an important cause of myocarditis and chronic cardiomyopathy and is accompanied by microvascular spasm and myocardial ischemia. We reported previously that infection of cultured endothelial cells with T. cruzi increased the synthesis of biologically active endothlein-1 (ET-1). In the present study, we examined the role of ET-1 in the cardiovascular system of CD1 mice infected with the Brazil strain of T. cruzi and C57BL/6 mice infected with the Tulahuen strain during acute infection. In the myocardium of infected mice myonecrosis and multiple pseudocysts were observed. There was also an intense vasculitis of the aorta, coronary artery, smaller myocardial vessels and the endocardial endothelium. Immunohistochemistry studies employing anti-ET-1 antibody revealed increased expression of ET-1 that was most intense in the endocardial and vascular endothelium. Elevated levels of mRNA for preproET-1, endothelin converting enzyme and ET-1 were observed in the same myocardial samples. Plasma ET-1 levels were significantly elevated in infected CD1 mice 10-15 days post infection. These observations suggest that increased levels of ET-1 are a consequence of the initial invasion of the cardiovascular system and provide a mechanism for infection-associated myocardial dysfunction.     

Cryptococcus neoformans capsule structure evolution in vitro and during murine infection

Cryptococcus neoformans capsule structure modifications after prolonged in vitro growth or in vivo passaging have been reported previously. However, nothing is known about the dynamics of these modifications or about their environmental specificities. In this study, capsule structure modifications after mouse passaging and prolonged in vitro culturing were analyzed by flow cytometry using the glucuronoxylomannan-specific monoclonal antibody E1. The capsule structures of strains recovered after 0, 1, 8, and 35 days were compared by using the level of E1-specific epitope expression and its cell-to-cell heterogeneity within a given cell population. In vitro, according to these parameters, the diversity of the strains was higher on day 35 than it was initially, suggesting the absence of selection during in vitro culturing. In contrast, the diversity of the strains recovered from the brain tended to decrease over time, suggesting that selection of more adapted strains had occurred. The strains recovered on day 35 from the spleen and the lungs had different phenotypes than the strains isolated from the brain of the same mouse on the same day, thus strongly suggesting that there is organ specificity for C. neoformans strain selection. Fingerprinting of the strains recovered in vitro and in vivo over time confirmed that genotypes evolved very differently in vitro and in vivo, depending on the environment. Overall, our results suggest that organ-specific selection can occur during cryptococcosis.     

T-dependent production and activation of mononuclear phagocytes during murine BCG infection

Mice infected with a high dose of viable Bacillus Calmette Guerin (BCG) intravenously offer an interesting model to study regulatory functions of T cells on hemopoiesis. The proposition that T lymphocytes may play such a regulatory role was tested in nu/nu and two genetically different strains of mice: while the hemopoiesis of C3H/He mice remained unchanged during BCG injection, that of infected C57BL/6 mice was rapidly and transiently modified towards increased production of phagocytes at the expense of the erythroid lineage. The number of BCG-specific T cells present in C57BL/6 bone marrow was 50-100 higher than that determined in C3H/He mice. Moreover, between day 0 and 5 of infection the majority of BCG-specific T cells in C57BL/6 animals were of the L3T4+ Lyt2- surface phenotype. An attempt was made to identify the nature of the T cell product(s) able to activate young bone marrow-derived macrophages to render them non-permissive to growth of BCG.     

Transient cytokeratin expression in skeletal muscle during murine embryogenesis.

Cytokeratin expression was investigated in paravertebral skeletal musculature of 10 d and 18 d old embryos as well as in adult NMRI-mice. The muscular nature of the evaluated tissue was evidenced by their expression of vimentin and desmin. Binding moieties for cytokeratin antibodies (polyclonal and monoclonal) could be demonstrated only in muscle cells of 10 d old embryos. Concerning subtypes, the mouse equivalents of the human cytokeratins Nos. 8, 18 and 19 could be made probable. The importance of the transient cytokeratin expression in murine embryonal skeletal muscle cells is emphasized, because this intermediate filament type is expressed in rhabdomyosarcomas too.     

Comparative temporospatial expression profiling of murine amelotin protein during amelogenesis

Tooth enamel is formed in a typical biomineralization process under the guidance of specific organic components. Amelotin (AMTN) is a recently identified, secreted protein that is transcribed predominantly during the maturation stage of enamel formation, but its protein expression profile throughout amelogenesis has not been described in detail. The main objective of this study was to define the spatiotemporal expression profile of AMTN during tooth development in comparison with other known enamel proteins. A peptide antibody against AMTN was raised in rabbits, affinity purified and used for immunohistochemical analyses on sagittal and transverse paraffin sections of decalcified mouse hemimandibles. The localization of AMTN was compared to that of known enamel proteins amelogenin, ameloblastin, enamelin, odontogenic ameloblast-associated/amyloid in Pindborg tumors and kallikrein 4. Three-dimensional images of AMTN localization in molars at selected ages were reconstructed from serial stained sections, and transmission electron microscopy was used for ultrastructural localization of AMTN. AMTN was detected in ameloblasts of molars in a transient fashion, declining at the time of tooth eruption. Prominent expression in maturation stage ameloblasts of the continuously erupting incisor persisted into adulthood. In contrast, amelogenin, ameloblastin and enamelin were predominantly found during the early secretory stage, while odontogenic ameloblast-associated/amyloid in Pindborg tumors and kallikrein 4 expression in maturation stage ameloblasts paralleled that of AMTN. Secreted AMTN was detected at the interface between ameloblasts and the mineralized enamel. Recombinant AMTN protein did not mediate cell attachment in vitro. These results suggest a primary role for AMTN in the late stages of enamel mineralization.     

Evaluation of thyroglobulin expression in murine reproductive organs during pregnancy

Citation Moravej A, Jeddi‐Tehrani M, Salek‐Moghaddam AR, Dokouhaki P, Ghods R, Rabbani H, Kazemi‐Sefat GE, Shahbazi M, Zarnani AH. Evaluation of thyroglobulin expression in murine reproductive organs during pregnancy. Am J Reprod Immunol 2010; 64: 97–103 Problem In pregnant women with antithyroglobulin antibody, prevalence of abortion is 2–4 fold higher compared to normal controls. Direct effect of such harmful autoantibodies on female reproductive organs may serve a role in pregnancy loss. Method of study Expression of thyroglobulin in decidua, placenta, and ovary of pregnant Balb/c mice ((Balb/c×Balb/c and Balb/c×C57BL/6) during early, middle, and late stages of pregnancy was evaluated. Expression of thyroglobulin was investigated in these tissues by semi‐quantitative RT‐PCR. In addition, polyclonal antithyroglobulin antibody was produced, and expression of thyroglobulin protein in aforesaid tissues was evaluated by immunohistochemistry and dot‐blot analysis. Results The results showed that thyroglobulin message is not expressed in placenta, decidua, or ovary in any stages of pregnancy. The same results were obtained at the protein level. Conclusion It is likely that antithyroglobulin antibodies have no direct detrimental effect on such organs in patients with thyroid autoimmunity suffering from recurrent abortion.     

Different chemokine expression in lethal and non-lethal murine West Nile virus infection

West Nile (WN) virus is a mosquito-borne flavivirus that can cause lethal encephalitis in humans and horses. The WN virus endemic in New York City (NY) in 1999 caused large-scale mortality of wild birds that was not evident in endemic areas in other parts of the world, and the pathogenesis of the WN virus strain isolated in NY (NY strain) appears to differ from that of previously isolated strains. However, the pathogenesis of NY strain infection remains unclear. This study examined CC (RANTES/CCL5, MIP-1 alpha/CCL3, MIP-1 beta/CCL4) and CXC (IP-10/CXCL10, B lymphocyte chemoattractant (BLC/CXCL13), and B cell- and monocyte-activating chemokine (BMAC/CXCL14)) chemokine expression during lethal NY strain and non-lethal Eg101 strain infection in mice. We found that the mRNA of the CC chemokines, RANTES, MIP-1 alpha, MIP-1 beta, and IP-10 was highly up-regulated in the brain of NY strain-infected mice. By contrast, BLC mRNA was not detected in either group of mice, and BMAC mRNA was highly up-regulated in late stage of infection with the non-lethal Eg101 strain relative to levels in NY strain-infected mice.     

Antigen presentation in virus infection.

Important recent advances have been made in our understanding of antigen processing of cytoplasmic antigens and presentation by class I molecules of the MHC. Peptide transporter-like molecules encoded within the MHC have been characterized and have, by transfection, corrected some of the presentation-mutant cell lines. The nature of peptide-MHC class I interactions has been clarified by further resolution of the HLA A2 and B27 crystals and elution of peptides. The differences between antigenicity and immunogenicity for viral antigens have been highlighted by studies in transgenic animals.