T cells cause acute immunopathology and are required for long-term survival in mouse adenovirus type 1-induced encephalomyelitis

Infection of adult C57BL/6 (B6) mice with mouse adenovirus type 1 (MAV-1) results in dose-dependent encephalomyelitis. Utilizing immunodeficient mice, we analyzed the roles of T cells, T-cell subsets, and T-cell-related functions in MAV-1-induced encephalomyelitis. T cells, major histocompatibility complex (MHC) class I, and perforin contributed to acute disease signs at 8 days postinfection (p.i.). Acute MAV-1-induced encephalomyelitis was absent in mice lacking T cells and in mice lacking perforin. Mice lacking alpha/beta T cells had higher levels of infectious MAV-1 at 8 days, 21 days, and 12 weeks p.i., and these mice succumbed to MAV-1-induced encephalomyelitis at 9 to 16 weeks p.i. Thus, alpha/beta T cells were required for clearance of MAV-1. MAV-1 was cleared in mice lacking perforin, MHC class I or II, CD4+ T cells, or CD8+ T cells. Our results are consistent with a model in which either CD8+ or CD4+ T cells are sufficient for clearance of MAV-1. Furthermore, perforin contributed to MAV-1 disease but not viral clearance. We have established two critical roles for T cells in MAV-1-induced encephalomyelitis. T cells caused acute immunopathology and were required for long-term host survival of MAV-1 infection.     

Distribution of Deoxyribonucleic Acid Complementary to the Ribonucleic Acid of Avian Myeloblastosis Virus in Tissues of Normal and Tumor-Bearing Chickens

(3)H-labeled 70S ribonucleic acid (RNA) from purified avian myeloblastosis virus (AMV) was used as a probe in deoxyribonucleic acid (DNA)-RNA hybridization experiments to detect the presence of DNA complementary to the AMV genome in various tissues from noninfected normal chickens and from chickens infected with AMV. There was a remarkable constancy in the average cellular concentration of virus-specific DNA found in every tissue from the same uninfected chicken, and even in different chickens from the same strain. In contrast, different tissues from chickens bearing AMV-induced kidney tumors (embryonal nephromas) revealed an unequal distribution in the average virus-specific DNA content per cell. The increase was limited to tumor cells and to tissues that contain target cells for AMV, i.e., red blood cells, kidney cells, and possibly leukocytes. The red blood cells from AMV-infected chickens suffering from acute myeloblastic leukemia, although producing no virus, contained as many viral genome equivalents per cell as did leukemic myeloblasts known to produce large quantities of AMV. An increased viral DNA content was observed in the target cells of chickens that did not show any sign of tumor formation 6 months after infection with AMV. This study demonstrates that vertically transmitted viral DNA is uniformly and stably distributed among all tissues of the offspring, but that horizontal infection after hatching results in an increase in viral DNA content only in some dividing, target tissues that may or may not give rise to neoplasias.     

Comparison of an avian osteopetrosis virus with an avian lymphomatosis virus by RNA-DNA hybridization.

Myeloblastosis-associated virus (MAV)-2(0), a virus which was derived from avian myeloblastosis virus and induced a high incidence of osteopetrosis, was compared with avian lymphomatosis virus 5938, a recent field isolate which induced a high incidence of lymphomatosis. The following information was obtained. (i) MAV-2(0) induced osteopetrosis, nephroblastoma, and a very low incidence of hepatocellular carcinoma. No difference was seen in the oncogenic spectrum of end point and plaque-purified MAV-2(0). (ii) 125I-labeled RNA sequences from MAV-2(0) formed hybrids with DNA extracted from osteopetrotic bone at a rate suggesting five proviral copies per haploid cell genome. The extent of hybridization of MAV-2(0) RNA with DNA from osteopetrotic tissue was more extensive (87%) than was observed in reactions with DNA from uninfected chicken embryos (52%). (iii) Competition of unlabeled viral RNA in hybridization reactions between the radioactive RNA from the two viruses and their respective proviral sequences present in tumor tissues showed that 15 to 20% of the viral sequences detected in these reactions were unshared. In contrast, no differences were detected in competition analyses of RNA sequences from the two viruses detected in DNA of normal chicken cells. (iv) MAV-2(0) 35S RNA was indistinguishable in size from avian lymphomatosis virus 5938 35S RNA by polyacrylamide gel electrophoresis.     

Sequences from myeloblastosis-associated virus MAV-2(O) and UR2AV involved in the formation of plaques and the induction of osteopetrosis, anemia, and ataxia.

Recombinant viruses were made between myeloblastosis-associated virus MAV-2(O) and UR2AV to examine the relationship between regions of the MAV-2(O) genome and disease induction. The env-long terminal repeat (LTR) portion of MAV-2(O), when substituted into UR2AV, was sufficient to induce osteopetrosis identical to that caused by the parent MAV-2(O). When this region was reduced to the gp37 and LTR of MAV-2(O), osteopetrosis more severe than that caused by the parent virus was induced. Recombinant viruses that contained all or part of the MAV-2(O) env gene in the absence of the MAV-2(O) LTR induced a severe, chronic anemia and late-onset osteopetrosis, leading to the conclusion that the MAV-2(O) LTR, in addition to env, was required for rapid induction of osteopetrosis. A viral recombinant, pEU, which contained the gp85 segment of UR2AV substituted into MAV-2(O), induced an ataxia/cerebellar dysfunction not seen during infection with the other chimeric or parent viruses. In vitro studies of the parent and recombinant viruses demonstrated that the ability to form plaques on chicken embryo fibroblasts correlated with the presence of the MAV-2(O) gp37 and LTR except for construct pEU. When the viruses were inoculated into 10-day-old chickens, chimeras containing the env-LTR of gp37-LTR region of MAV-2(O) induced severe regenerative anemia similar to that induced by MAV-2(O). pEU was the exception, suggesting that the unique configuration of this chimera is responsible for its unusual pathogenic properties.     

The effect of alterations in myc gene expression on B cell development in the bursa of Fabricius

Infection of 18-day embryonic bursal lymphocytes with a v-myc-containing retrovirus leads directly to a polyclonal proliferation of surface immunoglobulin-positive (slg+) cells in the bursa of Fabricius detected four weeks after hatching. These v-myc-expressing bursal cells repopulate the follicles of chemically ablated bursae more efficiently than total normal 18-day embryonic bursal cells. In contrast, comparable normal bursal cells lose the ability to repopulate follicles by four weeks. Bursal lymphocytes expressing either a retroviral v-myc or a c-myc gene deregulated by adjacent retroviral integration retain the ability of embryonic bursal lymphocytes to diversify their immunoglobulin light chain genes. These results suggest that retroviral deregulation of myc expression during avian B cell development induces outgrowth of a population of cells with the cardinal phenotypic characteristics of bursal stem cells.     

Persistence of Marek''s disease virus in a subpopulation of B cells that is transformed by avian leukosis virus, but not in normal bursal B cells.

Previous studies have described an augmentation of avian leukosis virus (ALV)-induced lymphoid leukosis in chickens that were coinfected with a serotype 2 Marek's disease virus (MDV) strain, SB-1. As a first step toward understanding the mechanism of this augmentation, we have analyzed the tropism of the MDV for the ALV-transformed B cell. After hatching, chickens were coinfected with ALV and a nonpathogenic strain of MDV, SB-1. Seventy primary and metastatic ALV-induced lymphomas that developed in chickens between 14 and 20 weeks of age were found, with only one exception, to carry SB-1 DNA. The MDV genome was maintained in cell lines derived from the tumors. However, MDV DNA could not be detected in nontransformed bursal B cells from chickens carrying ALV lymphomas. Moreover, during and after the lytic phase of MDV infection, SB-1 DNA was near or below the level of detection in bursal cells, suggesting that MDV most likely infects only a small subpopulation of bursal cells. By contrast, ALV-transformed B cells from MDV-free chickens could be persistently infected with MDV in vitro. These findings indicate that ALV lymphoma cells, unlike nontransformed bursal B cells, are susceptible to persistent MDV infection and can serve as a reservoir of MDV that can potentially influence the physiology of the transformed cell.     

Comparison of an Avian Osteopetrosis Virus with an Avian Lymphomatosis Virus by RNA-DNA Hybridization

Myeloblastosis-associated virus (MAV)-2(0), a virus which was derived from avian myeloblastosis virus and induced a high incidence of osteopetrosis, was compared with avian lymphomatosis virus 5938, a recent field isolate which induced a high incidence of lymphomatosis. The following information was obtained. (i) MAV-2(0) induced osteopetrosis, nephroblastoma, and a very low incidence of hepatocellular carcinoma. No difference was seen in the oncogenic spectrum of end point and plaque-purified MAV-2(0). (ii) ¹²⁵I-labeled RNA sequences from MAV-2(0) formed hybrids with DNA extracted from osteopetrotic bone at a rate suggesting five proviral copies per haploid cell genome. The extent of hybridization of MAV-2(0) RNA with DNA from osteopetrotic tissue was more extensive (87%) than was observed in reactions with DNA from uninfected chicken embryos (52%). (iii) Competition of unlabeled viral RNA in hybridization reactions between the radioactive RNA from the two viruses and their respective proviral sequences present in tumor tissues showed that 15 to 20% of the viral sequences detected in these reactions were unshared. In contrast, no differences were detected in competition analyses of RNA sequences from the two viruses detected in DNA of normal chicken cells. (iv) MAV-2(0) 35S RNA was indistinguishable in size from avian lymphomatosis virus 5938 35S RNA by polyacrylamide gel electrophoresis.     

The Role of Mouse Adenovirus Type 1 Early Region 1A in Acute and Persistent Infections in Mice

Mouse adenovirus type 1 (MAV-1) early region 1A (E1A) viral mutants were used to determine the importance of this region in pathogenesis and establishment of a persistent infection in the natural host. Lethal dose analysis with adult male Swiss outbred mice revealed a significant reduction in virulence for all of the E1A mutants. During acute infections with 10⁵ PFU of virus, an E1A null mutant, pmE109, was found in the same organs (brain, spleen, and spinal cord) and the same cell types (endothelial cells and mononuclear cells in lymphoid tissue) as wild-type virus. Another null mutant, pmE112, was detected in the same organs but in lower numbers. However, when mice were given a lower dose, 1 PFU, pmE109 and pmE112 reached none of the target organs analyzed by 14 days postinfection (p.i.). The absence of E1A did not hinder the ability of MAV-1 to establish a persistent infection. Viral nucleic acid was detected by PCR amplification or in situ hybridization in the kidneys, brains, spleens, and prefemoral lymph nodes of mice infected with wild-type or mutant virus up to 55 weeks p.i. The brain, spleen, and lymph node are recognized sites of acute viral infection but are previously unrecognized sites for MAV-1 persistence. Evidence for the potential reactivation of persistent MAV-1 infections is also presented.     

成体干细胞向肝细胞分化

成体干细胞跨越胚层限制分化为其他胚层来源的细胞,对揭示不同胚层细胞间相互分化的生物学意义和机制具有重要学术价值,并可以为临床细胞移植治疗开辟新的途径,从而成为当前研究的热点之一。综述了近年来肝源性卵圆细胞、成肝细胞、骨髓源干细胞和其他成体干细胞跨越分化为肝细胞的研究现状与进展,以及卵圆细胞、成肝细胞等的分离鉴定,表面标志、生物学特征和跨越分化机制,并对成体干细胞在肝脏疾病细胞治疗上的应用前景作了展望。     

BEN, a novel surface molecule of the immunoglobulin superfamily on avian hemopoietic progenitor cells shared with neural cells.

BEN is a novel molecule of the immunoglobulin superfamily that we previously identified by means of a monoclonal antibody on neural cell populations during avian development and epithelial cells of the bursa of Fabricius. In this paper, we describe the expression of BEN by hemopoietic cells during ontogeny. In the thymus, BEN is expressed as early as E9, and from E12 until just after hatching 30-60% of thymocytes are BEN positive. Thus the cells expressing BEN are immature thymocytes and not yet differentiated T cells. In the spleen, BEN expression parallels the myelopoietic activity. It is present on 75% of splenocytes during embryonic development and falls rapidly to 20% of cells during the first week after hatching when the spleen is becoming a secondary lymphoid organ. BEN is also found on a large proportion (about 80% positive cells) of bone marrow cells during ontogeny. Post hatching, BEN is present on 40-50% of bone marrow cells. The population of BEN-positive cells in the bone marrow includes myeloid and erythroid progenitor cells, identified by their ability to form colonies in vitro. BEN expression is lost as progenitor cells proliferate and differentiate to develop mature colonies in the clonal assay. Mature myeloid cells, such as macrophages, granulocytes, thrombocytes, and erythrocytes do not express the BEN antigen. Taken together, these data demonstrated that BEN is a stage-specific rather than a lineage-specific differentiation antigen expressed by immature hemopoietic cells.     

HIV-1 tracing method of systemic viremia in vivo using an artificially mutated virus pool

The appearance of human immunodeficiency virus type 1 (HIV-1) plasma viremia is associated with progression to symptomatic disease and CD4⁺ T cell depletion. To locate the source of systemic viremia, this study employed a novel method to trace HIV-1 infection in vivo. We created JRCSFξnef, a pool of infectious HIV-1 (strain JR-CSF) with highly mutated nef gene regions by random mutagenesis PCR and infected this mutated virus pool into both Jurkat-CCR5 cells and hematopoietic stem cell-transplanted humanized mice. Infection resulted in systemic plasma viremia in humanized mice and viral RNA sequencing helped us to identify multiple lymphoid organs such as spleen, lymph nodes, and bone marrow but not peripheral blood cells as the source of systemic viremia. Our data suggest that this method could be useful for the tracing of viral trafficking in vivo.     

Developmental Endocrinology of the Dipnoan, Neoceratodus forsteri

The development of the pineal, pituitary and thyroid glandsof the extant lungfish, Neoceratodus forsteri, are being studiedboth morphologically and functionally. This paper presents datafrom hatching to 40–52 weeks for a standardised seriesof lungfish, bred at Macquarie University. At hatching, thepineal comprises a single organ attached to the roof of thediencephalon, with well-developed photoreceptor, supportingand ganglion cells. The photoreceptors gradually degenerate,giving way to secretory cells which contain electron dense granules.These latter are immunoreactive to melatonin antibodies anddigestable with protease. The pituitary at hatching comprisesa hollow ball of cells lying beneath the infundibular regionof the hypothalamus. Ultrastructurally, four cell types canbe distinguished by cytoplasmic granule size after the firstfour weeks of development posthatching. By 20 weeks, a furtherthree cell types are recognisable. Inununogold labelling hasidentified corticotropes and melanotropes at four weeks and,at 20 weeks, prolactin cells, thyrotropes and somatotropes alsocan be identified. The thyroid is only just apparent at hatching,containing 2–3 follicles. Thenumbers of follicles increasesgradually, and variably between animals, with age. Iodine uptakein methimazole-treated animals did not exceed that of controlsat any of the three stages tested, indicating a lack of feedbackcontrol between thyroid hormones and pituitary thyrotropes at,or before, 40 weeks of age. Thyroid hormone receptors in theliver at 40 weeks are predominantly immunoreactive to humanTRac antibodies. These findings taken together suggest that,up to 40 weeks post hatching, lungfish development is equivalentto amphibian premetamorphic development. This would be consistentwith lungfish neoteny, but cannot be taken as evidence for neotenyuntil confirmed at later stages of development     

Neonatal tolerance induction in the thymus to MHC-class II-associated antigens. III. Significance of hemopoietic stem cells for induction and maintenance of Mls tolerance by continuous supply of tolerance-inducing nonlymphocytes

The role of hemopoietic stem cells and other cell types in the induction and maintenance of immunologic tolerance in the thymus was investigated by intravenous injection of Mls-semi-allogeneic cells into newborn mice less than 24 hr after birth. Mls-specific tolerance was induced by inoculation of peritoneal cells and thymus cells, and the tolerant state was compared with that induced by bone marrow cells which had hemopoietic stem cell activity and were able to create a stable chimera in both central and peripheral lymphoid organs. When peritoneal or thymus cells were injected, the level of tolerance attained was proportional to the number of cells injected, though peritoneal cells were 20 times as effective as thymus cells. In vivo functions of tolerance-inducing cells and their immediate precursors were radiosensitive and belonged to a Thy-1-, nylon-wool-nonadherent (probably non-B), weakly Sephadex G-10-adherent cell population. Tolerance induced by peritoneal cell injections was transient, starting to terminate within the first 2 weeks of life, while tolerance caused by bone marrow cell injections persisted through more than 6 weeks. Such transient tolerance induced by the former became long-lasting when followed by an additional injection of bone marrow cells, which did not cause thymic lymphocyte chimerism. All data indicated that bone marrow stem cells were engaged in tolerance induction and maintenance by continuously supplying tolerance-inducing nonlymphocytes.     

Avian leukosis virus infection: analysis of viremia and DNA integration in susceptible and resistant chicken lines

Avian leukosis viruses induce lymphoid leukosis, a lymphoma which develops within the bursa of Fabricius several months after virus infection. Chickens from the Hyline SC and FP lines are, respectively, susceptible and resistant to avian leukosis virus-induced lymphoid leukosis. We examined plasma and cellular DNA obtained from avian leukosis virus-infected chickens for the presence of viremia and integrated viral sequences to determine whether the extent of virus infection is comparable in individuals of both lines. A less than twofold difference in the frequency of viremia was detected between chickens of the two different lines. Although the analysis of plasma samples, which were obtained at different times postinfection, demonstrated that the duration of viremia was comparable in both susceptible and resistant chickens, the onset of the viremia observed in susceptible chickens generally preceded by 1 week that observed in resistant chickens. Moreover, integrated viral sequences were detected in approximately 90% of the SC and 40% of the FP chickens. The appearance of infectious virus in the plasma was, in general, associated with the presence of integrated viral sequences in both the bursal cells and the erythrocytes obtained from the same chicken. The presence of both the viremia and the integrated viral DNA sequences was transient, suggesting a mechanism for the elimination of virus-infected cells in both susceptible and resistant chickens. Furthermore, at 5 weeks postinfection no integrated exogenous viral sequences were detected in splenic lymphocytes obtained from either chicken line, regardless of whether these chickens were viremic or had integrated viral sequences detectable in other tissues. Our results indicate that extensive avian leukosis virus replication occurs in approximately 50% of the FP and 100% of the SC chickens. Although it appears that the viral infection spreads more quickly in the SC chickens, our results afford no obvious explanation of the resistance to the development of lymphoma exhibited by FP chickens.     

Susceptibility to acute mouse adenovirus type 1 respiratory infection and establishment of protective immunity in neonatal mice

There is an incomplete understanding of the differences between neonatal immune responses that contribute to the increased susceptibility of neonates to some viral infections. We tested the hypothesis that neonates are more susceptible than adults to mouse adenovirus type 1 (MAV-1) respiratory infection and are impaired in the ability to generate a protective immune response against a second infection. Following intranasal infection, lung viral loads were greater in neonates than in adults during the acute phase but the virus was cleared from the lungs of neonates as efficiently as it was from adult lungs. Lung gamma interferon (IFN-γ) responses were blunted and delayed in neonates, and lung viral loads were higher in adult IFN-γ(-/-) mice than in IFN-γ(+/+) controls. However, administration of recombinant IFN-γ to neonates had no effect on lung viral loads. Recruitment of inflammatory cells to the airways was impaired in neonates. CD4 and CD8 T cell responses were similar in the lungs of neonates and adults, although a transient increase in regulatory T cells occurred only in the lungs of infected neonates. Infection of neonates led to protection against reinfection later in life that was associated with increased effector memory CD8 T cells in the lungs. We conclude that neonates are more susceptible than adults to acute MAV-1 respiratory infection but are capable of generating protective immune responses.     

Defective bursa regeneration after irradiation of young thymectomized chickens

The ability of the bursa of Fabricius to regenerate after gamma-irradiation and bone marrow reconstitution was examined in chickens thymectomized (TX) immediately after hatching. Irradiation (2 X 500 R) 3 weeks after hatching was followed by impaired bursa regeneration, as judged both by bursa/body weight ratios and by bursa follicle development 3-6 weeks later in TX as compared to control birds. Germinal center formation in the spleen was deficient, and immune responses to sheep erythrocytes (SRBC) and B. abortus (BA) were moderately reduced in the TX as compared to control birds irradiated at 3 weeks but not in TX birds irradiated at 5 weeks of age.     

Distribution of Mouse Adenovirus Type 1 in Intraperitoneally and Intranasally Infected Adult Outbred Mice

In situ nucleic acid hybridization and immunohistochemistry were used to determine the histological localization of mouse adenovirus type 1 (MAV-1) during acute infection of adult mice infected either intraperitoneally or intranasally with 1,000 PFU of wild-type virus. Organ samples were collected from days 1 to 17 postinfection for the intraperitoneally infected mice and from days 1 to 13 for the intranasally infected mice. Endothelial cells of the brain and spinal cord showed extensive evidence of MAV-1 infection. Endothelial cells in lungs, kidneys, and other organs were also positive for MAV-1, indicating a widespread involvement of the systemic circulation. The presence of viral nucleic acid and/or antigen was also demonstrated in lymphoid tissue. The spleens, Peyer’s patches, and peripheral lymph nodes showed positive staining at various times postinfection in mice infected by either route. Virus-infected cells in the spleen exhibited a stellate shape and were localized to the red pulp and germinal centers, suggesting that they are cells of the mononuclear phagocytic system.