Interleukin-6 and retroperitoneal fibromatosis from SRV-2-infected macaques with simian AIDS.

We investigated the role of Interleukin-6 (IL-6) as an autocrine growth factor for the retroperitoneal fibromatosis (RF) cells present in macaques infected with the simian retrovirus type 2 (SRV-2). Elevated levels of IL-6 were found in serum of SRV-2 antibody-positive macaques, ascites from RF-positive animals, and RF cell line culture media. IL-6 mRNA levels increased approximately five-fold in RF cells incubated with exogenous SRV-2. In RF cells, SRV-2 functions to increase IL-6 mRNA and protein production and presumably serves as autocrine growth factor.     

Type D SRV-2 virus-specific CD8+ and CD4- CD8- T cells that regulate virus-induced T cell proliferation in Celebes macaques

These studies defined SRV-2 envelope peptides 96-102, 127-152, and 233-249 as T cell epitopes that induce significant T cell proliferation. Peripheral blood lymphocytes of Celebes macaques (Macaca nigra) exposed to SRV-2 and currently virus^- antibody⁺, cultured with SRV-2 virus show strongly suppressed T cell responses and have two immunoregulatory T cell populations.     

Immunocytochemistry of Kaposi's sarcoma-like tumor cells from pigtailed macaques with simian AIDS

Some macaques infected with SRV-2 developed SAIDS and RF, a Kaposi's sarcoma (KS)-like tumor. We investigated the immunophenotypic markers of this SAIDS-associated retroperitoneal fibromatosis (RF). RF tumor is characterized by proliferation of spindle cells accompanying inflammatory cell infiltrates, fibroblasts, and endothelial cells. RF spindle cells in tumor tissues revealed several immunobiologic characteristics similar to vascular smooth-muscle cells or myofibroblasts based on positive immunoreactivity of smooth-muscle α-actin and desmin. The majority of cultured RF spindle cells also expressed specific markers for vascular smooth muscle. These results suggest that the RF spindle cells are derived from vascular smooth-muscle cells. Furthermore, RF tissues and cells were persistently infected with SRV-2, which may play an important role in viral etiology of AIDS-associated neoplasm in this macaque model.     

Long-term protection of macaques against high-dose type D retrovirus challenge after immunization with recombinant vaccinia virus expressing envelope glycoproteins

Recombinant vaccinia virus expressing the envelope proteins of type D retrovirus-Washington (SRV-2/W) was used to immunize macaques against SRV-2 infection. Four immunized macaques which had resisted a prior low-dose challenge were rechallenged with a high dose (10” infectious particles) of SRV-2 two years after being immunized. All four non-immunized control macaques became infected, but the four vaccinated animals resisted this intravenous challenge, as determined by the inability to detect SRV-2 in peripheral blood mononuclear cells and by the lack of seroconversion to new viral antigens.     

Monoclonal antibodies to type D retrovirus (SRV-2)

Monoclonal antibodies (MoAbs) to the major gag core protein p27 and a viral protein p44 of type D retrovirus (SRV-2) were produced and used in the detection of SRV-2 antigens in infected Raji cells and in tissues from macaques with simian acquired immunodeficiency syndrome (SAIDS) and retroperitoneal fibromatosis (RF). Anti-p44 MoAb showed inhibition of syncytium formation by both SRV-1- and SRV-2-infected Raji cells.     

Low rates of transmission of SRV-2 and STLV-I to juveniles in a population of Macaca fascicularis facilitate establishment of specific retrovirus-free colonies

Background  Prevalence of simian retrovirus-2 (SRV-2) and simian T lymphotropic virus type I (STLV-I), was unknown in 337 captive cynomolgus macaques.
Methods and Results  Molecular assays identified 29% of animals as SRV-2 mono-infected, 4% of animals as STLV-I mono-infected and 9% of animals as dual-infected. Of 108 juvenile animals, 83% were SRV-2-negative and no juvenile animal was STLV-I-positive. A subsequent study of juvenile macaques over a period of 2.5 years detected no STLV-I and 10 SRV-2 infections, six of which occurred between testing and day of colony formation. The study also highlighted that an anti-SRV-2 serological response does not presuppose infection. Tissue reservoirs of latent SRV-2 were not identified in suspected SRV-2 infections.
Conclusions  Low transmissibility of the viruses present in the parental cohort and improved knowledge of the host response to SRV-2 has facilitated the creation of specific-retrovirus-free colonies of cynomolgus macaques.     

Serologic and virologic analysis of type D simian retrovirus infection in a colony of Celebes black macaques (Macaca nigra)

Celebes macaques were tested for type D simian retrovirus (SRV) infection. SRV infection was first detected in one serum sample collected during 1980. By 1983, 32 of 46 monkeys (70%) were infected. Serotyping of the SRV isolates determined that 0/26 of the isolates were SRV-1; 24/26 were SRV-2; 1/26 was SRV-5; and 1/26 could not be typed. Restriction endonuclease mapping confirmed the SRV-2C and SRV-5 isolates. In addition, two SRV-2C variants were detected.     

Protein of macaques against infection with simian type D retrovirus (SRV-1) by immunization with recombinant vaccinia virus expressing the envelope glycoproteins of either SRV-1 or Mason-Pfizer monkey virus (SRV-3).

Rhesus macaques were immunized with live vaccinia virus recombinants expressing the envelope glycoproteins (gp70 and gp22) of simian type D retrovirus (SRV), serotype 1 or 3. All of the animals immunized with either the SRV-1 env or the SRV-3 env vaccinia virus recombinant developed neutralizing antibodies against the homologous SRV. In addition, both groups developed cross-reactive antibodies and were protected against an intravenous live-virus challenge with SRV-1. The four control animals immunized with a vaccinia virus recombinant expressing the G protein of respiratory syncytial virus were not protected against the same SRV-1 challenge. Although SRV-1 and SRV-3 immune sera showed cross-neutralization, they failed to neutralize a separate, more distantly related serotype, SRV-2, in an in vitro assay. These findings are consistent with the known degree of serologic and genetic relatedness of these three SRV strains.     

Vesicular stomatitis virus-simian retrovirus type 2 vaccine protects macaques from detectable infection and B-cell destruction

Natural infection with simian retrovirus (SRV) has long been recognized in rhesus macaques (RMs) and may result in an AIDS-like disease. Importantly, SRV infections persist as a problem in recently imported macaques. Therefore, there is a clear need to control SRV spread in macaque colonies. We developed a recombinant vesicular stomatitis virus (VSV)-SRV vaccine consisting of replication-competent hybrid VSVs that express SRV gag and env in separate vectors. The goal of this study was to assess the immunogenicity and protective efficacy of the VSV-SRV serotype 2 vaccine prime-boost approach in RMs. The VSV-SRV vector (expressing either SRV gag or env) vaccines were intranasally administered in 4 RMs, followed by a boost 1 month after the first vaccination. Four RMs served as controls and received the VSV vector alone. Two months after the boost, all animals were intravenously challenged with SRV-2 and monitored for 90 days. After the SRV-2 challenge, all four controls became infected, and viral loads (VLs) ranged from 10(6) to 10(8) SRV RNA copies/ml of plasma. Two animals in the control group developed simian AIDS within 7 to 8 weeks postinfection and were euthanized. Anemia and weight loss were observed in the remaining controls. During acute infection, severe B-cell depletion and no significant changes in T-cell population were observed in the control group. Control RMs with greater preservation of B cells and lower VLs survived longer. SRV-2 was undetectable in vaccinated animals, which remained healthy, with no clinical or biological signs of infection and preservation of B cells. Our study showed that the VSV-SRV vaccine is a strong approach for preventing clinically relevant type D retrovirus infection and disease in RMs, with protection of 4/4 RMs from SRV infection and prevention of B-cell destruction. B-cell protection was the strongest correlate of the long-term survival of all vaccinated and control RMs.     

Proliferative lifespan is conserved after nuclear transfer

Cultured primary cells exhibit a finite proliferative lifespan, termed the Hayflick limit. Cloning by nuclear transfer can reverse this cellular ageing process and can be accomplished with cultured cells nearing senescence. Here we describe nuclear transfer experiments in which donor cell lines at different ages and with different proliferative capacities were used to clone foetuses and animals from which new primary cell lines were generated. The rederived lines had the same proliferative capacity and rate of telomere shortening as the donor cell lines, suggesting that these are innate, genetically determined, properties that are conserved by nuclear transfer.     

Maternal transmission of type D simian retrovirus (SRV-2) in pigtailed macaques

Pregnant macaques were used as a natural model for maternal-infant transmission of SRV-2 retrovirus. Fifty-one pregnant females were placed into one of four virus/antibody groups. Nonviremic mothers produced 100% virus-negative offspring at birth. In contrast, viremic mothers produced offspring which were 17% virus-negative and 83% virus-positive at birth. SRV-2 infection occurred principally in utero by the transplacental route. Infants born to viremic mothers exhibited low birth weight, prematurity, high perinatal death, and increased incidence of SAIDS.     

Establishment of tendon-derived cell lines exhibiting pluripotent mesenchymal stem cell-like property

Development of the musculoskeletal system requires coordinated formation of distinct types of tissues, including bone, cartilage, muscle, and tendon. Compared to muscle, cartilage, and bone, cellular and molecular bases of tendon development have not been well understood due to the lack of tendon cell lines. The purpose of this study was to establish and characterize tendon cell lines. Three clonal tendon cell lines (TT-E4, TT-G11, and TT-D6) were established using transgenic mice harboring a temperature-sensitive mutant of SV40 large T antigen. Proliferation of these cells was significantly enhanced by treatment with bFGF and TGF-beta but not BMP2. Tendon phenotype-related genes such as those encoding scleraxis, Six1, EphA4, COMP, and type I collagen were expressed in these tendon cell clones. In addition to tendon phenotype-related genes, expression of osteopontin and Cbfal was observed. These clonal cell lines formed hard fibrous connective tissue when implanted onto chorioallantoic membrane in ovo. Furthermore, these cells also formed tendon-like tissues when they were implanted into defects made in patella tendon in mice. As these tendon cell lines also produced fibrocartilaginous tissues in tendon defect implantation experiments, mesenchymal stem cell properties were examined. Interestingly, these cells expressed genes related to osteogenic, chondrogenic, and adipogenic lineages at low levels when examined by RT-PCR. TT-G11 and TT-E4 cells differentiated into either osteoblasts or adipocytes, respectively, when they were cultured in cognate differentiation medium. These observations indicated that the established tendon cell line possesses mesenchymal stem cell-like properties, suggesting the existence of mesenchymal stem cell in tendon tissue.     

Autologous B lymphoblastoid cell lines and long-term cultured T cells as stimulators in the mixed lymphocyte reaction: analysis of the role of HLA class II antigens as stimulatory molecules

Human activated T cells, long-term cultured in the presence of interleukin 2 (IL 2), were compared with autologous Epstein Barr virus-transformed B lymphoblastoid cell lines for expression of human leukocyte (HLA)-HLA-DR and -DQ antigens and for ability to induce proliferative responses in autologous and allogeneic lymphocytes. Immunofluorescence analysis performed with a panel of monoclonal antibodies (mAb) specific for HLA-DR or -DQ antigens did not reveal any significant difference in the expression of HLA-DR antigens but revealed reduced expression of HLA-DQ antigens on two out of four T cell lines tested. No obvious difference could be detected in the two-dimensional gel electrophoretic profile of HLA-DR and -DQ beta-chains synthesized by the autologous pairs of B and T cell lines. In contrast with previous reports, the IL 2-dependent cell lines consistently induced alloproliferative responses in standard 6-day mixed lymphocyte cultures; however, these responses were severalfold lower than those elicited by the autologous B lymphoid lines. Both anti-HLA-DR and anti-HLA-DQ mAb blocked the proliferative responses induced by the B cell lines but did not affect those generated by the T cell lines, suggesting that the latter cells induce T lymphocyte activation via a mechanism independent of HLA-DR or -DQ antigen expression on their surface. Addition of IL 2 to the mixed cultures with B cell lines as stimulators did not affect the outcome of the proliferative responses but partially or completely reversed the blocking activity of the mAb. In contrast, IL 2 significantly enhanced the alloproliferation induced by the T lymphoblastoid cell lines, and the anti-HLA class II mAb partially antagonized this effect. Taken together, these data suggest that unlike the HLA-DR and -DQ gene products on B cells, those on IL 2-dependent long-term cultured T cells do not play a direct or primary stimulatory role in the mixed lymphocyte reaction; the reduced levels of alloproliferation induced by the T cell lines are, at least in part, due to a defective production of endogenous IL 2 by the responder lymphocytes rather than to a defective expression of IL 2 receptors by the alloproliferative T cell subset; and the anti-HLA class II mAb in these cultures act only at the responder cell level, since they can efficiently block the enhancement of T cell proliferation triggered by exogenous IL 2, but not the proliferative responses induced by T cell lines in standard conditions.     

Virus load and sequence variation in simian retrovirus type 2 infection

The natural history of type D simian retrovirus (SRV) infection is poorly characterized in terms of viral load, antibody status, and sequence variation. To investigate this, blood samples were taken from a small cohort of mostly asymptomatic cynomolgus macaques (Macaca fascicularis), naturally infected with SRV type 2 (SRV-2), some of which were followed over an 8-month period with blood taken every 2 months. Provirus and RNA virus loads were obtained, the samples were screened for presence of antibodies to SRV-2 and neutralizing antibody titers to SRV-2 were assayed. env sequences were aligned to determine intra- and intermonkey variation over time. Virus loads varied greatly among cohort individuals but, conversely, remained steady for each macaque over the 8-month period, regardless of their initial levels. No significant sequence variation was found within an individual over time. No clear picture emerged from these results, which indicate that the variables of SRV-2 infection are complex, differ from those for lentivirus infection, and are not distinctly related to disease outcome.     

Simian retrovirus-D serotype 1 (SRV-1) envelope glycoproteins gp70 and gp20: expression in yeast cells and identification of specific antibodies in sera from monkeys that recovered from SRV-1 infection.

The gp70 and transmembrane gp20 envelope proteins of simian retrovirus-D serotype 1 (SRV-1) were expressed in Saccharomyces cerevisiae as fusion proteins with human superoxide dismutase (SOD). Expression of the SOD-gp70 and SOD-gp20 sequences yielded fusion proteins of 52 and 29 kilodaltons, respectively. The yeast-expressed SRV-1 envelope proteins were used in an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies in the sera of rhesus macaques that recovered from SRV-1. Sera from 47 of 49 such monkeys tested positive for antibodies to the SOD-gp70 fusion protein, while 45 of 49 reacted positively to SOD-gp20. None of 26 SRV-1-nonexposed monkeys tested positive in either ELISA. Monkeys immunized with the recombinant SRV-1 gp20 and gp70 proteins made good ELISA and Western blot (immunoblot) antibodies to whole SRV-1. This antibody was not neutralizing in vitro, however.     

Immortalization of macrophages from mouse bone marrow and fetal liver

Fresh bone marrow (BM)-derived cells infected with the J2 recombinant retrovirus (carrying v-myc and v-raf/mil oncogenes) grow as immortal cell lines belonging to the monocytic lineage. BM cells cultured for 24 h in conventional medium are no longer able to grow following infection with the J2 virus. We investigated whether specific growth factors affected the proliferative response of BM cells to the J2 virus. If the BM cells were cultured for 24 h in the presence of concanavalin A or CSF-1 and then infected with the J2 virus, immortalization of BM cells was observed. Under these conditions, the cell lines that we obtained were shown to belong to the monocytic lineage. We investigated whether target cells for the J2 virus existed in other hematopoietic organs. We observed J2-induced proliferation in fetal liver (FL) but not in spleen or thymus. The cells proliferating in the FL had macrophage characteristics during the early passages. However, some macrophage markers were lost upon extensive in vitro culture. We conclude that we have identified conditions in which J2 virus consistently and selectively stimulates the growth of macrophages from murine bone marrow and a wider range of hematopoietic cells from fetal liver.     

Elongated cells derived from rat mammary cuboidal epithelial cell lines resemble cultured mesenchymal cells in their pattern of protein synthesis

Two-dimensional gel electrophoresis has been used to identify polypeptide patterns characteristic of rat mammary cuboidal epithelial cells or mesenchyme-derived cells. Elongated cells and cell lines derived from cloned cuboidal epithelial cells in culture possess a polypeptide pattern which resembles that of the cultured mesenchymal cells rather than that of the cuboidal epithelial cells from which they were derived. These elongated converts also resemble cultured mesenchymal cells in possessing a Triton-insoluble matrix in which vimentin and not prekeratin predominates.     

Evidence for mesenchymal-epithelial transition associated with mouse hepatic stem cell differentiation

Mesenchymal-epithelial transition events are related to embryonic development, tissue construction, and wound healing. Stem cells are involved in all of these processes, at least in part. However, the direct evidence of mesenchymal-epithelial transition associated with stem cells is unclear. To determine whether mesenchymal-epithelial transition occurs in liver development and/or the differentiation process of hepatic stem cells in vitro, we analyzed a variety of murine liver tissues from embryonic day 11.5 to adults and the colonies derived from hepatic stem/progenitor cells isolated with flow cytometry. The results of gene expression, immunohistochemistry and Western blot showed that as liver develops, the expression of epithelial markers such as Cytokeratin18 and E-cadherin increase, while expression of mesenchymal markers such as vimentin and N-cadherin decreased. On the other hand, in freshly isolated hepatic stem cells, the majority of cells (65.0%) co-express epithelial and mesenchymal markers; this proportion is significantly higher than observed in hematopoietic cells, non-hematopoietic cells and non-stem cell fractions. Likewise, in stem cell-derived colonies cultured over time, upregulation of epithelial genes (Cytokeratin-18 and E-cadherin) occurred simultaneously with downregulation of mesenchymal genes (vimentin and Snail1). Furthermore, in the fetal liver, vimentin-positive cells in the non-hematopoietic fraction had distinct proliferative activity and expressed early the hepatic lineage marker alpha-fetoprotein. CONCLUSION: Hepatic stem cells co-express mesenchymal and epithelial markers; the mesenchymal-epithelial transition occurred in both liver development and differentiation of hepatic stem/progenitor cells in vitro. Besides as a mesenchymal marker, vimentin is a novel indicator for cell proliferative activity and undifferentiated status in liver cells.     

Inhibition of in vitro T cell activation by corneal endothelial cells.

Cells and tissues of the anterior uvea and aqueous humor express activities which inhibit immune responses. These activities include soluble factors such as TGF-beta and uncharacterized cell surface interactions. Relatively little is known regarding the immunologic activities of corneal endothelium, despite its potentially important role in contributing to the immune privilege of the anterior chamber and the high success rate of corneal transplantation. In this report, in vitro studies of cultured rat corneal endothelial (CE) cells were done using S-antigen-specific LEW rat T cell lines, or S-antigen-specific T cell hybridomas, to examine the immunologic capabilities of CE cells. Monolayers of LEW rat CE cells were unable to present antigen or a mitogen, Con A, to T cell lines or hybridomas as assessed by the lack of a proliferative response or IL-2 secretion. Furthermore, the CE cells exerted a potent inhibitory effect when added to in vitro proliferation assays of T cell lines stimulated with antigen or Con A. When T cells were preactivated on conventional antigen presenting cells and then transferred to wells containing CE cells, their proliferation was not inhibited. Although CE cells inhibited activation of T cell lines and hybridomas, they did not inhibit the growth of T cell hybridomas or CTLL cells, nor did the CE cells adversely affect the viability of resting T cells cultured on CE monolayers. The inhibitory effect was reversible as preincubation of T cells on CE cells for up to 6 days followed by washes restored T cell responsiveness when assayed on splenocytes. The inability to stimulate proliferative responses was not affected by preincubation of the CE cells with lymphokines which increase MHC antigen expression. The inhibition observed in these assays was not MHC-restricted as CE cells from both LEW and BN rats were equally inhibitory. CE cells from rabbits and cats were also potent inhibitors of T cell activation, suggesting that the mechanism is evolutionarily conserved. The mechanism of inhibition of CE cells is unknown at this time.