Tinjil island, a natural habitat breeding facility of simian retrovirus-free Macaca fascicularis.

Institut Pertanian Bogor (Bogor Agricultural University) has established a collaborative agreement with the Indonesian Ministry of State for Population and Environment and the United States Primate Research Consortium, consisting of the University of Washington Regional Primate Research Center (UW-RPRC), the Oregon Regional Primate Research Center (ORPRC), and the Bowman Gray School of Medicine at the Wake Forest University, to populate and manage a breeding facility of longtailed macaques (Macaca fascicularis) on Tinjil island, a 6 km2 island off the southern coast of West Java, Indonesia. Screening protocols have been established to select only simian retrovirus (SRV)-free animals for the colony. Animals originating in either West Java or Sumatra were individually caged and screened over a period of 3-5 months for the presence of SRV and tuberculosis. Whole blood specimens were taken from seronegative animals for virus isolation. Two months after the first screening, all negative animals were retested for SRV antibody and virus isolation. All animals remaining negative after this testing procedure and which have at least four consecutive negative TB tests were transported to the island. To date, 1,306 animals have been screened with 478 released to the island, and at least 750 babies were born on the island. Three batches of progeny of 45-50 juveniles each have been retrieved from the island, and are being used in AIDS-related research projects. © 1994 Wiley-Liss, Inc.

Verminous vasculitis, pneumonia and pulmonary infarction in a cynomolgus monkey after treatment with ivermectin.

An apparently healthy cynomolgus monkey (Macaca fascicularis) died 2 hours after routine inhalation anesthesia and implantation of a femoral catheter. Gross necropsy findings included patchy raised areas of severe pulmonary hemorrhage and consolidation. Filarioid nematodes (Edesonfilaria malayensis) were located in pulmonary blood vessels and in numerous 0.1-2 cm fibrous cysts on the pleural surfaces of the lungs, pericardium, diaphragm, retroperitoneum, and in the urinary bladder wall. Microscopic lesions included verminous vasculitis, pulmonary infarcts and pneumonia. Many of the nematodes were more necrotic than the surrounding host tissue. During quarantine, 17 days before surgery, the monkey had been given a single dose of ivermectin (200 micrograms/Kg, intramuscular) as an anthelminthic for gastrointestinal nematodes. It is postulated that many of the filarioid nematodes were killed by this treatment. These parasitic emboli caused pulmonary infarction and the severe inflammatory reaction. The resulting pulmonary disease compromised pulmonary function and contributed to death after anesthesia. This complication should be considered if monkeys possibly harboring filarioid nematodes are treated with ivermectin.

Retroperitoneal fibromatosis and acquired immunodeficiency syndrome in macaques. Pathologic observations and transmission studies.

A peculiar fibroproliferative syndrome called retroperitoneal fibromatosis (RF) has been observed in Macaca nemestrina, Macaca mulatta, Macaca fascicularis, and Macaca fuscata at the Washington Regional Primate Research Center. RF is characterized by the aggressive proliferation of highly vascular fibrous tissue subjacent to the peritoneum covering the ileocecal junction and associated mesenteric lymph nodes. In the early, proliferative phase of the disease, most of the fibroblastlike cells contain Factor VIII-related antigen. Two syndromes have been recognized: localized, in which fibroproliferative lesions occur only in solitary nodules; and progressive, in which fibromatosis occurs throughout the abdominal cavity. RF-affected monkeys often develop a simian acquired immunodeficiency syndrome (SAIDS) with severe thymic and lymphoid atrophy, chronic enterocolitis, and wasting. Experimental intraperitoneal inoculation with suspensions of RF tissue in two separate experiments resulted in the development of SAIDS in 5 of 16 and RF-SAIDS in 3 of 16 macaques. RF associated with SAIDS appears to be an excellent model for the Kaposi's sarcoma associated with AIDS.

Normal immunologic response to a neoantigen, bacteriophage phiX-174, in baboons with long-term lymphohematopoietic reconstitution from highly purified CD34+ Lin- allogeneic marrow cells.

The CD34 antigen is thought to be expressed by hematopoietic stem cells in adult humans and nonhuman primates. We present data that baboons transplanted with highly purified allogeneic CD34+ marrow cells devoid of detectable mature and immature T and B lymphocytes and myeloid cells, isolated from sex-mismatched mixed lymphocyte culture (MLC) nonreactive siblings, have maintained stable lymphohematopoietic engraftment with donor cells for greater than 4.9, greater than 6.0, and 5.0 years. Cytogenetic analysis of unfractionated marrow and peripheral blood cells at multiple time points after transplantation show virtually all donor cells in two animals and stable mixed chimerism in the third. We used polymerase chain reaction to show that colony-forming unit-granulocyte-macrophage, burst-forming unit-erythroid, and high proliferative potential colony-forming cells (HPP-CFC) were virtually all of donor origin in two animals and present at lower levels in the stable mixed chimera. CD20+ B-lymphoblastoid cell lines derived by Herpesvirus Papio transformation of peripheral blood cells were virtually all donor in two animals and 50% donor in the mixed chimera. CD4+ and CD8+ T cells and neutrophils purified from the peripheral blood of the two female animals also were all donor-derived. To assess immunologic function after transplantation, we immunized the three long-term chimeric animals and two normal control animals with bacteriophage phiX-174, a neoantigen that requires the interaction of antigen-presenting cells, T lymphocytes, and B lymphocytes to mount a normal antibody response. Experimental and control animals, when immunized with bacteriophage, had similar serum Ig levels. The experimental and control animals generated similar titers of antibacteriophage antibodies after primary and secondary immunizations with evidence of amplification and class switching. These findings further support the hypothesis that the CD34+ antigen is expressed on hematopoietic stem cells that can mediate stable long-term lymphohematopoiesis in vivo and, importantly, that normal immunologic function can be reconstituted in vivo after transplantation of the highly purified CD34+ Lin- cells alone.

A primate model for chancroid.

Adult pigtailed macaques (Macaca nemestrina) were evaluated for their usefulness as a primate model for chancroid. To initiate infection, 10(7)-10(8) cfu of Haemophilus ducreyi were inoculated into the foreskins of 5 adult males and into the vaginal labia of 4 adult females. Lesions developed in the male macaques that were similar in appearance, histopathologic changes, and progression to those of human disease, including the development of ulcers 6-12 days after infection. In addition, H. ducreyi could be recovered from the lesions up to 20 days after inoculation, humoral antibodies were induced beginning 1 week after inoculation, and inguinal lymphadenopathy was noted in 4 of the 5 males. None of the 4 female macaques inoculated with the same preparation of live H. ducreyi developed comparable lesions. Thus, experimental chancroid in adult male macaques closely resembles human disease and should be useful for future studies of the pathogenesis of chancroid.

Rapid engraftment by peripheral blood progenitor cells mobilized by recombinant human stem cell factor and recombinant human granulocyte colony-stimulating factor in nonhuman primates.

We have previously shown that administration of low-dose recombinant human stem cell factor (rhSCF) plus recombinant human granulocyte colony-stimulating factor (rhG-CSF) to baboons mobilizes greater numbers of progenitor cells in the blood than does administration of rhG-CSF alone. The purpose of the present study was to determine whether marrow repopulating cells are present in the blood of nonhuman primates administered low-dose rhSCF plus rhG-CSF, and if present, whether these cells engraft lethally irradiated recipients as rapidly as blood cells mobilized by treatment with rhG-CSF alone. One group of baboons was administered low-dose rhSCF (25 micrograms/kg/d) plus rhG-CSF (100 micrograms/kg/d) while a second group received rhG-CSF alone (100 micrograms/kg/d). Each animal underwent a single 2-hour leukapheresis occurring the day when the number of progenitor cells per volume of blood was maximal. For baboons administered low-dose rhSCF plus rhG-CSF, the leukapheresis products contained 1.8-fold more mononuclear cells and 14.0-fold more progenitor cells compared to the leukapheresis products from animals treated with rhG-CSF alone. All animals successfully engrafted after transplantation of cryopreserved autologous blood cells. In animals transplanted with low-dose rhSCF plus rhG-CSF mobilized blood cells, we observed a time to a platelet count of > 20,000 was 8 days +/- 0, to a white blood cell count (WBC) of > 1,000 was 11 +/- 1 days, and to an absolute neutrophil count (ANC) of > 500 was 12 +/- 1 days. These results compared with 42 +/- 12, 16 +/- 1, and 24 +/- 4 days to achieve platelets > 20,000, WBC > 1,000, and ANC > 500, respectively, for baboons transplanted with rhG-CSF mobilized blood cells. Animals transplanted with low-dose rhSCF plus rhG-CSF mobilized blood cells had blood counts equivalent to pretransplant values within 3 weeks after transplant. The results suggest that the combination of low-dose rhSCF plus rhG-CSF mobilizes greater numbers of progenitor cells that can be collected by leukapheresis than does rhG-CSF alone, that blood cells mobilized by low-dose rhSCF plus rhG-CSF contain marrow repopulating cells, and finally that using a single 2-hour leukapheresis to collect cells, the blood cells mobilized by low-dose rhSCF plus rhG-CSF engraft lethally irradiated recipients more rapidly than do blood cells mobilized by rhG-CSF alone.

Recombinant human ligand for MPL, megakaryocyte growth and development factor (MGDF), stimulates thrombopoiesis in vivo in normal and myelosuppressed baboons.

Megakaryocyte growth and development factor (MGDF) is a ligand for c-mpl and a member of the hematopoietic growth factor superfamily. Recombinant murine MGDF specifically stimulates thrombopoiesis in mice. Recombinant human (rHu) MGDF stimulates megakaryocytic differentiation of baboon CD34+ marrow cells in vitro. Therefore, we determined the in vivo biological effects of rHuMGDF administered to normal baboons in the absence and presence of myelosuppression with 5-fluorouracil (5-FU). rHuMGDF was administered to normal baboons as a single s.c. injection at doses of 1, 10, 25 and 50 micrograms/kg/day for 10 days and, as a control, heat-inactivated MGDF was administered at a dose of 10 micrograms/kg/day. Platelet counts were markedly increased in all animals administered native rHuMGDF but not in animals given heat-inactivated rHuMGDF. Platelet counts began to increase between three and six days after starting rHuMGDF administration and the maximum average increases were 1.7-, 3.4-, 5.1- and 4.0-fold above baseline in animals administered 1, 10, 25 and 50 micrograms/kg/day, respectively. Maximum platelet counts were reached between 7 and 10 days after starting rHuMGDF and maintained for four days after the last dose. Thereafter, platelet counts decreased, reaching stable pretreatment values between 11 and 14 days after the last dose of rHuMGDF. No changes in red cell mass, peripheral blood white blood cell counts or differentials were observed during rHuMGDF treatment. For animals administered 10, 25 and 50 micrograms/kg/day of rHuMGDF, megakaryocytes increased more than threefold in marrow, were markedly enlarged, and had increased numbers of lobes. Overall marrow cellularity remained unchanged, as did red cell and white cell morphology. No marrow fibrosis was detected. Progenitor cells were not increased in marrow but did increase modestly in the peripheral blood, associated with increased numbers of CD34+ cells in the circulation. Following a single dose of 5-FU (120 mg/kg) animals were given either saline or pegylated (PEG) rHuMGDF (25 micrograms/kg/day) for 14 days. Platelet counts recovered to baseline by 13.8 +/- 1.8 days for PEG-rHuMGDF-treated baboons compared with 16.8 +/- 0.6 days for saline treated controls. Marrow biopsies revealed more rapid recovery of overall marrow cellularity and megakaryocytes in PEG-rHuMGDF-treated animals compared with controls. Thus, rHuMGDF specifically stimulates thrombopoiesis in normal and myelosuppressed baboons. rHuMGDF may be useful for stimulating thrombopoiesis in humans in clinical settings after myelosuppression.

The ligand for c-kit, stem cell factor, stimulates the circulation of cells that engraft lethally irradiated baboons.

Recombinant human stem cell factor (SCF), the ligand for c-kit, has been shown to stimulate increased numbers of hematopoietic progenitor cells of multiple types to circulate in the blood of baboons, but it was not known if the cells stimulated to circulate by SCF contained cells capable of engrafting and rescuing lethally irradiated baboons. Peripheral blood mononuclear cells (PBMNC) were collected by leukapheresis from four untreated control baboons and from three baboons on the 10th or 11th day of treatment with SCF (200 micrograms/kg/d). All animals were transplanted with 1.00 to 1.04 x 10(8)/kg of cryopreserved autologous PBMNC after treatment with a single dose of 1,020 cGy total body irradiation (TBI). Three animals were transplanted with PBMNC that had been collected during SCF treatment, 24 to 38 days after the last dose of SCF. Rapid trilineage engraftment was documented by bone marrow biopsy in all three. The mean time to a total white blood cell count (WBC) > or = 500/microL, WBC > or = 1,000/microL, and an absolute neutrophil count (ANC) > or = 500/microL was 15 +/- 3 (mean +/- SD), 19 +/- 1, and 19 +/- 2 days, respectively. Two animals remain alive with stable engraftment more than 180 and 245 days posttransplant. The third died of sepsis 32 days posttransplant with a hypercellular marrow showing trilineage engraftment. The surviving animals were transfusion independent by 10 and 59 days posttransplant. Four control animals were transplanted with PBMNC collected in the absence of SCF stimulation. One was treated for 11 days with SCF (200 micrograms/kg/d) after PBMNC were collected. This animal was transplanted 25 days after the last dose of SCF. None of the four control animals engrafted and they died 13, 16, 28, and 38 days posttransplant with marrow aplasia. Treatment with SCF stimulates the circulation of cells that engraft and rescue lethally irradiated baboons. The characteristics of the transplantable cells present in the circulation are now amenable to direct study.

Retroperitoneal fibromatosis and acquired immunodeficiency syndrome in macaques: clinical and immunologic studies.

A simian acquired immunodeficiency syndrome (SAIDS) associated with retroperitoneal fibromatosis (RF) has been observed in several species of macaque at the Washington Regional Primate Research Center. Clinical signs were recurrent diarrhea, weight loss, mesenteric lymphadenopathy, and opportunistic infections. Most affected macaques in the later stages of illness showed marked immunodeficiency. Response of peripheral blood mononuclear cells to mitogens was impaired significantly. There was sharply depressed primary and secondary antibody response to the T-cell dependent antigen, bacteriophage phi X174. Affected monkeys did not switch from IgM to IgG antibody following a secondary immunization, as did normal macaques. Twenty-four (67%) of 36 affected animals with progressive RF or deteriorated stages of illness had hypoproteinemia and hypoalbuminemia. Quantitative serum immunoglobulins of 23 cases showed that eight (35%) had hypogammaglobulinemia, six (26%) had hypergammaglobulinemia, and the remainder (39%) were within the normal range. Opportunistic infections were predominantly bacterial pathogens. Type D retrovirus appeared to be closely associated with RF-affected macaques (12/12 or 100%). The case fatality rate (including animals sacrificed after prolonged illness) was 98%. The leading cause of death was due directly to RF lesions in 43%, to enterocolitis in 36%, septicemia in 12%, amyloidosis in 5%, and malignant lymphoma (2%). Clinical, immunologic and pathologic changes reveal an acquired immunodeficiency syndrome that has many similarities to human AIDS. SAIDS and RF may be a useful model for studying human AIDS.

CD34+ marrow cells, devoid of T and B lymphocytes, reconstitute stable lymphopoiesis and myelopoiesis in lethally irradiated allogeneic baboons.

CD34+ cells devoid of detectable mature and immature T and B lymphocytes, expressing the CD2, CD10, and CD20 antigens, were isolated from marrows of three pairs of sex-mismatched, mixed lymphocyte culture (MLC) nonreactive, sibling baboons. Reciprocal transplants were performed between members of each pair, using the sex chromosomes, identified by standard cytogenetic techniques, as markers of the transplanted cells. Five animals from these three pairs were transplanted with 0.6 to 2.1 x 10(6)/kg of isolated cryopreserved and/or fresh isolated cells that were greater than 95% to 97% CD34+. Before transplantation, animals were treated with either single (920 or 1,020 cGy) or split (700 cGy x 2) dose total body irradiation. All animals engrafted with donor cells, as demonstrated by cytogenetic analysis of bone marrow metaphase cells 4 weeks after transplantation, with days to white blood cell count (WBC) greater than 500 being 19 +/- 2, to WBC greater than 1,000 23 +/- 2, to absolute neutrophil count greater than 500 24 +/- 3, and to platelets greater than 20,000 30 +/- 7. Three animals died of infectious-related complications at 34, 42, and 109 days after transplantation with evidence of host and donor cells (mixed chimerism) in marrow. Two animals remain alive and healthy more than 545 and 455 days after transplantation with stable mixed chimerism in marrow and blood. For these two animals, cytogenetic analysis of granulocyte/macrophage and erythroid colonies derived from marrow precursors between weeks 25 and 42 posttransplant showed evidence of mixed chimerism. Cytogenetic studies of CD2+ T cells and CD20+ B cells isolated from blood of these two animals between weeks 21 and 51 posttransplant showed the presence of mixed chimerism in both lymphocyte populations. Thus, isolated allogeneic CD34+ marrow cells devoid of detectable mature and immature T and B lymphocytes can engraft and reconstitute stable long-term myelopoiesis and lymphopoiesis in lethally irradiated baboons. These results are consistent with the hypothesis that CD34+ marrow cells contain pluripotent hematopoietic stem cells capable of fully reconstituting lymphohematopoiesis in the transplanted host.

A c-kit ligand, recombinant human stem cell factor, mediates reversible expansion of multiple CD34+ colony-forming cell types in blood and marrow of baboons.

The ligand for the human c-kit, recombinant human stem cell factor (SCF), was administered to baboons at doses of 200, 100, 50, 25, and 10 micrograms/kg/d. SCF induced a dose-dependent expansion of hematopoietic colony-forming cells (CFC) of multiple types in both blood and marrow, including colony-forming unit (CFU) granulocyte-monocyte, burst-forming unit-erythroid, CFU-MIX, and high proliferative potential-CFC. These changes were associated with a dose-dependent leukocytosis, involving all leukocyte lineages, a reticulocytosis, and increases in marrow cellularity. At 200 micrograms/kg/d of SCF, CFC in blood were increased 10-fold to greater than 100-fold. This correlated with an increased frequency of CD34+ cells in blood. The frequency of CFC in blood approached that of marrow in some animals. These changes were reversed within 7 to 14 days of stopping SCF. The results of these studies suggest a role for the c-kit ligand in stimulating the expansion of multiple CFC types in blood and marrow for potential therapeutic purposes.

In vivo synergy between recombinant human stem cell factor and recombinant human granulocyte colony-stimulating factor in baboons enhanced circulation of progenitor cells.

Recombinant human stem cell factor (rhSCF) and recombinant human granulocyte colony-stimulating factor (rhG-CSF) are synergistic in vitro in stimulating the proliferation of hematopoietic progenitor cells and their precursors. We examined the in vivo synergy of rhSCF with rhG-CSF for stimulating hematopoiesis in vivo in baboons. Administration of low-dose (LD) rhSCF (25 micrograms/kg) alone did not stimulate changes in circulating WBCs. In comparison, administration of LD rhSCF in combination with rhG-CSF at 10 micrograms/kg or 100 micrograms/kg stimulated increases in circulating WBCs of multiple types up to twofold higher than was stimulated by administration of the same dose of rhG-CSF alone. When the dose of rhG-CSF is increased to 250 micrograms/kg, the administration of LD rhSCF does not further increase the circulating WBC counts. Administration of LD rhSCF in combination with rhG-CSF also stimulated increased circulation of hematopoietic progenitors. LD rhSCF alone stimulated less of an increase in circulating progenitors, per milliliter of blood, than did administration of rhG-CSF alone at 100 micrograms/kg. Baboons administered LD rhSCF together with rhG-CSF at 10, 100, or 250 micrograms/kg had 3.5- to 16-fold higher numbers per milliliter of blood of progenitors cells of multiple types, including colony-forming units granulocyte/macrophage (CFU-GM), burst-forming unit-erythroid (BFU-E), and colony-forming and burst-forming units-megakaryocyte (CFU-MK and BFU-MK) compared with animals given the same dose of rhG-CSF without rhSCF, regardless of the rhG-CSF dose. The increased circulation of progenitor cells stimulated by the combination of rhSCF plus rhG-CSF was not necessarily directly related to the increase in WBCs, as this effect on peripheral blood progenitors was observed even at an rhG-CSF dose of 250 micrograms/kg, where coadministration of LD rhSCF did not further increase WBC counts. Administration of very-low-dose rhSCF (2.5 micrograms/kg) with rhG-CSF, 10 micrograms/kg, did not stimulate increases in circulating WBCs, but did increase the number of megakaryocyte progenitor cells in blood compared with rhG-CSF alone. LD rhSCF administered alone for 7 days before rhG-CSF did not result in increased levels of circulating WBCs or progenitors compared with rhG-CSF alone. Thus, the synergistic effects of rhSCF with rhG-CSF were both dose- and time-dependent. The doses of rhSCF used in these studies have been tolerated in vivo in humans.(ABSTRACT TRUNCATED AT 400 WORDS)

Recombinant human stem cell factor, a c-kit ligand, stimulates hematopoiesis in primates.

Recombinant human stem cell factor (SCF) is homologous with recombinant rat SCF (rrSCF) and is a ligand for c-kit. We determined the influence of SCF on hematopoiesis in vitro and in vivo in baboons. In vitro, SCF alone stimulated little growth of hematopoietic colony-forming cells from baboon marrow, but did increase the number of colonies formed in response to erythropoietin (Epo), interleukin-3 (IL-3), and granulocyte-macrophage colony-stimulating factor (GM-CSF). In vivo, SCF caused an increase in the peripheral blood of the number of erythrocytes, neutrophils, lymphocytes, monocytes, eosinophils, and basophils. In marrow, it caused an increase in marrow cellularity and in the absolute number of colony-forming unit-granulocyte-monocyte (CFU-GM) and burst-forming unit-erythroid (BFU-E) in marrow following infusion of SCF. The in vivo stimulation of multiple lymphohematopoietic lineages corroborates previous in vitro studies and suggests a potentially important clinical role for SCF.