Diffusion Chambers In Mice Research Articles

Development of a recombinant vaccine against human onchocerciasis

ABSTRACT Introduction Human onchocerciasis caused by the filarial nematode parasite Onchocerca volvulus remains a major cause of debilitating disease infecting millions primarily in Sub-Saharan Africa. The development of a prophylactic vaccine, along with mass drug administration, would facilitate meeting the goal of onchocerciasis elimination by 2030. Areas covered Models used to study immunity to Onchocerca include natural infection of cattle with Onchocerca ochengi and O. volvulus infective third-stage larvae implanted within diffusion chambers in mice. A vaccine, comprised of two adjuvanted recombinant antigens, induced protective immunity in genetically diverse mice suggesting that it will function similarly in diverse human populations. These antigens were recognized by immune humans and also induced protective immunity against Brugia malayi. We describe the development of a fusion protein composed of the two vaccine antigens with the plan to test the vaccine in cows and non-human primates as a prelude to the initiation of phase 1 clinical trials. Expert opinion The adjuvanted O. volvulus vaccine composed of two antigens Ov-103 and Ov-RAL-2 was shown to be consistently effective at inducing protective immunity using multiple immune mechanisms. The vaccine is ready for further evaluation in other animal models before moving to clinical trials in humans.

IL-4- and IL-5-dependent protective immunity to Onchocerca volvulus infective larvae in BALB/cBYJ mice.

A significant reduction in challenge worm survival occurred when BALB/cBYJ mice were vaccinated against Onchocerca volvulus infective third stage larvae (L3) by using irradiated O. volvulus L3. Challenge infections consisted of L3 implanted in diffusion chambers, which were used as a means to contain, and thus efficiently recover, the larvae from the host. The goal of the present study was to describe the mechanism of immune-mediated killing of O. volvulus L3 in diffusion chambers in mice. Direct contact between host cells and parasites was required for killing of larvae in immunized hosts. To define the mechanism of immune-mediated killing in this system, the time of influx of cells and cytokines into the infection site was compared with the time challenge infections were killed. The only cell type that was found to increase in diffusion chambers in immunized mice was eosinophils; maximal levels of eosinophils were coincident with the time of parasite killing. IL-5 was found in diffusion chambers of immunized mice coincident with the time of parasite killing; IL-5 was not found in diffusion chambers recovered from control mice. Significant levels of IFN-gamma were absent in the diffusion chambers of both groups. Immunized mice were treated with mAb to eliminate IL-5 or IL-4 to assess the role these cytokines or their by-products play in larval killing. Elimination of either IL-5 or IL-4 significantly reduced the protective effects of vaccination against larval O. volvulus.

Radioprotective potential of primitive hematopoietic precursors forming colonies in diffusion chambers in mice

The purpose of this study was to determine the radioprotective ability of primitive hematopoietic precursors which form colonies in diffusion chambers in mice (CFU-D). Thirty-two lethally irradiated female ICR mice were injected with 5 to 7 male ICR mouse bone marrow-derived CFU-D colonies each. Fourteen of these mice survived over 30 days and were sacrificed at intervals up to a year. As a control, 20 lethally irradiated female ICR mice received cells from intercolony areas. All of these mice died before day 20. DNA samples obtained from hematopoietic organs and liver from 8 sacrificed mice were analyzed for the presence of CFU-D colony-derived cells. Only in 1 ICR mouse was CFU-D colony origin DNA detected by Southern analysis in all hematopoietic organs: bone marrow, spleen, thymus and lymph nodes. In 6 mice, only selected hematopoietic organs were repopulated by CFU-D colony-derived cells as judged by Southern analysis. In some of these mice, the remaining hematopoietic organs contained small CDU-D-derived cell populations which could be detected by more sensitive polymerase chain reaction (PCR). In 1 mouse, the presence of CFU-D-derived cells in all hematopoietic organs was only demonstrated by PCR. These findings suggest that lethally irradiated mice can be rescued by CFU-D-derived daughter cells. They appear to have the potential to give rise to clones containing lymphoid and myeloid cells in all hematopoietic organs, at least temporarily. Thus, it can be concluded that CFU-D represents a very primitive hematopoietic precursor cell with radioprotective capability.

Potentiality of the Murine Colony-Forming Cells Detected by an In Vivo Diffusion Chamber Culture System

AbstractCulture of a mixture of bone marrow cells with and without T6 chromosome marker in diffusion chambers in mice yielded colonies (CFU-DG) containing cells of a single karyotype, suggesting clonality. Injection of individual CFU-DG colonies into lethally irradiated mice resulted in increased spleen colony formation on day 12 (CFU-S). The possibility of endogenous origin was excluded by demonstrating the presence of T6 marker in both CFU-DG and CFU-S colonies in karyotypically normal hosts. These findings suggest that the cells giving rise to granulocytic colonies in diffusion chambers also can give rise to multipotential hemopoietic cells.

Potentiality of the murine colony-forming cells detected by an in vivo diffusion chamber culture system

Culture of a mixture of bone marrow cells with and without T6 chromosome marker in diffusion chambers in mice yielded colonies (CFU- DG) containing cells of a single karyotype, suggesting clonality. Injection of individual CFU-DG colonies into lethally irradiated mice resulted in increased spleen colony formation on day 12 (CFU-S). The possibility of endogenous origin was excluded by demonstrating the presence of T6 marker in both CFU-DG and CFU-S colonies in karyotypically normal hosts. These findings suggest that the cells giving rise to granulocytic colonies in diffusion chambers also can give rise to multipotential hemopoietic cells.

Separation by velocity sedimentation of human haemopoietic precursors forming colonies in vivo and in vitro cultures.

Cells which give rise to granulocyte-macrophage colonies under the influence of peripheral blood white cells (CFU-c (WBC] and Mo T cell conditioned medium (CFU-c (Mo] sedimented at a faster rate than the cells which form mixed erythroid-granulocytic colonies in methylcellulose in vitro (CFU-mix) and granulocytic (CFU-dg) and megakaryocytic (CFU-dm) colonies in diffusion chambers in mice. Despite identical peak sedimentation rate for the two CFU-c populations, sedimentation profiles suggest that they are heterogeneous with respect to size. A proportion of CFU-c (Mo) may be identical with CFU-dg and CFU-mix. Sedimentation profiles for cells which give rise to mixed colonies in vitro (CFU-mix) and to granulocytic colonies in diffusion chambers in cyclophosphamide pretreated mice (CFU-dg (CY] and in Mo conditioned medium treated mice (CFU-dg (Mo] were similar. On the average CFU-dm sedimented somewhat slower than CFU-dg. These and other observations suggesting a close relationship between CFU-dg and multipotential haemopoietic precursors are discussed.

The role of polyamine biosynthesis in hematopoietic precursor cell proliferation in mice

Under the influence of a selective irreversible inhibitor of ornithine decarboxylase (ODC), DL-alpha-difluoromethylornithine (DFMO), early hematopoiesis was enhanced. In the bone marrow, the absolute number of cells that give rise to spleen colonies in lethally irradiated mice (CFU-S), granulocytic colonies in diffusion chambers in mice (CFU-DG), and granulocyte-monocyte colonies in agar in vitro (CFU-C) was increased 2–4 fold. This could be abrogated by administration of putrescine, confirming the association of the stimulatory effect with polyamine biosynthesis most likely via depression of ornithine decarboxylase activity and subsequent synthesis of putrescine. Analysis of cell cycle characteristics by 3H-TdR suicide technique demonstrated that the proportion of CFU-S, CFU-DG, and CFU-C in S-phase was significantly increased. Additionally, the stimulatory effect was reflected by enhanced colony formation in diffusion chambers implanted intraperitoneally in mice receiving DFMO. This could also be eliminated by treatment of the host animal with putrescine, again suggesting that polyamine biosynthesis plays an important role at the early stages of hematopoiesis in vivo. Effect of DFMO on colony formation in vitro (CFU- C) was inhibitory and not reversible with putrescine. It could be partially eliminated by aminoguanidine, which neutralizes diamine oxidase present in fetal calf serum used in the CFU-C assay. These data suggest that the effect of DFMO in vitro was nonspecific.

The Role of Polyamine Biosynthesis in Hematopoietic Precursor Cell Proliferation in Mice

Under the influence of a selective irreversible inhibitor of ornithine decarboxylase (ODC), DL-alpha-difluoromethylornithine (DFMO), early hematopoiesis was enhanced. In the bone marrow, the absolute number of cells that give rise to spleen colonies in lethally irradiated mice (CFU-S), granulocytic colonies in diffusion chambers in mice (CFU-DG), and granulocyte-monocyte colonies in agar in vitro (CFU-C) was increased 2-4 fold. This could be abrogated by administration of putrescine, confirming the association of the stimulatory effect with polyamine biosynthesis most likely via depression of ornithine decarboxylase activity and subsequent synthesis of putrescine. Analysis of cell cycle characteristics by 3H-TdR suicide technique demonstrated that the proportion of CFU-S, CFU-DG, and CFU-C in S-phase was significantly increased. Additionally, the stimulatory effect was reflected by enhanced colony formation in diffusion chambers implanted intraperitoneally in mice receiving DFMO. This could also be eliminated by treatment of the host animal with putrescine, again suggesting that polyamine biosynthesis plays an important role at the early stages of hematopoiesis in vivo. Effect of DFMO on colony formation in vitro (CFU-C) was inhibitory and not reversible with putrescine. It could be partially eliminated by aminoguanidine, which neutralizes diamine oxidase present in fetal calf serum used in the CFU-C assay. These data suggest that the effect of DFMO in vitro was nonspecific.

Human T-Lymphocyte Products Stimulate Human Hemopoietic Progenitor Cell Proliferation in Diffusion Chambers In Vivo

Abstract Human myeloid colony formation in diffusion chambers in mice (CFU-DG) was enhanced following administration of a human T-cell-line-derived conditioned medium (Mo). The Mo cell line also elaborates activities stimulating human myeloid colony formation in vitro in agar (CSF) and potentiating erythroid colony formation in vitro in methylcellulose (EPA). Depletion of CSF from Mo conditioned medium by heat inactivation or gel exclusion chromatography did not affect CFU-DG formation. EPA and CFU-DG stimulating activities are heat stable and have approximately the same molecular weight. Culture of human bone marrow cells in diffusion chambers in mice for 4 days under the influence of Mo conditioned medium resulted in significant increment of BFU-E and CFU-DG as judged by subculture of diffusion chamber contents. No effect on CFU-C could be detected.

Human T-lymphocyte products stimulate human hemopoietic progenitor cell proliferation in diffusion chambers in vivo

Human myeloid colony formation in diffusion chambers in mice (CFU-DG) was enhanced following administration of a human T-cell-line-derived conditioned medium (Mo). The Mo cell line also elaborates activities stimulating human myeloid colony formation in vitro in agar (CSF) and potentiating erythroid colony formation in vitro in methylcellulose (EPA). Depletion of CSF from Mo conditioned medium by heat inactivation or gel exclusion chromatography did not affect CFU-DG formation. EPA and CFU-DG stimulating activities are heat stable and have approximately the same molecular weight. Culture of human bone marrow cells in diffusion chambers in mice for 4 days under the influence of Mo conditioned medium resulted in significant increment of BFU-E and CFU-DG as judged by subculture of diffusion chamber contents. No effect on CFU-C could be detected.

Responsiveness of a human myelogenous leukemia cell line (KG-1) to humoral factors in vivo

A human acute myelogenous leukemia cell line (KG-1) was used to study the humoral regulation of leukemic cell proliferation in diffusion chambers implanted in mice. The mice were treated with several agents known to enhance normal granulopoiesis. Leukemic cell proliferation was measured by total cell numbers and in vivo colony formation in plasma clot. Greatest leukemic cell growth was observed when the host mice were treated with cyclophosphamide. Administration of glucan or endotoxin stimulated growth to a significant but lesser degree. Pretreatment of the mice had no agreeable effect on leukemic cell maturation. To determine whether enhanced leukemic cell proliferation correlated with colony-stimulating activity (CSA) levels in the host animals, sera were collected daily and assayed in agar cultures using mouse bone marrow and KG-1 cells as targets. Sera from endotoxin-treated mice were found to be superior to sera from mice injected with cyclophosphamide in supporting in vitro colony formation. We conclude that there are humoral factor(s) other than CSA which are important in stimulating human leukemic cell growth in diffusion chambers in mice.

Antibody-Dependent Complement-Mediated Killing of Schistosomula in Intraperitoneal Diffusion Chambers in Mice

Abstract The present investigations were designed to determine the biologic role of the different host defense mechanisms against schistosomiasis mansoni that are independent of direct cell-parasite contact. Schistosomula were placed in Millipore chambers of 0.22-µm pore size and were implanted i.p. into immune and nonimmune age- and sex-matched control mice. Significant degree of mortality was detected on recovery of the implanted schistosomula at 24 hr; the mean dead organisms in immune animals was 30 ± 4% as compared to 10 ± 2% in control mice (p < 0.01). This effect on the implanted schistosomula into immune mice was confirmed by injecting these organisms into normal recipients and by recovering those that mature to adult worms. The ability of immune mice to kill significant numbers of implanted schistosomula was dependent on the intensity of infection and developed only after 8 weeks, which parallels the onset of protective immunity in vivo. Adoptive transfer of immune sera to normal recipients resulted in a mean of 23 ± 2% dead schistosomula in the recipient mice, which represents approximately 65% of the protection detected in the donor animals. Depletion of the eosinophils or neutrophils in the immune mice did not alter their ability to kill significant numbers of the implanted schistosomula. Cobra venom factor treatment, on the other hand, resulted in abrogation of the significant schistosomula killing detected in immune mice or in animals adoptively transferred with immune serum. These results demonstrate that specific acquired immunity to schistosomiasis in vivo is partially mediated by humoral factors and is dependent on the presence of complement. The Millipore chambers may help elucidate the significance of the many mechanisms which have already been described for host immune responses against the multicellular parasite Schistosoma mansoni.

Induction of 8-azaguanine- and ouabain-resistant mutants by cyclophosphamide and 1-(pyridyl-3)-3,3-dimethyltriazene in Chinese hamster cells cultured in diffusion chambers in mice

A host-mediated assay is described for induction of 8-azaguanine-resistant (azg r) and ouabain-resistant (oua rr) mutants in Chinese hamster V79 cells cultured in diffusion chambers (DC) in C3H mice. Injection of the hosts with the indirect mutagen/carcinogen cyclophosphamide (CPP) or 1-(pyridyl-3)-3,3-dimethyltriazene (PyDT) caused a dose-dependent increase in mutation frequency at the loci of azg r and oua r in the V79 target cells. Plating efficiency of V79 cells in DC in mice was decreased depending upon the dose of CPP or PyDT given to the hosts. In addition, the relationship between expression time and mutation frequency was examined and discussed. The data support the use of this system as an effective screening procedure for suspected environmental mutagens or carcinogens, especially those that need to be metabolically activated in vivo.

Sister chromatid exchange induced by promutagens/carcinogens in Chinese hamster cells cultured in diffusion chambers in mice.

SummaryA system for in vivo analysis of sister chromatid exchange (SCE) was described. It involved culturing Chinese hamster V-79 cells in diffusion chambers (DC) in mice. Treatment of the hosts with repeated injections of 5-bromodeoxyuridine permitted the demonstration of differentially stained metaphases in the V-79 target cells after using the fluorescence plus Giemsa technique. Thus, it was possible to determine the number of SCE's under in vivo conditions. Induction of SCE in V-79 cells in vivo were studied following treatment with seven known promutagens/carcinogens: Cyclophosphamide, 1-(pyridyl-3)-3,3-dimethyltriazene, dimethylnitrosamine, diethylnitrosamine, benzo(α)-pyrene, 7,12-dimethylbenz(α)anthracene, 3-methylcholanthrene and three nonmutagens/noncarcinogens: perylene, pyrene, and anthracene. With the exception of DEN, all the promutagens/carcinogens caused a significant increase in the frequency of SCE. DEN was effective at the highest dose studied only after pretreatment with Aroclor 1254, ...

Sister chromatid exchanges and growth inhibition induced by the flame retardant tris(2,3-dipromopropyl) phosphate in Chinese hamster cells.

The effects of the flame retardant tris(2,3-dibromopropyl) phosphate (Tris-BP) on growth, sister chromatid exchanges (SCE), and chromosome aberrations of Chinese hamster V79 cells cultured either in vitro or in diffusion chambers (DC) implanted into mice were studied. Tris-BP caused a dose- and time-dependent reduction of cell growth as measured by colony-forming activities. A significant dose-dependent increase in SCE was observed in V79 cells either in the cultures treated with Tris-BP or in DC in mice given injections of the chemical. In contrast, Tris-BP in V79 cells in culture, in V79 cells in DC in mice, in two human lymphoid cell lines, or in mouse bone marrow cells in vivo did not significantly increase chromosome aberrations.

Diffusion chamber cultures for mutagenic and carcinogenic assays

A modified host-mediated assay for testing mutagenicity or carcinogenicity of environmental compounds was described. It involved culturing human lymphoid cells or Chinese hamster V79 cells in diffusion chambers (DC) in mice. A chemical under test was injected into the hosts. Induction of chromosome aberrations, sister chromatid exchanges (SCE), and gene mutations at the loci of 8-azaguanine and ouabain resistance (azgr, ouar) in the target cells were used as indicators. This assay system takes into account the activation or deactivation of a substance, sensitivity, economy in time and cost, and human relevance. The growth potential of cells from ten human lymphoid cell lines and V79 cells in DC in mice were studied. Cells from three human lines failed to grow, while the other seven human lines and V79 cells proliferated well. The growth of human cells in DC in mice differed among the seven lines. For induction of chromosome aberrations, SCE and azgr or ouar mutants in the target cells in DC in mice, the following known promutagens or carcinogens were used: cyclophosphamide (Cy), ifosfamide (If), 1-(pyridyl-3)-3,3-dimethyltriazene (PyDT), dimethylnitrosamine (DMN), diethylnitrosamine (DEN), benzo(α) pyrene (BP), 7,12-dimethylbenz(α)anthracene (DMBA), and 3-methylcholanthrene (MCA). Three noncarcinogenic analogs, pyrene (Py), anthracene (An), and perylene (Pr) were used as negative controls. A dose-related increase in chromosome aberrations was observed in six human lines in DC in mice injected with various doses of Cy. A dose-dependent increase in the number of SCE/cell was observed in two human lines in DC in mice treated with Cy. For V79 cells in DC in mice, the number of SCE/cell at various doses of Cy, If, PyDT, DMN, DEN, BP, DMBA, MCA, Py, An, and Pr were examined. All substances except DEN, Py, Pr, and An caused a significant increase of SCE. Induction of azgr and ouar mutants in V79 cells in DC in mice by PyDT, Cy, and If was studied. A dose-dependent decrease in plating efficiency and a dose-dependent increase in mutation frequency were observed for all three chemicals.

In vitro suppression of normal granulocytic stem cells by inhibitory activity derived from human leukemia cells.

Extracts and conditioned medium of bone marrow and blood cells from patients with acute leukemia were assayed for activity against granulocyte-monocyte colony-forming cells in agar (CFU-C). Cell extracts from 76 of 85 patients reproducibly inhibited colony formation of normal CFU-C by 28–90%. Leukemia cell-derived Inhibitory activity (LIA) was at a maximum In medium conditioned for 3–4 days with leukemia cells. LlA was against CFU-C during S-phase and had no Influence on production of colony-stimulating activity (CSA) by CSA-produclng cells. No evidence of CSA Inactivation was obtained. LlA was thus different from the previously reported PMN-derived Inhibitory activity which only Inhibits CSA production. Most of the crude LIA was lost at 37° C within 5 hours, but 10 minutes of Incubation with CFU-C resulted in maximum inhibition. Size and cell morphology of colonies surviving LIA were not Influenced. Inhibition of CFUC by leukemia cell extract was observed after 6–8 days but not after 14 days of Incubation. Velocity sedimentation experiments demonstrated that this observation was not due to a reversible effect on sensitive CFU-C. Colonies scored after 14 days were derived from CFU-C not sensitive at the time of treatment but were inhibited by multiple additions of leukemia cell extract. Human marrow cells that form granulocyte colonies In fibrin clot diffusion chambers In mice and pre-CFU-C in suspension culture In vitro were not Inhibited by single and multiple additions of leukemia cell extract. Extracts from normal hematopoietic cells did not inhibit exogenously stimulated colony formation. The results suggest that Inhibition was not nonspecifically toxic and may explain suppression of normal hematopoiesis associated with acute leukemia.

Sister chromatid exchanges in human lymphoid cell lines cultured in diffusion chambers in mice: A host-mediated assay system for mutagenicity

A host-mediated assay system for evaluating mutagenic potential of a compound was described. It involved culturing cells from human lymphoid lines in diffusion chambers and implanted into mice. Induction of sister chromatid exchanges (SCE) in the human target cells by injection of the model compound cyclophosphamide to the hosts was used as an index. The numbers of SCE/cell was significantly increased in a dose-dependent manner.

Sister chromatid exchanges induced by cyclophosphamide in V79 cells cultured in diffusion chambers in mice

Chinese hamster cells V79 were cultured in diffusion chambers (DC) and implanted into mice. An exponential growth was observed from the 2nd to 4th day after implantation. The maximum growth was reached on the 6th day. After that, cell growth and viable cell counts decreased. Three days after implantation of DC with V79 cells, the hosts received 6 hourly injections of 0.2 ml of 5-bromodeoxyuridine (BUdR) solution at concentrations of 0.125 to 1.0 × 10 −2 M. DC were removed for chromosome and sister-chromatid exchanges (SCE) analyses 24 h after the first BUdR injection. The frequency of metaphases with differentially stained chromatids, with aberrations, and the number of SCE per cell increased with BUdR dose. The frequency of metaphases with differentially stained chromatids was also positively correlated with the duration of BUdR exposure or the number of hourly injections of BUdR-solution. The effects of cyclophosphamide (CY) in V79 cells in DC in mice were studied. Injections of CY at 2.5, 5, 10 and 15 μg per gram of body weight to the hosts caused an increase in the number of SCE per cell in a linear manner. The results from this study indicate that V79 cells cultured in DC in mice may provide a potential test system for mutagenicity.

The fine structure of monoclonal Hodgkin cells cultured in diffusion chambers

Pleural effusion cells from two patients with stage IV Hodgkin's disease have been cultured continuously in diffusion chambers in mice and studied by electron microscopy after a culture period exceeding 100 days. Cell identity and monoclonal growth in culture has been documented by marker chromosomes (Hossfeld and Schmidt, 1978). These cultured cells grow in close connection, projecting pseudopode-like processes into the intercellular spaces. Most nuclei are lobulated. They always are of low electron density with a norrow rim of condensed chromatin confined to the nuclear membrane. One large prominent nucleolus and up to four smaller nucleoli are found. Nuclear pockets in case 1 and deep cytoplasmic invaginations into the nuclear area in both cases frequently occur. In the cytoplasm, besides microtubuli and fibrils, the Golgi apparatus and mitochondria are the predominant organelles. Most mitochondria appear to be dilated containing fragmented cristae. Free ribosomes and polysomal aggregates are randomly distributed. The ratio nucleoplasm:cytoplasm, on the average, is 0.7 in both cases and the cell diameters lie distinctly above those of lymphocytes. At the electron microscope level these cultured monoclonal cells of Hodgkin's disease are not distinguishable from those described in genuine Hodgkin material. Their probable origin and apparent relation to true histiocytic lymphoma cells will be discussed.