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1.
Colony-stimulating activities (CSA) are potent granulopoietic stimulators in vitro. Using clonogenic assay techniques, we analyzed the degree to which mononuclear phagocytes and T lymphocytes cooperate in the positive (production/release of CSA) and feedback (inhibition of CSA production/release) regulation of granulopoiesis. We measured the effect of lactoferrin (a putative feedback regulator of CSA production) on CSA provision in three separate assay systems wherein granulocyte colony growth of marrow cells from 22 normal volunteers was stimulated by (a) endogenous CSA-producing cells in the marrow cells suspension, (b) autologous peripheral blood leukocytes in feeder layers, and (c) medium conditioned by peripheral blood leukocytes. The CSA-producing cell populations in each assay were varied by using cell separation techniques and exposure of isolated T lymphocytes to methylprednisolone or to monoclonal antibodies to surface antigens and complement. We noted that net CSA production increased more than twofold when a small number of unstimulated T lymphocytes were added to monocyte cultures. Lactoferrin's inhibitory effect was also T lymphocyte dependent. The T lymphocytes that interact with monocytes and lactoferrin to inhibit CSA production are similar to those that augment CSA production because their activities are neither genetically restricted not glucocorticoid sensitive, and both populations express HLA-DR (Ia-like) and T3 antigens but not T4 or T8 antigens. These findings are consistent with results of our studies on the mechanism of lactoferrin's inhibitory effect with indicate that mononuclear phagocytes produce both CSA and soluble factors that stimulate T lymphocytes to produce CSA, and that lactoferrin does not suppress monocyte CSA production, but does completely suppress production or release by monocytes of those factors that stimulate T lymphocytes to produce CSA. We conclude that mononuclear phagocytes and a subset of T lymphocytes exhibit important complex interactions in the regulation of granulopoiesis.  相似文献   

2.
Monocyte tumor cell line PU5-1.8 does not normally produce colony-stimulating activity (CSA) required by granulocyte and macrophage progenitors to proliferate and mature in agar. However, CSA is induced in the culture line by as little as 10 ng/ml endotoxic lipopolysaccharide (LPS), with maximum CSA production and release to the medium between 2 and 3 days of incubation. Derived lipid A, but not alkali-treated LPS, is also active. Induction requires RNA and protein synthesis, but is not blocked by mitomycin C or Colcemid. Other inducers of CSA include Mycobacterium Bacillus Calmette-Guérin, tuberculin protein preparation purified protein derivative, zymosan, and phorbol myristate. All inducing agents are specific inhibitors of the monocyte tumor cell proliferation in vitro. Latex beads, another macrophage-activating agent, are rapidly phagocytosed by PU5-1.8 cells, but neither inhibit growth nor induce CSA.  相似文献   

3.
A recently developed micro agar culture system has been optimized for the in vitro growth of human B-lymphocytes. Enriched B-lymphocytes from the peripheral blood of normal individuals were suspended in an agar layer, above which a liquid overlayer containing 20% PHA-LCM, 2-ME and Prot A or LPS as stimulants was added. Two morphologically distinguishable colony types were observed using this culture technique: diffusely proliferating colonies (Type I) were found within the agar layer, and round, compact colonies (Type II) appeared to leave the agar layer and continue growth in the liquid overlayer. For both colony types a linear relationship was demonstrated between the number of seeded cells and the number of formed colonies. The appearance of the two colony types in vitro was not concurrent, and they exhibited differing sensitivity to mitogen concentration and to the type of serum used as additive to the culture medium. The implications of clonal in vitro cultivation of lymphocytes, both normal and pathological, are discussed.  相似文献   

4.
Polyurethane films that contained various amounts of zinc diethyldithiocarbamate (ZDEC) and zinc dibutyldithiocarbamate (ZDBC) were prepared as standard reference materials (SRM). Using three cell lines of V79, L929, and Balb/3T3 cells, the cytotoxicity of the dithiocarbamates and the SRM films were compared by agar diffusion assay, filter diffusion assay, neutral red assay, cell growth assay, and colony assay. Among these in vitro cytotoxicity tests, colony assay was found to be the most sensitive method for detecting the cytotoxicity. The cytotoxic potentials of extracts from SRM films correlated well with the concentrations of ZDEC or ZDBC involved in SRM. When various rubber materials including SRM and surgical rubber latex materials were tested, cytotoxic potentials of these extracts were also correlated with the inflammatory tissue capsule thickness in short-term implantation tests. On the basis of these results, the SRM is judged to be useful for validating test sensitivity, and comparing the correlation between in vitro and in vivo responses.  相似文献   

5.
A 22-year-old female with cyclic neutropenia was studied. Her bone marrow cells showed high colony-forming activity in soft agar through the cycle, though there were slight fluctuations in the number and the shape of colonies. On the other hand, the peak of urinary colong-stimulating activity (CSA) occurred at the neutropenic stage. The dialyzed serum showed two peaks of CSA, i.e., at the peak and the valley of the neutrophil count, although the undialyzed serum showed only one peak at the neutroenic stage. It is suggested on the basis of these data that humoral factors may play a role in maintaining the neutrophil cycle.  相似文献   

6.
Osteogenesis by chondrocytes from growth cartilage of rat rib.   总被引:17,自引:0,他引:17  
Chondrocytes were isolated from growth and resting cartilage of rat rib and cultivated in vitro. The cultivated chondrocytes were placed in Millipore diffusion chambers, which were then implanted into the abdominal cavities of rats for several weeks and prepared for histological analysis. The results indicate that growth cartilage cells have a remarkable osteogenic potential, even after cultivation in vitro, whereas resting cartilage cells show no osteogenic activity. However, growth cartilage cells alone do not form new bone but require the participation of certain host cells to initiate osteogenic differentiation.  相似文献   

7.
In vitro studies were performed in two patients with B-cell chronic lymphocytic leukemia who developed pure red cell aplasia (CLL-PRCA). During the active phase of their red cell aplasia, there was a marked reduction in the numbers of erythroid colony-forming units (CFU-E). Unfractionated sera or separated IgG fractions from these patients did not impair CFU-E proliferation from either autologous or allogeneic marrows. Increased numbers of T lymphocytes were present in marrow aspirates of these patients. Analysis of these T cells indicated that 90 and 35%, respectively, bore Fc receptors for IgG (T gamma cells). Removal of T cells by E-rosetting techniques augmented CFU-E growth in CLL-PRCA 10-fold. Similar treatment of normal marrows did not cause similar enhanced growth of CFU-E. Co-cultures of marrow T cells or T gamma cells obtained during the active phase of CLL-PRCA suppressed CFU-E growth from autologous or allogeneic marrows. After achieving drug-induced remission of the PRCA, marrow T cells were no longer inhibitory. In contrast, BFU-E (erythroid burst-forming units) or granulocyte proliferation in diffusion chambers were not suppressed by CLL-PRCA T cells. These findings suggest that the development of PRCA in B-cell CLL may result from suppression of CFU-E proliferation by T gamma cells.  相似文献   

8.
背景:有研究表明端粒酶活性抑制剂不仅能抑制或杀死肾癌细胞,而且对决定肾癌发生发展的干细胞也有作用.目的:观察无血清悬浮培养的肾癌干细胞表面标志CD133及端粒酶活性的表达情况,并与含血清培养的肾癌细胞作比较.设计、时间及地点:细胞学体外观察,于2008-0612009-02在江苏大学完成.材料:手术切除肾癌周围新鲜的正常肾组织标本由江苏大学附属医院提供,肾癌干细胞株OS-RC-2由上海中科院细胞库提供.方法:取处于对数生长期的肾癌干细胞OS-RC-2,胰酶消化后离心弃上清,悬浮于含EGF、bFGF的DMEM,F12无血清培养基中,调整细胞浓度为2×105L-1进行接种,在37℃、体积分数为5%的CO2培养箱中悬浮培养.以含血清培养的肾癌细胞及正常肾组织细胞作为对照.主要观察指标:倒置显微镜下观察细胞生长情况,流式细胞仪检测肾癌干细胞CD133及CD34的表达,采用TRAP实时定量检测肾癌干细胞端粒酶活性.结果:肾癌干细胞接种于无血清培养基后,细胞呈圆形悬浮于培养液中:2 d后有细胞球生成,每个细胞球含3~8个细胞,折光性较强;7 d后细胞球明显增多,体积增大,为规则的圆形或卵圆形.无血清悬浮培养的肾癌干细胞球CD133+CD34-率明显高于含血清培养的肾癌细胞CD133+CD34-率,正常肾组织细胞未测出有CD133及CD34的表达,3者间比较差异有显著性意义(F=328.25,P<0.05).肾癌干细胞球和肾癌细胞端粒酶活性均高于正常肾组织细胞(F=278.74,P<0.05),而前2者间端粒酶活性无明显差异.结论:与含血清培养的肾癌细胞相比,无血清悬浮培养的肾癌干细胞表面标志CD133呈明显高表达,二者端粒酶活性均高于正常肾组织.  相似文献   

9.
Colony-stimulating activity (CSA) is essential for in vitro differentiation of bone marrow cells into colonies of granulocytes and mononuclear cells. While blood monocytes and macrophages are a major source of CSA, recent studies have indicated that CSA may be produced by lymphocytes responding to immunologic stimulation. Lymphocytes, purified from spleens and thymuses of mice by glass wool columns, were incubated in CMRL-1066 medium with fetal calf serum in vitro. Lymphocytes from the thumus and spleen released CSA when cultured in vitro, with peak levels of CSA observed after 7 days of incubation. Stimulation of cultures with phytohemagglutinin, concanavalin A, or pokeweed mitogen resulted in a 2-5-fold increase in CSA release, with peak levels of CSA released after 4 days of incubation. Thymus-dependent lymphocytes were responsible for the release of CSA from unstimulated and mitogen-stimulated cultures, since the incubation of these cultures with rabbit anti-mouse T cell sera abolished their ability to release CSA. Anti-mouse B cell sera had no effect on the ability of lymphocyte cultures to release CSA. These studies suggest that thymocytes and thymus-derived lymphocytes can release CSA in vitro and may be responsible for the increase in CSA observed in certain immunologic reactions.  相似文献   

10.
Neonatally thymectomized mice were implanted intraperitoneally at 7 days of age with Millipore diffusion chambers containing either embryonic or neonatal thymus tissue. Mice which received either empty diffusion chambers or no further treatment following neonatal thymectomy served as controls. In contrast to these controls, most of the mice implanted with thymus-filled chambers gained weight satisfactorily, did not develop a wasting syndrome, and had the capacity to produce serum antibodies in response to sheep erythrocytes and to reject allogeneic skin grafts. Lymphoid follicles were present in the lymph nodes, spleen, and intestinal tract of the implanted mice but most still showed some diminution in the population of lymphocytes in both blood and tissues. Control thymectomized mice had markedly depleted lymphoid tissues and low peripheral blood lymphocyte levels. The tissue recovered after 1 to 2 months from the diffusion chambers showed only epithelial-reticular cells but no lymphoid cells. It is suggested that a humoral factor produced by the thymus epithelial-reticular complex may be responsible for endowing lymphoid cells with immunological competence.  相似文献   

11.
The in vitro interaction of polyaspartic acid (PAA) with aminoglycosides was evaluated using double diffusion in agar, dialysis chambers and changes in the optical density of test solutions. The results document a reversible, presumably electrostatic interaction that is optimized at a pH of approximately 5.0, by the absence of proteins over 800 Da, and at a 20:1 or 10:1 molar ratio of aminoglycoside to PAA. The PAA-aminoglycoside complex lost antibacterial activity and the ability to inhibit pronase E enzymatic activity. These results allow generation of a hypothesis as to the mechanism whereby PAA prevents aminoglycoside experimental nephrotoxicity.  相似文献   

12.
Endothelial cells have been shown to produce granulopoietic colony stimulating activity (CSA) under the regulatory control of a humoral factor, MRA produced by blood monocytes. An endothelial cell-derived granulopoietic inhibitory factor has also been described. To further define these apparently paradoxical observations, human bone marrow mononuclear cells were co-cultured with umbilical cord derived endothelial cells in a plasma clot system in vitro. To enhance the sensitivity of the assay for growth effects attributable to the endothelial cells (or their products) alone, an exogenous source of CSA (e.g. a peripheral blood leukocyte feeder layer) was not used. On day ten of culture, less than or equal to 1% endothelial cells markedly stimulated the growth of early granulocyte progenitors (large diaminofluorine positive (DAF+) GM-CFUc) (p less than .01) and a linear dose response relationship was confirmed (p less than .001). Late granulocyte progenitors (DAF+ clusters) were coincidently suppressed by less than or equal to 2% endothelial cells (p less than .01). No effect of endothelial cells on intermediate progenitors (small GM-CFUc) was demonstrated at any concentration. Similar effects were observed with the addition of 5% to 30% endothelial conditioned medium (ECM) (p less than .01). When cohort cultures were evaluated serially, suppression of clusters was observed by day four and stimulation of large GM-CFUcs by day six. These varied effects on different stages of granulocyte differentiation suggest that endothelial cell derived CSA(S) may be of biological relevance in the regulation of granulopoiesis.  相似文献   

13.
The influence of postinflammatory rat serum containing granulocytic stimulating activity (GSA) on the process of spontaneous regeneration of hemopoietic tissue was studied and compared to the effect of colony stimulating activity (CSA) in CBA mice in the regenerative phase of hemopoiesis after aplasia induced by a sublethal dose of cyclophosphamide (Cy). One hour after Cy application, mice were injected with 0.2 ml of GSA or CSA. Then 6, 12, 24 and 48 hr later the changes within bone marrow and spleen CFU - GM, morphologically recognizable hemopoietic cells and white blood cells (WBC) were followed. GSA stimulated spontaneous regeneration of granulocytic cells, specifically increasing the number of morphologically recognizable proliferative granulocytes continuously during 48 hr. The number of bone marrow (at the 6th hr) and splenic (until the 24th hr) CFU - GM were also increased under the influence of GSA. Under the same experimental conditions in vivo, CSA did not induce any changes within bone marrow granulocytic cells, but the increase in splenic CFU - GM was observed until the 12th hr. The differences in the action of GSA and CSA on the process of spontaneous regeneration of granulocytic cells in vivo in Cy treated mice indicate that these 2 factors play different roles in the regulation of granulopoiesis.  相似文献   

14.
Results previously reported using the Wet Disc Topical Antimicrobial Assay (WDA) suggested that adding nystatin (NY) to a 0.5% mafenide acetate (MA) suspension or genitourinary irrigant (double antibiotic [DAB]) to expand their antimicrobial activity to include Candida sp. antagonized the antibacterial effect of MA but not DAB. We use DAB solution as described by the authors of the previous study, also, but we use a 5% commercially available mafenide acetate solution instead of the in-house prepared 0.5% mafenide acetate suspension that they used. Further, we use both the WDA and the Agar Well Diffusion Topical Antimicrobial Assay (AWDA) to test topical antimicrobials at this institution. In light of the previously reported results, this study 1) examined whether adding nystatin to DAB or the 5% mafenide acetate solution used at this institution caused any interference in the ability of these substances to migrate through the agar matrices and cause zones of growth inhibition in the two test assay systems and 2) compared the assessment of microbial susceptibility (by very precise definition) between the two systems. The addition of nystatin did not interfere with the ability of either DAB or mafenide acetate to migrate through the agar matrices and cause clear zones. However, on the assessment of susceptibility a significantly larger number of organisms were judged susceptible using the AWDA than the WDA. We believe that the disparity is caused by a large difference in agar diffusion kinetics between the two assays. Therefore, we recommend the AWDA rather than the WDA for susceptibility studies.  相似文献   

15.
Fluconazole is an azole agent with primarily fungistatic activity in standard in vitro susceptibility tests. The present study was undertaken to develop a diffusion chamber model system in mice in order to study the in vivo effects of prolonged fluconazole treatment on Candida albicans. Chambers containing 100 C. albicans yeast cells were implanted subcutaneously on the flanks of C57BL/6 mice and were then retrieved 6 or 14 weeks later (after fluconazole treatment for 4 or 12 weeks, respectively). Leukocyte counts demonstrated that implantation of the chambers did elicit an inflammatory response but that only small numbers of inflammatory cells were able to enter the chamber interior. Treatment with fluconazole at 10 mg/kg of body weight/day for 12 weeks not only reduced the numbers of viable organisms within the chambers compared to those in untreated mice (mean +/- standard deviation of log(10) CFU of 0.7 +/- 1.2 versus 2.3 +/- 2.0; P < 0.001 by the Bonferroni test) but also increased the numbers of chambers that became sterile over the treatment period (14 of 16 versus 6 of 19; P = 0.0009 by the chi-square test). However, treatment for only 4 weeks had minimal effects on the numbers of chamber CFU, and none of the chambers became sterile during this period. Distribution of retrieved organisms between interior fluid and the chamber filters was approximately equal in all the treatment groups. This model system appears to be useful for evaluating the effects of antifungal drugs over prolonged periods in vivo. Its use in the present study demonstrates that fluconazole can increase the rate of sterilization of C. albicans foci that are protected from the host's inflammatory response.  相似文献   

16.
A squamous cell carcinoma of 33-yr-old patient who developed marked leukocytosis and hypercalcemia was transplanted into nude mice in which more marked leukocytosis and hypercalcemia also developed. This tumor (LJC-1-JCK) produced a colony-stimulating factor (CSF) and formed a cyst in the tumor from which a CSF-producing cell line (T3M-1) was established. The CSF causes predominantly formation of granulocytic colonies in addition to macrophage colonies. Bone-resorbing activity (BRA) was detected in the cystic fluid and was eluted as two separate peaks with proteins of an apparent molecular weight of 30,000-50,000 and 10,000-20,000. Colony-stimulating activity (CSA) was eluted at an apparent 30,000 mol wt. The conditioned medium of the T3M-1 cells also contained a BRA with an apparent 14,000 mol wt, whereas CSA eluted at an apparent 30,000 mol wt. PTH, epidermal growth factor, transforming growth factor-alpha, prostaglandin Es, and vitamin D could not account for the powerful BRA. In contrast to CSA, BRA was not inactivated by trypsin and more stable at 70 degrees C. When T3M-1 cells were transplanted into nude mice, marked hypercalcemia developed in addition to granulocytosis. Our findings suggest that the tumor produces and secretes a powerful BRA in vivo and in vitro, which is different from CSA in terms of molecular weight, heat stability, and trypsin treatment. We speculate that the synergistic action of CSF that stimulates macrophage colony formation and recruits osteoclast precursors, and BRA, which stimulates mononuclear phagocytes and/or osteoclasts were responsible for a marked increase in osteoclastic bone resorption and humoral hypercalcemia in the patient.  相似文献   

17.
This study investigates the proliferative and osteogenic role of marrow stromal/osteoprogenitor cells in the development of the cortical bone deficit in ovariectomized (OVX) female rats. In vitro, clonal growth of marrow stromal cells from OVX rats was significantly impaired (vs. sham-operated controls). Yet in vivo, cells from sham-operated and OVX rats had equal osteogenic potential in several in vivo experimental situations, such as in intraperitoneally implanted millipore diffusion chambers and in intramuscular implants of marrow plus osteoinductive bone matrix (composite grafts). Long-term (6 mo) dihydrotachysterol (DHT) treatment of OVX rats enhanced their in vitro proliferative potential and clonal growth, as well as their osteogenic expression in composite grafts. The observation that the in vivo osteogenic performance of OVX rat marrow stromal cells was normal at extraosseous sites suggests that the mechanisms leading to osteopenia may involve an abnormality in cell-matrix interactions.  相似文献   

18.
The extravascular kinetics of ceftizoxime were studied both in an in vitro kinetic model and in an in vivo rabbit model. Visking tubing chambers were used in both models to provide extravascular spaces with large or small volumes and surface areas, but identical surface area/volume ratios. Four rabbits, each implanted with two large Visking chambers and four small chambers, received 50 mg of ceftizoxime per kg intramuscularly every 3 h for eight doses. In the in vitro model, 80 mg of ceftizoxime was infused over 30 min every 3 h for eight doses. Intravascular and extravascular spaces were sampled in both models after the eighth dose. Ceftizoxime had similar intravascular kinetics in both models, i.e., the peak levels, the peak-to-trough fluctuations, and the half-life were comparable. The area under the curve (AUC) for the extravascular spaces was also similar in the two models. Large and small chambers having identical surface area/volume ratios demonstrated identical kinetics. The extravascular Visking chamber spaces achieved equilibrium with the intravascular spaces in both models, i.e., the AUC for the extravascular spaces was the same (P > 0.2) as that for the serum (rabbit model) or the test chamber (in vitro model). This study illustrates (i) that our modified in vitro model is a potentially valid model for studying extravascular kinetics; (ii) that extravascular spaces with identical surface area/volume ratios show similar penetration kinetics with a freely diffusible drug, such as ceftizoxime, despite differences in size; and (iii) that the Visking chamber extravascular-space model permits the free diffusion of the antimicrobial agent and reaches equilibrium (equivalent AUC) with the intravascular space.  相似文献   

19.
Long-term culture and cloning of nontransformed human B lymphocytes   总被引:4,自引:7,他引:4       下载免费PDF全文
B lymphocyte-enriched cell populations cultured with mitogens in initial suspension cultures formed colonies in soft agar when the same mitogenic agent was present in the lower layer of a two-layer soft agar system. Colony formation depended upon the presence of T cells in the initial culture, and was optimal after an initial 72-h culture with phytohemagglutinin (PHA; 12.5 microliters/ml), pokeweed mitogen (PWM; 2.5 micrograms/ml), or protein A (10 micrograms/ml). The colonies could be picked from the agar and propagated by feeding every 3 d with medium supplemented with a growth factor-containing tissue culture supernate. The growth factor-containing supernate was prepared by stimulating pools of human peripheral blood mononuclear cells for 72 h with PHA or PWM. The lines propagated in this manner were membrane Ig+, lacked sheep erythrocyte rosette-forming ability, and did not ingest latex. They lacked the Epstein-Barr nuclear antigen (EBNA) and had 46 chromosomes. Such lines have been propagated for over 1 yr. One line (BL1) was subjected to limiting dilution cloning and a line, BL1.1, was prepared that contained 96% lambda-bearing cells and no kappa-bearing cells. This line was also EBNA negative. This procedure can thus be used to prepare and clone long-term lines of nontransformed human B lymphocytes.  相似文献   

20.
We have examined bacterial determinants that influence beta-lactam activity in Haemophilus influenzae cells cultivated in a system that reproduces in vivo growth conditions. Bacteria grown in diffusion chambers were recovered from the peritoneal cavities of rats, and their cell properties were compared with those of bacteria grown in broth cultures by various tests performed in vitro. The rate of peptidoglycan synthesis was measured as the incorporation of [14C]alanine into cell wall material in the presence of chloramphenicol. The total incorporation of [14C]alanine into peptidoglycan was markedly increased in cells grown in rats prior to the assay but was efficiently reduced by the beta-lactams. The extent of cross-linking was lower in the peptidoglycan of in vivo-grown bacteria, as estimated by sodium dodecyl sulfate- to trichloroacetic acid-insoluble radioactive cell wall material ratios. A whole-cell labeling assay with 125I-penicillin was used to characterize the penicillin-binding proteins (PBPs). Four PBPs showed a striking reduction in the binding of the labeled penicillin in cells grown in rats. Such changes resembled the PBP alterations seen in beta-lactamase-negative clinical strains that were resistant to the beta-lactams. Although ampicillin and moxalactam showed delayed inhibitory activities in vitro for cells collected from rats, cells recovered from beta-lactam-treated rats showed evidence of antibiotic effectiveness (binding of the beta-lactams to PBPs in vivo and altered morphology), and the killing of cells exposed to antibiotics in broth or in peritoneal fluid was equally good. Finally, the frequencies of spontaneous resistance or tolerance to ampicillin or moxalactam were estimated, and there was no significant difference for in vitro- or in vivo-grown cells. These data demonstrated that the cultivation of H. influenzae in animals created changes in PBPs and the overall peptidoglycan metabolism. Such alterations did not impair the bactericidal activities of the beta-lactams, although they resulted in delayed bacterial inhibition, a phenomenon that may have important consequences in antibiotherapy.  相似文献   

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