Classification of enterotoxins on the basis of activity in cell culture.

Two cell culture systems were used in a study of the biological properties of several bacterial enterotoxins in vitro. By means of one model, in which HeLa cell monolayers were used, cytotoxic effects, interms of detachment of cells from a glass surface due to cell death, were assayed. By means of the second model, activation of the adenyl cyclase-cyclic adenosine 3', 5'-monophosphate (AMP) system, in terms of increased steroidogenesis by Y-1 adrenal cells (an effect which we have termed cytotonic), was assayed. None of the four toxins manifested both properties. Rather there was a clear segregation into two cytotoxic enterotoxins (Shigella dysenteriae type 1 and Clostridium perfingens) and two cytotonic products (Vibrio cholerae and Escherichia coli). These data raise the possibility that some enterotoxins may not interact with the adenyl cyclase-cyclic AMP system; this possibility has also been suggested by studies of the toxin of S. dysenteriae type 1 in the rabbit small bowel. These cell culture systems may therefore serve as convenient models for the study of the mechanism of action of both classes of enterotoxin.     

Coordinated assembly of multisubunit proteins: oligomerization of bacterial enterotoxins in vivo and in vitro.

In this paper we study the assembly, in vivo and in vitro, of a family of hexameric, heat-labile enterotoxins produced by diarrheagenic bacteria. The toxins, which consist of an A subunit and five B subunits, are assembled by a highly coordinated process that ensures secretion of the holotoxin complex. We show that (i) oxidation of cysteine residues in the B subunits is a prerequisite step for in vivo formation of B-subunit pentamers, (ii) reduction of dissociated B subunits in vitro abolishes their ability to reassemble, (iii) the kinetics of B-pentamer assembly in vivo can be mimicked under defined conditions in vitro, (iv) A subunits cannot associate with fully assembled B pentamers in vitro, and (v) A subunits cause an approximately 3-fold acceleration in the rate of B-subunit pentamerization in vivo, implying that A subunits play a coordinating role in the pathway of holotoxin assembly. The last finding is likely to be of general significance, since it provides a mechanism for preferentially excluding or favoring certain intermediates in the assembly of multisubunit proteins.     

In vitro studies on the relative potency of bronchodilator agents

This study describes a rapid in vitro assay for the order of potency of bronchodilator drugs using specific binding of (−)-[³H] dihydroalprenolol ([³H]DHA) to rat lung membranes. Under linear conditions with respect to tissue, specific binding of [³H]DHA showed saturability, rapid kinetics of association and dissociation of radioligand, and sterospecificity. Nanomolar (nM) concentrations for 50% inhibition (IC₅₀±SE) for the bronchodilator drugs examined were as follows: albuterol, 1485±170; isoproterenol, 136±53; procaterol, 162±28; terbutaline, 3310±934; and zinterol, 51±8.3. A comparison of binding studies using rat lung tissue membranes and similar preparations of rat heart and skeletal muscle demonstrated that lung tissue had 7 to 8 times more receptor sites (B_max) for [³H]DHA than heart or skeletal muscle. Adenyl cyclase activit of the rat lung membrane preparation almost doubled in the presence of (−)-isoproterenol. Displacement of specific (³H)DHA binding in membrane preparations may provide useful data for evaluating bronchodilator compounds.     

In vitro culture of primary plasmacytomas requires stromal cell feeder layers.

Attempts to grow primary murine plasmacytomas in vitro have, to date, been largely unsuccessful. In this study, we demonstrate that long-term in vitro growth of primary plasmacytomas is accomplished by using feeder layers composed of stromal cells from the initial site of plasmacytomagenesis. The early neoplastic lines established in this manner are dependent on physical contact with the stromal layer, which is mediated in part by CD44, for growth and survival. The stromal cells provide at least two stimuli for the plasma cells, one being interleukin 6 and the second, of unknown nature, resulting from direct physical interaction that cannot be replaced by soluble factors. These plasma cell lines have been passaged for as long as 20 months yet still maintain characteristics associated with primary plasmacytomas as they will grow in vivo only in pristane-primed animals, indicating a continued dependence on the pristane-induced microenvironment characteristic of early-stage tumors. The ability to grow primary plasmacytomas in culture and maintain their "primary" properties provides a model system for detailed analysis of early events in plasma cell tumor progression involving neoplastic cells completely dependent on physical contact with a stromal feeder layer for survival and expansion.     

丙型肝炎病毒体外感染MT-2细胞的研究

目的建立体外丙型肝炎病毒感染的ＭＴ－２细胞模型。方法用ＨＣＶＲＮＡ阳性的丙型肝炎患者血清感染人淋巴细胞株ＭＴ－２以ＨＣＶＲＮＡ阴性血清作对照，用ＲＴ－ＰＣＲ分析ＨＣＶ在体外细胞培养中的复制状态。结果用ＨＣＶ阳性血清感染的ＭＴ－２细胞在培养第７天开始检出ＨＣＶＲＮＡ，并一直持续阳性至第２８天；培养至第１０天可测出ＨＣＶ复制中间体，于第１８天消失。用ＨＣＶ阴性血清感染的ＭＴ－２细胞未检出ＨＣＶＲＮＡ。扩增产物经Ｓｏｕｔｈｅｒｎ转移后用ＨＣＶ探针杂交证实ＨＣＶ特异性序列。结论ＨＣＶ可在ＭＴ—２中短期复制。     

In vitro and in vivo potency of insulin analogues designed for clinical use.

Analogues of human insulin designed to have improved absorption properties after subcutaneous injection have been prepared by recombinant DNA technology. Five rapidly absorbed analogues, being predominantly in mono- or di-meric states in the pharmaceutical preparation, and a hexameric analogue with very low solubility at neutral pH and slow absorption, were studied. Receptor binding assays with HEP-G2 cells showed overall agreement with mouse free adipocyte assays. Two analogues, B28Asp and A21Gly + B27Arg + B30Thr-NH2, had nearly the same molar in vitro potency as human insulin. Another two showed increased adipocyte potency and receptor binding, B10Asp 194% and 333% and A8His + B4His + B10Glu + B27His 575% and 511%, while B9Asp + B27Glu showed 29% and 18% and the B25Asp analogue only 0.12% and 0.05% potency. Bioassays in mice or rabbits of the analogues except B25Asp showed that they had the same in vivo potency as human insulin 1.00 IU = 6.00 nmol. Thus the variation had the same in vivo potency as human insulin 1.00 IU = 6.00 nmol. Thus the variation in in vivo potency reflects the differences in receptor binding affinity. Relative to human insulin a low concentration is sufficient for a high affinity analogue to produce a given receptor complex formation and metabolic response. In conclusion, human insulin and analogues with markedly different in vitro potencies were equipotent in terms of hypoglycaemic effect. This is in agreement with the concept that elimination of insulin from blood and its subsequent degradation is mediated by insulin receptors.     

Paneth cell differentiation in the developing intestine of normal and transgenic mice.

Paneth cells represent one of the four major epithelial lineages in the mouse small intestine. It is the only lineage that migrates downward from the stem-cell zone located in the lower portion of the crypt of Lieberkühn to the crypt base. Mature Paneth cells release growth factors, digestive enzymes, and antimicrobial peptides from their apical secretory granules. Some of these factors may affect the crypt stem cell, its transit-cell descendants, differentiating villus-associated epithelial lineages, and/or the gut microflora. We used single and multilabel immunocytochemical methods to study Paneth cell differentiation during and after completion of gut morphogenesis in normal, gnotobiotic, and transgenic mice as well as in intestinal isografts. This lineage emerges coincident with cytodifferentiation of the fetal small intestinal endoderm, formation of crypts from an intervillus epithelium, and establishment of a stem-cell hierarchy. The initial differentiation program involves sequential expression of cryptdins, a phospholipase A2 (enhancing factor), and lysozyme. A dramatic increase in Paneth cell number per crypt occurs during postnatal days 14-28, when crypts proliferate by fission. Accumulation of fucosylated and sialylated glycoconjugates during this period represents the final evolution of the lineage's differentiation program. Establishment of this lineage is not dependent upon instructive interactions from the microflora. Transgenic mice containing nucleotides -6500 to +34 of the Paneth cell-specific mouse cryptdin 2 gene linked to the human growth hormone gene beginning at its nucleotide +3 inappropriately express human growth hormone in a large population of proliferating and nonproliferating cells in the intervillus epithelium up to postnatal day 5. Transgene expression subsequently becomes restricted to the Paneth cell lineage in the developing crypt. Cryptdin 2 nucleotides -6500 to +34 should be a useful marker of crypt morphogenesis and a valuable tool for conducting gain-of-function or loss-of-function experiments in Paneth cells.     

In vitro gamma irradiation of leukemic cells in mice, rats, and guinea pigs.

In vitro gamma irradiation of virus-induced (Gross) mouse leukemia cells at doses of 350-1600 rads (1 rad = 0.01 gray) had no effect on their ability to induce leukemia, usually within 2 weeks, after transplantation into syngeneic mice. However, when cells irradiated at doses of 2000-20,000 rads were transplanted, they induced leukemia after a latency period exceeding 2.5 months, similar to the results observed in mice inoculated with filtered mouse leukemia extracts. Similar results were also obtained after irradiation of leukemic cells derived from rats in which leukemia had been induced by rat-adapted mouse leukemia virus. Apparently, gamma irradiation at a dose of, or exceeding, 2000 rads, inhibits the ability of mouse and rat leukemic cells to induce leukemia after transplantation into syngeneic hosts; however, it does not inactivate the virus carried by such cells nor prevent it from inducing leukemia. [In previous experiments, doses of more than 4,500,000 rads were needed to inactivate the passage A (Gross) leukemia virus carried in either mouse or rat leukemic cells.] In vitro gamma irradiation of L2C guinea pig leukemic cells at doses of 750-2500 rads had no apparent effect on their ability to induce leukemia after transplantation effect on their ability to induce leukemia after transplantation into strain 2 guinea pigs. However, irradiation at doses of 3250-20,000 rads inactivated their ability to do so. The morphology of mouse, rat, and guinea pig leukemic cells and the virus particles present in such cells was not affected by irradiation at doses of 20,000 rads.     

Leukaemogenic potency of WEHI-3B cells grown in vitro or in leukaemic mice.

In order to quantify contaminating leukaemia inducing cells in blood or bone marrow from WEHI-3B bearing BALB/c mice (injected with 10(5) WEHI-3B suspension culture cells), in vitro colony forming leukaemia cells (CFU-L) and survival rate and/or survival time of mice transplanted with cells from WEHI bearing mice or suspension culture were correlated. ED(50) (inoculum inducing leukaemia in 50% of the animals) was 109 CFU-L (33-361) from suspension culture cells. Three weeks after initiation of leukaemia 2 x 10(5) BM or 0.5-2.0 x 10(6) blood cells induced 100% mortality of recipients. After mobilisation with CY or CY and G-CSF, the same amount of blood or BM cells did not induce leukaemia in recipients. A significant negative correlation was found between the survival time of leukaemic mice and the log number of CFU-L inoculated from in vivo sources. In terms of CFU-L cells, leukaemia induction to BM or WBC obtained 3 weeks after leukaemia induction were more potent; those from BM or WBC also obtained at 3 weeks but after mobilisation were less potent inducers than those from suspension culture. These data suggest that CFU-L and leukaemogenic cells are associated, but not identical.     

In vitro colony-forming cells in the marrow of leukemic and preleukemic mice.

In vitro colony-forming cells (CFUc) were evaluated in preleukemic and leukemic AK mice. Increased concentrations of CFUc were found in normal appearing marrow of superinfected animals 2-3 wk prior to the onset of lymphoma. CFUc were present in marrows of mice with virus-accelerated, spontaneous, and transplanted lymphoma. CFUc concentration was often increased in mice with advanced disease and marrow replacement by lymphoblasts. When calculated on the basis of CFUc per normal marrow cells, this increase was marked. The colonies developed only in the presence of added colony-stimulating activity (CSA) and had the morphologic features of colonies from normal marrow. Leukemic cells did not form colonies. Lymphoma cells, from virus accelerated, spontaneous and transplanted lymphoma, did not produce CSA in feeder layers or conditioned medium. Leukemic and nonleukemic AK bone were found to produce similar small amounts of CSA. These studies showed that the preleukemic state as well as marrow replacement by lymphoblasts resulted in increased marrow CFUc's. No evidence for increased local production of CSA by lymphoblasts or the marrow microenvironment was found to account for this.     

In vitro erythropoietin assay based on erythroid colony formation in fetal mouse liver cell culture

Critical studies were made on erythroid colony formation from cultured fetal mouse liver cells in an attempt to develop a simple and sensitive erythropoietin (Epo) assay procedure. The maximum colony formation was observed 24 h after plating of the cells when an evident dose-response relation was found for Epo added. The colony forming ability decreased steadily as the gestational age of the fetus advanced and was gradually lost by postnatal days 10-11. By morphological and cytochemical criteria almost all the colonies were found to be erythroid. 59Fe-labelling experiments revealed a fairly good correlation between the colony number and 59Fe incorporation into both cells and haem. Dose-response curves for plasma were parallel to the Epo standard curve. Based on these findings we developed a procedure which could measure as little as 0.4 mU of Epo without requiring 59Fe. Using this method, plasma Epo titres were determined in 16 normal and 69 anaemic subjects.     

Spatial organization of bacterial flora in normal and inflamed intestine： A fluorescence in situ hybridization study in mice

AIM: To study the role of intestinal flora in inflammatory bowel disease (IBD). METHODS: The spatial organization of intestinal flora was investigated in normal mice and in two models of murine colitis using fluorescence in situ hybridization. RESULTS: The murine small intestine was nearly bacteria-free. The normal colonic flora was organized in three distinct compartments (crypt, interlaced, and fecal), each with different bacterial compositions. Crypt bacteria were present in the cecum and proximal colon. The fecal compartment was composed of homogeneously mixed bacterial groups that directly contacted the colonic wall in the cecum but were separated from the proximal colonic wall by a dense interlaced layer. Beginning in the middle colon, a mucus gap of growing thickness physically separated all intestinal bacteria from contact with the epithelium. Colonic inflammation was accompanied with a depletion of bacteria within the fecal compartment, a reduced surface area in which feces had direct contact with the colonic wall, increased thickness and spread of the mucus gap, and massive increases of bacterial concentrations in the crypt and interlaced compartments. Adhesive and infiltrative bacteria were observed in inflamed colon only, with dominant Bacteroides species. CONCLUSION: The proximal and distal colons are functionally different organs with respect to the intestinal flora, representing a bioreactor and a segregation device. The highly organized structure of the colonic flora, its specific arrangement in different colonic segments, and its specialized response to inflammatory stimuli indicate that the intestinal flora is an innate part of host immunity that is under complex control.     

In vitro angiogenic potency in human microvascular endothelial cells derived from myocardium, lung and skin

Human microvascular endothelial cells derived from myocardium (HCMEC), lung (HPMEC) and foreskin (HDMEC) showed different angiogenic potency when cultivated in their original growth media provided by the distributors. In order to standardize microenvironmental conditions in an all-in-one assay of angiogenesis the aim of this study was to find one optimal growth medium for the endothelial cells derived from the different organs. Therefore each endothelial cell type was cultivated under identical conditions in the different original growth media as well as in several media formulations of the original growth media. Results reveal that even if cultivated in the same growth medium under exactly the same cultivation conditions--over a prolonged time period of 60 days--the endothelial cells still showed different angiogenic potency. This is due to a combination of extrinsic factors, i.e. the isolation procedure and in particular the growth medium, as well as to intrinsic differences between cells of diverse origin.     

人脐带血浆细胞样树突状细胞亚群的体外诱导及鉴定

目的建立浆细胞样树突状细胞(pDC)的体外培养方法。方法采集2014年6月-2015年2月于湖南中医药大学第一附属医院就诊的健康产妇脐带血40 ml,分离脐带血单个核细胞(CBMC),用含重组人FMS样酪氨酸激酶3配体(Flt3-L)100ng/ml、白细胞介素(IL)3 10 ng/ml的RPMI1640完全培养基连续培养7 d,每隔1天半换液。第8天加入2μg/ml的CpG ODN,24 h后收集细胞行流式检测,同时检测pDC培养上清干扰素(IFN)α水平。在培养细胞的第1、3、5、7、8天,观察培养孔中的pDC形态特征变化。结果 CBMC培养2 h后可见圆形扁平细胞布满视野,24 h后细胞开始贴壁,细胞质伸展开,体积有所增大,圆形,透亮,并可见散在的小集落形成;培养第3~4天,细胞体积较前继续增大,多数为圆形,部分细胞表面可见小的突起,并可见少量梭形、蝌蚪状、星形或其他不规则形的细胞,培养液中集落较前明显增多、增大;培养第5~8天,集落数量及集落内细胞数逐渐减少,而培养液中圆形或具有小突起的悬浮细胞逐渐增多。流式细胞仪检测发现CD123、BDCA-2、BDCA-4均表达阳性的细胞,该细胞为pDC。在培养过程中,pDC比例不断增加,其在培养开始时仅占CBMC总数的1.08%,第4天升至5.32%,第8天时达到高峰,为19.8%。培养第8天时,pDC培养上清IFNα水平达(11 302.61±1745.31)pg/ml。结论以人CBMC为培养初始细胞,利用Flt3-L与IL-3联合,可成功体外诱导出pDC。     

Maturation of the rat small intestine at weaning: changes in epithelial cell kinetics, bacterial flora, and mucosal immune activity.

The relationship between maturation of the small intestine and change in mucosal immune activity was examined in the DA rat during the weaning period from 12 to 30 days. Two stages of jejunal maturation were observed: an initial stage of morphological development and crypt proliferation (days 12 to 22), followed by a period of stabilisation (days 24 to 30). By day 22 of the initial phase, villi increased principally in width but not in length, crypt length increased, and crypt cell production rate increased from 0.5 (day 12) to 11.1 (day 22) cells/crypt/hour. Various measures of mucosal immune activity showed a biphasic response. Up to days 20 to 22, the weight of the mesenteric lymph node increased seven-fold (p less than 0.0001), counts of jejunal eosinophils and goblet cells increased 3- (p less than 0.0001) and 19-fold (p less than 0.0001) respectively, and mean serum rat mucosal mast cell protease II, released from mucosal mast cells, increased from 24 (day 12) to 313 (day 22) ng/ml (p less than 0.0001). After day 22, mesenteric lymph node weight stabilised, eosinophil count stabilised and goblet cells decreased, serum rat mucosal mast cell protease II decreased three-fold (p less than 0.0001), and mean jejunal count of intraepithelial lymphocytes increased from 26 (day 22) to 54 (day 24) cells per mm of muscularis mucosae (p less than 0.0001), before stabilising. These results demonstrated a close association between maturation of the small intestine and change in activity of the mucosal immune system.