KINETICS OF HAEMOPOIETIC RECOVERY IN ENDOTOXIN-TREATED MICE

Kinetics of mouse spleen colony forming units were studied after intra-peritoneal injection of 1 μ/g body weight bacterial endotoxin S. typhosa. When these mice were used as unirradiated and sublethally irradiated donors, it was possible to study the effect of the endotoxin injection upon the cells. Use of the treated mice as irradiated recipients of normal cells gave information about the host effect. In treated unirradiated mice, the total nucleated cell and the CFU counts were disturbed, and 2 days later a large fraction of the CFU were found in the DNA synthesis (S) phase. This meant that injection of endotoxin generated factors affecting the kinetics of the CFU and triggering the resting CFU into the proliferative cycle. If then the mice were given supralethal irradiation and used as recipients of normal bone marrow cells, more CFU seeded to the spleen as compared to normal recipients; but the dip and the growth rate of the CFU were not changed. Hence the endotoxin-generated factors had been eliminated in 2 days. A total body sublethal irradiation by 400 rad X-ray 2 days after endotoxin injection reduced the post-irradiation dip in the recovery curve of the CFU, indicating that though the factors affecting the cell kinetics had been eliminated, the cycling CFU behaved like a growing population. During the first week, the growth rate of the CFU remained the same as in control irradiated mice. The growth rate of the spleen CFU of the endotoxin-treated mice slowed down during the second week, and their self-replicating ability was low. Fluctuations in the DNA synthesizing fraction of the spleen CFU suggested a variability in the ratio of the length of the S phase and the cell generation time.     

KINETICS OF GROWTH OF HAEMOPOIETIC COLONY CELLS IN AGAR

Cell kinetic parameters of mouse granulocytic and mononuclear cells growing in colonies in agar cultures have been measured. Analysis of flash and continuous labelling studies with ³H-thymidine together with determinations of colony size, growth fraction and mitotic indices, gave the following values for the phases of the cell cycle: G₁= 6·3 1·6 hr, S = 5·8 ± 1·4 hr, G₂= 1·7 ± 0·1 hr and M = 0·7 ± 0·1 hr (42 ± 8 min). No difference in the cell cycle parameters of granulocytic and mononuclear cells were found in this study.
Colonies of different size from cultures of the same age group had similar labelling indices, indicating that the size of a colony is not a function of the rate of proliferation of cells in the colony. Rather, variation in colony size is probably representative of an initial delay in the onset of colony development.     

KINETICS OF MURINE HAEMOPOIETIC CELL PROLIFERATION IN DIFFUSION CHAMBERS

Murine bone marrow cells were cultured in cell impermeable diffusion chambers in the abdominal cavities of mice. The kinetics of granulocyte and macrophage formation were studied by stathmokinetic and autoradiographic techniques. During the period of most rapid growth of proliferative granulocytes, their generation time and its different phases were: t _c∽ 8 hr, t _G1∽ 1·5 hr, t _s∽ 5·5 hr, t _G2∽ 0·7 hr and t _M∽ 0·25 hr.
The generation time of macrophages and their precursors was approximately 8 hr. Formation of macrophages was significantly reduced when chamber inoculum was increased, as judged by ³H-TdR labelling index.     

CELL KINETICS IN DIFFUSION CHAMBERS: SURVIVAL, RESUMPTION OF PROLIFERATION, AND MATURATION RATE OF MURINE HAEMOPOIETIC CELLS

Bone marrow cells and blood leucocytes from mice have been cultured in vivo in diffusion chambers. Granulocytes and macrophages were formed in the chambers, whereas lymphocytes and mature end cells were gradually lost.
The proliferation, differentiation and death of cultured cells were quantified by total and differential cell counting, by scintillation counting and by radio-autographic evaluation of ³H-thymidine incorporation. Hydroxyurea and vinblastine were used as cytotoxic agents.
Ordinarily 40–60% of the cells inoculated could not be recovered after a few hours in culture. Proliferative cells resumed DNA synthesis shortly after implantation, and the rate of synthesis did not decline on further culturing. Steady-state progenitor cells had a pre-replicative lag period of about 18 hr, whereas regenerating progenitors started to synthesize DNA after about 14 hr. After 72 hr of culturing newly formed segmented granulocytes were detected. Maturation from the myelocyte to the segmented granulocyte stage lasted about 18 hr. Eosinophilic granulocytes had a long life span in the chambers, whilst the segmented neutrophils were rapidly eliminated after a life span estimated to be about 2 days.     

HAEMOPOIETIC STEM CELL (CFU) KINETICS IN THE CULTURE

Haemopoiesis continued for over 2 months in organ culture of embryonal mouse liver, and haemopoietic stem cells (CFU_s) capable of DNA-synthesis were found in it all that time. Between the 10th and 40th day the number of stem cells in the culture was sustained in a steady state. Both in normal and in regenerating adult bone marrow haemopoiesis ceased within a short time in the culture. Induction of proliferation in haemopoietic stem cells combined with undamaged or improved micro-environment resulted in a little better maintenance of CFU_s in the adult bone marrow culture, The results are discussed in the light of current concepts of haemopoietic stem cell regulation.     

ABILITY OF THYMIC LYMPHOCYTES TO ALTER CFU KINETICS IN RADIATION CHIMERAS

It is known that the poor colony-forming ability of B6 bone marrow transplanted into B6D2F₁ hybrids can be improved if B6 lymphocytes are given in addition. It was recently reported that the augmenting lymphocytes decrease the doubling time of differentiating hemopoietic cells. to determine whether thymus cells alter the self-renewal of CFUs in this parent F₁ combination, retransplan-tation and ³H-thymidine ‘suicide’ were employed as methods to determine the cell-division rate. We have observed that in the presence of thymocytes, parental bone marrow cells are seeded more efficiently in the spleen, and the lag phase of the CFUs growth curve is shortened. However, thymic lymphocytes do not increase the slope of the exponential growth phase of CFUs.     

鼻NK／T细胞淋巴瘤间质清道夫受体A的表达及意义

目的 观察清道夫受体A（scavenger receprorA,SR-A）在鼻NK／T细胞淋巴瘤间质中的表达,探讨其意义。方法 对鼻NK／T细胞淋巴瘤,鼻B细胞淋巴瘤以及鼻部炎症的石蜡标本进行HE染色和SP免疫组织化学染色检测SR-A的表达。结果 SR-A蛋白在鼻NK／T细胞淋巴瘤,鼻B细胞淋巴瘤和鼻部炎症中阳性率分别为92．7％,29．2％和6．25%。统计学分析发现,鼻NK／T细胞淋巴瘤中SR-A的表达与B细胞淋巴瘤和炎症中SR-A的表达均有显著差异（P〈0．001）。结论 SR-A可能在鼻NK／T细胞淋巴瘤的诊断及鉴别诊断中有一定的参考价值。     

喉癌间质肥大细胞的形态学特点及分布意义的探讨

应用免疫组织化学和透射电镜技术对 45例喉鳞癌组织 (其中 35例取癌中心为癌组 ,另 10例取癌旁组织为癌旁组 )中的肥大细胞 (MC)进行研究。结果 :1.癌旁组织中有丰富的 MC浸润而癌间质中 MC脱颗粒明显 ;2 .阿尔辛蓝·藏红 (AB· S)染色 ,二组 MC的组化性质显示出了差异 ;3.喉癌细胞增殖细胞核抗原 (PCNA)的阳性表达与 MC分布之间存在一定的相关性 ;4.癌间质 MC属类胰蛋白酶及糜蛋白酶 (TC)型 ,常与癌细胞有紧密接触 ,并可见 MC颗粒脱向癌细胞。结果提示 :MC与癌细胞关系密切 ,可能有抑制癌细胞增殖的倾向。     

SOME COMPARISONS OF THE KINETIC PROPERTIES OF FEMORAL AND SPLENIC HAEMOPOIETIC STEM CELLS

A slightly different radiosensitivity is confirmed between colony forming units (CFU) in the femur as opposed to those of the spleen. The recovery pattern of the CFU in the femur and in the spleen following sublethal doses of radiation can be markedly different, depending on the radiation dose, particularly concerning the postirradiation ‘dip’. The turnover state of splenic CFU appears to be greater in normal animals than that of the femoral CFU. Hypertransfusion doubles the splenic CFU by the fourth day, while only marginally affecting femoral CFU. Erythropoietin, while increasing the CFU turnover significantly in both femur and spleen of the polycythaemic animal, also increases femoral but not splenic CFU.     

前体化合物与诱导子对紫杉醇和紫杉烷类化合物合成的调控作用

添加Ｄ,Ｌ－β－苯丙氨酸和苯丙氨酸没有显著促进紫杉醇的合成,乙酸和苯甲酸对紫杉醇的合成有抑制作用,Ｃ１３位侧链合成的前体含量并不是合成紫杉醇的限制性因素。１￣３ｍｇ／Ｌ的α－亚麻酸没有促进紫杉醇和紫杉烷类化合物的合成。茉莉酮酸显著地促进了紫杉醇的合成,５ｍｇ／Ｌ茉莉酮酸处理的紫杉醇含量达到１３４μｇ／ｇＤＷ,是对照的９倍。     

KINETIC PROPERTIES OF HAEMOPOIETIC STEM CELLS

A comparison of the exocolonizing and autorepopulating tests for haemopoietic stem-cell assay indicate that the ‘overshoot’in splenic colony formation, observed 12–14 days after 150 rad total-body radiation (TBR), only occurs with the auto-repopulation assay. The explanation is that the priming dose of 150 rad increases the absolute seeding rate of stem cells from the marrow. A seeding rate significantly greater than normal can ‘take’only if the spleen is available—it can expand and accommodate stem cells while the bone marrow cannot. If, however, the absolute number of colony-forming cells are decreased in the femur, a relative increase in seeding rate can take place even in the splenectomized animal. Evidence is presented concerning the different turnover states of exo- and autorepopulating stem cells (CFU) and those responsible for erythropoietic response (ERC), and the precursors of agar colony-formers.     

CFU-C YIELDS FROM THE HAEMOPOIETIC TISSUES OF NORMAL AND LEUKAEMIC RFM MICE

Spleen and bone marrow cells from normal and leukaemic RFM mice have been assayed for numbers of colony forming cells in soft agar (CFU-C). The fluctuations in CFU-C yield observed during the development of myeloid leukaemia are similar to the results from in vitro experiments set up to test a model, and are not incompatible with the idea that interaction between normal and leukaemic cells may modify the yield of CFU-C under the present conditions of culture. Colonies grown from leukaemic spleen and bone marrow cells appear to be derived from the residual population of normal haemopoietic cells within the leukaemic mouse.     

DIFFERENT ACTION OF SUICIDAL DOSES OF TRITIATED THYMIDINE AND HYDROXYUREA ON MURINE HAEMOPOIETIC CELLS

In order to gather information on the factors that cause the different action of suicidal doses of tritiated thymidine (³H-TdR) and of hydroxyurea on murine stem cells, the incorporation of ³H-TdR into DNA of bone marrow and spleen cells has been studied. Continuous death of labelled cells after suicidal ³H-TdR is indicated by a more pronounced decline of total DNA-bound radioactivity in bone marrow and spleen cells compared to that in control animals which had received tracer doses of ³H-TdR. Extensive and rapid loss of DNA-bound radioactivity occurred in ³H-TdR labelled animals after hydroxyurea treatment indicating an instantaneous and highly effective killing of labelled cells. After double labelling of DNA with ³H-TdR and ¹²⁵iodo-deoxyuridine (¹²⁵I-UdR), the decline of the ratio of DNA-bound ¹²⁵I to DNA-bound ³H after suicidal ³H-TdR indicates prolonged tritium reutilization. Following hydroxyurea, reutilization was completed within the first 12 hr after drug administration. These findings explain in part the slow recovery of different stem cell compartments after suicidal ³H-TdR on the basis of protracted tritium reutilization as compared to the fast recovery which follows the rapid action of hydroxyurea.     

ANALYSIS OF PROLIFERATION AND DIFFERENTIATION OF FOETAL GRANULOCYTE-MACROPHAGE PROGENITOR CELLS IN HAEMOPOIETIC TISSUE*

Analysis of in vitro colony formation in agar cultures of foetal haemopoietic tissues of eight mammalian species has shown that granulocyte-macrophage progenitor cells are present in foetal liver, yolk sac, marrow and spleen in numbers approaching the incidence in adult marrow. Such characteristics as buoyant density, growth rate and differentiation served to distinguish foetal from adult colony forming cells (CFCs). Cell cycle analysis performed by exposing haemopoietic cells to high doses of tritiated thymidine in vitro showed that foetal CFC proliferation in species of short gestation (rabbit, rat, mouse) approached or exceeded that observed in adult marrow. In contrast, in species of long gestation (human, monkey, calf, lamb, guinea-pig) a period of variable duration was observed when foetal liver CFCs entered a non-cycling G₀ or blocked G₁ phase. In these species foetal liver CFCs were found to be proliferating actively early in gestation and following the non-cycling phase again re-entered a proliferative state associated with onset of active granulopoiesis in foetal marrow and possible migration of CFC from liver to marrow. These results indicate the existence of granulocyte-macrophage progenitor populations displaying foetal characteristics and adapted to particular stages of haemopoietic development, a situation which closely parallels that reported for erythropoiesis.     

MOBILIZATION OF B AND T LYMPHOCYTES AND HAEMOPOIETIC STEM CELLS BY POLYMETHACRYLIC ACID AND DEXTRAN SULPHATE

The leucocytosis which can be evoked by the polyanions dextran sulphate (DS), polymethacrylic acid (PMAA) and the copolymer of PMAA and styrene (PMAA—STYR) was studied in mice. After intravenous administration of these polyanions peak numbers of leucocytes were found in the peripheral blood 3 hr after injection. All three types of polyanions increased the number of lymphocytes, granulocytes and monocytes. Dose—response studies revealed that the nature of the polyanion determined the degree of leucocyte mobilization. The most potent mobilizer was found to be DS. This polyanion could evoke a six-fold increase of the number of peripheral blood leucocytes. By means of the membrane fluorescence technique it could be demonstrated that optimal doses of DS, PMAA and PMAA—STYR mobilized both B and T lymphocytes. The ratio between the number of B and T cells mobilized was greater for DS than for the other two polyanions. Intravenous injection of DS, PMAA and PMAA—STYR also increased the number of circulating haemopoietic stem cells (CFU-S). The most potent stem cell mobilizer appeared to be PMAA—STYR. This polyanion evoked a twenty-five-fold increase in the number of CFU-S.     

KINETICS OF THE SWELLING OF CELLS AND TISSUES

The rate of swelling of Arbacia eggs in dilute sea water, studied by Lillie and by Lucke and McCutcheon, may be expressed by the formulæ derived for the rate of increase in volume of a solution enclosed in a collodion sac. The rate of swelling of slices of carrot in distilled water, measured by Stiles and Jørgensen, may be expressed by the equation derived previously for the swelling of similarly shaped blocks of gelatin.     

NUCLEAR INCORPORATION OF RADIOACTIVE DNA PRECURSORS AND PROGRESSION OF CELLS THROUGH S

Bone marrow blast cells of nine children with untreated acute leukaemia (five lymphoid, three myeloid, one monocytic), myeloid precursor cells of a haema-tologically normal child and thoracic duct lymph cells of a patient with sclerodermia were pulse-labelled in vitro with tritiated thymidine and/or tritiated cytidine. Combined radioautographic and cytophotometric techniques were used for the determination of the median nuclear size and the median grain count of labelled cells in different segments of S. It was found that the median grain count reached maximum values in the second or third quarter of S in all cell populations studied, and that the variation, during S, of the median grain count appeared to be independent of the median DNA synthesis times of the cell types investigated. In six cell populations a clear-cut inverse relation existed between the number and the median grain count of labelled cells in different segments of S. In three populations this relation was less apparent and in two it was not found at all.     

AN ICAM-1 LIKE CELL ADHESION MOLECULE IS RESPONSIBLE FOR CD34 POSITIVE HAEMOPOIETIC STEM CELLS ADHESION TO BONE-MARROW STROMA

The microenvironment in the haematopoietic organs plays an important role in regulating and sustaining differentiation and self-renewal of haematopoietic stem cells. Although crucial for stem cell maintenance and homing, the stromal cell—stem cell interactions are poorly understood. Here we show that an ICAM-like molecule is responsible for stem cell adhesion to stromal cellsin vitro. The molecule was characterized by a monoclonal antibody 3E₁₀. Immunoblotting results indicated that the molecule had an electrophoretic mobility equal to that of intercellular cell adhesion molecule-1 (ICAM-1). Binding inhibition assays, however, showed that inhibition of binding of enriched CD34 cells by 3E₁₀was more prominent in comparison with that of ICAM-1.     

FAP在乳腺癌间质中的表达及其与微血管密度的关系

目的观察乳腺各组良恶性病变中FAP表达变化及其与微血管密度(MVD)的关系。方法本文应用免疫组织化学,Western Blotting实验方法观察FAP在乳腺腺病、乳腺纤维腺瘤、乳腺非浸润癌(导管原位癌),乳腺浸润性导管癌及MCF-7-CCC-HPF-1、MDA-MB-231-CCC-HPF-1共培养模型中的表达变化并探讨FAP与微血管密度(MVD)的关系。结果FAP在乳腺腺病、乳腺纤维腺瘤间质中表达阴性,在乳腺非浸润癌(导管原位癌)、乳腺浸润性导管癌间质中高表达;FAP在细胞系MDA-MB-231中不表达,在共培养模型MCF-7-CCC-HPF-1,MDA-MB-231-CCC-HPF-1中均有表达;FAP表达与MVD相关(P<0.05)。结论FAP可能作为判定乳腺良恶性病变的指标之一,且可能在促进乳腺癌浸润、生长及转移中发挥一定作用。     

EFFECTS OF MELANOPROTEINS AND THEIR PRECURSORS ON CELL PROLIFERATION THE PROBLEM OF SELECTIVITY

The actions of melanin and its precursors on mitotic frequencies, cell division and ³H-thymidine incorporation in protokaryotic and eukaryotic cells are studied. It was also suggested that the binding of melanin precursors with proteins in the melanosomes is a way of scavenging a cytotoxia activity.