THE PREDOMINANT ROLE OF THE SPLEEN IN LYMPHOCYTE RECIRCULATION

Lymphocyte recirculation through the isolated pig spleen was studied by means of a perfusion system which kept the organ alive for a prolonged period of time. By changing the perfusate to a leucocyte-enriched or cell-free perfusate and taking serial arterial and venous samples, the numbers of lymphocytes which homed to or were released from the spleen were measured. In all experiments more lymphocytes homed than were released per minute. There was no apparent difference when autologous or allogeneic cells were used. The number of lymphocytes released depended on the number of lymphocytes homed previously. During the phase of constant release up to 3-3 × 10⁶ lymphocytes were released per gram spleen per minute. From these values it can be extrapolated that up to 270 × 10⁹ lymphocytes recirculate through the isolated pig spleen per day. Based on kinetic data from other species it is estimated that in the entire pig a total number of 300–400 × 10⁹ lymphocytes recirculate per day. Thus, it can be concluded that the spleen is the most important organ for lymphocyte recirculation in the pig.     

THE PREDOMINANT ROLE OF THE SPLEEN IN LYMPHOCYTE RECIRCULATION

Autologous blood lymphocytes from three normal pigs were labelled with ³H-uridine and retransfused before and after splenectomy. Frequent samples for up to 150 min after retransfusion were evaluated autoradiographically to determine the rate of disappearance of labelled lymphocytes from the blood. In one pig retransfusion was performed before and after sham-splenectomy. In all preoperative experiments the pattern of disappearance of labelled lymphocytes was very similar. After a first rapid decline (halving time on average 8 min) a short rise of the labelling index was observed from 10 to 15 min after retransfusion. Then a second more gradual decrease of labelled lymphocytes followed. The mean halving time during this period was less than 32 min. From 60 min onwards the labelling index remained nearly constant. Retransfusions performed 3 days after splenectomy revealed only one nearly constant decline of the labelling index (halving time on average 129 min). After sham-splenectomy the pattern of disappearance was similar to the preoperative experiment. One hour after the end of retransfusion the labelling index had decreased by three-quarters of the initial value in normal pigs and by only one-third in the splenectomized ones. These results indicate that in the pig the total rate of recirculation is at least 4 times faster with the spleen in situ than without the spleen.     

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.     

EFFECTS OF ENVIRONMENTAL TEMPERATURE ON HAEMOPOIETIC STEM CELLS IN THE TAILS OF MICE

Using the endogenous spleen colony assay method of Till & McCulloch (1963), the numbers of haemopoietic stem cells present in the bone marrow in the tails of mice were estimated under different environmental temperatures. Compared to animals kept at 22–26°C, mice transferred to and kept at 36.5°C showed a doubling of colony-forming units in the tail in 1–4 weeks. Exposing them to 8°C caused a significant depopulation to approximately one-third in 3–4 weeks. By transferring the mice from one temperature extreme to another these changes could be reversed. Tail marrow depleted of viable stem cells by X-irradiation was repopulated within approximately 3 weeks in animals kept at room temperature or above but this process was inhibited in the cold.     

PROPERTIES OF HAEMOPOIETIC STEM CELLS IN PHENYLHYDRAZINE TREATED MICE

Recovery of erythropoiesis was fast in Balb/c mice irradiated 700 R 5 days after initiation of phenylhydrazine treatment and took place predominantly in the spleen, which showed numerous large frequently confluent endogenous colonies. Post irradiation phenylhydrazine induced anaemia did not accelerate recovery of erythropoiesis; it did, however, produce a slight but significant rise in endogenous colony formation.
Radiosensitivity of spleen CFU-S from phenylhydrazine treated mice was similar to that of CFU-S in normal mouse spleen.
Spleen CFU-S in mice 5 days after initiation of phenylhydrazine treatment were sensitive to the lethal action of Hydroxyurea, while bone marrow CFU-S were not.
The self-renewal capacity of CFU-S in the endogenously repopulated spleen of phenylhydrazine pretreated 700 R X-irradiated mice was low when compared to that of spleen exogenously repopulated by cells from normal mouse bone marrow, normal and phenylhydrazine treated mouse spleen. CFU circulating in blood of phenylhydrazine treated mice had a low self-renewal capacity.
The marked strain differences in self-renewal capacity of spleen CFU-S, and of the capacity of spleen CFU-S to increase by proliferation are discussed.     

A COMPARATIVE STUDY OF THE REPOPULATING POTENTIAL OF GRAFTS FROM VARIOUS HAEMOPOIETIC SOURCES: CFU REPOPULATION

The growth rate of the CFU populations in spleens and femora has been studied in irradiated mice injected with cell suspensions, containing equivalent number of CFU, from various sources. The doubling times are shown to be dependent upon the source of the cells. Grafts of bone marrow, spleen and foetal liver cells produced doubling times in the spleen of approximately 25, 19 and 16 hr respectively. Grafts of marrow-derived and spleen-derived spleen colony cells both gave rise to CFU doubling times lower than those of the corresponding primary grafts (approx. 33 and 26 hr respectively in the spleen). In the case of both bone marrow and spleen grafts the CFU population growth was shown to be independent of the size of the graft. A hypothesis is advanced which invokes at least a dual population of CFU, having different doubling times, different seeding capacities in the haemopoietic tissues following i.v. injection and present in different proportions in the various haemopoietic tissues.     

THE ENHANCEMENT OF HAEMOPOIETIC STEM CELL RECOVERY IN IRRADIATED MICE BY PRIOR TREATMENT WITH CYCLOPHOSPHAMIDE

Studies are reported of the enhancement of stem cell recovery following whole body irradiation as a result of prior administration of cyclophosphamide. It is shown that the much larger enhancement of regeneration observed for the hosts own surviving stem cells, compared to the regeneration of injected bone marrow stem cells, is due to the different numbers of stem cells initiating the regeneration in conjunction with the time course of stem cell regeneration. The results show that the environmental changes produced by cyclophosphamide greatly enhance haemopoietic recovery even though at the dose used this agent is relatively toxic to stem cells. Furthermore it has been shown that the level of stem cell regeneration is nearly independent of the γ-ray dose in the range 3–8 gray (300–800 rad). If human bone marrow should respond similarly it follows that regeneration produced by cytotoxic drugs administered prior to radiation embodies a considerable safety factor as far as recovery of the haemopoietic system is concerned.     

LOCATION OF THE G0-PHASE IN THE CELL CYCLE OF THE MOUSE HAEMOPOIETIC SPLEEN COLONY FORMING CELLS

Experiments in mice on the fraction of haemopoietic stem cells in S-phase after irradiation indicated that a large fraction of the cells resting in G₀ will enter S-phase after a very short interval of time.
After excluding alternative explanations it must be concluded that cells in G₀ have completed all preparations for going into S-phase or, in other words, that the localization of these G₀ cells in relation to other phases of the cell cycle must be between G₁ and S-phase.     

THE FINE STRUCTURE OF LIPOFUSCIN AGE PIGMENT IN THE NERVOUS SYSTEM OF AGED MICE

An examination of the topographic distribution of lipofuscin pigment granules with the light and electron microscope revealed either smaller and randomly "dispersed" or larger and more complex "clustered" pigment configurations in the cytoplasm of neurons in the dorsal ganglia and ventral spinal cord of 24-month old male mice. Qualitative comparisons revealed no major differences in shape, size, complexity, density, orientation, and cytologic distribution of the pigment bodies in motor and sensory neurons. In general, when the pigment granules were quite numerous within the 2 types of cells, they were smaller in size (~lµ), had a dense homogeneous matrix with few bands or lamellae, and were uniformly distributed throughout the cytoplasm. In contrast, when the pigment configurations were less in number, they were usually larger in size (~3µ), had a more complex internal banded structure, and appeared more localized within the cell. Examination of the bands revealed a repeating pattern of ~70 A. The bands appeared to fuse, forming hexagonal arrays of linear densities intersecting at an angle of approximately 120° in some regions of the pigment bodies. Structural similarities suggested that the striated membranous bands may be composed of phospholipids.     

PREVENTION OF GRAFT VERSUS HOST REACTION BY INCUBATION OF LYMPHOID CELLS WITH A SPLENIC EXTRACT (NOT AFFECTING THE REPOPULATION OF THE HAEMOPOIETIC TISSUE)

An alcohol extract of calf spleen possessing the in vivo characteristics of a lymphoid chalone has already been shown to prevent or to control the acute graft versus host reaction (GVHR) in lethally irradiated F₁ recipients, when given respectively to the parental cell donors or to the F₁ recipients. The present experiments demonstrate the efficacy of the incubation of the donor cells with this extract on the prevention of GVHR, and shows that it selectively inactivates cells responsible for it.     

外源性线粒体DNA在细胞内的重建(简报)

近年来发现人类多种神经肌肉疾病存在线粒体电子传递链(electron transport chain,ETC)缺陷。由于线粒体在遗传上受核基因和线粒体基因双重控制,给确定ETC缺陷的来源造成困难。转线粒体DNA技术是线粒体同无线粒体DNA的细胞(ρ°cells)融合,形成转线粒体DNA细胞系(mtDNA-transferred cell line,也称cytoplasmic hybrids,简称cybrids),使病人的线粒体DNA(mito-     

AGE DEPENDENCE OF THE NUMBER OF THE STEM CELLS IN HAEMOPOIETIC TISSUES OF RATS

The number and concentration of haemopoietic stem cells in the femoral bone marrow and spleen of Wistar rats of different ages were investigated. Stem cells were assayed by the spleen colony technique in irradiated rat recipients. The ability of the recipient spleen to harvest transplanted tissue as a macroscopic colony was found to be dependent on the recipient's age. Changes with senescence were observed also in the concentration and the size of the stem cell compartment both in the marrow and spleen. No differences were demonstrated in the seeding of transplanted colony-forming units into the spleen of recipients of 1 and 4 months of age. A rats-mice strain difference in the effect of senescence on the haemopoietic stem cells is discussed.     

THE EFFECT OF FETUIN AND OF SPLEEN EXTRACTS ON HEMATOPOIETIC PRECURSORS AND ON DIFFERENTIATED HEMATOPOIETIC CELLS IN IRRADIATED MICE

Injection of extracts from normal mouse spleen tissue into irradiated mice enhance the rate of regeneration of colony forming units (CFU-S) in the femoral marrow. This effect was most pronounced when spleen extract was injected between 24 hr before and 24 hr after the time of irradiation, and was observed only during the first week after a single injection of extract. Another result of injecting spleen extract was an immediate and transient decrease in the marrow cellularity and particularly in the number of mature myeloid cells in the marrow. Fetuin produced comparable effects on the rate of regeneration of CFU-S and on the numbers of mature myeloid cells in the marrow. On the basis of these results it is tempting to speculate that injection of spleen extracts and of fetuin primarily cause a rapid depletion of the marrow's granulocyte reserve. This in turn releases the precursor cell compartment from the inhibitory effects of cell–cell interaction and results in an acceleration of the rate of CFU-S regeneration. It is equally plausible that factors present in spleen extract and in fetuin cause a depletion of the marrow granulocyte reserve and, by an unrelated mechanism, directly accelerate the rate of regeneration of CFU-S.     

蓖麻蚕对鼠生殖系统雌激素样效应的研究

目的研究蓖麻蚕雌蛾乙醇提取物(EEFM)的雌激素效应,以寻找理想的ERT药源并提高蓖麻蚕的综合利用价值.方法根据每日药物及剂量处理,将雌性小鼠随机分为模型对照组,正常对照组,乙烯雌酚组,EEFM低剂量(EEFM-L,0.2 g/kg·d-1)组,EEFM高剂量(EEFM-H,0.4 g/kg·d-1)组.测定实验鼠的子宫、免疫器官脏器指数、阴道角化细胞发生率及POD活性.结果EEFM两个剂量对未成年雌性小鼠、成年去势雌性小鼠子宫作用效应均达到显著或极显著.EEFM对去势成年雌性小鼠角化细胞发生率的影响约为乙烯雌酚(DES)组的2/3(EEFM-H77.20%,DES95.7%),效果极显著.结论EEFM对实验鼠具有显著的雌激素效应,对免疫器官元不良影响(DES组显著降低实验鼠胸腺重量),有可能成为雌激素替代疗法(ERT)的理想药源物质.     

交感神经系统对切除迷走神经大鼠的胃酸分泌作用的观察

与迷走神经完整大鼠比较,迷走神经切除(VT)大鼠胃酸分泌显著减少(从2.67到1.14μEq/10min),约两个月恢复到术前水平。在迷走神经完整大鼠,异丙肾上腺素(IS,10μg/min)显著地刺激酸分泌,去甲肾上腺素(NA,5μg/min)则抑制酸分泌。在 VT 三月后的大鼠,IS 抑制酸分泌,NA 则显著地刺激酸分泌。这种变化的生理意义可能是交感神经系统代偿丧失了的迷走泌酸机能。IS 和 NA 增加大鼠心率,心得安(25μg/min)显著地减少它,而酚妥拉明(25μg/min)没有明显的作用;IS、NA、心得安或酚妥拉明对迷走神经完整、VT即刻和三月后的大鼠心率的影响均无差异,表明 VT 后交感神经系统调节胃酸分泌的作用的变化并非循环改变的结果。