Cell proliferation in the bone marrow, thymus and spleen of mice studied by continuous, in vivo bromodeoxycytidine labelling and flow cytometric analysis

Abstract We have applied the technique of labelling dividing cells with bromodeoxyuridine (BrdUrd) in combination with in vivo continuous labelling, propidium iodide (PI) staining for DNA content, and flow cytometric analysis, for the determination of cell proliferation in bone marrow, thymus and spleen of mice. The percentage of BrdUrd labelled cells increased as a function of exposure time in a tissue specific manner for each of the three tissues. Thymus and bone marrow had cell populations which exhibited different kinetics for the accumulation of label: (1) those that cycled and became labelled within 2–3 days (88% in 2 days for bone marrow, 84% in 3 days for thymus); (2) those that cycled during the remainder of the 6 day infusion period (11% of bone marrow and 13% of thymus cells); and (3) those that did not cycle during the 6 day period studied (<2% of bone marrow and 3% of thymus cells). In contrast, the spleen exhibited a slower, constant accumulation of labelled cells. After six days of infusion a large proportion of spleen cells (50%) had not become labelled. These results suggest that a larger proportion of spleen cells are long lived than indicated by other methods. We also have found that the period of labelling with BrdUrd extended several days beyond the period of infusion. This method will be very useful in studying perturbations of cell populations induced in mice exposed to toxic agents.     

Cell proliferation in the bone marrow, thymus and spleen of mice studied by continuous, in vivo bromodeoxycytidine labelling and flow cytometric analysis

We have applied the technique of labelling dividing cells with bromodeoxyuridine (BrdUrd) in combination with in vivo continuous labelling, propidium iodide (PI) staining for DNA content, and flow cytometric analysis, for the determination of cell proliferation in bone marrow, thymus and spleen of mice. The percentage of BrdUrd labelled cells increased as a function of exposure time in a tissue specific manner for each of the three tissues. Thymus and bone marrow had cell populations which exhibited different kinetics for the accumulation of label: (1) those that cycled and became labelled within 2-3 days (88% in 2 days for bone marrow, 84% in 3 days for thymus); (2) those that cycled during the remainder of the 6 day infusion period (11% of bone marrow and 13% of thymus cells); and (3) those that did not cycle during the 6 day period studied (less than 2% of bone marrow and 3% of thymus cells). In contrast, the spleen exhibited a slower, constant accumulation of labelled cells. After six days of infusion a large proportion of spleen cells (50%) had not become labelled. These results suggest that a larger proportion of spleen cells are long lived than indicated by other methods. We also have found the period of labelling with BrdUrd extended several days beyond the period of infusion. This method will be very useful in studying perturbations of cell populations induced in mice exposed to toxic agents.     

The primary role of murine bone marrow in the production of natural killer cells. A cytokinetic study

The normal steady state production of natural killer (NK) cells in the bone marrow and spleen was characterized with cytokinetic technics. We developed a protocol to enrich for NK cells in bone marrow and demonstrate that target binding can be used as a criterion for marrow NK cells if nonspecifically "sticky" cells are eliminated. The selected population of B cell-depleted bone marrow lymphoid cells was comprised mainly of lymphocytes, of which 80% were NK-1.1+. B cell-depleted bone marrow lymphocytes that bound to YAC-1 could be characterized as two populations on the basis of morphology and proliferative status: large, proliferating target-binding cells (TBC), of which 25% were in S phase of the mitotic cycle, and small postmitotic TBC. Pulse and chase studies indicated that the small TBC in bone marrow were derived from an immediate proliferating precursor, presumably the large TBC, which were, in turn, derived from a precursor population that was more rapidly proliferating. In contrast, few if any splenic TBC were labeled after a 30-min pulse with [3H]TdR and significant numbers of labeled TBC did not appear in the spleen until 2 or more days after the pulse label. Surprisingly, some of the splenic TBC were relatively long lived and survived 2 mo or longer. These studies are the first to directly characterize the production of NK cells in situ in normal marrow. We demonstrate that the marrow is the primary site of production of NK cells and that little, if any, proliferation of NK cells occurs in the periphery of unstimulated mice. The data suggest the existence in the bone marrow of at least three compartments in the NK lineage: a rapidly proliferating NK precursor population, a less rapidly proliferating population of large TBC, and a population of small postmitotic TBC.     

Endocytosis of lactate dehydrogenase isoenzyme M4 in rats in vivo. Experiments with enzyme labelled with O-(4-diazo-3,5-di[125I]iodobenzoyl)sucrose.

1. Pig lactate dehydrogenase isoenzyme M4 was labelled with O-(4-diazo-3,5-di[125I]iodobenzoyl)sucrose and injected intravenously into rats. Previous work has shown that this label does not influence the clearance of the enzyme (half-life about 26 min) and that it is retained within the lysosomes for several hours after endocytosis and breakdown of the protein [De Jong, Bouma & Gruber (1981) Biochem. J. 198, 45--51]. 2. The distribution of the radioactivity over a large number of tissues was determined 2 h after injection. A high percentage of the injected dose was found in liver (41%), spleen (10%) and bone including marrow (21%). 3. Autoradiography indicated uptake of the enzyme mainly by Kupffer cells of the liver, by spleen macrophages and by bone marrow macrophages. 4. Liver cells were isolated 1 h after injection of the enzyme. Kupffer cells, endothelial cells and parenchymal cells were found to endocytose the enzyme at rates corresponding to 4230, 35 and 25 ml of plasma/day per g of cell protein, respectively. 5. Previous injection of carbon particles greatly reduced the uptake of the enzyme by liver and spleen, but the uptake by bone marrow was not significantly changed.     

Stimulation of monocyte precursors in vivo by an extract from Listeria monocytogenes.

A water-soluble monocytosis-producing activity (MPA) extracted from Listeria monocytogenes was found to stimulate proliferation of promonocytes in vivo. Mice were pulse-labelled for 2 h with tritiated thymidine ([3H]TdR) at various times after intraperitoneal injection of MPA. Autoradiography of bone marrow cells revealed an increased labelling index of promonocytes of MPA-treated mice which was maximum 8 h after the MPA injection. Mice labelled with [3H]TdR 8 h after MPA injection developed a monocytosis at the expected time (peak at 48 h) and the blood monocytes were found to be highly labelled. Both the generation time of monocyte precursors and the halftime of blood monocytes were found to be shorter than the corresponding values in control mice.     

MIGRATION OF SMALL LYMPHOCYTES IN ADULT MICE DEMONSTRATED BY PARABIOSIS

Parabiotic BALB/C mice were used to study the traffic of small lymphocytes in immunological mature but unchallenged mice. By giving ³H-thymidine (³H-TdR) injections to only one member (A) of a pair by preventing the escape of the radioactive isotope to the other member (B), the kinetics of newly-formed cells was followed. Less than 10% labelled small lymphocytes were found in the peripheral lymphoid tissues of both A and B members, while the thymusses and bone marrows of A members showed labelling percentages up to 70% in this period. Hardly any labelled cells gained entrance into the thymus while a detectable number was found in the bone marrows of B members. Results from pairs set up to follow migration of long-lived lymphocytes revealed that labelled cells detected 4–5 weeks after injections were equilibrated between the peripheral tissues and the bone marrows of the partners. Very few labelled cells were seen in the thymic medulla and none were observed in the thymic cortex, germinal centres or medullary cords of lymph nodes from any B member. It was concluded that short-lived small lymphocytes are formed primarily in the thymus and bone marrow and the migration of these cells is limited in adult animals. Furthermore, the vast majority of long-lived small lymphocytes are freely recirculating, and these cells gain entrance to and are normal residents in the bone marrow.     

Immediate bromodeoxyuridine labelling of unseparated human bone marrow cells ex vivo is superior to labelling after routine laboratory processing

It is important to evaluate the proliferation of bone marrow cells in several disease conditions and during treatment of patients with for example cytokines. Labelling with bromodeoxyuridine (BrdUrd), immunocytochemical staining with anti-BrdUrd antibody and analysis by flow cytometry provides a reliable and reproducible technique for estimation of the fraction of cells that incorporated BrdUrd into DNA during S-phase. We have compared immediate BrdUrd labelling of unseparated bone marrow cells with the previously used labelling in the laboratory after routine separation of the mononuclear cells. Bone marrow aspirates from seven lymphoma patients without bone marrow involvement were studied with these two methods. We found higher BrdUrd labelling indices (LI) in the mononuclear cells, when cells were labelled immediately. A large variation in LI was found between patients. Our results suggest that ex vivo BrdUrd labelling of bone marrow cells should be performed immediately after aspiration and before separation, because these data are closer to values reported from in vivo labelling with BrdUrd.     

The predominant role of the spleen in lymphocyte recirculation. II. Pre- and postsplenectomy retransfusion studies in young pigs.

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.     

Are all lymphoid blasts in the cell cycle? DNA synthesis in the lymphoid tissues of the dog

Labelling index after one or repeated intravenous injections of 3H-thymidine was measured for various subpopulations of lymphatic cells in different canine lymphoid compartments and correlated with cell morphology. High doses of tritiated thymidine were injected and exposure times of up to 211 days were used. The labelling indices of lymphoid blasts were comparable in all tissues investigated. Labelling index varied from 100% in immunoblasts to 4% in small-sized lymphocytes. Approximately 80% of immunoblasts were labelled 1 h after 3H-thymidine application and 100% labelling was obtained after 12 h repetitive 3H-thymidine labelling. In contrast with medium-sized and large lymphocytes, immunoblasts seem to be rapidly proliferating cells in the dog with almost no Go cells.     

Are all lymphoid blasts in the cell cycle?

Labelling index after one or repeated intravenous injections of 3H-thymidine was measured for various subpopulations of lymphatic cells in different canine lymphoid compartments and correlated with cell morphology. High doses of tritiated thymidine were injected and exposure times of up to 211 days were used. The labelling indices of lymphoid blasts were comparable in all tissues investigated. Labelling index varied from 100% in immunoblasts to 4% in small-sized lymphocytes. Approximately 80% of immunoblasts were labelled 1 h after 3H-thymidine application and 100% labelling was obtained after 12 h repetitive 3H-thymidine labelling. In contrast with mediumsized and large lymphocytes, immunoblasts seem to be rapidly proliferating cells in the dog with almost no G_o cells. Supported by the Deutsche Forschungsgemeinschaft DFG Grant SFB 112     

Plasma clearance and endocytosis of cytosolic malate dehydrogenase in the rat.

1. Pig heart cytosolic malate dehydrogenase was radiolabelled with O-(4-diazo-3,5-di-[125I]iodobenzoyl)sucrose and intravenously injected into rats. Enzyme activity and radioactivity were cleared from plasma identically, with first-order kinetics, with a half-life of about 30 min. 2. The tissue distribution of radioactivity was determined at 2 h after injection. All injected radioactivity was recovered from the tissues. A high percentage of the injected dose was found in liver (37%), spleen (6%) and bone including marrow (19%). 3. Radioactivity in liver and spleen increased up to 2 h after injection and subsequently declined, with a half-life of about 20 h. 4. After differential fractionation of liver, radioactivity was largely found in the mitochondrial and lysosomal fraction. 5. Liver cells were isolated 1 h after injection of labelled enzyme. We found that Kupffer cells, endothelial cells and parenchymal cells had endocytosed the enzyme at rates corresponding to 2725, 94 and 63 ml of plasma/day per g of cell protein respectively. 6. Radioautography indicated that in spleen and bone marrow the enzyme is mainly taken up by macrophages. 7. Internalization of the enzyme by liver, spleen and bone marrow was saturable. This indicates that the enzyme is taken up in these tissues by adsorptive endocytosis. 8. The present results closely resemble those obtained previously for the mitochondrial isoenzyme of malate dehydrogenase and for lactate dehydrogenase M4. Since those enzymes are positively charged at physiological pH, whereas cytosolic malate dehydrogenase is negative, net charge cannot be the major factor determining the rate of uptake of circulating enzymes by reticuloendothelial macrophages, as has been suggested in the literature [Wachsmuth & Klingmüller (1978) J. Reticuloendothel. Soc. 24, 227-241].     

Effect over time of in-vivo administration of the polysaccharide arabinogalactan on immune and hemopoietic cell lineages in murine spleen and bone marrow.

Current evidence indicates an immunostimulating role for complex carbohydrates, i.e., polysaccharides, from several plant sources. In the present work, we determined the specific in vivo effects, with time of administration, of one such compound, a neutral arabinogalactan from larch not only on immune (lymphoid) cells, but also on natural killer (NK) lymphoid cells, as well as a variety of other hemopoietic cells in both the bone marrow and spleen of healthy, young adult mice. The latter were injected daily (i.p.) with arabinogalactan (500 microg in 0.1 ml pH 7.2 phosphate buffered saline-PBS) for 7 or 14 days. Additional, aged (1 1/2-2 yr) mice were similarly injected for 14 days only. Control mice were given the PBS vehicle in all cases, following the above injection regimen. Animals from all groups were sampled 24 h after the final injection and the immune and hemopoietic cell populations in the bone marow and spleen were assessed quantitatively. The results indicated that immediately following either 7 or 14 days of arabinogalactan administration to young, adult mice, lymphoid cells in the bone marrow were significantly decreased (p < 0.004; p < 0.001, respectively) relative to controls but remained unchanged at both time intervals in the spleen. NK cells, after 7 days of arabinogalactan exposure, were also decreased significantly in the bone marrow (p < 0.02), but unchanged in the spleen. After 14 days' exposure to the polysaccharide, NK cells in the bone marrow had returned to normal (control) levels, but were increased in the spleen (p < 0.004) to levels greater than 2-fold that of control. Among other hemopoietic cell lineages, none was influenced in the bone marrow or spleen by one-week administration of arabinogalactan; however, after two-week exposure, precursor myeloid cells and their mature (functional) progeny (granulocytes), were significantly reduced in the spleen (p < 0.043; p < 0.006, respectively), as were splenic monocytes (p < 0.001). These lineages in the bone marrow, however, remained steadfastly unaltered even after 14 days of continuous exposure to the agent. Of the vast cascade of cytokines induced in the presence of this polysaccharide, it appears that immunopoiesis- and hemopoiesis-inhibiting ones are most prevalent during at least the first two weeks of daily exposure.     

Radioprotection of mice with interleukin-1: relationship to the number of erythroid and granulocyte-macrophage colony-forming cells

This report presents the results of an investigation of changes in the number of erythroid and granulocyte-macrophage colony-forming cells (GM-CFC) that had occurred in tissues of normal B6D2F1 mice 20 h after administration of a radioprotective dose (150 ng) of human recombinant interleukin-1 (rIL-1). Neutrophilia in the peripheral blood and changes in the tissue distribution of GM-CFC demonstrated that cells were mobilized from the bone marrow in response to rIL-1 injection. For example, 20 h after rIL-1 injection marrow GM-CFC numbers were 80% of the numbers in bone marrow from saline-injected mice. Associated with this decrease there was a twofold increase in the number of peripheral blood and splenic GM-CFC. Also, as determined by hydroxyurea injection, there was an increase in the number of GM-CFC in S phase of the cell cycle in the spleen, but not in the bone marrow. Data in this report suggest that when compared to the spleen, stimulation of granulopoiesis after rIL-1 injection is delayed in the bone marrow. Also, the earlier recovery of GM-CFC in the bone marrow of irradiated mice is not dependent upon an increase in the number of GM-CFC at the time of irradiation.     

Plasma clearance and endocytosis of mitochondrial malate dehydrogenase in the rat.

1. Pig mitochondrial malate dehydrogenase was labelled with 125I and intravenously injected into rats. Enzyme activity and radioactivity were cleared from plasma identically, with first-order kinetics, with a half-life of only 7 min. 2. Radioactivity accumulated in liver, spleen, bone (marrow) and kidneys, reaching maxima of 3 1, 4, 6 and 9% of the injected dose respectively, at 10 min after injection. 3. Our data allow us to calculate that in the long run 59, 5, 11 and 13% of the injected dose is taken up and subsequently broken down by liver, spleen, bone and kidneys respectively. 4. Differential fractionation of liver showed that the acid-precipitable radioactivity was mainly present in the lysosomal and microsomal fractions, suggesting that the endocytosed protein is transported via endosomes to lysosomes, where it is degraded. 5. Radioautography of liver and spleen suggested that the labelled protein was taken up by macrophages of the reticuloendothelial system. 6. Mitochondrial malate dehydrogenase is probably internalized in liver, spleen and bone marrow by adsorptive endocytosis, since uptake of the enzyme of these tissues is saturable.     

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 CELL RENEWAL, CELL MIGRATION AND CELL LOSS IN THE HAIRLESS MOUSE DORSAL EPIDERMIS

Forty hairless mice were given injections of tritiated thymidine every 4th hour during 10 days. At 24 hr intervals groups of four mice were killed. The numbers of labelled basal and differentiating cells were determined by autoradiography with a stripping film technique. To determine the background activity skin sections from uninjected control mice were subjected to the same stripping film procedure. Another group of hairless mice was given one single pulse labelling with tritiated thymidine. The number of labelled mitoses was scored for 12 hr after the injection. At 10, 12 and 15 hr after the injection, the numbers of labelled basal and differentiating cells were also determined. A mathematical model of cell population kinetics in the epidermis has been suggested. The results of different simulations on this model were compared with the observed results. The curve of mean grain counts under continuous labelling increased from day to day with two well-defined plateaux. The percentage of all labelled cells increased rapidly up to the 3rd day, and thereafter the curves gradually flattened off. When basal cells and differentiated cells were considered separately the labelling index of the basal cells increased rapidly for the first 3 days and then flattened off at the 100% level on the 5th day. The labelling index of the differentiating cells was low during the first 3–4 days. Then a steep increase in the percentage of labelled differentiating cells was seen, but the curve flattened off again close to the 100 % level after the 7th day. The labelled mitosis curve had its maximum 5 hr after the thymidine injection. The curve fell again to almost zero at 12 hr. Ten, 12 and 15 hr after the injection, 6, 7 and 7% respectively of the labelled cells were found in the spinous layer. It was concluded that three grains over each nucleus could be used as lower limit for considering a cell as labelled. On this basis, tritiated thymidine injections every 4th hour can be considered as continuous labelling.     

Biochemical and morphological modifications in rabbit Achilles tendon during maturation and ageing.

1. The plasma clearance of intravenously injected 125I-labelled mitochondrial malate dehydrogenase (half-life 7 min) was not influenced by previous injection of suramin and/or leupeptin (inhibitors of intralysosomal proteolysis). 2. Pretreatment with both inhibitors considerably delayed degradation of endocytosed enzyme in liver, spleen, bone marrow and kidneys. 3. The tissue distribution of radioactivity was determined at 30 min after injection, when only 3% of the dose was left in plasma. All injected radioactivity was still present in the carcass. The major part of the injected dose was found in liver (49%), spleen (5%), kidneys (13%) and bone, including marrow (11%). 4. Liver cells were isolated 15 min after injection of labelled enzyme. We found that Kupffer cells and parenchymal cells had endocytosed the enzyme at rates corresponding to 9530 and 156 ml of plasma/day per g of cell protein respectively. Endothelial cells do not significantly contribute to uptake of the enzyme. 5. Uptake by Kupffer cells was saturable, whereas uptake by parenchymal cells was not. This suggests that these cell types endocytose the enzyme via different receptors. 6. Previous injection of carbon particles greatly decreased uptake of the enzyme by liver, spleen and bone marrow.     

Ultraviolet B irradiation induces epidermal regeneration with rapidly cycling cells

The left flank of hairless mouse skin was irradiated with a minimal erythema dose of ultraviolet B (UVB) light at 297 nm (25 mJcm-2), while the right flank served as untreated control. The alterations in epidermal growth kinetics induced by this UVB dose were studied with the percentage of labelled mitoses (PLM) technique during the period of increased proliferation. Thirty hours after irradiation, when a large cohort of cells appears in S phase, each animal was injected intra-peritoneally with 50 microCi tritiated thymidine [( 3H]-TdR). The number of labelled basal and suprabasal cells, as well as their localization in epidermis were registered in histological sections at short intervals up to 48 h after the [3H]-TdR pulse. Labelled mitoses were also counted in the same specimens. The results showed a four-fold increase of the high initial number of labelled cells in UVB-exposed epidermis within 18 h of the pulse injection, and a six-fold increase after 36 h. In control epidermis, where the starting value of the labelling index was much lower, there was only a three to four-fold increase in the number of labelled cells during the period studied. The PLM and the labelling index data were consistent with an average cell cycle time of approximately 10-12 h for UVB-exposed cells, in contrast to about 30 h for the fastest cycling population in control epidermis. The PLM curve also indicated a prolonged S phase duration in UVB-exposed epidermis compared with controls. In addition, labelled cells were seen in the suprabasal layer as early as 6 h after the [3H]-TdR injection and within 36 h labelled cells had reached the outermost layer of nucleated cells, indicating a reduced transit time through epidermis. The present study shows that a minimal erythema dose of UVB light at 297 nm induced a period of increased transit time through the S phase, combined with rapid cell proliferation, leading to an overall shortening of the epidermal cell cycle time. The cohort of cells labelled with [3H]-TdR 30 h after irradiation seemed to proceed as a wave of partially synchronized cells through the cell cycle for more than two rounds, which is comparable with the cell kinetic perturbations observed in regenerating mouse epidermis.     

Cell proliferation kinetics of the bile duct epithelium of the rat

Abstract. Cell proliferation kinetics of the extrahepatic bile duct were studied by flash and cumulative labelling methods and immunohistochemical techniques. We compared the cell kinetics of the epithelium of the intra- and extra-pancreatic bile ducts and of the bile duct of the ampulla in rats administered intraperitoneally with bromodeoxyuridine (BrdUrd). After a single injection of BrdUrd (flash labelling), labelled cells appeared in the lower portion of the downgrowths of the epithelium in the intra-and extra-pancreatic bile ducts. A gradual accumulation of the labelled cells at the surface epithelium was observed during the cumulative labelling. After cumulative labelling the labelled cells gradually decreased in number and were finally confined to the degenerative cell zone of the surface epithelium 30 days later. Similarly, after a single injection of BrdUrd, the labelled cells in the bile duct of the ampulla appeared at the lower half of the crypt from where they migrated to the upper portion during cumulative labelling. These findings indicate that epithelial cells of the bile duct are renewed at the lower portion of the downgrowths of the epithelium, or crypt, and shed from the surface epithelium or upper portion of the fold. The labelling indices reached 23.83 ± 7.47% in the intra-pancreatic bile duct, 14.74 ± 7.99% in the extra-pancreatic bile duct and 43.42 ± 4.40% in the bile duct of the ampulla at the end of 70 h cumulative labelling. The fluctuating values of the labelling index were higher in the bile duct of the ampulla than in the intra- or extra-pancreatic bile ducts. These results indicate that the bile-duct epithelium undergoes a slower renewal rate than the other parts of the gastrointestinal tract, and that the renewal time of the epithelial cells is shorter at the bile duct of the ampulla than at the intra- or extra-pancreatic bile ducts.     

Studies on the regulation of lymphocyte production in the murine thymus and some effects of a crude thymus extract.

Cell proliferation in the murine thymus was studied in vivo under normal conditions and from 0 to 24 hr after a single injection of a water-soluble extract from mouse thymus, mouse spleen, and mouse skin. The thymus extract reduced during the first 24 hr the mitiotic activity 40%; the spleen extract had a weaker inhibitory effect. The skin extract had no such effect. The thymus extract and spleen extract inhibited the flux of cells into the S phase 0-8 hr after the injection of the extract. Initial labelling index was also reduced in this period. Eight hours after injection of the thymus or spleen extracts the inhibited cells initiated DNA synthesis. The rate of progression of blast cells through the cell cycle was normal 24 hr after the injection of the extracts. It was deduced from the analysis that the thymus extract inhibits processes triggering G0/G1 cells into DNA synthesis, the inhibition of G2 efflux being of minor importance. Finally a model for the regulation of proliferating thymic blast cells and the emigration of small lymphocytes from the thymus is proposed.