Proliferation kinetics of plasma cells and of normal haemopoietic cells in multiple myeloma

Abstract. DNA synthesis time ( T _s) and ³H thymidine (TdR) labelling index (LI) of bone marrow (BM) myelomatous plasma cells (PC) and of the residual haemopoietic cell population (RHCP) were measured by in vitro quantitative ¹⁴C-TdR autoradiography in five patients with multiple myeloma (MM) in different phases of disease (three at presentation and two at relapse) and in one patient with solitary extra-osseous myeloma. One other patient with plasma cell leukaemia (PCL) was studied during an initial relapse phase and later during the leukaemic terminal phase. PC T _s was 18.8 ± 3.7 (from 13.3 to 25.0) hr and PC LI was 2.5 ± 1.8%; (from 1.0 to 6.3%). In the case of PCL, circulating PC had a T _s of 14.4 hr and a LI of 3.1. From these experimental measurements, the fractional turnover rate (FTR—percentage of cells produced per unit time) and the potential doubling time ( T _d) of BMPC were calculated assuming that all BMPC were in a steady-state at the time of the study. BMPC FTR was 3.53 ± 2.3% cells per day (from 1.2 to 6.72) and BMPC T _d was 46.8 ± 27.5 days (from 15.0 to 75.4). Comparison with results obtained in BM blasts of children with acute lymphoblastic leukaemia (ALL) indicated that BMPC had a lower proliferative activity ( P < 0.001), although BMPC T _s was not significantly different. In two patients a tumour doubling time of 6 and 13 months was determined by clinical follow up. Comparison of this parameter with T _d showed a cell loss factor of more than 90% in both patients.
Kinetic data relative to RHCP showed slight variations with respect to those found in normal subjects, with a general tendency towards a prolongation of T _s and a reduction of LI.     

RESPONSE OF MOUSE BONE MARROW COLONY FORMING UNITS IN DIFFERENT STAGES OF THE CELL CYCLE TO IN VITRO INCUBATION WITH MITOMYCIN-C

A short-term in vitro method was employed to study the Mitomycin-C sensitivity of normal mouse bone marrow CFU without triggering the G₀-phase cells into the proliferative cycle. Comparison was made of the toxicities of the drug against cells in different phases of the cell cycle including G₀. Mitomycin-c killed CFU both in and out of the S-phase. No significant difference could be found between its toxicities against normal and proliferating CFU; along the exponential part of the survival curve 1·6 μg/ml concentration of the drug reduced survival to 10%. Although in the normal bone marrow only a few CFU are in the S-phase and are killed by the agent, presence of the sensitive G₀ cells produce a significant amount of non-S-phase mortality. Among the proliferating CFU population the non-S-phase lethality is less due to the absence of G₀ cells. About 75% of the S-phase cells are killed after incubation with 1 μg/ml drug; outside the S-phase, the lethality is about 40–50%. The studies indicate that the G₀ cells which are situated near the G₁-S boundary are almost as sensitive to the drug as other non-S-phase cells like G₁ cells. The clinical significance of the findings is 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 RELATIONSHIP BETWEEN SPLEEN COLONY PRODUCTION AND SPLEEN CELLULARITY

The fraction, f, of injected spleen colony-forming cells which can be recovered from the spleen of a radiated mouse has been determined at various times up to 24 hr following the initial cell injection. the cellularity of the spleen at the time of assay was also measured and compared with the calculated f number. the linear relationship between these two parameters indicated that over the period of 2-24 hr the number of CFU/10⁶ spleen cells was constant, both falling in a parallel fashion. A further experiment using non-irradiated W/W^v mice in which the spleen size did not change showed the same f number at the end of this period as at 2 hr. It is concluded that CFU are expelled from the spleen during its post-irradiation contraction thus leading to the apparent fall in f number. It is also concluded that a more realistic f number is obtained by assaying the splenic CFU content 24 hr after the primary cell transfer rather than 2 hr.     

MULTIPLICATION OF HAEMOPOIETIC COLONY FORMING UNITS (CFU) IN MICE AFTER X-IRRADIATION AND BONE MARROW TRANSPLANTATION

Kinetics of the multiplication of haemopoietic CFUs was studied in lethally irradiated mice receiving various numbers of syngeneic bone marrow cells. After transplantation of a small number of bone marrow cells, the growth rate of CFU in femoral bone marrow appeared to decrease after about 10 days after transplantation, before the normal level of CFU in the femur was attained. In the spleen it was found that the overshoot which was observed about 10 days after transplantation of a large number of bone marrow cells is smaller or absent when a small number of cells is transplanted. Experiments dealing with transplantation of 50 x 10⁶ bone marrow cells 0, 4 or 10 days after a lethal irradiation indicated that the decline in growth rate of CFUs about 10 days after irradiation could not be attributed to environmental changes in the host.
The results are explained by the hypothesis that a previous excessive proliferation of CFUs diminishes the growth rate thereafter. This hypothesis is supported by experiments in which 50 x 10⁶ bone marrow cells derived from normal mice or from syngeneic chimaeras were transplanted. The slowest growth rate was observed when bone marrow that had been subjected to the most excessive proliferation in the weeks preceding the experiment was transplanted.     

Exogenous bone marrow cells do not rescue non-irradiated mice from acute renal tubular damage caused by HgCl2, despite establishment of chimaerism and cell proliferation in bone marrow and spleen

Abstract.   Objective : Various studies have shown that bone marrow stem cells can rescue mice from acute renal tubular damage under a conditioning advantage (irradiation or cisplatin treatment) favouring donor cell engraftment and regeneration; however, it is not known whether bone marrow cells (BMCs) can contribute to repair of acute tubular damage in the absence of a selection pressure for the donor cells. The aim of this study was to examine this possibility. Materials and methods : Ten-week-old female mice were assigned into control non-irradiated animals having only vehicle treatment, HgCl₂-treated non-irradiated mice, HgCl₂-treated non-irradiated mice infused with male BMCs 1 day after HgCl₂, and vehicle-treated mice with male BMCs. Tritiated thymidine was given 1 h before animal killing. Results : Donor BMCs could not alleviate non-irradiated mice from acute tubular damage caused by HgCl₂, deduced by no reduction in serum urea nitrogen combined with negligible cell engraftment. However, donor BMCs could home to the bone marrow and spleen and display proliferative activity. This is the first report to show that despite no preparative myeloablation of recipients, engrafted donor BMCs can synthesize DNA in the bone marrow and spleen. Conclusions : Exogenous BMCs do not rescue non-irradiated mice from acute renal tubular damage caused by HgCl₂, despite establishment of chimerism and cell proliferation in bone marrow and spleen.     

THE INFLUENCE OF GENETIC RESISTANCE ON CFU GROWTH KINETICS IN SPLEEN AND FEMUR

An impaired colony formation of C57BL marrow cells transplanted into F_t (C57BL x CBA) mice was observed. In accordance with the literature this phenomenon has been designated as ‘genetic resistance’. Studies to elucidate the mechanism of the genetic resistance demonstrated that the multiplication phase of the CFU growth curve started in the semi-isogeneic combination about 48 hr later than in the isogeneic combination. In the spleen this resulted in a lower ‘dip’. For the spleen as well as for the femur similar CFU doubling times were found during the multiplication phase when both transplantation combinations were compared. Furthermore the percentage of CFU in S-phase (assessed with the ³H-TdR suicide technique) during the first days after transplantation were similar in both combinations. When the spleen was removed 5–6 months before irradiation and bone marrow transplantation was performed the growth curve of parental CFU in the femur was identical with the growth curve of isogeneic CFU (no delay was observed). These results are discussed and a few theories explaining the observations are proposed.     

The role of photosynthesis/light relationships in determining lower depth limits of Characeae in South Island, New Zealand lakes

1. The maximum depth of colonization of aquatic macrophytes ( Z _c) was investigated in eighteen South Island, New Zealand lakes. The downward attenuation coefficient for photosynthetically active radiation ( K _d(PAR)) was calculated and the spectral characteristics of the lakes determined with a spectroradiometer.
2. Characean algae dominated the deepest communities in sixteen of the study lakes.
3. Z _c was significantly related to K _d(PAR) by the relationship Z _c = 4.5/ K _d– 2.2.
4. From measurements of the photosynthetic properties of Chara corallina (Kl. ex Willd., em R.D.W.) and incident radiation over the course of a year we calculated the depth at which daily net photosynthesis would be equal to zero for each day of the year. An annual average of this depth was significantly related to Z _c with an r 2 of 0.86.
5. Correcting K _d(PAR) for spectral quality and taking into account the potential absorption spectrum of a characean meadow did not improve the relationships.
6. We suggest that relationships established between K _d(PAR) and Z _c of characean algae in South Island, New Zealand lakes can be explained to a great extent by light limitation of photosynthesis.     

DNA SYNTHESIS TIME IN LEUKAEMIC CELLS AS MEASURED BY THE DOUBLE LABELLING AND THE PERCENTAGE LABELLED MITOSES METHODS

Values of T _s provided by the double labelling method have been compared with those given by the percentage labelled mitoses curve for blast cells in the peripheral blood of a patient with plasma cell leukaemia and of rats bearing a transferable acute leukaemia. the double labelling method was carried out giving the first label (³H-thymidine) in vivo and the second label (¹⁴C-thymidine) in vitro with several values for the interval between the two labels. T _s was calculated by fitting regression lines to the results obtained. Data for percentage labelled mitoses were analysed by computer. For the plasma cell leukaemia values of T _s= 17.1 ± 7.0 hr and T _s= 19.8 ± 3.4 hr, and for the rat leukaemia values of 8.7 ± 1.7 hr and 9.0 ± 1.7 hr (7.1 hr corrected for exponential growth) were obtained from the percentage labelled mitoses and double labelling methods respectively. It is concluded that the double labelling method is valid for the study of cell proliferation in leukaemic blast cells.     

The role of photosynthesis/light relationships in determining lower depth limits of Characeae in South Island, New Zealand lakes

1. The maximum depth of colonization of aquatic macrophytes ( Z _c) was investigated in eighteen South Island, New Zealand lakes. The downward attenuation coefficient for photosynthetically active radiation ( K _d(PAR)) was calculated and the spectral characteristics of the lakes determined with a spectroradiometer.
2. Characean algae dominated the deepest communities in sixteen of the study lakes.
3. Z _c was significantly related to K _d(PAR) by the relationship Z _c = 4.5/ K _d– 2.2.
4. From measurements of the photosynthetic properties of Chara corallina (Kl. ex Willd., em R.D.W.) and incident radiation over the course of a year we calculated the depth at which daily net photosynthesis would be equal to zero for each day of the year. An annual average of this depth was significantly related to Z _c with an r 2 of 0.86.
5. Correcting K _d(PAR) for spectral quality and taking into account the potential absorption spectrum of a characean meadow did not improve the relationships.
6. We suggest that relationships established between K _d(PAR) and Z _c of characean algae in South Island, New Zealand lakes can be explained to a great extent by light limitation of photosynthesis.     

THE ROLE OF THE SPLEEN IN THE REPOPULATION OF THE HAEMOPOIETIC SYSTEM OF HEAVILY-IRRADIATED MICE

The respective role of the spleen or of the bone marrow in the regeneration of the haemopoietic progenitor compartment of heavily-irradiated mice has been investigated. Splenectomy was used to this end in animals injected with exogenous isogenic cells or regenerating from endogenous spleen or marrow cells. Analysis of the data as a function of time shows that the presence of the spleen affects marrow CFU repopulation only at the early post-irradiation stages. The expansion of the marrow progenitor pool proceeds, however, rather independently of the spleen and marrow CFU remain eventually as the main source of haemopoietic cells in the surviving mice. Thus the reaction of the spleen may be envisaged as a fast, important but transient contribution to the overall haemopoietic function of heavily-irradiated animals.     

Effect of graft versus host reaction on cell cycle time in neonatal mouse jejunum

Abstract. The duration of cell cycle parameters in control mouse jejunum has been compared with that found following induction of a graft-versus-host reaction (GvHR) during the first 3 weeks of postnatal life.
Values for t_c , and t_G1 were found to decrease progressively during normal development: estimates for the whole crypt column in 21-day-old mice were approximately half to one quarter those found 6 days after birth 12.1 ± 0.5 hr and 24.2 ± 0.3 hr for t_c ; 2.8 ± 0.3 hr and 12.1 ± 0.3 hr for t_G1 respectively; (means ± SE). t_S and t_G2 were found to remain approximately constant during this period of neonatal development.
Injecting foreign spleen cells into 3-day-old mice produced no effect on crypt cell proliferation or cell cycle parameters measured 3 days later. GvHR mice studied 8 days after spleen cell injection, however, showed both an increase in crypt cell proliferation and decreases in the values for t_c and t_G1 , to levels similar to those normally found in 21-day-old control animals ( t_c 12.4 ± 0.4 hr and t_G1 5.4 ± 0.4 hr for 11-day-old GvHR mice). The possible mechanism leading to these changes is discussed.
The ability of GvHR to stimulate cell proliferation is used in the present work to test the hypothesis that the total number of cell divisions taking place after birth determines the temporal sequence of changes in disaccharidase content produced during neonatal development.     

KINETICS OF THE INNER ENAMEL EPITHELIUM IN THE ADULT RAT INCISOR DURING ACCELERATED ERUPTION

Inner enamel epithelial (IEE) cell production was compared in accelerated and normal eruption (control). Each group consisting of thirty rats received 1 μCi/g tritiated thymidine. The animals were sacrificed at short time intervals up to 14 hr after injection. The excised incisors were cut mid-sagittally and processed autoradiographically.
The fast growing incisor produces twice as many cells as the control. Increased cell production is achieved in two ways: proliferative pool expansion (by 25.5%) and generation time ( t _c) shortening to 16 hr ( t _c= 23 hr in the control). Generation time shortening resulted mainly from a diminution in t _g1= 8.6 hr ( t _g1= 14.1 hr in the control) and t _s which equaled 5.5 hr ( t _s= 7 hr in control). Mitotic times which equaled 0.4 hr and t _g2, 1.5 hr were identical in both groups.
The eruption/IEE cell production ratio equals 1.1 in both groups.     

ESTIMATION OF THE KINETIC PARAMETERS OF UNIDIRECTIONAL TRANSPORT ACROSS THE BLOOD-BRAIN BARRIER

Abstract— The unidirectional transport of metabolic substrates from blood to brain may be defined in terms of Michaelis-Menten saturable ( K m, V _max) and non-saturable ( K _d) components of influx. Various computation procedures have been previously reported to estimate the kinetic parameters when an intracarotid injection technique is used. Transformations of the influx data which allow linear plots to obtain estimates were compared with estimates obtained directly from a best fit on a least means squares criterion for both experimental and simulated data. Large discrepancies were apparent between the various estimates of the kinetic parameters when an equal weight was given to transformed data. For pyruvate (21-day-old rats), K _m, values varied between 1.02 and 6.25 mM and V _max varied between 0.68 and 2.30 μmol g⁻¹ min⁻¹. The estimates were almost equivalent when pyruvate data was re-analysed using a weighting scheme based on the finding that the absolute value of the S.D. of influx increased in proportion to influx. It is recommended that estimates of kinetic parameters be obtained by an iterative, non-linear least squares method to fit appropriately weighted data directly.     

Effect of syngeneic lymphocytes, T immunodeficiency and the genotype of bone marrow donors on the differentiation of early and late splenic colonies

The formation of "early" (5-8 days) and "late" (12-14 days) colonies in spleen of lethally irradiated syngeneic or hybrid recipients after transplantation of bone marrow cells has been studied. The differentiation pattern did not depend on bone marrow cell donor's genotype and the donor-recipient combination. Erythroid to granulocyte colonies ratio (E/G) equals 2. Change of direction of bone marrow colony-forming units (CFU) differentiation has the same pattern at different stages of colony-formation. Under the influence of antigen-stimulated lymphocytes the granulopoiesis (E/G 0.3-0.5) dominanted. The thymectomy of adult animals leads to a predominant formation of erythroid colonies (E/G 3.5-5.1). When T-immunodeficiency is reversed with syngeneic lymphocytes, the differentiation of CFU is normalized at all stages of colony-formation. The process of differentiation of haemopoietic precursors, that form "early" and "late" colonies, is under T-lymphocyte control.     

THE KINETICS OF GRANULOSA CELLS IN DEVELOPING FOLLICLES IN THE MOUSE OVARY

This investigation describes the kinetics of the granulosa cells in medium-sized follicles type 3b, 4 and 5a in ovaries of 28-day-old Bagg mice. the method of labelling with ³H-thymidine followed by high resolution autoradiography is used in the experimental work, which consist of determining percentage labelled mitosis (PLM-) and continuous labelling (CL-) curves. In order to analyse the data by computer two alternative hypotheses A and B are set up. Both include the assumptions of no cell loss, exponential growth and a resting compartment Q. In hypothesis A cells from Q re-enter the mitotic cycle via the normal DNA-synthesis compartment S_p. Hypothesis B includes beside compartment S_p a special DNA-synthesis compartment S_q where only cells from Q are synthesizing DNA, and these cells re-enter the mitotic cycle via the G₂ compartment. the mean transit time in S_q is considered to be longer than the mean transit time in S_q. On the basis of the hypothesis mathematical expressions for the PLM- and CL-curves are obtained, and by means of a computer the theoretical curves are fitted to the experimental values: thereby all relevant cell kinetical parameters are estimated. Hypothesis B seems to give the best fit between the theoretical and experimental curves. the estimated parameters are: mean cycle times, μ_c= (56.1 hr, 56.1 hr and 22.3 hr for type 3b, 4 and 5a respectively), doubling times, T _D= (96.4 hr, 118.6 hr and 59.1 hr) and the proportion of cells in Q, p _Q = (0.60, 0.71 and 0.69).     

VARIATION IN THE DURATION OF MITOSIS IN THE CRYPTS OF LIEBERKUHN OF THE RAT; A CYTOKINETIC STUDY USING VINCRISTINE

The variation in the duration of mitosis ( t _m) with cell position in the small intestinal crypts of the adult rat has been measured by a stathmokinetic technique using vincristine. The value for the whole crypt column was 0.43 hr, or 26 min. At the bottom of the crypt t _m was in excess of 1 hr, but rapidly decreased and throughout the greater part of the proliferative compartment was between 0.40 and 0.50 hr. At the top of the proliferative compartment an increase in t _m was demonstrated.
If the value of 0.43 hr for the whole crypt column is correct, then one argument for postulating the formation of metabolic DNA during differentiation in the small bowel epithelium of the rat becomes invalid. Variations in t _m within the crypt have been shown to increase the values of cell velocity obtained from cumulative birth rate diagrams. Finally further evidence has been presented for the existence of a slowly dividing subpopulation of cells at the base of the crypt. These cells may be important in crypt repopulation after damage with phase specific anti-tumour drugs.     

Fluctuations in the tympanic membrane temperatures of non-restrained captive giant anteaters and southern tamanduas

Members of the family Myrmecophagidae (i.e. anteaters) show a variety of anatomical and behavioural adaptations to deal with their low-energy diet; for example, they all have low body temperatures in comparison with other eutherian mammals. In this study, we investigated the tympanic membrane temperatures ( T _mt) of two giant anteaters and three southern tamanduas, housed in captivity and exposed to natural climatic variations in temperature, using an infrared thermometer. Additionally, we measured external dorsal temperature ( T _d), air temperature ( T _a), substrate temperature ( T _s) and whether the subject was active or not. To understand the effect of time of day on these variables, we recorded them, on the hour, over four 24-h cycles for each animal during which the subjects were non-restrained within their enclosures. The results show that both giant anteaters and southern tamanduas allow their T _mt to reduce between 4.0 and 6.5 °C when they are sleeping. Furthermore, linear regressions between T _mt and T _a or T _s showed that the giant anteaters were much more affected by T _a and T _s than the southern tamanduas. Both species also showed higher T _mt when active (comparing subjects active and inactive at the same T _a). Both species appear to use shallow torpor during a normal 24-h cycle probably as a means to economize energy. The torpor in giant anteaters occurred during the night when asleep, whereas in the southern tamanduas it occurred at any time of day when asleep. The giant anteaters appeared to be more directly affected by environmental temperature than the southern tamanduas.     

Dual biological function of a T-cell-derived murine immunoenhancing factor.

Murine enhancing factor (MEF), derived from the culture fluid of mixtures of histoincompatible spleen cells, was found to have two apparently different, but perhaps closely related, biological activities. First, MEF can functionally replace T cells in nonspecifically augmenting the anti-sheep erythrocyte plaque-forming cell response of T-cell-depleted, mouse splenic B-cell cultures. Second, the mediator acts similarly to colony stimulating factor from human urine in promoting the formation of colony-forming units (CFU) in soft agar bone marrow cell cultures. This latter function of MEF was manifest in the absence of detectable increases in the level of incorporation of [³H]thymidine by cultured bone marrow cells. Morphologically, the cells comprising the CFU were macrophage-like in appearance. The data suggest that MEF may function as a differentiation signal for the maturation of antigen-activated B lymphocytes into immunoglobulin-secreting cells, as well as for the modulation of hematopoietic or granulopoietic macrophage stem cells into mature, functional macrophages.     

THE LYMPHOCYTES OF CHRONIC LYMPHOCYTIC LEUKEMIA: THEIR PROLIFERATION AND CELL CYCLE KINETICS

Lymphocytes obtained from CLL patients exhibited a delayed and reduced response to PHA when cultured in diffusion chambers. DNA synthesis (8–10 hr) and general time (15–19 hr) of the late-developing CLL blasts were consistent with normal values ( T _s: 8–10 hr; T _c: 14–17 hr). However, the G₂ period of CLL blasts seemed more variable, and their mitotic index during the response at 5–6 days was 30–50% of the values determined for normal blasts during their peak response at 2–3 days.