- Email: [email protected]
Clonogenic potential in vitro of proliferative and quiescent leukemic cells
L e u k e m k Re.watch. Vol. -l. No. 2. pp. 245 248 ~" Pergamon Press Ltd.. 19811. Printed in Great Britain
0145 2126/80/0401--0245 $02.00/0
CLONOGENIC POTENTIAL PROLIFERATIVE AND QUIESCENT
V/TRO OF LEUKEMIC CELLS*
IN
HARVEY D. PRE1SLER Department of Medicine A Rosewell Park Memorial Institute 666 Elm Street Buffalo, NY 14263, U.S.A. (Received 30 Januao' 1979. Accepted 30 November 1979) Abstract--Leukemic cells obtained from 6 patients with acute nonlymphocytic leukemia were subjected to centrifugal elutriation to obtain subpopulations of kinetically active and kinetically quiescent leukemic cells for study. The clonogenic potential in agar in vitro of these subpopulations of leukemic cells was compared and it was found that only cells obtained from the kinetically active subpopulations were able to clone in ritro. This observation raises the possibility that, in contradistinction to currently held views, kinetically quiescent leukemic cells may not be able to resume active proliferation. If this is also the case in rh,o, then growth kinetic-models of leukemic cells population expansion cannot resort to a presumed re-entry of quiescent leukemic cells into cycle to account for the apparent descrepancy between observed proliferative rates of leukemic cells and the expansion of the leukemic cell compartment. Key words: Ctongenicity, proliferative and quiescent, elutriation.
INTRODUCTION HUMAN ACUTE leukemia cell populations are kinetically heterogeneous, consisting of a minority of actively proliferating cells and a majority of cells which are kinetically quiescent [1, 31. Making use of the differences in size between these leukemic cell subpopulations, we have previously reported that centrifugal elutriation can be used to separate these subpopulations for study [71. We would now like to report that these two subpopulations also differ in their ability to proliferate in vitro in agar. MATERIALS A N D METHODS Bone marrow cells were obtained from 5 patients and peripheral blood from one patient with acute nonlymphocytic leukemia (ANLL). Sodium citrate was used as the anticoagulant and the erythrocytes were lysed using hypotonic ammonium chloride solution. The leukemia cells were separated by centrifugal elutriation into different sized subpopulations and the proportions of cells in S phase estimated from DNA histograms using the method of Krishan [41. The unseparated leukemic cell population as well as the subpopulations containing the smallest and largest size cells were studied. Since elutriation provides a series of subpopulations which differ in mean cell size, we decided to study the two extremes--that is, the subpopulation containing the smallest sized cells and the subpopulation containing the largest sized cells. When possible, intermedfated sized cell populations (cell populations containing mixtures of large, small, and intermediate sized cells) were also studied. The populations under study were classified morphologically fusing Wright-Giemsa stained specimens), their aH-TdR labeling indices, and their clonogenicity in ritro in agar were determined. As previously described [6] the soft agar method was used to clone the cells. One-hundrod thousand cells were suspended in 0.85 ml of 0.3 °/.;agar and layered over a previously solidified 2.5 ml layer of 0.5 ~ agar. The lower layer of agar contained colony-stimulating factors (20~/ov/v) obtained from a human tissue culture cell line [6]. Cultures were set up in quintiplicate and incubated in a humidified atmosphere containing 5 ~ CO 2 at 37°C. The number of clusters was counted at 7 days and ten clusters were picked from each of 3 plates, smeared on slides and stained with Wright-Giemsa to permit morphologic evaluation of the cells growing in the clusters. The two remaining plates were kept for 14 days to determine if longer growth times would lead to an alteration in the production of clusters by the various subpopulations of cells.
RESULTS The clinical characteristics of the patients whose leukemic cells were studied is given in Table 1. Five of the patients were previously untreated and one patient was in her first relapse. *This study was supported by U.S.P.H.S. Grants No. CA-24162 and No. CA-5834. 245
246
HARVEY D. PREISLER TABLE 1. PATIENT CHARACTERISTICS Pt.
Age
1Q 2(if' 3Q 40" 5Q 6~
70 53 34 58 70 22
Dx AML* AMMoLt AEL --*AML AML AML AMMoL
WBC 136,000 156,000 8,000 73,700 78,500 70,100
Status Prev. Untreated Prey. Untreated 1st Relapse Prev. Untreated Prey. Untreated Prev. Untreated
* Acute Myelocytic Leukemia tAcute Myelomonocytic Leukemia ~.At initial presentation patient had acute erythroleukemia. She was treated, was in complete remission for l year and on relapse had acute myelocytic leukemia.
In 5 cases, bone marrow leukemic cells were studied, while in one case peripheral blood leukemic cells were studied. In this study, as in our previous report, we obtained a population of small-sized cells containing few if any cells synthesizing DNA and a subpopulation of large-sized cells which contained a high proportion of cells synthesizing DNA. In accordance with these thymidine labeling indices and with the studies of other investigators [ 1, 2, 8 ], we have disignated these subpopulations as kinetically quiescent and kinetically active. Table 2 describes the characteristics of the leukemic cell populations which were studied. The proportion of myeloblasts were comparable in virtually all of the subpopulations. Considering the leukemic cells studied on a patient-by-patient basis, in each study, save one, the labeling index of the unseparated leukemic cell population was intermediate between that of the highly proliferative and the quiescent cell populations. In each case, two subpopulations of leukemic cells were obtained by elutriation in which the labeling indices of the separated subpopulations differed from 5 to 50 fold. Table 2 also reports the clonogenic potential in agar of the unseparated leukemic cells as well as that of the unseparated subpopulations. Growth in vitro in every instance was limited to cluster formation with most clusters limited to 8-12 cells but the sizes ranged from 4 to 20 cells per cluster. The distribution of cluster size did not vary between the unseparated population and the various subpopulations. Clusters contained abnormal appearing myeloblasts, promyelocytes or macrophages. There was no evidence of more mature elements. In every instance, the cell subpopulations with the highest labeling indices produced more clusters than the subpopulations with lowest labeling indices. In fact, in four out of six instances, the subpopulations with the lowest labeling indices failed to produce clusters. In the two cases in which subpopulations were cultured whose labeling index was intermediate between that of the actively proliferating and the quiescent cell subpopulations, the cloning efficiency of these kinetically intermediate subpopulations was intermediate as well. Continued culture of the plates for up to 14 days simply resulted in the disappearance of clusters. The suicide indices of the unseparated leukemic cells as well as the separated subpopulations were determined in 3 cases. In each instance, the suicide index markedly exceeded the corresponding labeling index. In two of three instances the subpopulation with the highest labeling index also had the highest suicide index. DISCUSSION The studies reported in this paper compared the clonogenic potential in vitro of kinetically active and kinetically quiescent myeloid leukemia cells. The clonogenic potential of these two leukemic cell subpopulations differed in that the kinetically active cell populations were
Clonogenicity of proliferative and quiescent leukemic cells
247
TABLE 2. PROLIFERATIVE AND GROWTH CHARACTERISTICS OF ELUTRIATOR SEPARATED LEUKEMIC CELLS ~o Myeloblasts + Promyelocytes ( ~ )
LI:~ ( ~ )
N o . Clusters Produced
SI§ (~)
Pre* a? b c
99 36 100 99
0.5 26 0 0.5
1 _+ 0 30 + 2 0 2.5 + 0,5
-----
2
Pre a b
59 95 100
18 47 1
37 + 10 25 +_ 4 0
34 54 --
3
Pre a b
96 83 100
18 5.3 1.2
377 + 43 551 +_ 111 69 + 3
56 85 62
4
Pre a b
91 72 82
1.8 22.2 4.8
10 ___ 1 38 + 2 0.5 + 0.5
----
5
Pre a b
89 89 91
2.5 22 0
0.3 + 0.3 7 + 1 0
----
6
Pre a b c
91 74 73 98
12 25 0.7 9.5
194 + 8 172 __+ 3 0 141 _ 2
75 57 -ND
Pt
Specimen
1
* Specimen prior to eluriation. t a, b, c--subpopulations of leukemic cells obtained by elutriation. 3 H T d R labeling index § 3 H T d R suicide index The studies were performed on leukemic cells obtained from bone marrow except in the case of patient N o . 1 whose peripheral blood was studied.
significantly more clonogenic than the quiescent cells. In fact, in 4 of 6 instances, the quiescent cell subpopulations failed to produce clusters. Of the two remaining quiescent specimens studied, one produced only 0.5 cluster/10 s cells and the other (patient No. 3) produced 69 clusters/105 cells. While production of 69 clusters appears to be substantial, it is only 12 ~o of the number of clusters produced by the kinetically active subpopulation obtained from the same marrow specimen. Taken together, the data indicated that at best, under the conditions used in these studies, the cloning efficiency of the quiescent leukemic cells was extremely low with less than 1 cell in 105 being capable of producing clusters in vitro. There is suggestive evidence that the clusters produced by the subpopulation of q uiescent leukemic cells might in fact be the result of a low level of contamination of this cell subpopulation by kinetically active cells. The thymidine labeling index of the two quiescent subpopulations which produced clusters was higher (1.2 and 4.8~) than that of the 4 quiescent subpopulations which failed to clone in vitro (L.I. 0 to 1 ~ ) , indicating greater numbers of actively cycling cells. More detailed analysis of the studies employing cells obtained from patient number 3 provides further support for the possibility of contamination of the quiescent cell population by actively proliferating cells. While the thymidine labeling index of the quiescent cell population obtained from this patient's marrow was only 1.2 ~o, the thymidine suicide index of the clonogenic cells present in the quiescent cell population was 62 ~0. This suicide index was not significantly different from that of the clonogenic cells in the unseparated marrow (56°~,) or of those present in the kinetically active leukemic cell subpopulation (85~o). This observation is compatible with the possibility that the clonogenicity of the quiescent cell population is due to contamination of this subpopulation of cells by small numbers of kinetically active cells.
248
HARVEY D. PREISLER
Serious consideration must be given to other possible explanations for the apparent lack of clonogenic potential in ~'itro of quiescent leukemic cells since the resumption of active proliferation by quiescent leukemic cells is believed to be essential for the maintenance of leukemic cell population size and may account for leukemic cell resistance to cycle specific chemotherapeutic agents. There is suggestive evidence that colony stimulating activity (CSA) derived from different tissue sources induce the ctonal proliferation in t,itro of distinct subpopulations of colony forming cells E5]. Hence, it is possible that under different conditions (use of a different CSA in vitro, or growth in vivo for example) the quiescent leukemic cells may be capable of clonal proliferation. Alternatively, it is possible that a population of cells exist which inhibit clonal proliferation by leukemic cells and that these inhibitory cells migrate with the kinetically quiescent cells during centrifugal eluatriation. This possibility is being addressed by mixing experiments which are currently in progress. In any event, the studies reported here demonstrate that under the conditions employed, only leukemic cell subpopulations containing actively proliferating cells are clonogenic in vitro. This observation is of particular significance since it calls into question the assertion that kinetically quiescent leukemic cells can resume active proliferation. Clearly further study of this question is indicated. Acknowledgement--The author would like to thank Ms. Irene Walczak. Ms. Kim Jankowski. Ms. Laura Arnold, and Mr. Gregory Christoff for their excellent technical assistance.
REFERENCES 1. CLARKSONI . O. (1969) Review of recent studies of cellular proliferation in acute leukemia. Natl. Cancer Inst. Monogr. 30. 81. 2. GAaUTTI V., PILERI A., TARCCO R. P. et al. ~1969) Proliferative potential of out-of-cycle leukaemic cells. Nature, Lond. 224, 275. 3. GAVOSTOF., PILERI A., BACH!C. et al. (1964) Proliferation and maturation defect in acute teukaemia cells. Nature. Lond. 203, 92. 4. KRISHANA. (1975) Rapid flow cytofluorometric analysis of mammalian cell cycle by propidium iodide staining. J. Cell Biol. 66, 188. 5. METCALFO. ~¢. MACDONALDH. R. (1975) Heterogeneity of in vitro colony-and cluster-forming cells in the mouse marrow: segration by velocity sedimentation. J. Cell. Physiol. 85, 643. 6. PRE1SLERH. D. & SHOHAM D. 0978) Comparison of tritiated thymidine labeling and suicide indices in acute nonlymphocytic leukemia. Cancer Res. 38, 3681. 7. PREISLERH. D., WALCZAKI. RENICK J. et al. (1977) Separation of leukemic marrow into proliferative and nonproliferative subpopulations by centrifugal elutriation. Cancer Res. 37, 3876. 8. SAUNDERSE. F. & MAUERA. M. (1969) Reentry of nondividing leukemic cells into a proliferative phase in acute childhood leukemia. J. Clin. Invest. 48, 1299.
0145 2126/80/0401--0245 $02.00/0
CLONOGENIC POTENTIAL PROLIFERATIVE AND QUIESCENT
V/TRO OF LEUKEMIC CELLS*
IN
HARVEY D. PRE1SLER Department of Medicine A Rosewell Park Memorial Institute 666 Elm Street Buffalo, NY 14263, U.S.A. (Received 30 Januao' 1979. Accepted 30 November 1979) Abstract--Leukemic cells obtained from 6 patients with acute nonlymphocytic leukemia were subjected to centrifugal elutriation to obtain subpopulations of kinetically active and kinetically quiescent leukemic cells for study. The clonogenic potential in agar in vitro of these subpopulations of leukemic cells was compared and it was found that only cells obtained from the kinetically active subpopulations were able to clone in ritro. This observation raises the possibility that, in contradistinction to currently held views, kinetically quiescent leukemic cells may not be able to resume active proliferation. If this is also the case in rh,o, then growth kinetic-models of leukemic cells population expansion cannot resort to a presumed re-entry of quiescent leukemic cells into cycle to account for the apparent descrepancy between observed proliferative rates of leukemic cells and the expansion of the leukemic cell compartment. Key words: Ctongenicity, proliferative and quiescent, elutriation.
INTRODUCTION HUMAN ACUTE leukemia cell populations are kinetically heterogeneous, consisting of a minority of actively proliferating cells and a majority of cells which are kinetically quiescent [1, 31. Making use of the differences in size between these leukemic cell subpopulations, we have previously reported that centrifugal elutriation can be used to separate these subpopulations for study [71. We would now like to report that these two subpopulations also differ in their ability to proliferate in vitro in agar. MATERIALS A N D METHODS Bone marrow cells were obtained from 5 patients and peripheral blood from one patient with acute nonlymphocytic leukemia (ANLL). Sodium citrate was used as the anticoagulant and the erythrocytes were lysed using hypotonic ammonium chloride solution. The leukemia cells were separated by centrifugal elutriation into different sized subpopulations and the proportions of cells in S phase estimated from DNA histograms using the method of Krishan [41. The unseparated leukemic cell population as well as the subpopulations containing the smallest and largest size cells were studied. Since elutriation provides a series of subpopulations which differ in mean cell size, we decided to study the two extremes--that is, the subpopulation containing the smallest sized cells and the subpopulation containing the largest sized cells. When possible, intermedfated sized cell populations (cell populations containing mixtures of large, small, and intermediate sized cells) were also studied. The populations under study were classified morphologically fusing Wright-Giemsa stained specimens), their aH-TdR labeling indices, and their clonogenicity in ritro in agar were determined. As previously described [6] the soft agar method was used to clone the cells. One-hundrod thousand cells were suspended in 0.85 ml of 0.3 °/.;agar and layered over a previously solidified 2.5 ml layer of 0.5 ~ agar. The lower layer of agar contained colony-stimulating factors (20~/ov/v) obtained from a human tissue culture cell line [6]. Cultures were set up in quintiplicate and incubated in a humidified atmosphere containing 5 ~ CO 2 at 37°C. The number of clusters was counted at 7 days and ten clusters were picked from each of 3 plates, smeared on slides and stained with Wright-Giemsa to permit morphologic evaluation of the cells growing in the clusters. The two remaining plates were kept for 14 days to determine if longer growth times would lead to an alteration in the production of clusters by the various subpopulations of cells.
RESULTS The clinical characteristics of the patients whose leukemic cells were studied is given in Table 1. Five of the patients were previously untreated and one patient was in her first relapse. *This study was supported by U.S.P.H.S. Grants No. CA-24162 and No. CA-5834. 245
246
HARVEY D. PREISLER TABLE 1. PATIENT CHARACTERISTICS Pt.
Age
1Q 2(if' 3Q 40" 5Q 6~
70 53 34 58 70 22
Dx AML* AMMoLt AEL --*AML AML AML AMMoL
WBC 136,000 156,000 8,000 73,700 78,500 70,100
Status Prev. Untreated Prey. Untreated 1st Relapse Prev. Untreated Prey. Untreated Prev. Untreated
* Acute Myelocytic Leukemia tAcute Myelomonocytic Leukemia ~.At initial presentation patient had acute erythroleukemia. She was treated, was in complete remission for l year and on relapse had acute myelocytic leukemia.
In 5 cases, bone marrow leukemic cells were studied, while in one case peripheral blood leukemic cells were studied. In this study, as in our previous report, we obtained a population of small-sized cells containing few if any cells synthesizing DNA and a subpopulation of large-sized cells which contained a high proportion of cells synthesizing DNA. In accordance with these thymidine labeling indices and with the studies of other investigators [ 1, 2, 8 ], we have disignated these subpopulations as kinetically quiescent and kinetically active. Table 2 describes the characteristics of the leukemic cell populations which were studied. The proportion of myeloblasts were comparable in virtually all of the subpopulations. Considering the leukemic cells studied on a patient-by-patient basis, in each study, save one, the labeling index of the unseparated leukemic cell population was intermediate between that of the highly proliferative and the quiescent cell populations. In each case, two subpopulations of leukemic cells were obtained by elutriation in which the labeling indices of the separated subpopulations differed from 5 to 50 fold. Table 2 also reports the clonogenic potential in agar of the unseparated leukemic cells as well as that of the unseparated subpopulations. Growth in vitro in every instance was limited to cluster formation with most clusters limited to 8-12 cells but the sizes ranged from 4 to 20 cells per cluster. The distribution of cluster size did not vary between the unseparated population and the various subpopulations. Clusters contained abnormal appearing myeloblasts, promyelocytes or macrophages. There was no evidence of more mature elements. In every instance, the cell subpopulations with the highest labeling indices produced more clusters than the subpopulations with lowest labeling indices. In fact, in four out of six instances, the subpopulations with the lowest labeling indices failed to produce clusters. In the two cases in which subpopulations were cultured whose labeling index was intermediate between that of the actively proliferating and the quiescent cell subpopulations, the cloning efficiency of these kinetically intermediate subpopulations was intermediate as well. Continued culture of the plates for up to 14 days simply resulted in the disappearance of clusters. The suicide indices of the unseparated leukemic cells as well as the separated subpopulations were determined in 3 cases. In each instance, the suicide index markedly exceeded the corresponding labeling index. In two of three instances the subpopulation with the highest labeling index also had the highest suicide index. DISCUSSION The studies reported in this paper compared the clonogenic potential in vitro of kinetically active and kinetically quiescent myeloid leukemia cells. The clonogenic potential of these two leukemic cell subpopulations differed in that the kinetically active cell populations were
Clonogenicity of proliferative and quiescent leukemic cells
247
TABLE 2. PROLIFERATIVE AND GROWTH CHARACTERISTICS OF ELUTRIATOR SEPARATED LEUKEMIC CELLS ~o Myeloblasts + Promyelocytes ( ~ )
LI:~ ( ~ )
N o . Clusters Produced
SI§ (~)
Pre* a? b c
99 36 100 99
0.5 26 0 0.5
1 _+ 0 30 + 2 0 2.5 + 0,5
-----
2
Pre a b
59 95 100
18 47 1
37 + 10 25 +_ 4 0
34 54 --
3
Pre a b
96 83 100
18 5.3 1.2
377 + 43 551 +_ 111 69 + 3
56 85 62
4
Pre a b
91 72 82
1.8 22.2 4.8
10 ___ 1 38 + 2 0.5 + 0.5
----
5
Pre a b
89 89 91
2.5 22 0
0.3 + 0.3 7 + 1 0
----
6
Pre a b c
91 74 73 98
12 25 0.7 9.5
194 + 8 172 __+ 3 0 141 _ 2
75 57 -ND
Pt
Specimen
1
* Specimen prior to eluriation. t a, b, c--subpopulations of leukemic cells obtained by elutriation. 3 H T d R labeling index § 3 H T d R suicide index The studies were performed on leukemic cells obtained from bone marrow except in the case of patient N o . 1 whose peripheral blood was studied.
significantly more clonogenic than the quiescent cells. In fact, in 4 of 6 instances, the quiescent cell subpopulations failed to produce clusters. Of the two remaining quiescent specimens studied, one produced only 0.5 cluster/10 s cells and the other (patient No. 3) produced 69 clusters/105 cells. While production of 69 clusters appears to be substantial, it is only 12 ~o of the number of clusters produced by the kinetically active subpopulation obtained from the same marrow specimen. Taken together, the data indicated that at best, under the conditions used in these studies, the cloning efficiency of the quiescent leukemic cells was extremely low with less than 1 cell in 105 being capable of producing clusters in vitro. There is suggestive evidence that the clusters produced by the subpopulation of q uiescent leukemic cells might in fact be the result of a low level of contamination of this cell subpopulation by kinetically active cells. The thymidine labeling index of the two quiescent subpopulations which produced clusters was higher (1.2 and 4.8~) than that of the 4 quiescent subpopulations which failed to clone in vitro (L.I. 0 to 1 ~ ) , indicating greater numbers of actively cycling cells. More detailed analysis of the studies employing cells obtained from patient number 3 provides further support for the possibility of contamination of the quiescent cell population by actively proliferating cells. While the thymidine labeling index of the quiescent cell population obtained from this patient's marrow was only 1.2 ~o, the thymidine suicide index of the clonogenic cells present in the quiescent cell population was 62 ~0. This suicide index was not significantly different from that of the clonogenic cells in the unseparated marrow (56°~,) or of those present in the kinetically active leukemic cell subpopulation (85~o). This observation is compatible with the possibility that the clonogenicity of the quiescent cell population is due to contamination of this subpopulation of cells by small numbers of kinetically active cells.
248
HARVEY D. PREISLER
Serious consideration must be given to other possible explanations for the apparent lack of clonogenic potential in ~'itro of quiescent leukemic cells since the resumption of active proliferation by quiescent leukemic cells is believed to be essential for the maintenance of leukemic cell population size and may account for leukemic cell resistance to cycle specific chemotherapeutic agents. There is suggestive evidence that colony stimulating activity (CSA) derived from different tissue sources induce the ctonal proliferation in t,itro of distinct subpopulations of colony forming cells E5]. Hence, it is possible that under different conditions (use of a different CSA in vitro, or growth in vivo for example) the quiescent leukemic cells may be capable of clonal proliferation. Alternatively, it is possible that a population of cells exist which inhibit clonal proliferation by leukemic cells and that these inhibitory cells migrate with the kinetically quiescent cells during centrifugal eluatriation. This possibility is being addressed by mixing experiments which are currently in progress. In any event, the studies reported here demonstrate that under the conditions employed, only leukemic cell subpopulations containing actively proliferating cells are clonogenic in vitro. This observation is of particular significance since it calls into question the assertion that kinetically quiescent leukemic cells can resume active proliferation. Clearly further study of this question is indicated. Acknowledgement--The author would like to thank Ms. Irene Walczak. Ms. Kim Jankowski. Ms. Laura Arnold, and Mr. Gregory Christoff for their excellent technical assistance.
REFERENCES 1. CLARKSONI . O. (1969) Review of recent studies of cellular proliferation in acute leukemia. Natl. Cancer Inst. Monogr. 30. 81. 2. GAaUTTI V., PILERI A., TARCCO R. P. et al. ~1969) Proliferative potential of out-of-cycle leukaemic cells. Nature, Lond. 224, 275. 3. GAVOSTOF., PILERI A., BACH!C. et al. (1964) Proliferation and maturation defect in acute teukaemia cells. Nature. Lond. 203, 92. 4. KRISHANA. (1975) Rapid flow cytofluorometric analysis of mammalian cell cycle by propidium iodide staining. J. Cell Biol. 66, 188. 5. METCALFO. ~¢. MACDONALDH. R. (1975) Heterogeneity of in vitro colony-and cluster-forming cells in the mouse marrow: segration by velocity sedimentation. J. Cell. Physiol. 85, 643. 6. PRE1SLERH. D. & SHOHAM D. 0978) Comparison of tritiated thymidine labeling and suicide indices in acute nonlymphocytic leukemia. Cancer Res. 38, 3681. 7. PREISLERH. D., WALCZAKI. RENICK J. et al. (1977) Separation of leukemic marrow into proliferative and nonproliferative subpopulations by centrifugal elutriation. Cancer Res. 37, 3876. 8. SAUNDERSE. F. & MAUERA. M. (1969) Reentry of nondividing leukemic cells into a proliferative phase in acute childhood leukemia. J. Clin. Invest. 48, 1299.