Incorporation of Purine Nucleosides in Cultured Fibroblasts from a Patient with Purine Nucleoside Phosphorylase Deficiency and Associated T-cell Immunodeficiency WYLIE G. BURKE,' SHI-HAN CHEN,' C. RONALD SCOTT ' AND ARTHUR J. AMMANN, JR.' ' Division of Genetics, Department of Pediatrics, University of Washington, Seattle, Washington 981 95 and Department ofPediatrics, University of California, Sail Francisco, California 94122
ABSTRACT Cultured skin fibroblasts from a patient with T-cell immune deficiency and an absence of purine nucleoside phosphorylase activity in red cells were assayed for their capacity to metabolize inosine and guanosine. The cultured fibroblasts were lacking activity of nucleoside phosphorylase and, compared to normal fibroblasts, could incorporate only 2%and 4% of 14C-inosineand 3H-guanosine, respectively, into acid precipitable material. Autoradiography visually confirmed the failure of the NP deficient cell line to incorporate the nucleosides into nuclear material. The physiological mechanism by which the deficiency of purine nucleoside phosphorylase causes T-cell dysfunction remains unclear. The immune deficiency diseases are a heterogeneous group of disorders characterized by increased susceptibility to infections. Many of these disorders are inherited as autosomal recessive or X-linked states (Stiehm, '73). The first evidence for a biochemical cause of an immune deficiency disorder was reported by Giblett e t al. ('72) who described two children with a severe form of combined immunodeficiency accompanied by absence of red cell adenosine deaminase (ADA). Subsequent studies have sustained the claim t h a t inherited ADA deficiency is one cause of combined immune deficiency (Meuwissen et al., '73). Furthermore, Giblett et al. ('75) have described another child in whom severely defect.ive T-cell function was associated with no measurable activity of red cell purine nucleoside phosphorylase (NP), a n enzyme which catalyzes the metabolic step just beyond that of ADA in the purine salvage pathway. One possible mechanism leading to defective immunity is a n inability of the lymphocyte to differentiate and replicate in response to an antigenic stimulus. A limiting factor in this response could be an inadequate level of nucleic acids required for replication. Such a n inadequacy could arise from a block in purine metabolism affecting either de novo synthesis, nucleotide salvage or both pathways. J. CELL. PHYSIOL.,92: 109-114.
To evaluate the role of nucleoside phosphorylase in the uptake of nucleosides for nucleic acid synthesis, we measured the incorporation of radioactive inosine and guanosine in fibroblasts established in culture from a patient with purine nucleoside phosphorylase deficiency. METHODS
A skin biopsy was obtained from the arm of the patient with nucleoside phosphorylase deficiency and viable fibroblasts established in culture. Normal skin fibroblasts were established in culture from persons who were healthy. Cultures were grown in DulbeccoVogt medium with 20%calf serum, penicillin and streptomycin. Each cell line was tested for mycoplasma (Kenny, '75) and found to be free of contamination. The cell suspensions were subcultured into a series of 25 cm2culture flasks (Falcon) a t a concentration of 1-2 x l o 5 cells per flask in the medium containing 20% calf serum. The following day the flasks were rinsed with saline and refed with medium containing no serum. The absence of serum is essential because of the presence of NP in fetal calf serum. 14C-labelled inosine (0.25 pclml a t a Received Aug. 31, '76. Accepted Dec. 13, '76.
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W G BCRKE, SHI-HAN CHEN, C R SCOTT AND A J AMMANN, J R
'
'
INOSINE
I
Time (hours)
G UANOSINE
2
Time (hours)
Figs. 1. 2 These figures demonstrate t h a t the incorporation of '1C-inosine and "-guanosine by a cell line lacking activity of purine nucleoside phosphorylase is markedly restricted. The maximum uptake of inosine and guanosine was 2% and 4%, respectively, of normal.
final concentration of 4 pM) or jH-inosine (1.0 p d m l a t a final concentration of 0.1 pM) was then added to each flask. To follow incorporation, duplicate flasks were collected and assayed a t varying times after addition of the appropriate radioisotope. Each flask was washed twice with saline and treated with 0.5 ml of SDS buffer (0.9%NaC1, 0.6% Tris, 0.03% EDTA, 0.5% sodium dodecylsulfate) for five minutes a t room temperature. After agitation of the flask, 0.1 ml of each sample was used for determination of protein according to the method of Lowry et al. ('51).The remainder of the sample was precipitated on ice with 0.2 ml 100% (wt/vol) trichloracetic acid for 20 minutes and then filtered through a Whatman GF/C glass-fiber filter. Filters were washed with cold 5% trichloracetic acid and 95%EtoH and counted in a liquid scintillation counter (Packard Model 3375) using a toluene scintillation fluid. For autoradiography, numerous cultures were subcultured onto sterilized cover slips in 25-cm2culture dishes. The day following subculture the cover slips with attached cells were washed and refed with medium containing no serum. Radioactive "-guanosine (1.0 pci/ml a t a final concentration of 0.4 pM) or 14C-inosine(1pci/ml a t a final concentration of 4 pM) was then added. After four hours of incubation, the medium was removed and the cover slips were washed twice with saline and once with 95% EtoH. They were then treated with 95% EtoH for 30 minutes a t room temperature and allowed to dry. The dried cover
slips were mounted on slides and prepared for autoradiography according to the method of Gartler and Burt ('641, with the exception that NTB-2 emulsion (Kodak) was used a t a 1:3 dilution for exposure of 3H labelling and undiluted for exposure of 14Clabelling. The purine nucleoside phosphorylase activity of each cell line was measured in duplicate a t 37" in 40 p1 of 50 mM sodium phosphate buffer, (pH 7.4) containing 200 pM (8-14C) inosine (1.3 pCi/mol) and 5 p1 of cellular extract. The reaction was stopped after 30 minutes by immersing the tubes in boiling water for 2 minutes. Aliquots (10 or 20 ~ 1 ) were chromatographed on thin layer silica gel (E.M. Laboratories. F-254) separating hypoxanthine and inosine with a solvent containing 94% n-butanol and 44% aqueous propionic acid in equal volumes. Spots of inosine and hypoxanthine were assayed for 14C activity in a Packard scintillation counter. Specific enzyme activity of NP was expressed in terms of the number of nanomoles of inosine converted to hypoxanthine per mg protein per hour, the latter being determined by the method of Lowry et al. ('51). RESULTS
The mean NP activity of nine control cultured cell lines was 719 n moles/mg proteidhr 124 (S.D.). No detectable enzyme activity was found in the cells cultured from the patient with red cell NP deficiency by this method. The apparent lack of NP activity in the patients' cultured fibroblasts was further
*
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W. G. BURKE. SHI-HAN CHEN, C. R. SCOTT AND A. J. AMMANN, J R
confirmed by their markedly reduced ability to incorporate inosine or guanosine into acid precipitable material. After six hours of exposure to the radioactively labelled nucleosides, the NP deficient cells had only 2% of inosine incorporation and 4% of guanosine incorporation when compared with the normal controls (figs. 1,2). For visual assessment of nucleoside incorporation in the fibroblasts, autoradiograms were prepared from the patient and from normal persons. The autoradiographs of normal cells (figs. 3A, 4A) showed dense grain patterns representing incorporation of "-guanosine and I4C-inosine, respectively. The greatest grain density appeared over the cell nuclei, consistent with purine incorporation into newly made DNA and RNA. By contrast, cells from the NP deficient patient showed little incorporated radioactivity (figs. 3B, 4B), indicating greatly reduced ability to form nucleic acid from either guanosine or inosine. DISCUSSION
In order for cells which are deficient in purine nucleoside phosphorylase activity to bypass their metabolic block the cells would have to phosphorylate guanosine and inosine via their respective kinases. Guanosine kinase, however, has never been demonstrated in mammalian tissue and there is doubt about inosine kinase. The existence of an inosine kinase has been suggested in Ehrlich ascitestumor cells (Pierre and Lepage, '68) and in white blood cells from a patient lacking HGPRT (Payne e t al., '70).The data from this study would indicate that both inosine kinase and guanosine kinase are essentially absent from human fibroblasts when evaluated a t low substrate concentrations. A similar conclusion was reached by Freidman and associates (Freidman et al., '69) in their study of nucleoside incorporation in a cell line which lacked hypoxanthine guanosine phosphoribosyl transferase (HGPRT). In ADA and NP deficiency the metabolic basis of the associated immune deficiency is not readily apparent. Greene and Chan ('73) originally suggested that increased cellular concentrations of adenosine caused pyrimidine starvation and cell death. Hovi and colleagues ('77) have suggested that inhibition of phosphoribosyl pyrophosphate (PRPP) synthetase, by accumulating adenosine nucleotides, is the most logical physiological mechanism t o explain the failure of lympho-
cyte function in ADA deficient patients. On the other hand, Wolberg et al. ('75) have proposed that in ADA deficiency there is a direct suppression of lymphocyte function by elevated levels of cyclic AMP. Although in this study we can demonstrate the inability of the NP deficient cell line t o salvage purine bases for nucleic acid formation, we doubt this is the direct cause of the defective immunity observed in patients with ADA or NP deficiency. If this were the mechanism, patients with HGPRT deficiency would have similar impaired immunity. Although Allison and coworkers ('75) have presented evidence that HGPRT deficiency is associated with mild impairment of B cell function, this aspect of the disease is not. clinically apparent. The single biochemical characteristic that ADA and NP deficient patients share is the inability to metabolize a purine nucleoside, either adenosine or inosine. This common characteristic suggests that accumulation of adenosine, inosine, guanosine or their related metabolites interferes with cell proliferation. Alternatively, a relative deficiency of ribose1-phosphate or hypoxanthine, products of the phosphorylase reaction, could be factors in preventing normal lymphocyte function. The answer to these alternative physiological mechanisms will need t o await the careful quantitation of intracellular purine metabolites. ACKNOWLEDGMENTS
The authors are grateful to Doctor Eloise Giblett for her helpfulness and advice in the preparation of the manuscript. This study was supported by Grants from The National Institutes of Health AI-12617, GM-15253 and HD-02274 and The National Foundation. Wylie G. Burke, Shi-Han Chen and C. Ronald Scott are Associates of the Child Development and Mental Retardation Center of the University of Washington. LITERATURE ClTED Allison, A. C., T. Hovi, R. W. E. Watts and A. D. B. Webster 1975 Immunological observations on patients with Lesch-Nyhan syndrome and on the role of de-novo purine synthesis in lymphocyte transformation. Lancet, 2: 11791182. Freidman, T., J. E. Seegmiller and J. H. Subak-Sharpe 1969 Evidence against the existence of guanosine and inosine kinases in human fibroblasts in tissue culture. Res., 56: 425-429. Gartler, S. M., and B. Burt 1964 Replication patterns of
PURINE NUCLEOSIDE PHOSPHORYLASE DEFICIENCY Bovine sex chromosomes in cell culture. Cytogenetics, 3: 135-142. Giblett, E. R., A. J. Ammann, R. Sandman, D. W. Wara and L. K. Diamond 1975 Nucleoside phosphorylase deficiency in a child with severely defective T-cell immunity and normal B-cell immunity. Lancet, 1: 1010-1013. Giblett, E. R., J. E. Anderson, F. Cohen, B. Pollara and H.J. Meuwissen 1972 Adenosine deaminase deficiency in two patients with severely impaired cellular immunity. Lancet, 2: 1067-1069. Greene, H., and T-S Chan 1973 Pyrimidine starvation induced by adenosine in fibroblasts and lymphoid cells: role of adenosine kinase. Science, 182: 836-837. Hovi, T., J. F. Smyth, A. C. Allison and S. Williams 1977 Role of adenosine deaminase in lymphocyte proliferation. Clin. Exp. Immun., 23: 395-403. Kenny, G. E. 1975 Rapid detection of mycoplasmata and non-culturable agents in animal cell cultures by uracil incorporation. Microbiology, 1975: 32-36. Lowry, 0. H., N. J. Rosebrough, A. L. Farr and R. J. Randall
113
1951 Protein measurement with the folin phenol reagent. J . Biol. Chem., 193: 265-275. Meuwissen, H. J., R. J. Pickering, B. Pollara and I. H. Poster 1977 Combined Immunodeficiency Diseases and Adenosine Deaminase Deficiency. Academic Press, Inc., N.Y. Payne, M. R., J. Dancis, P. H. Berman and M. E. Balis 1970 Inosine kinase in leucocytes of Lesch-Nyhan patients. Exp. Cell. Res., 59: 489-490. Pierre, K.J.,and G. A. Lepage 1968 Formation of inosine5-mono-phosphate by a kinase in cell-free extracts of Ehrlich ascites cell in vitro. Pro. SOC.Exp. Biol. Med., 127: 432-440. Stiehm, E. R. 1973 Immunodeficiency disorders: general considerations. In: Immunologic Disorders in Infants and Children. Stiehm and Fulginiti, eds. Saunders Co., Philadelphia, London, Toronto, pp. 145-167. Wolberg, G., J. P. Zimmerman, K. Hiemstra, M. Winston and L-C. Chu 1975 Adenosine inhibition of lymphocytemediated cytolysis: possible role of cyclic adenosine monophosphate. Science, 187: 957-959.
ABSTRACT Cultured skin fibroblasts from a patient with T-cell immune deficiency and an absence of purine nucleoside phosphorylase activity in red cells were assayed for their capacity to metabolize inosine and guanosine. The cultured fibroblasts were lacking activity of nucleoside phosphorylase and, compared to normal fibroblasts, could incorporate only 2%and 4% of 14C-inosineand 3H-guanosine, respectively, into acid precipitable material. Autoradiography visually confirmed the failure of the NP deficient cell line to incorporate the nucleosides into nuclear material. The physiological mechanism by which the deficiency of purine nucleoside phosphorylase causes T-cell dysfunction remains unclear. The immune deficiency diseases are a heterogeneous group of disorders characterized by increased susceptibility to infections. Many of these disorders are inherited as autosomal recessive or X-linked states (Stiehm, '73). The first evidence for a biochemical cause of an immune deficiency disorder was reported by Giblett e t al. ('72) who described two children with a severe form of combined immunodeficiency accompanied by absence of red cell adenosine deaminase (ADA). Subsequent studies have sustained the claim t h a t inherited ADA deficiency is one cause of combined immune deficiency (Meuwissen et al., '73). Furthermore, Giblett et al. ('75) have described another child in whom severely defect.ive T-cell function was associated with no measurable activity of red cell purine nucleoside phosphorylase (NP), a n enzyme which catalyzes the metabolic step just beyond that of ADA in the purine salvage pathway. One possible mechanism leading to defective immunity is a n inability of the lymphocyte to differentiate and replicate in response to an antigenic stimulus. A limiting factor in this response could be an inadequate level of nucleic acids required for replication. Such a n inadequacy could arise from a block in purine metabolism affecting either de novo synthesis, nucleotide salvage or both pathways. J. CELL. PHYSIOL.,92: 109-114.
To evaluate the role of nucleoside phosphorylase in the uptake of nucleosides for nucleic acid synthesis, we measured the incorporation of radioactive inosine and guanosine in fibroblasts established in culture from a patient with purine nucleoside phosphorylase deficiency. METHODS
A skin biopsy was obtained from the arm of the patient with nucleoside phosphorylase deficiency and viable fibroblasts established in culture. Normal skin fibroblasts were established in culture from persons who were healthy. Cultures were grown in DulbeccoVogt medium with 20%calf serum, penicillin and streptomycin. Each cell line was tested for mycoplasma (Kenny, '75) and found to be free of contamination. The cell suspensions were subcultured into a series of 25 cm2culture flasks (Falcon) a t a concentration of 1-2 x l o 5 cells per flask in the medium containing 20% calf serum. The following day the flasks were rinsed with saline and refed with medium containing no serum. The absence of serum is essential because of the presence of NP in fetal calf serum. 14C-labelled inosine (0.25 pclml a t a Received Aug. 31, '76. Accepted Dec. 13, '76.
109
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W G BCRKE, SHI-HAN CHEN, C R SCOTT AND A J AMMANN, J R
'
'
INOSINE
I
Time (hours)
G UANOSINE
2
Time (hours)
Figs. 1. 2 These figures demonstrate t h a t the incorporation of '1C-inosine and "-guanosine by a cell line lacking activity of purine nucleoside phosphorylase is markedly restricted. The maximum uptake of inosine and guanosine was 2% and 4%, respectively, of normal.
final concentration of 4 pM) or jH-inosine (1.0 p d m l a t a final concentration of 0.1 pM) was then added to each flask. To follow incorporation, duplicate flasks were collected and assayed a t varying times after addition of the appropriate radioisotope. Each flask was washed twice with saline and treated with 0.5 ml of SDS buffer (0.9%NaC1, 0.6% Tris, 0.03% EDTA, 0.5% sodium dodecylsulfate) for five minutes a t room temperature. After agitation of the flask, 0.1 ml of each sample was used for determination of protein according to the method of Lowry et al. ('51).The remainder of the sample was precipitated on ice with 0.2 ml 100% (wt/vol) trichloracetic acid for 20 minutes and then filtered through a Whatman GF/C glass-fiber filter. Filters were washed with cold 5% trichloracetic acid and 95%EtoH and counted in a liquid scintillation counter (Packard Model 3375) using a toluene scintillation fluid. For autoradiography, numerous cultures were subcultured onto sterilized cover slips in 25-cm2culture dishes. The day following subculture the cover slips with attached cells were washed and refed with medium containing no serum. Radioactive "-guanosine (1.0 pci/ml a t a final concentration of 0.4 pM) or 14C-inosine(1pci/ml a t a final concentration of 4 pM) was then added. After four hours of incubation, the medium was removed and the cover slips were washed twice with saline and once with 95% EtoH. They were then treated with 95% EtoH for 30 minutes a t room temperature and allowed to dry. The dried cover
slips were mounted on slides and prepared for autoradiography according to the method of Gartler and Burt ('641, with the exception that NTB-2 emulsion (Kodak) was used a t a 1:3 dilution for exposure of 3H labelling and undiluted for exposure of 14Clabelling. The purine nucleoside phosphorylase activity of each cell line was measured in duplicate a t 37" in 40 p1 of 50 mM sodium phosphate buffer, (pH 7.4) containing 200 pM (8-14C) inosine (1.3 pCi/mol) and 5 p1 of cellular extract. The reaction was stopped after 30 minutes by immersing the tubes in boiling water for 2 minutes. Aliquots (10 or 20 ~ 1 ) were chromatographed on thin layer silica gel (E.M. Laboratories. F-254) separating hypoxanthine and inosine with a solvent containing 94% n-butanol and 44% aqueous propionic acid in equal volumes. Spots of inosine and hypoxanthine were assayed for 14C activity in a Packard scintillation counter. Specific enzyme activity of NP was expressed in terms of the number of nanomoles of inosine converted to hypoxanthine per mg protein per hour, the latter being determined by the method of Lowry et al. ('51). RESULTS
The mean NP activity of nine control cultured cell lines was 719 n moles/mg proteidhr 124 (S.D.). No detectable enzyme activity was found in the cells cultured from the patient with red cell NP deficiency by this method. The apparent lack of NP activity in the patients' cultured fibroblasts was further
*
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W. G. BURKE. SHI-HAN CHEN, C. R. SCOTT AND A. J. AMMANN, J R
confirmed by their markedly reduced ability to incorporate inosine or guanosine into acid precipitable material. After six hours of exposure to the radioactively labelled nucleosides, the NP deficient cells had only 2% of inosine incorporation and 4% of guanosine incorporation when compared with the normal controls (figs. 1,2). For visual assessment of nucleoside incorporation in the fibroblasts, autoradiograms were prepared from the patient and from normal persons. The autoradiographs of normal cells (figs. 3A, 4A) showed dense grain patterns representing incorporation of "-guanosine and I4C-inosine, respectively. The greatest grain density appeared over the cell nuclei, consistent with purine incorporation into newly made DNA and RNA. By contrast, cells from the NP deficient patient showed little incorporated radioactivity (figs. 3B, 4B), indicating greatly reduced ability to form nucleic acid from either guanosine or inosine. DISCUSSION
In order for cells which are deficient in purine nucleoside phosphorylase activity to bypass their metabolic block the cells would have to phosphorylate guanosine and inosine via their respective kinases. Guanosine kinase, however, has never been demonstrated in mammalian tissue and there is doubt about inosine kinase. The existence of an inosine kinase has been suggested in Ehrlich ascitestumor cells (Pierre and Lepage, '68) and in white blood cells from a patient lacking HGPRT (Payne e t al., '70).The data from this study would indicate that both inosine kinase and guanosine kinase are essentially absent from human fibroblasts when evaluated a t low substrate concentrations. A similar conclusion was reached by Freidman and associates (Freidman et al., '69) in their study of nucleoside incorporation in a cell line which lacked hypoxanthine guanosine phosphoribosyl transferase (HGPRT). In ADA and NP deficiency the metabolic basis of the associated immune deficiency is not readily apparent. Greene and Chan ('73) originally suggested that increased cellular concentrations of adenosine caused pyrimidine starvation and cell death. Hovi and colleagues ('77) have suggested that inhibition of phosphoribosyl pyrophosphate (PRPP) synthetase, by accumulating adenosine nucleotides, is the most logical physiological mechanism t o explain the failure of lympho-
cyte function in ADA deficient patients. On the other hand, Wolberg et al. ('75) have proposed that in ADA deficiency there is a direct suppression of lymphocyte function by elevated levels of cyclic AMP. Although in this study we can demonstrate the inability of the NP deficient cell line t o salvage purine bases for nucleic acid formation, we doubt this is the direct cause of the defective immunity observed in patients with ADA or NP deficiency. If this were the mechanism, patients with HGPRT deficiency would have similar impaired immunity. Although Allison and coworkers ('75) have presented evidence that HGPRT deficiency is associated with mild impairment of B cell function, this aspect of the disease is not. clinically apparent. The single biochemical characteristic that ADA and NP deficient patients share is the inability to metabolize a purine nucleoside, either adenosine or inosine. This common characteristic suggests that accumulation of adenosine, inosine, guanosine or their related metabolites interferes with cell proliferation. Alternatively, a relative deficiency of ribose1-phosphate or hypoxanthine, products of the phosphorylase reaction, could be factors in preventing normal lymphocyte function. The answer to these alternative physiological mechanisms will need t o await the careful quantitation of intracellular purine metabolites. ACKNOWLEDGMENTS
The authors are grateful to Doctor Eloise Giblett for her helpfulness and advice in the preparation of the manuscript. This study was supported by Grants from The National Institutes of Health AI-12617, GM-15253 and HD-02274 and The National Foundation. Wylie G. Burke, Shi-Han Chen and C. Ronald Scott are Associates of the Child Development and Mental Retardation Center of the University of Washington. LITERATURE ClTED Allison, A. C., T. Hovi, R. W. E. Watts and A. D. B. Webster 1975 Immunological observations on patients with Lesch-Nyhan syndrome and on the role of de-novo purine synthesis in lymphocyte transformation. Lancet, 2: 11791182. Freidman, T., J. E. Seegmiller and J. H. Subak-Sharpe 1969 Evidence against the existence of guanosine and inosine kinases in human fibroblasts in tissue culture. Res., 56: 425-429. Gartler, S. M., and B. Burt 1964 Replication patterns of
PURINE NUCLEOSIDE PHOSPHORYLASE DEFICIENCY Bovine sex chromosomes in cell culture. Cytogenetics, 3: 135-142. Giblett, E. R., A. J. Ammann, R. Sandman, D. W. Wara and L. K. Diamond 1975 Nucleoside phosphorylase deficiency in a child with severely defective T-cell immunity and normal B-cell immunity. Lancet, 1: 1010-1013. Giblett, E. R., J. E. Anderson, F. Cohen, B. Pollara and H.J. Meuwissen 1972 Adenosine deaminase deficiency in two patients with severely impaired cellular immunity. Lancet, 2: 1067-1069. Greene, H., and T-S Chan 1973 Pyrimidine starvation induced by adenosine in fibroblasts and lymphoid cells: role of adenosine kinase. Science, 182: 836-837. Hovi, T., J. F. Smyth, A. C. Allison and S. Williams 1977 Role of adenosine deaminase in lymphocyte proliferation. Clin. Exp. Immun., 23: 395-403. Kenny, G. E. 1975 Rapid detection of mycoplasmata and non-culturable agents in animal cell cultures by uracil incorporation. Microbiology, 1975: 32-36. Lowry, 0. H., N. J. Rosebrough, A. L. Farr and R. J. Randall
113
1951 Protein measurement with the folin phenol reagent. J . Biol. Chem., 193: 265-275. Meuwissen, H. J., R. J. Pickering, B. Pollara and I. H. Poster 1977 Combined Immunodeficiency Diseases and Adenosine Deaminase Deficiency. Academic Press, Inc., N.Y. Payne, M. R., J. Dancis, P. H. Berman and M. E. Balis 1970 Inosine kinase in leucocytes of Lesch-Nyhan patients. Exp. Cell. Res., 59: 489-490. Pierre, K.J.,and G. A. Lepage 1968 Formation of inosine5-mono-phosphate by a kinase in cell-free extracts of Ehrlich ascites cell in vitro. Pro. SOC.Exp. Biol. Med., 127: 432-440. Stiehm, E. R. 1973 Immunodeficiency disorders: general considerations. In: Immunologic Disorders in Infants and Children. Stiehm and Fulginiti, eds. Saunders Co., Philadelphia, London, Toronto, pp. 145-167. Wolberg, G., J. P. Zimmerman, K. Hiemstra, M. Winston and L-C. Chu 1975 Adenosine inhibition of lymphocytemediated cytolysis: possible role of cyclic adenosine monophosphate. Science, 187: 957-959.
