Limited effect of erythrocyte and plasma infusions in adenosine deaminase deficiency

October 1978 The Journal o f P E D I A T R I C S

597

Limited effect of erythrocyte and plasma infusions in adenosine deaminase deficiency A lO-month-old child with a profound deficiency of adenosine deaminase and severe combined bnmunodefieienc.~, was treated for a period of ! 7 months with red cell and plasm a transfi~sions containh~g normal amounts of the deficient enz)'me. Following each transfusion, the plasm a adenosine, red cell and I)vnphoc)'te A TP, urinary ade.nine., and urinary deox)'adenosine decreased transiently. During this period, the absolute blood lymphocyte count rose and a limited increased in the response of the l)'mphocytes to PIIA-P was observed. Delayed h)'persensitivity skin tests remained negative during (he transfusion periods. A quantitative elevation of serum Onm.u.noglobulins occurred, but specific antibody formation ~'as not elicited. In contrast to a prerious report of successfid therapy of ADA deficiency with red cell and plasma infusions, this patient responded poorly to enz)vne replacement therapy. The difference may be related to a tnore profound enz).tne deficiency in our patient.

Frank C. Schmalstieg, M.D., Ph.D.,* Gordon C. Mills, Ph.D., J. Arly Nelson, Ph.D., Linda T. M a y , M.D., A r m o n d S. Goldman, M.D., and Randall M. Goidblum, M.D., Galveston, T e x a s

SEVERE DEFICIENCY in red cell and lymphocyt e adenosine deaminase is an autosomal recessive disease which leads to severe dysfunction i n thymic-dependent lymphocytes (T lymphocytes or T cells), variable deficiencies in thymic-independen!lyinphocytes .(B lymphQcytes or B cells), and, in some instances, charactei'istic skeletal abnormalities. The resultant severe combined immunodeFrom the Departments of Pediatrics, Pharmacology and Human Biological Chemistry and Genetics, The University of Texas Medical Branch. Supported in part by the following: National Institutes of llealth, Grant No. DHEW RR-00073-14, General Clinical Research Centers Branch, "Division of Research Facilities and Resources; Grant No. RO1CA-17057; Grant No. D H E W 06-S-000170-09,~ DHEW Research Grant 5T32-GM-02204; a grant from Multidisciplinary Research Program in Mental llealth, UTMB, Galveston; and a grant from The National Foundatr of Dhnes No. 6-130. Dr. Schmalstieg was the recipient of Th.eJeanne B. Kempner Scholarship A ward; Drs. May and Schmalstiegqre recipients. of The James IV..McLaughlin Post-DoctoralFellowship, The Universityof Texas Medical Branch, Galveston. *Reprint address: Division of lmmunology and Allerg),;. Department of Pediatrics, The Universityof Texas Medical Branch. Gah'eston, TX 77JJO.

0022-3476/78/100597+07500.70/0 9 1978 The C. V. Mosby Co.

ficiency is invariably fatal unless the immune system is reconstituted. The genetic and clinical findings in this disease are well characterizedy 6 but the metabolic abnormalities which are responsible for the dysfunction of the lymphocytes have not been entirely elucidated. We have recently measure d some of the key purine and pyrimidine metabolites in the red cells and lymphocytes of an ADAAbbreviations used ADA: adenosine deaminase severe combined immunodeficiency SCID: PHA-P: phytohemagglutinin erythrocyte E:. HPLC: 9high pressure liquid chromatography ATP: adenosine 5'-triphosphate AMP: adenosine 5'-monophosphate deficient patient with SCiD. T. 8 Our findings suggested the possibility of treating this disease with exogenous ADA in the form of human red cells and plasma. We were encouraged by a report of success with this type of treatment in another child with this disorder. 9 We, therefore, conducted an extensive trial of enzyme replacement therapy with human red cells and plasma transfusions in a child with ADA deficiency.

Vol. 93, No. 4, pp. 597-603

598

Schmalstieg et al.

MATERIALS

AND METItODS

Case presentation. The results of clinical, immunologic, and metabolic investigations of this 27-month-old white boy with ADA deficiency and SCID have been previously described,'. 8 The ADA activity in both red cells and lymphocytes remained at less than 1% of normal. Since no suiiable bone marrow donor was found, we chose to begin ADA replacement therapy in the form of red cell transfusions at age 10 months. The child was maintained in reverse but nonsterile isolation. Guidelines for human reasearch were in accord with the Declaration of Helsinki of the World Medical Association and the research was approved by the Institutional }tuman Research Committee. Transfusion. Glycerol-treated blood and fresh frozen plasma used in these transfusions had been frozen at least three weeks. Irradiation was not used after the first transfusion since very few viable lymphocytes could be recovered after thawing and deglyceration , and these cells could not be stimulated by phytohemagglutinin as measured by the uptake of 3H-thymidine. Our present practice is to examine all blood preparations for viable lymphocytes prior to infusion and not transfuse blood that contains viable lymphocytes. Beginning at age 10 months, partial exchange transfusions were performed by removing 10 ml/kg of venous blood from the patient and immediately infusing 10 to 15 ml/kg of packed red cells through another vein. Since the patient's blood type was A +, O + red cells were administered so that the donor cells in the blood could be quantitated? ~This procedure was generally carried out on two consecutive days and resulted in a total transfusion of 20 to 30 ml/kg (40 to 50% exchange). For plasma infusions, 15 ml/kg of the thawed plasma was given over a six-hour period. Four of the 18 transfusions included concurrent plasma infusions. Graft-vs-host disease was not evidenced through 18 transfusions. Immunologic studies. Blood lymphocytes were cultured with PHA-P (3 #g/ml) or concanavalin A (100 /~g/ml) (Difco Laboratories, Detroit, Mich.) for three days." The extent of stimulation was determined by measuring the incorporation of ~iI-ihymidine b y these lymphocyte.s. E-rosettes with sheep red blood cells were formed and counted accordingto the method of Jondal et al." Serum immunoglobulins were quantitated by the Mancini method '~ using commerclal plates and standards (Behring Diagnostics, Somerville, N.J.). Class specific antibodies against a pool of Escherichia coli 0 antigens and Candida albicans were determined by an enzyme-linked immunosorbent assay technique." Delayed hypersensitivity was assessed with intradermal tests to Candida albicans (1:200; 1:100; 1:50 ~,/v) (Hollister-Stier, Dallas, Texas) and strep-

Tile Journal of Pediatrics October 1978

tokinase-streptodornase (1:I0 v/v) (Lederle Laboratories, Pearl River, N.Y.) and with epicutaneous sensitization with 2,4-dinitrochlorobenzene (Eastman-Kodak Laboratories, Rochester, N.Y.). Adenosine deaminase determinations. ADA activity in red cell lysates Was determined by the method of Kalckar." Activity in lymphocyte sonicates and plasma was measured by following the production o f uric acid at 295 nm. This was accomplished by coupling the ADA reaction to reactions catalysed by added nucleoside phosphorylase and xanthine oxidase (Sigma Chem'ical Co., St. Louis, Mo.). '6 Appropriate corrections were made for exogenous ADA in these enzyme preparations. ADA activity in intact red cells was determined by an ammonia liberation technique." In certain instances ADA activity in normal red.cell lysates was determined in plasma free media (0.05M phosphate-buffered saline, 0.154M NaCI, pH 7.4) and in the patient's plasma at a substrate concentration of approximately 1.8/.tM. The conversion of adenosine ([8"C] adenosine, 54 /.tCi/mole) (New England Nuclear, Boston, Mass.) t ~ inosine and hypoxanthine was followed by the appearance of radioactivity in these two compounds. Adenosine, inosine, and hypoxanthine were separated by one dimensional thin layer chromatography on PEI-cellulose (Baker Chemical Co., Phillipsburg, N.J.) using the solvent system, t-butyl alcohol:ammonia:water (50:1:49). Correction for endogenous adenosine in the patient's plasma was made from the concentration detected by ItPLC. Adenosine triphosphate assaY. Protein-free lysates of red blood cells and lymphocytes were prepared as previously described.'. 8 ATP in red ceil extracts was then quantitated by an enzymatic assay~ TM A fluorescent modification of this procedure was used for measuring lymphocyte ATP. This was accomplished bY following t.he oxidation of NADH (Sigma Chemical Co., St. Louis, Mo.) fluorometrically ~vith an Aminco-Bowman spectroph0tofluorometer at an excitation wavelength of 365 nm and an emission wavelength of 470 nm. In certain instances, lymphocyt e ATP was also measured by HPLC. s The two methods agreed within _ 10%. Adenosine and adenine in plasma and red cells. Protein~ free extract.s were prepared as previ'~usly described.'. Two nmoles per milliliter of tubercidin (P & L Biochemica!s, Milwaukee, Wis.) were then added to each sample to serve as an internal standard. Nucleotides were removed by. adding 0.2 gm of moist Dowex AGI-X2 anion exchange resin.(pH 5, acetate f o r m ) p e r milliliter of supernatani and equilibrating for 20 minutes with occasional shaking. The anion exchange resin was then removed with a millipore filter. The etheno derivatives'of

Volume 93 Number 4

the adenine containing compounds were made according to Mills et al. TAfter four extractions of excess chloroacetaldehyde with water-saturated ether, the samples were subjected to HPLC using a reverse phase column (Waters Associates, Milford, Mass.) (u-bondapak, C ' , 4 mm • 30 mm)." The elution conditions were as follows: buffer A0.05M potassium Phosphate, pH 7.5, and buffer B- 0.05M potassium phosphate, pH 7.5 and 207o (v/v) methanol. A 15-minute linear gradient (A ~ B) was performed. Peaks were detected with a Schoeffel Model FS970 fluorescence detector equipped with a KV389 emission filter after excitation at 280 nm. Absolute amounts were calculated on the basis of peak areas. The results were corrected for recovery by using the inte/nal standard, tubercidin. UrinaD" adenine and deoxyadenosine. Adenine containing compounds* in the urine were assayed both by cation exchange chromatography on AG50-X4 as previously described, T and by HPLC of the etheno derivatives as described above.

Erythroc)'te and plasma il~tsions in A DA deficiency

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Fig. 1. Donor red cell survival and ADA activity after transfusion. Each data point ___SEM includes values from three or more different transfusions.- - = % Donor red cells; . . . . ADA activity; * = transfusion.

RESULTS Red cell adenosine deaminase activity. The percentage of donor red cells and A D A activity was plotted versus time after transfusions (Fig. 1). ADA and donor red cells decreased at a similar rate. Most of the A D A activity was gone by one month after transfusion. The addition of the patient's plasma to lysates of normal red cells did not alter the activity of these cells at adenosine concentrations ranging from 1/tM to 1 mM. Red cells from the patient at various times after transfusion had comparable A D A activity in both intact and lysed cell preparations. Immunologic studies. For this analysis, each transfusion period was arbitrarily defined as 21 days. Because blood sampling was necessarily limited, data points were not obtainable for each day. Therefore, for statistical purposes each 21-day course was divided into seven 3-day periods. Data from 14 transfusions are presented in Table? ~ Absolute blood lymphocyte numbers increased to a maximum (P < 0.01) after nine to 12 days, while the monocyte count reached a maximum at six to nine days posttransfusion (P < 0.01). There were no significant changes in neutrophil counts or proportion of E-rosetting blood lymphocytes following the transfusions. The uptake o P H thymidine into PtlA-P-stimulated lymphocytes reached a *Dr. A. Simmonds of Guy's Hospital, London, called our attention to the acid lability of deoxyadenosine. We found that deoxyadenosine was converted to adenine by the cation exchange resin, tl + form, with the conversion occurring when either plt 5 acetate buffers or ttCI were used for elution. When columns with the sample added were left standidg overnight prior to buffer elution, deoxyadenosine was converted quantitatively to adenine. Therefore, values for adenine in the previous paper' represent deoxyadenosine plus adenine. The trichloroaeetie acid protein precipitation procedurealso converts a portion (15 to 25%) of deoxyadenosine to adenine.

maximum at 12 to 15 days (P < 0.01). The stimulation index also reached its highest value at 12 to 15 days, but this increase was not statistically significant. Before transfusions the serum immunoglobulin levels were decreased (lgG, 52 mg/dl; lgA, < 4.5 mg/dl; IgM, 7 mg/dl). They increased during transfusion therapy to normal levels (IgG, 710 mg/dl; lgM, 168 mg/dl; IgA, 85 mg/dl); however, serum antibodies to Candida albicans or E. coli O, though detectable, remained less than I% of that found in a pool of normal human serum despite infections and intestinal colonization with these organisms. During the transfusion periods, the rate of linear growth and weight gain remained retarded and there was no evidence ofdevelopment of lymphoid tissue or delayed hypersensitivity elicited by intracutaneous Candida albicans or streptokinase-streptodornase antigen or epicutaneous 2,4-dinitrochlorobenzene. Intermittent candidiasis of the mouth and perineum persisted during this period. In addition, the patient developed osteomyelitis involving the upper and lower extremities and pelvis. Acid-fast bacilli were demonstrated in a biopsy of a femoral lesion. The pathogen was not further characterized since it did not grow in cultl~re. The infection was treated with isoniazid, rifampin, or streptomycin for nine months. The infection has not recurred for one year since completion of this therapy. The patient, currently 39 months of age, is maintained in a clean, isolated environment, and is receiving pooled human gammaglobulin and trimethoprim-sulfamethoxazole (5 mg/kg/day, trimethoprim; 25 mg/kg/d.ay, sulfamethoxazole). On this regimen he has been free of serious infection.

600

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The Journal of Pediatrics October 1978

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Fig. 2B. The effect of red blood cell and plasma transfusions on erythrocyte ATP. Data from nine transfusions have been combined. Each data point • SEM includes values from at least three independent transfusions. Several red blood cell transfusions included simultaneous plasma infusions. Lymphocyte and red cell A T P during transfusion. The effect of transfusion on lymphocyte and red cell ATP levels is seen in Fig. 2, A and B. A large but transient decrease ill lymphocyte A T P occurred at six to 10 days after transfusions (P < 0.05). Althougil the lymphocyte

Fig. sine Y= (r =

3. Relationship between red cell ATP and plasma adenoconcentration. The data were fitted to the relationship, aeBX, where Y = plasma adenosine and X = red cell ATP 0.62). *If these two points are ignored, r = 0.87.

ATP decreased to one-third the initial value, it was never normal ( N 600 nmoles/10 ~ cells).;' A similar but more, modest decrease in red cell ATP concentration for the same 6- to 10-day period was also found (P < 0.05) (normal N 1300/~mole/liter of ceils).' More than 75% of the transferred A D A activity remained two weeks after transfusion (Fig. I), but by 12 to 14 days both red cell and lymphocyte A T P returned to pretransfusion levels. Adenosine and adenine in plasma and red ceils. Adenosine and adenine were determined simultaneously by P. separating the etheno derivatives of these compounds by HPLC. The average pretransfusion level of adenosine in plasma was approximately 1 ~M. A relationship between the concentration of red cell ATP and plasma adenosine was found (Fig. 3). Trace amounts of adenine were detected occasionally in the child's plasma and were not correlated with time after transfusion. Although no adenosine was found in red cells, large amounts o f adenine were present. Before transfusion, the red cell adenine was 95.7 __+ 12.7 ~moles/liter of cells. After treatment was begun, adenine decreased to 26.0 _+ 2.0 p.moles/liter of cells (P < 0.01). Urinary adenine and deoxyadenosine. Both adenine and deoxyadertosine were evident in urine by HPLC; however, only adenine was detected when the separations were performed on cation exchange columns using HCI as an eluant. The amounts of these two components in urine decreased after each transfusion, and then gradually increased again (Fig. 4). However, the excretion never returned to the level noted prior to the first transfusion. The ratio of adenine to deoxyadenosine in five samples of urine collected before and after treatment was 0.55 and 0.72, respectively.

Vohmw 93 Number 4

Er)'throc)'te and plasma infusions in A DA deficiency

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Fig. 4. Effect of transfusions on excretion of adenine plus deoxadenosine in urine of the ADA-deficient child. All excretion values are expressed as percent of uric acid excretion _ SD. For statistical purposes, the urine samples ",,,ere divided into three groups as follows: group 1, pretransfusion, Ade + dAdo excretion = 5.84 +_. 1.86% (n = 7); group 2, days 1 through 10 post-transfusion, Ade + dAdo = 0.75 • 0.32% (n = 12); and group 3, days I 1 through 30 posttransfusion, Ade + dAdo excretion = !.66 • 0.87% (n = 16). Probability values were: group I compared to group 2 or to group 3, P < 0.001; group 2 compared to group 3, P = 0.002. Table. Effect o f packed red cell infusion on immunologic measurements

Da)'s after infusion

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2,229 1,338 -940 654 3,225* 684 -!,511.7

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l~ffect of red cell transfusion on blood leukocytes. Data summarized from 17 transfusions were averaged for seven three-day periods after transfusion. Differences of these periods from the composite mean ",,.'eretested using the Dunnett method?~ *P < O.Ol. DISCUSSION It has been suggested that A D A deficiency m a y be amenable to treatment with exogenous A D A in the form of transfusions of normal h u m a n red cells. T. 9 The rationale for this therapy is based on the hypothesis that accumulation o f adenosine secondary to the block in purine metabolism causes the immunodeficiency. Certain experimental evidence favors this hypothesis. (1) Plasma adenosine concentration (1 to 2 FM) in our patient approached that which is inhibitory to lymphocyte function in vitro in the presence o f A D A inhibitors. "~:'=~(2) In at least two patients with this disease, large (approximately tenfold) increases in lymphocyte A T P concentrations. have been demonstrated, s, 9 In addition, the patient in this report and another reported case tr had increases in red cell ATP before transfusion. These increases in cellular A T P concentrations are likely due to the. action o f adenosine kinase (K= ~ I FM) in the presence o f adeno-

sine and an intact glycolytic pathway. (3) The subject of this investigation also had a fourfold elevation in lymphocyte cyclic AMP. ~ It is not known whether this is a consequence o f increased substrate A T P or a direct effect of adenosine on adenyl cyclase. =6 Presumably, the increased intracellular nucleotide concentrations coul.d inhibit lymphocyte function, although the mechanism remains unknown. 22-~s If in.creased nucleotide concentrations secondary to increased plasma adenosine concentrations are responsible for the i m m u n e defect, exogenous A D A could theoretically repair the defect by deaminating the adenosine, which readily crosses cell membranes. Polmar et al d found that the immune response o f a 7-month-old child with A D A deficiency was restored in large part by red cell transfusions. T h a t child differed from our patient in that the A D A activity in his lymphocytes (6% of normal) was considerably higher than our patient's. (less than I%).

602

Schmalstieg et al.

Despite many red cell and plasma transfusions performed over a year and a half, there was comparatively little change in our patient's immune status. Certain immunologic and biochemical changes occurred, however, after transfusion. ~H-thymidine incorporation in PHA-P-stimulated blood lymphocytes increased somewhat, and the absolute numbers o f blood lymphocytes and monocytes increased approximately twofold (Table). -"~ Large but transient decreases in lymphocyte and red cell ATP occurred during the transfusion periods (Fig. 2). Decreases in the transfused A D A activity (Fig. !) were not sufficient to explain the transient nature of the reduction in ATP concentration. In addition, no inhibitors of ADA activity were found in the plasma. It is possible that the partial correction o f the metabolic defect was not sufficient to restore lymphocyte function, even though on at least one occasion the lymphocyte ATP remained at approximately 3,000 nmoles/107 cells for more than one week. It is also possible that A D A is necessary in the thymus and other lymphoid organs for the development of the lymphoid system. ' ' If this is the case, the exogenous ADA may not have been effective at these sites. Failure to restore lymphocyte function in vivo with exogenous A D A in the form of red cells or plasma is consistent with the limited in vitro effect of calf intestine ADA on the patient's lymphocytes? These in vivo and in vitro effects of exogenous A D A were in contrast to the case reported by Polmar et al. 9 A correlation between plasma adenosine level and red cell ATP concentration was found. Caution must be used in interpreting these results. Although adenosine added to whole blood is rapidly utilized,'8. -~ we have found that the concentration of adenosine in plasma as determined by HPLC remained constant at room temperature for at least 20 minutes after venipuncture. It is possible that adenosine in plasma is a result of a steady state or is a measurement artifact and may not accurately reflect the adenosine concentration at other organ sites. The deoxyadenosine noted in urine is presumably a consequence of DNA breakdown to the constituent nucleotides, followed by a nucleotidase catalyzed conversion of dAMP to deoxyadenosine. Since A D A is required to convert deoxyadenosine to deoxyinosine, the failure of the ADAdeficient child to convert deoxyadenosine to uric acid is readily explained. Deoxyadenosine kinase (purified from calf thymus) s~ has a high Km for deoxyadenosine (K,~ 0.7 raM), and this may minimize the reutilization of deoxyadenosine for nucleotide synthesis. Adenine and deoxyadenosine in the urine, expressed as percent of uric acid, decreased after beginning red cell transfusions. To ascertain whether these changes in adenine and deoxyadenosine reflected.a.lterations in uric

The Journal of Pediatrics October 1978

acid excretion, we made the following additional evaluations. When the levels of adenine plus deoxyadenosine were expressed as percent of total cationic ultraviolet absorbing compounds and as percent of creatinine excretion, the results were similar to those shown in Fig. 4. In contrast to adenine in urine, adenine in red ceils decreased only slightly with transfusion. Although we could not demonstrate deoxyadenosine in red cells, the source of the adenine in these cells might also be deoxyadenosine. It appears that some patients with ADA-deficient SCID may not be helped by red cell or plasma transfusion. The efficacy of transfusion may be dictated by the amount o f residual cellular ADA activity. Apparently, the biochemical effect of transfused red cells in this patient was not sustained long enough or was not substantial enough to enhance lymphocyte function. The role o f adenine and deoxyadenosine in the pathogenesis of this disease remains unknown, but it is noteworthy that both deoxyinosine and deoxyguanosine were increased in the urine of a patient with nucleoside phosphorylase deficiency,~' an enzyme deficiency that is also associated with a severe T cell defect. The role of deoxynucleosides and deoxynucleotides in the immune response is currently being investigated in our laboratory. ADDENDUM Recently, it was reported that deoxy-ATP was greatly elevated in erythrocytes of certain ADA-deficient patients and that this elevation might be important in the pathogenesis of the immunodeficiency (Proc Natl Acad Sci 75:472, 1978; J Biol Chem 263:!619, 1978). We were unable to confirm this finding in our patient by chemical, enzymatic or chromatographic means. We thank Ms. Elizabeth J. Finger for her capable secretarial and technical graphic assistance in the preparation of this manuscript; Ms. Katherine Newkirk for technical assistance; professional staff of the Clinical Research Center for their excellent patient care; and Dr. Rodney N. Dotson for the patient referral. REFERENCES

1. Giblett ER, Anderson JE, Cohen F, Pollara 13, and Meuwissen H J: Adenosine-deaminase deficiency in two patient~ with severely impaired cellular immunity, Lancet 2:!067, 1972. 2. Dissing J, and Knudsen B: Adenosine-deaminase deficiency and combined immunodeficiency syndrome, Lancet 2:1316, 1972. 3. Ochs HD, Yount JE, Giblett ER, Chert S-H, Scott CR, and Wedgwood RJ: Adenosine-deaminase deficiency and severe combined immunodeficiency syndrome, Lancet 1:1393, 1973. 4. Yount J, Nichols P, Ochs HD, ttammar SP, Scott CR, Chen

Volume 93 Number 4

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S-H, Gib,lett ER, and Wedgwood R J: Absence of erythrocyte adenosine deaminase associated with severe combined immun0deficiency, J PEDxA'rR84:173, 1974. Meuwissen tlJ, Pollara B, and Pickering R J: Combined immunodeficiency disease associated with adenosine deaminase deficiency, J PFrnATR86:169, 1975. Parkman R, Gelfand EW, Rosen FS, Sanderson A, and ttirschhorn R: Severe combined immun0deficiency and adenosine deaminase deficiency, N Engl J Med 292:714, 1975. Mills GC, Schmalstieg FC, Trimmer KB, Goldman AS, and Goldblum RM: Purine metabolism in adenosine deaminase deficiency, Proc Nail Acad Sci USA 73:2867, 1976. Schmalstieg FC, Nelson JA, Mills GC, Monahan T M , Goldman AS, and Goldblum RM: Increased purine nucleo7 tides in adenosine deaminase-deficient lymphocytes, J PEDtx'rR 91:48, 1977. Polmar SH, Stern RC, Schwartz AL, Wctzler E M, Chase PA, and Hirschhorn R:. Enzyme replacement therapy for adenosine deaminase deficiency and severe combined immunodeficiency, N Engl J Med 295:!337, 1976. Ashby W: The determination of the life of transfused blood corpuscles in man, J Exp Med 29:267, 1919. Junge U, Hoekstra J, Wolfe L, and Deinhardt F: Microtechnique for quantitative evaluation of in vitro lymphocyte transformation, Clin Exp Immunol 7:431, 1970. Jondal M, tlolm G, and Wigzell H: Surface markers on human T and B lymphocytes. I. A large population of lymphocytes forming nonimmune rosettes with sheep red blood cells, J Exp Med 136!207, 1972. Mancini G, Carbonera AO, and Heremans JF: Immunochemical quantitation of antigens bY single radial immunodiffusion, immunochemistry 2:235, 1965. Engvall E, and Pearlman P: Enzyme-linked immunosorbent assay, ELISA III. Quantitation of specific antibodies9 by enzyme labelled anti-immunoglobulin in antigen coated tubes, J Immunol 109:129, 1972. Kalckar HM: Differential spectroph0tometry of purine compounds by means of specific enzymes. III. Studies of the enzymes of purine metabolism, J Biol Chem .167:461, 1947. Ilirschhorn R, and Levytska V: Alterations in isozymes of adenosine deaminase during stimulation of human peripheral blood lymphocytes, Cell Immunol 12:387, 1974. 9 Agarwal RP, Crabtree CW, Parks RE Jr, Nelson JA, Keightley R, Parkman R, Rosen FS, Stern RC, and Polmar SH: Purine nucleoside metabolism in th e erythrocytes of patients with adenosine deaminase deficiency and severe combined immunodeficiency, J Clin Invest 57:!025, 1976. Adams H: Adenosine 5'-triphosphate, determination with phosphoglycerate kinase, in Bergmeyer tlU, editor: Meth-

Erythrocyte and plasma it~tsions in A DA deficiency

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ods of enzyme analysis, New York, 1963, Academic Press, Inc., pp 539-543. Kuttesch JF, Schmalstieg FC, and Nelson JA: Analysis of adenosine and other adenine compounds in patients with immunodeficiencydiseases, J Liquid Chromatography 1:97, 1978. Dunnett CW: A multiple comparisons procedure for comparing several treatments with a control, J Am Stat Assoc 50:1096, 1955. Scholar EM, Brown PR, Parks RE Jr, and Calabresi P: Nucleotide profiles of the formed elements of the blood determined by high pressure liquid chromatography, Blood 41:927, 1973. Carson DA, and Seegmiller JE: Effect of adenosine deaminase inhibition upon human lymphocyte blastogenesis, J Clin Invest 57:274, 1976. Hovi T, Smyth JF, Allison AC, and Williams SC: Role of adenosine deaminase in lymphocyte proliferation, Ctin Exp Immunol 23:395, 1976. Fox IH, Keystone EC, Gladman DD, Moore M, and Cane D: Inhibition of mitogen mediated lymphocyte blastogenesis by adenosine, lmmunol Commun 4:419, 1975. Wolberg G, Zimmerman TP, Hiemstra K, Winston M, and Chu L-C: Adenosine inhibition of lymphocyte-mediated cytolysis: Possible role of cyclic adenosine monophosphate, Science 187:957, 1975. Clark RB, Gross R, Su Y-F, and Perkins JP: Regulation of adenosine 3':5'-monophospbate content in human astrocytoma cells by adenosine and the adenine nucleotides, J Biol Chem 249:5296, 1974. Ballet J-J, lnsel R, Merler E, and Rosen FS: Inhibition of maturation of human precursor lymphocytes by coformycin, an inhibitor of the enzyme adenosine deaminase, J Exp Med 143:1271, 1976. Kolassa N, and Pfleger K: Adenosine uptake by erythrocytes of man, rat and guinea-pig and its inhibition by hexobendine and dipyridamole, Biochem Pharmacol 24:154, 1975. van Belle H: Uptake and deamination of adenosine by blood. Species differences, effect of pH, ions, temperature and metabolic inhibitors, Biochim Biophys Acta 192:124, 1969. Krygier U, and Momparler RL: Mammalian deoxynucleoside kinases. III. Deoxyadenosine kinase: Inhibition by nucleotides and kinetic studies, J Biol Chem 246:2752, 1971. Stoop JW, Zegers BJM, Ilendrickx GFM, Siegenbeek van Heukelom Ltl, Staal GEJ, de Bree PK, Wadman SK, and Ballieux RE: Purine nucleoside phosphorylase deficiency associated with selective ceUular immunodeficiency, N Engl J Med 296:651, 1977.