- Email: [email protected]
Immune functions in children treated with biosynthetic growth hormone
420
Clinical and laboratory observations
REFERENCES
1. Hayes-Allen MC. Obesity and short stature in children with myelomeningocele. Dev Med Child Neurol 1972;14(suppl 27):59-64. 2. Rosenblum MF, Finegold DN, Charney EB. Assessment of stature of children with myelomeningocele and usefulness of arm-span measurement. Dev Med Child Neurol 1983;25:33842. 3. Rimoin DL. Genetic disorders of the pituitary gland. In: Emery AEH, Rimoin DL, eds. Principles and practice of medical genetics. New York: Churchill Livingston, 1983:1134-51. 4. Hamill PVV. NCHS growth curves for children birth to 18 years: United States. Hyattsville, Md.: National Center for Health Statistics, 1977. DHEW publication No. 78-1650. (Vital and health statistics; series 11; No. 165, pp 20-3. 5. Maresh MM. Linear growth of long bones of limbs from infancy through adolescence. Am J Dis Child 1955;89:725-42. 6. Greulich WW, Pyle SI. Radiographic atlas of skeletal development of the hand and wrist. Stanford, Calif.: Stanford University Press, 1959. 7. Tanner JM. Growth at adolescence. Oxford: Blackwell Scientific Publications, 1962:33. 8. Gil-Ad I, Topper E, Laron Z. Oral clonidine as a growth hormone stimulation test. Lancet 1979;2:278-9.
The Journal of Pediatrics September 1989
9. Lin T, Kirkland RT, Sherman BM, Kirkland JL. Twentyfour-hour studies of growth hormone secretion in normal and short children and their lack of predictive value in growth hormone therapy [Abstract]. Pediatr Res 1988;23:280A. 10. Belt-Niedbala BJ, Ekval S, Cook C, Oppenheimer S, Wessel J. Linear growth measurement: a comparison of single armlength and arm-span. Dev Med Child Neurol 1986;28:319-24. 11. Reigel DH. Tethered spinal cord. Concepts Pediatr Neurosurg 1983;4:142-64. 12. Leonard CO, Freeman JM. Spina bifida: a new disease. Pediatrics 1981;68:136-7. 13. Greene SA, Frank M, Zachmann M, Prader M. Growth and sexual development in children with meningomyelocele. Eur J Pediatr 1985;144:146-8. 14. Meyer S, Landau H. Precocious puberty in myelomeningocele patients. J Pediatr Orthop 1984;4:28-31. 15. Costin G, Kaufman FR. Growth hormone secretory patterns in children with short stature.. J PEDIATR 1987;110:362-8. 16. Hammock MK, Milhorat TH, Baron IS. Normal pressure hydrocephalus in patients with myelomeningocele. Dev Med Child Neurol 1976;18(suppl 37):55-68. 17. Grumback MM. Growth hormone therapy and the short end of the stick. N Engl J Med 1988;319:238-40.
Immune functions in children treated with biosynthetic growth hormone Joseph A. Church, MD, G e r t r u d e Costin, MD, and Judith Brooks, Bs, MT From the Divisions of Allergy-Clinical Immunology, Endocrinology and Metabolism, and Research Immunology and Bone Marrow Transplantation, Childrens Hospital of Los Angeles and the University of Southern California School of Medicine, Los Angeles
Suppression of selected immune functions has been reported in children treated with pituitary-derived I and biosynthetiC, 3 growth hormone. These studies suggested that G H therapy was associated with transiently decreased C D 4 + ("helper") T cell and B cell numbers, T cell mitogen responses, and in vitro IgM production. However, the significance of these changes was challenged by A m m a n n and Sherman, 4 who emphasized the transient nature of the demonstrated abnormalities, the lack of clinical evidence for immune deficiency, the variable nature of immunologic tests, and the suggestion from animal studies that G H enhances immune functions. Our study was undertaken to determine prospectively Submitted for publication Jan. 5, 1989; accepted Mar. 28, 1989. Reprint requests: Joseph A. Church, MD, Division of AllergyClinical Immunology, Childrens Hospital of Los Angeles, P.O. Box 54700, Terminal Annex, Los Angeles, CA 90054. 9/22/12737
the effects of biosynthetic G H therapy on specific and nonspecific T cell and B cell functions. METHODS
Patients. Children who were candidates for G H therapy were eligible for participation in the study. Informed cpm GH Hib LPR PHA PWM
Counts per minute Growth hormone
Haemophilus influenzae type b Lymphoproliferative response Phytohemagglutinin Pokeweed mitogen
consent was obtained from all parents and from children 12 years of age and older before initiation of G H treatment. The study group consisted of 13 patients (eight boys) whose ages ranged from 3.5 to 14.5 years (mean 11.3 years). Ten patients had G H deficiency, either idiopathic
Volume 115 Number 3
Clinical and laboratory observations
421
T a b l e I. Peripheral blood lymphocyte analyses before and during treatment with biosynthetic G H
Baseline
'I
3
6
9
12
Normal laboratory values
62 _+ 11 1467 • 699
65 • 8 1157 • 416
65 + 11 1729 + 537
58 + 18 1315 • 535
69 +_ 10' 1480 • 514
76 • 7* 1748 + 438
60 + 12 1456 + 291
Month of treatment
CD3 + cells Percent Per rnm 3 CD4 § cells Percent Per rnm3 CD8 + cells Percent Per mm 3 CD4+/CD8 + B 1§ cells Percent Per mm 3
34 • 15 792 • 496
36 + 11 964 • 306*
36 • 8 1022 • 295*
37 • 11 784 • 309
37 • 11 790 • 335
44 +-- 10" 990 • 295*
36 + 10 873 • 240
22 • 7 500 +_ 268 1.74 _+ 0.91
24 _+ 8 593 • 228 1.64 • 0.80
23 • 9 615 + 235 1.86 + 0.99
20 • I0 437 + 278 2.64 + 2.44
22 _+ 9 485 • 233 1.93 _+ 0.97
27 + 6 649 • 267 1.70 • 0.64
23 • 7 557 + 170 1.70 + 1.0
4 • 3 87 • 58
7 + 4* 167 • 72*
4+ 2 111 • 38*
6 • 5* 108 • 41
7 + 6* 157 • 123"
6+3 139 -+ 58*
6• 146 • 97
All values represent group means _+SD. *Indicates significantlyhigher than baseline (p ~ 0.05).
(six) or secondary to cranial radiation therapy (four), and three patients had significantly short stature associated with Noonan syndrome (two) or Turner syndrome (one). Biosynthetic GH preparations. Two preparations of biosynthetic G H produced with recombinant D N A technology were used: H u m a t r o p e (Eli Lilly and Company, Indianapolis) in 10 patients and Protropin (Genentech, South San Francisco, Calif.) in three patients. The hormone was administered in three to six subcutaneous injections weekly, except in one patient who received three intramuscular injections weekly. Clinical monitoring. During G H therapy the following were recorded at each assessment visit: number of infectious episodes requiring antibiotic therapy, estimated number of days with fever (temperature >38.5~ and number of days Of school missed because of any presumed infection. Immunologic studies. Immunologic studies were performed before (baseline) and 1, 3, 6, 9, and 12 months after initiation of G H therapy. Complete blood cell counts were used to calculate absolute granulocyte counts and absolute lymphocyte counts. Peripheral blood lymphocyte subsets were quantitated with commercially available monoclonal antibodies and analysis with an EPICS V Fluorescent Activated Cell Sorter (Coulter Corp., Hialeah, Fla.). The antibody OKT-3 (Ortho Diagnostic Systems Inc., Raritan, N.J.) or Leu-4 (Becton Dickinson Immunocytometry Systems, Mountain View, Calif.) was used to identify C D 3 + T ceils; O K T - 4 or Leu-3 was used to identify the C D 4 + helper T ceIi subset; OKT-8 or Leu-2 was used to identify the C D 8 + "suppressor" T cell subset; and B1 (Coulter) was used to identify B
cells. In vitro lymphocytic responses to phytohemagglutinin, pokeweed mitogen, tetanus toxoid antigen, and Candida antigen were assayed w i t h a standard microculture technique in which tritiated thymidine was used. Very briefly, aliquots of peripheral blood mononuclear cells, separated by density gradient, were placed in microtiter wells. W e added P H A , P W M , tetanus toxoid antigen, or Candida antige n to triplicate wells and incubated for 3 days (PHA, P W M ) or 6 days (tetanus, Candida). Tritiated thymidine was then added to each well. After 2 4 hours the tritiated thymidine-labeled D N A was extracted and the radioactivity quantitated in a beta scintillation counter as counts per minute per well. For data analysis, L P R s to mitogens an d specific antigens were expressed as the log of the difference between counts per minute in stimulated cell cultures and unstimulated cell cultures (log• Acpm). A t the initial visit, antibody levels to tetanus toxoid antigen and polyribosyl phosphate (the polysaccharide antigen of Haemophilus influenzae type b) were measured with standard enzyme-linked immunosorbent assays. Patients who had nonprotective levels of antibodies to either antigen (<0.10 I U / m l for tetanus toxoid; <100 n g / m l for polyribosyl phosphate) were immunized at the 1-month visit (while receiving biosynthetic G H ) with standard tetanus toxoid (tetanus toxoid absorbed, Wyeth Laboratories Inc., Philadelphia) or Hib vaccine (HibImune, Lederle Labs, Pearl River, N.Y.), respectively. Antibody levels were remeasured at the 3-month visit. A normal response was considered to be an increase in antibody Ievets into the respective protective ranges. Lymphocyt e subset analyses, lymphopr01iferative responses, and antibody levels were measured in the
4 22
Clinical and laboratory observations
The Journal of Pediatrics September 1989
T a b l e II. Mitogen- and antigen-induced lymphoproliferative responses before and during treatment with biosynthetic G H Month of treatment Stimulant
PHA PWM Tetanus toxoid Candida antigen
Baseline
5.09 4.36 3.64 3.99
-+ .30 + .48 _+ 1.41 _+ .61
1
4.70 4.39 4.02 4.02
+ .34* + .45 +_ .91 _+ .56
3
5.03 4.46 4.17 4.29
+ .26 + .25 + .78 _+ .71
6
4.92 4.19 4.43 3.94
+ .43 _+ .58 -+ .43 _+ .71
9
4.86 3.92 4.23 3.82
_ + + _
12
.45* .77 .78 .66
4.75 4.40 4.52 3.94
___ .49 + .16 • .50 _+ .90
Normal laboratory values
>4.70 >4.60 >3.30 >3.30
All values represent group means of iogto A cpm +_ SD. *Values significantlylower than baseline (p < 0.05).
laboratory of Dr. Robertson Parkman, Division of Research Immunology and Bone Marrow Transplantation. Childrens Hospital of Los Angeles. RESULTS Ten patients were followed for 1 year. one for 9 months. one for 6 months, and one for 3 months. O f 13 patients, 12 responded to biosynthetic G H with an increase in growth rate. The mean growth was 8.0 _+ 2.1 cm (mean _ SD); range 4.0 to 11.0 cm) in the 10 patients followed for a full year. One patient with an advanced bone age at the start of treatment did not respond to treatment, which was discontinued after 6 months: one patient decided to interrupt therapy after 3 months. The 13 children had six episodes of infection requiring antibodies and 15 days of fever in 10 episodes of illness. Seven children missed a total of 36 days of school during seven illnesses (six upper respiratory tract infections and one case of infectious mononucleosis syndrome). This last patient, a 15-year-old girl with Noonan syndrome, was given two courses of antibiotics and missed 14 days of school: she was not studied during the illness. N o other patients had an infection with a herpesvirus. The number of days of school missed, 3.3 days per patient per year. was less than the average 11 days per student per year in Los Angeles (Helen Hale, MD, Director, Student Medical Services, Los Angeles Unified School District: personal communication. Dee. 8. 1987). The mean percentages and absolute numbers of circulating granulocytes were not significantly different from baseline at any time measured (data not shown). As indicated in Table I, the percentage of CD3 § cells (T cells) was moderately increased over baseline at 9 and 12 months of G H treatment. Similarly. levels of CD4 + ceils (helper T cells) and CD8 § cells (suppressor T cells) were increased at 12 months. Assessment of absolute numbers of T cell subsets revealed significantly increased numbers of C D 4 cells at 1. 3, and 12 months. The C D 4 + / C D 8 + (helper/ suppressor) cell ratios were never significantly different
from baseline. The C D 4 + / C D 8 + ratios in individual patients remained stable throughout the study. In vitro LPRs to P H A were significantly decreased at 1 and 9 months (Table I!). Nine of 10 patients for whom data were available had a decre~ised P H A L P R 1 month after starting GH. Five of these patients had P H A LPRs below normal laboratory values. A t 3, 6, and 12 months, mean P H A LPRs values were normal. A t 3 months one patient had a persistently low P H A , L P R , and at 6 months decreased P H A LPRs were noted in two of the five patients who had decreased P H A L P R s at 1 month. Mean P W M LPRS were not decreased significantly at any time. Like P H A LPRs, P W M L P R s showed individual variations, but these were not consistently seen at any specific time or in any individual patient. The LPRs to specific antigens, tetanus toxoid and Candida antigen, remained normal throughout the study. Two patients had a decreased tetanus-specific LPRs on one occasion; four other patients had decreased Candidaspecific LPRs on one occasionl The timing of these decreases was variable. Of the 13 patients studied, seven had protective levels of antibodies to tetanus toxoid (>0.1 I U / m l ) and nine had protective levels of antibodies to Hib (>100 ng/ml). Nine patients received 10 immunizations: five tetanus toxoid, three Hib, and one tetanus toxoid plus Hib. Each of the 10 immunizations resulted in a significant antibody response into the normal protective range (data not shown). DISCUSSION Numerous immune-neuroendocrine interactions are likely to h a v e physiologic consequences? For example, Kiess et al. 6 found depressed natural killer cell activity in patients with G H deficiency caused by a hypothalamic deficit of GH-releasing hormone; short-term administration of GH-releasing hormone failed to eliminate this abnormality. Although isolated G H deficiency or dysfunction has not been associated with T cell or B cell deficiency, such immunologic dysfunction has been reported after
Volume 115 Number 3
treatment with pituitary-derivedI and biosynthetic2,3 GH. Rapaport et a12 described suppression of immune functions in eight GH-deficient children treated with pituitaryderived GH. Althoug h erythrocyte, circulating granulocyte, and serum immunoglobulin levels were not affected by GH, helper/suppressor T cell ratio, percentage of circulating B cells, and in vitro mitogen responses were significantly decreased; these abnormalities were generally transient. Bozzola et al. 2 reported similar T cell subset abnormalities without alteration in serum immun0globulin levels in children receiving biosynthetic GH and depressed in vitro IgM production before and after 3 to 6 months of therapy. Most recently, Rapaport et al. 3 described studies in children treated with biosynthesis GH and noted decreased percentages of circulating B ceils and in vitro mitogen responses. In none of these studies was evidence of clinical immune deficiency described. A recent report from Japan described an increased occurrence of leukemia in patients treated with GH. 7 A review of all available data concluded that there may be a small increase in leukemia incidence with GH treatment of GH-deficient childrenS; immune system abnormalities in GH-deficient patients was one of several possible explanations offered for this occurrence. Rogers et al. 9 offered the additional possibility of a direct effect of GH on resting lymphoid cells and subsequent malignant transformation. In contrast, Arslanian et al. t~concluded that GH therapy is probably not associated with an increased rate of brain tumor recurrence. Clayton et al. tl indicated that GH therapy did not increase the relapse rates of medulloblastoma, glioma, and leukemia. In none of the studies cited was an increased incidence of infectious complications identified in GH-treated patients. Our results support the conclusion that G H therapy is not associated with a clinically significant increase in the frequency or severity of infections in GH-deficient children. As previously documented,2 GH therapy does not appear to have an adverse effect on the number of circulating granulocytes. In contrast to previous studies, 1-3 we did not observe consistent Suppression of T cell or B cell functions in any of the systems measured. The percentage and absolute numbers of circulating CD3 + T cells, CD4 + helper T cells, CD8 § suppressor T cells, and B1 + B cells were intermittently increased during treatment with biosynthetic GH; the CD4+/CD8 § ratio remained stable. All measurements of lymphocyte subpopulations were within normal laboratory ranges. The timing of previously reported changes with regard to duration of G H therapy has not been consistent. In our study PHA LPRs were decreased at 1 and 9 months of treatment but did not appear to reflect general suppression of lymphocyte responsiveness because
Clinical and laboratory observations
423
PWM and antigen-specific responses remained stable throughout the study. All patients immunized with tetanus toxoid or polyribosyl phosphate generated significant and protective levels of antibodies. The maintenance of antigen-specific responsiveness in our study strongly indicates that clinically significant T and B cell functions are retained by GH-deficient patients during biosynthetic GH therapy, even though four of the patients had received potentially immunosuppressive therapy (cranial irradiation) for a variety of cancers. As suggested by Ammann and Sherman4 in reference to other studies, the variability in our results, including the significantly decreased PHA responses, may reflect the variable nature of immunologic tests .when these are performed sequentially. Alternatively, GH deficiency may be associated with enhanced PHA responsiveness, which is then suppressed after G H replacement. Why this phenomenon would be apparent only with PHA and not with other lymphocyte stimulants remains unclear. REFERENCES
1. Rapaport R. Oleske J. Ahdieh H. Solomon S. Delfaus C, Denny T. Suppression of immune function in growth hormone-deficient children during treatment with human growth hormone. J PEDTATR1986;109:434-9. 2. Bozzola M, Valtorta A, Maghnie M, et at. Influence of biosyntheticgrowth hormone(GH) therapy on the humoral and cell-mediatedimmunity of GH-deficientchildren. Presented at the International Congress on Advances in Growth Hormone and Growth Factor Research, Milan, Sept. 28-30, 1987. 3. Rapaport R, Peterson B, Skuza KA, He!m M, Goldstein S. Biosynthetic methiony!-human growth hormone (BMHGH) treatment affects immune functions (IF) in growth hormonedeficient children. Pediatr Res 1987;21:252A. 4. Ammann AJ, Sherman BM. Effect of growth hormone therapy on immune function. J P~DIATR1987;10:663-4. 5. Besedovsky HO, Del Rey A, Sorkin E. Immune-neuroendocrine interactions. J Immunol 1985;135:750s-4s. 6. Kiess W, Malozowski S, Gelato M, et al. Lymphocyte subset distribution and natural killer activity in growth hormone releasing hormone. Clin Immunol Immunopathol 1988;48:8594. 7. Watanabe S, Tsunematsu Y, Fujimoto J, Komiyama A. Leukaemia in patients treated with growth hormone. Lancet 1988;1:1159. 8. Fisher DA, Job J-C, Preece M, UnderwoodLE. Leukaemia in patients treated with growth hormone. Lancet 1988;1:115960. 9. Rogers PC, Komp D, Rogol A, Sabio H. Possible effects of growth hormone on development of acute lymphoblastic leukaemia. Lancet 1977;2:434-5. 10. Arslanian SA, Becker DJ, Lee PA, Drash AL, Foley TP. Growth hormone therapy and turner recurrence findingsin children with brain neoplasms and hypopituitarism. Am J Dis Child 1985;139:347-50. 11. Clayton PE, Shalet SM, Gattamaneni HR, Price DA. Does growth hormone cause relapse of brain tumors? Lancet 1987;1:711-13.
Clinical and laboratory observations
REFERENCES
1. Hayes-Allen MC. Obesity and short stature in children with myelomeningocele. Dev Med Child Neurol 1972;14(suppl 27):59-64. 2. Rosenblum MF, Finegold DN, Charney EB. Assessment of stature of children with myelomeningocele and usefulness of arm-span measurement. Dev Med Child Neurol 1983;25:33842. 3. Rimoin DL. Genetic disorders of the pituitary gland. In: Emery AEH, Rimoin DL, eds. Principles and practice of medical genetics. New York: Churchill Livingston, 1983:1134-51. 4. Hamill PVV. NCHS growth curves for children birth to 18 years: United States. Hyattsville, Md.: National Center for Health Statistics, 1977. DHEW publication No. 78-1650. (Vital and health statistics; series 11; No. 165, pp 20-3. 5. Maresh MM. Linear growth of long bones of limbs from infancy through adolescence. Am J Dis Child 1955;89:725-42. 6. Greulich WW, Pyle SI. Radiographic atlas of skeletal development of the hand and wrist. Stanford, Calif.: Stanford University Press, 1959. 7. Tanner JM. Growth at adolescence. Oxford: Blackwell Scientific Publications, 1962:33. 8. Gil-Ad I, Topper E, Laron Z. Oral clonidine as a growth hormone stimulation test. Lancet 1979;2:278-9.
The Journal of Pediatrics September 1989
9. Lin T, Kirkland RT, Sherman BM, Kirkland JL. Twentyfour-hour studies of growth hormone secretion in normal and short children and their lack of predictive value in growth hormone therapy [Abstract]. Pediatr Res 1988;23:280A. 10. Belt-Niedbala BJ, Ekval S, Cook C, Oppenheimer S, Wessel J. Linear growth measurement: a comparison of single armlength and arm-span. Dev Med Child Neurol 1986;28:319-24. 11. Reigel DH. Tethered spinal cord. Concepts Pediatr Neurosurg 1983;4:142-64. 12. Leonard CO, Freeman JM. Spina bifida: a new disease. Pediatrics 1981;68:136-7. 13. Greene SA, Frank M, Zachmann M, Prader M. Growth and sexual development in children with meningomyelocele. Eur J Pediatr 1985;144:146-8. 14. Meyer S, Landau H. Precocious puberty in myelomeningocele patients. J Pediatr Orthop 1984;4:28-31. 15. Costin G, Kaufman FR. Growth hormone secretory patterns in children with short stature.. J PEDIATR 1987;110:362-8. 16. Hammock MK, Milhorat TH, Baron IS. Normal pressure hydrocephalus in patients with myelomeningocele. Dev Med Child Neurol 1976;18(suppl 37):55-68. 17. Grumback MM. Growth hormone therapy and the short end of the stick. N Engl J Med 1988;319:238-40.
Immune functions in children treated with biosynthetic growth hormone Joseph A. Church, MD, G e r t r u d e Costin, MD, and Judith Brooks, Bs, MT From the Divisions of Allergy-Clinical Immunology, Endocrinology and Metabolism, and Research Immunology and Bone Marrow Transplantation, Childrens Hospital of Los Angeles and the University of Southern California School of Medicine, Los Angeles
Suppression of selected immune functions has been reported in children treated with pituitary-derived I and biosynthetiC, 3 growth hormone. These studies suggested that G H therapy was associated with transiently decreased C D 4 + ("helper") T cell and B cell numbers, T cell mitogen responses, and in vitro IgM production. However, the significance of these changes was challenged by A m m a n n and Sherman, 4 who emphasized the transient nature of the demonstrated abnormalities, the lack of clinical evidence for immune deficiency, the variable nature of immunologic tests, and the suggestion from animal studies that G H enhances immune functions. Our study was undertaken to determine prospectively Submitted for publication Jan. 5, 1989; accepted Mar. 28, 1989. Reprint requests: Joseph A. Church, MD, Division of AllergyClinical Immunology, Childrens Hospital of Los Angeles, P.O. Box 54700, Terminal Annex, Los Angeles, CA 90054. 9/22/12737
the effects of biosynthetic G H therapy on specific and nonspecific T cell and B cell functions. METHODS
Patients. Children who were candidates for G H therapy were eligible for participation in the study. Informed cpm GH Hib LPR PHA PWM
Counts per minute Growth hormone
Haemophilus influenzae type b Lymphoproliferative response Phytohemagglutinin Pokeweed mitogen
consent was obtained from all parents and from children 12 years of age and older before initiation of G H treatment. The study group consisted of 13 patients (eight boys) whose ages ranged from 3.5 to 14.5 years (mean 11.3 years). Ten patients had G H deficiency, either idiopathic
Volume 115 Number 3
Clinical and laboratory observations
421
T a b l e I. Peripheral blood lymphocyte analyses before and during treatment with biosynthetic G H
Baseline
'I
3
6
9
12
Normal laboratory values
62 _+ 11 1467 • 699
65 • 8 1157 • 416
65 + 11 1729 + 537
58 + 18 1315 • 535
69 +_ 10' 1480 • 514
76 • 7* 1748 + 438
60 + 12 1456 + 291
Month of treatment
CD3 + cells Percent Per rnm 3 CD4 § cells Percent Per rnm3 CD8 + cells Percent Per mm 3 CD4+/CD8 + B 1§ cells Percent Per mm 3
34 • 15 792 • 496
36 + 11 964 • 306*
36 • 8 1022 • 295*
37 • 11 784 • 309
37 • 11 790 • 335
44 +-- 10" 990 • 295*
36 + 10 873 • 240
22 • 7 500 +_ 268 1.74 _+ 0.91
24 _+ 8 593 • 228 1.64 • 0.80
23 • 9 615 + 235 1.86 + 0.99
20 • I0 437 + 278 2.64 + 2.44
22 _+ 9 485 • 233 1.93 _+ 0.97
27 + 6 649 • 267 1.70 • 0.64
23 • 7 557 + 170 1.70 + 1.0
4 • 3 87 • 58
7 + 4* 167 • 72*
4+ 2 111 • 38*
6 • 5* 108 • 41
7 + 6* 157 • 123"
6+3 139 -+ 58*
6• 146 • 97
All values represent group means _+SD. *Indicates significantlyhigher than baseline (p ~ 0.05).
(six) or secondary to cranial radiation therapy (four), and three patients had significantly short stature associated with Noonan syndrome (two) or Turner syndrome (one). Biosynthetic GH preparations. Two preparations of biosynthetic G H produced with recombinant D N A technology were used: H u m a t r o p e (Eli Lilly and Company, Indianapolis) in 10 patients and Protropin (Genentech, South San Francisco, Calif.) in three patients. The hormone was administered in three to six subcutaneous injections weekly, except in one patient who received three intramuscular injections weekly. Clinical monitoring. During G H therapy the following were recorded at each assessment visit: number of infectious episodes requiring antibiotic therapy, estimated number of days with fever (temperature >38.5~ and number of days Of school missed because of any presumed infection. Immunologic studies. Immunologic studies were performed before (baseline) and 1, 3, 6, 9, and 12 months after initiation of G H therapy. Complete blood cell counts were used to calculate absolute granulocyte counts and absolute lymphocyte counts. Peripheral blood lymphocyte subsets were quantitated with commercially available monoclonal antibodies and analysis with an EPICS V Fluorescent Activated Cell Sorter (Coulter Corp., Hialeah, Fla.). The antibody OKT-3 (Ortho Diagnostic Systems Inc., Raritan, N.J.) or Leu-4 (Becton Dickinson Immunocytometry Systems, Mountain View, Calif.) was used to identify C D 3 + T ceils; O K T - 4 or Leu-3 was used to identify the C D 4 + helper T ceIi subset; OKT-8 or Leu-2 was used to identify the C D 8 + "suppressor" T cell subset; and B1 (Coulter) was used to identify B
cells. In vitro lymphocytic responses to phytohemagglutinin, pokeweed mitogen, tetanus toxoid antigen, and Candida antigen were assayed w i t h a standard microculture technique in which tritiated thymidine was used. Very briefly, aliquots of peripheral blood mononuclear cells, separated by density gradient, were placed in microtiter wells. W e added P H A , P W M , tetanus toxoid antigen, or Candida antige n to triplicate wells and incubated for 3 days (PHA, P W M ) or 6 days (tetanus, Candida). Tritiated thymidine was then added to each well. After 2 4 hours the tritiated thymidine-labeled D N A was extracted and the radioactivity quantitated in a beta scintillation counter as counts per minute per well. For data analysis, L P R s to mitogens an d specific antigens were expressed as the log of the difference between counts per minute in stimulated cell cultures and unstimulated cell cultures (log• Acpm). A t the initial visit, antibody levels to tetanus toxoid antigen and polyribosyl phosphate (the polysaccharide antigen of Haemophilus influenzae type b) were measured with standard enzyme-linked immunosorbent assays. Patients who had nonprotective levels of antibodies to either antigen (<0.10 I U / m l for tetanus toxoid; <100 n g / m l for polyribosyl phosphate) were immunized at the 1-month visit (while receiving biosynthetic G H ) with standard tetanus toxoid (tetanus toxoid absorbed, Wyeth Laboratories Inc., Philadelphia) or Hib vaccine (HibImune, Lederle Labs, Pearl River, N.Y.), respectively. Antibody levels were remeasured at the 3-month visit. A normal response was considered to be an increase in antibody Ievets into the respective protective ranges. Lymphocyt e subset analyses, lymphopr01iferative responses, and antibody levels were measured in the
4 22
Clinical and laboratory observations
The Journal of Pediatrics September 1989
T a b l e II. Mitogen- and antigen-induced lymphoproliferative responses before and during treatment with biosynthetic G H Month of treatment Stimulant
PHA PWM Tetanus toxoid Candida antigen
Baseline
5.09 4.36 3.64 3.99
-+ .30 + .48 _+ 1.41 _+ .61
1
4.70 4.39 4.02 4.02
+ .34* + .45 +_ .91 _+ .56
3
5.03 4.46 4.17 4.29
+ .26 + .25 + .78 _+ .71
6
4.92 4.19 4.43 3.94
+ .43 _+ .58 -+ .43 _+ .71
9
4.86 3.92 4.23 3.82
_ + + _
12
.45* .77 .78 .66
4.75 4.40 4.52 3.94
___ .49 + .16 • .50 _+ .90
Normal laboratory values
>4.70 >4.60 >3.30 >3.30
All values represent group means of iogto A cpm +_ SD. *Values significantlylower than baseline (p < 0.05).
laboratory of Dr. Robertson Parkman, Division of Research Immunology and Bone Marrow Transplantation. Childrens Hospital of Los Angeles. RESULTS Ten patients were followed for 1 year. one for 9 months. one for 6 months, and one for 3 months. O f 13 patients, 12 responded to biosynthetic G H with an increase in growth rate. The mean growth was 8.0 _+ 2.1 cm (mean _ SD); range 4.0 to 11.0 cm) in the 10 patients followed for a full year. One patient with an advanced bone age at the start of treatment did not respond to treatment, which was discontinued after 6 months: one patient decided to interrupt therapy after 3 months. The 13 children had six episodes of infection requiring antibodies and 15 days of fever in 10 episodes of illness. Seven children missed a total of 36 days of school during seven illnesses (six upper respiratory tract infections and one case of infectious mononucleosis syndrome). This last patient, a 15-year-old girl with Noonan syndrome, was given two courses of antibiotics and missed 14 days of school: she was not studied during the illness. N o other patients had an infection with a herpesvirus. The number of days of school missed, 3.3 days per patient per year. was less than the average 11 days per student per year in Los Angeles (Helen Hale, MD, Director, Student Medical Services, Los Angeles Unified School District: personal communication. Dee. 8. 1987). The mean percentages and absolute numbers of circulating granulocytes were not significantly different from baseline at any time measured (data not shown). As indicated in Table I, the percentage of CD3 § cells (T cells) was moderately increased over baseline at 9 and 12 months of G H treatment. Similarly. levels of CD4 + ceils (helper T cells) and CD8 § cells (suppressor T cells) were increased at 12 months. Assessment of absolute numbers of T cell subsets revealed significantly increased numbers of C D 4 cells at 1. 3, and 12 months. The C D 4 + / C D 8 + (helper/ suppressor) cell ratios were never significantly different
from baseline. The C D 4 + / C D 8 + ratios in individual patients remained stable throughout the study. In vitro LPRs to P H A were significantly decreased at 1 and 9 months (Table I!). Nine of 10 patients for whom data were available had a decre~ised P H A L P R 1 month after starting GH. Five of these patients had P H A LPRs below normal laboratory values. A t 3, 6, and 12 months, mean P H A LPRs values were normal. A t 3 months one patient had a persistently low P H A , L P R , and at 6 months decreased P H A LPRs were noted in two of the five patients who had decreased P H A L P R s at 1 month. Mean P W M LPRS were not decreased significantly at any time. Like P H A LPRs, P W M L P R s showed individual variations, but these were not consistently seen at any specific time or in any individual patient. The LPRs to specific antigens, tetanus toxoid and Candida antigen, remained normal throughout the study. Two patients had a decreased tetanus-specific LPRs on one occasion; four other patients had decreased Candidaspecific LPRs on one occasionl The timing of these decreases was variable. Of the 13 patients studied, seven had protective levels of antibodies to tetanus toxoid (>0.1 I U / m l ) and nine had protective levels of antibodies to Hib (>100 ng/ml). Nine patients received 10 immunizations: five tetanus toxoid, three Hib, and one tetanus toxoid plus Hib. Each of the 10 immunizations resulted in a significant antibody response into the normal protective range (data not shown). DISCUSSION Numerous immune-neuroendocrine interactions are likely to h a v e physiologic consequences? For example, Kiess et al. 6 found depressed natural killer cell activity in patients with G H deficiency caused by a hypothalamic deficit of GH-releasing hormone; short-term administration of GH-releasing hormone failed to eliminate this abnormality. Although isolated G H deficiency or dysfunction has not been associated with T cell or B cell deficiency, such immunologic dysfunction has been reported after
Volume 115 Number 3
treatment with pituitary-derivedI and biosynthetic2,3 GH. Rapaport et a12 described suppression of immune functions in eight GH-deficient children treated with pituitaryderived GH. Althoug h erythrocyte, circulating granulocyte, and serum immunoglobulin levels were not affected by GH, helper/suppressor T cell ratio, percentage of circulating B cells, and in vitro mitogen responses were significantly decreased; these abnormalities were generally transient. Bozzola et al. 2 reported similar T cell subset abnormalities without alteration in serum immun0globulin levels in children receiving biosynthetic GH and depressed in vitro IgM production before and after 3 to 6 months of therapy. Most recently, Rapaport et al. 3 described studies in children treated with biosynthesis GH and noted decreased percentages of circulating B ceils and in vitro mitogen responses. In none of these studies was evidence of clinical immune deficiency described. A recent report from Japan described an increased occurrence of leukemia in patients treated with GH. 7 A review of all available data concluded that there may be a small increase in leukemia incidence with GH treatment of GH-deficient childrenS; immune system abnormalities in GH-deficient patients was one of several possible explanations offered for this occurrence. Rogers et al. 9 offered the additional possibility of a direct effect of GH on resting lymphoid cells and subsequent malignant transformation. In contrast, Arslanian et al. t~concluded that GH therapy is probably not associated with an increased rate of brain tumor recurrence. Clayton et al. tl indicated that GH therapy did not increase the relapse rates of medulloblastoma, glioma, and leukemia. In none of the studies cited was an increased incidence of infectious complications identified in GH-treated patients. Our results support the conclusion that G H therapy is not associated with a clinically significant increase in the frequency or severity of infections in GH-deficient children. As previously documented,2 GH therapy does not appear to have an adverse effect on the number of circulating granulocytes. In contrast to previous studies, 1-3 we did not observe consistent Suppression of T cell or B cell functions in any of the systems measured. The percentage and absolute numbers of circulating CD3 + T cells, CD4 + helper T cells, CD8 § suppressor T cells, and B1 + B cells were intermittently increased during treatment with biosynthetic GH; the CD4+/CD8 § ratio remained stable. All measurements of lymphocyte subpopulations were within normal laboratory ranges. The timing of previously reported changes with regard to duration of G H therapy has not been consistent. In our study PHA LPRs were decreased at 1 and 9 months of treatment but did not appear to reflect general suppression of lymphocyte responsiveness because
Clinical and laboratory observations
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PWM and antigen-specific responses remained stable throughout the study. All patients immunized with tetanus toxoid or polyribosyl phosphate generated significant and protective levels of antibodies. The maintenance of antigen-specific responsiveness in our study strongly indicates that clinically significant T and B cell functions are retained by GH-deficient patients during biosynthetic GH therapy, even though four of the patients had received potentially immunosuppressive therapy (cranial irradiation) for a variety of cancers. As suggested by Ammann and Sherman4 in reference to other studies, the variability in our results, including the significantly decreased PHA responses, may reflect the variable nature of immunologic tests .when these are performed sequentially. Alternatively, GH deficiency may be associated with enhanced PHA responsiveness, which is then suppressed after G H replacement. Why this phenomenon would be apparent only with PHA and not with other lymphocyte stimulants remains unclear. REFERENCES
1. Rapaport R. Oleske J. Ahdieh H. Solomon S. Delfaus C, Denny T. Suppression of immune function in growth hormone-deficient children during treatment with human growth hormone. J PEDTATR1986;109:434-9. 2. Bozzola M, Valtorta A, Maghnie M, et at. Influence of biosyntheticgrowth hormone(GH) therapy on the humoral and cell-mediatedimmunity of GH-deficientchildren. Presented at the International Congress on Advances in Growth Hormone and Growth Factor Research, Milan, Sept. 28-30, 1987. 3. Rapaport R, Peterson B, Skuza KA, He!m M, Goldstein S. Biosynthetic methiony!-human growth hormone (BMHGH) treatment affects immune functions (IF) in growth hormonedeficient children. Pediatr Res 1987;21:252A. 4. Ammann AJ, Sherman BM. Effect of growth hormone therapy on immune function. J P~DIATR1987;10:663-4. 5. Besedovsky HO, Del Rey A, Sorkin E. Immune-neuroendocrine interactions. J Immunol 1985;135:750s-4s. 6. Kiess W, Malozowski S, Gelato M, et al. Lymphocyte subset distribution and natural killer activity in growth hormone releasing hormone. Clin Immunol Immunopathol 1988;48:8594. 7. Watanabe S, Tsunematsu Y, Fujimoto J, Komiyama A. Leukaemia in patients treated with growth hormone. Lancet 1988;1:1159. 8. Fisher DA, Job J-C, Preece M, UnderwoodLE. Leukaemia in patients treated with growth hormone. Lancet 1988;1:115960. 9. Rogers PC, Komp D, Rogol A, Sabio H. Possible effects of growth hormone on development of acute lymphoblastic leukaemia. Lancet 1977;2:434-5. 10. Arslanian SA, Becker DJ, Lee PA, Drash AL, Foley TP. Growth hormone therapy and turner recurrence findingsin children with brain neoplasms and hypopituitarism. Am J Dis Child 1985;139:347-50. 11. Clayton PE, Shalet SM, Gattamaneni HR, Price DA. Does growth hormone cause relapse of brain tumors? Lancet 1987;1:711-13.