COMMUNICATIONS IN BRIEF’
This section is suitable for reporting cases, results of therapeutic trials, and descriptions of new procedures or instruments. Reports should be limited to seven hundred words and two references. Use of an illustration or table requires a proportionate reduction in total words.
Table I. T and B cell determinations at birth and at six weeks in four erythroblastotic infants whose mothers were treated with promethazine hydrochloride
The effect of promethazine hydrochloride on fetaJ and maternal lymphocytes JOHN
P.
GUSDON,
JR.,
HERBST,
M.D.
GLENN
A.
M.A.
Department of Medicine Carolina
of Obstetrics and Gynecoloa, of Wahe Forest University,
At birth Bowman Gray School Winston-Salem, North
T 7 * Mean
has been involved in the study of promethazine hydrochloride and its use in erythroblastosis for many years. Our interest in this drug was originally aroused by the report by Bierme-Alie-Enjalbert’ of a possible amelioration of the effects of erythroblastosis in 1 I patients treated with that drug during pregnancy. The possibility that its ability to ameliorate this immunologically mediated disease by an immunosuppressive capability was studied by us in patients and in the laboratory. Our most recent publication reviews our prior work and documents the in vitro inhibition, by promethazine hydrochloride, of the ability of macrophages of the newborn infant to form rosettes with anti-D opsonized red blood cells.’ We have previously shown that fetal red blood cell destruction in erythroblastosis caused by the Rh antigen was not accomplished directly by the antibody and that the anti-D was not complement-fixing. In addition, we have also documented the ability of promethazine hydrochloride to inhibit lymphocyte-mediated functions in vivo in animals. The first paper we published concerning the effectiveness of promethazine hydrochloride in the treatment of erythroblastosis involved only 15 patients. A subsequent paper involving more than 40 patients is in preparation. However, we have maintained a concern regarding the effects which promethazine hydroTHIS
LABORATORY
Reprint requests: School of Medicine Salem, North Carolina
Dr. John of Wake 27103.
Six weeks
l1
B
T
B
6 ?c 1.5
41 * 5.1
14 k 4.8
? 1 standard
deviation.
chloride might have on the mother and child. The action of promethazine hydrochloride in ameliorating the effects of erythroblatosis is probably not solely due to its immunosuppressive activities. However, the documented immunosuppressive activity of this drug engenders some concern over the long-term effects of administration of such a drug to both the mother and fetus. Therefore, we have studied some aspects of immunologic function in both the mother and baby at the time of birth and later. We have studied T and B lymphocytes in pregnancies complicated by erythroblastosis. All studies were conducted on patients at the North Carolina Baptist Hospital, Winston-Salem, North Carolina. Lymphocyte preparations and T and B cell determinations were performed according to methods outlined at a World Health Organization workshop.3 A modification 01 those methods was used only where indicated. The results of T and B cell determinations done on the cord blood of erythroblastototic infants are presented in Table I. Using the standard methods of lymphocyte separation, we found an apparently significant suppression of T and B cells at birth in these infants. However, in the process of gradient separation of the lymphocytes from the cord blood samples, we noticed that the cell layer which collected at the Ficollhypaque interface was red. This was in distinct contrast to the color of lymphocyte preparations made from
P. Gusdon, Bowman Gray Forest University, Winston-
730
Communications
blood collected from their respective mothers at the time of delivery. Those preparations were always white. We studied these cells carefully and found a significant contamination of the lymphocytes by nucleated red ceils. When blood from these children was again studied six to eight weeks later with the same methods, we found that the T and B cells had apparently returned to normal. Smears had been made for differential analysis from the cord blood of those erythroblastotic children. We found a range of 89 to 120 nucleated red cells per 100 white cells in those infants. By six weeks of age, the reticulocyte count in these inf-ants had returned to normal levels of 4 per cent or less. coincident with the elevation to apparently normal levels of the T and B cells. It was not until the fifth erythroblastotic infant that we finally found an effective technique by which we could remove the nucleated red cells from the lymphocyte preparations without negativeIy affecting the lymphocytes. We found that the simple modification of adding 2 ml, of distilled water to the lymphocyte pellet, dispersing the cells for twenty seconds by pipetting, and then immediatelv adding a salt solution of sufficient strength to biing the molarity of the solution to O.l.?hi resulted in lysis of the nucleated red cells only, with retained functional lymphocytes. Cord blood was obtained at the time of delivery of this erythroblastotic infant, and the lymphocytes from the Ficollhypdque gradient interface were separated into two halves. Half of these cells were processed in the usual fashion, and half were water lysed. T and B cell determinations were then accomplished in the standard fashion on both samples. The sampIe that was studied without removal of. nucleated red blood cells was read as being 6 per cent T cells and 4 per cent B cells while t.he other aliquot gave 41 per cent T cells and 15 per cent B cells. All treated mothers who were studied (five) were fomld to have normal adult levels of both T and B cells (54 ? 6 r’s and 15 ? 3 B’s). There was no apparent effect of the promethazine hydrochloride on maternal lymphocyte numbers. 0ur first impression that the low levels of T and B cells found in the erythroblastotic newborn infant might be clue to the promethazine hydrochloride treatment was erroneous. When contaminating nucleated red cells were removed from the lymphocyte preparation, the levels of T and B cells in the patients studied so far appeared to be normal. In a recvnt paper by Rubinstein and associates,’ the authors have taken d situation with two variables. the disease pj-ocess erythrobiastosis and the treatment promethazine hydrochloride. and, we believe, erroneously attributed the low levels of T and B lymphocytes to the treatment modality.4 It appears that the disease process and the infants’ response to it rather than the treatment are really at fault. However, we are continuing to study the patients we have treated.
in brief
731
REFERENCES
Bierme- Alie-Enjalbert, S.: Les formes graveh %I utero” de la maladie hemohtique perinatale par incompatabilite rhesus, Thesis, Toulouse, 1967, Centrt: Regional de Transfusion Sanguine et &Hematologie. Gusdon. 1. P.. Ir.. Caudle. M. R.. Herbst. G. A.. and IannuzLi, N. P.: Phagocytosis and erythroblas~osis. I. Modification of the neonatal response by promethaxine hydrochloride, AM. J. OBSTET. GYNECOL. 1% 224, 1976. Aiuti, F., et al.: IULS report of a WHO sponsored workshop on identification, enumeration and isolation OFB and T lymphocytes from human peripheral blc>od, Clin. Immunol. Immunopathol. 3: 584, 1975. Rubinstein A., eidelman, A. I., Melamed, J., Gartner, L M., Kandall, S.. and Schulman, H.: Possible effect uf maternal promethazine therapy on neonatal in1m~~nolo+c f\lnclions, J. Pediatr. 89: 136, 1976.
Hormonal evaluation of premature rnenarche produced by a follicular cyst RALPH G. WlELAND, M.D RAFAEL BENDEZG, M.D. M.dRVlN C, HALLBERG. %l.S. PETER TANG, M.D. KENNETH WEBSTER, M.D.
ov A RK A N follicular cysts have been reported as a cause of precocious pseudopuberty.’ The autonomicity of the tumor has been a matter of debate, mainly because of persistence or recurrence of the precocity after puncture of the cyst and in very rare instances after removal~ Sensitive assay techniques for gonadotropins and steroid hormones now make it possible to delineate the hormonal profile of these individuals. We have had the opportunity to study a case of precocious pseudopuberty associated with a follicular cyst. Serial determinations of serum gonadotropins, estradiol, and testosterone h the peripheral venous blood and of estradiol in the ovarian vein and cystic fluid for the first time confirm the impression of autonomous estrogen production. The patient, a 4-2/12-year-old girl, was admitted because of moderate vaginal bleeding of 1 week’s duration. Two days before admission the mother had noticed slight enlargement of the nipples. The patient was the product of an 8 month twin pregnancy. She and her twin brother had previously normal development. On examination slight nipple enlargement was noted. No raf( au lait spots were observed. The labia majwa were prominent. The clitoris was norn~al. The rugae Supported in part by a General Research %pport Grant from the Xational Institutes of Heal& Reprint requests: Dr. Ralph G, Wieland. Saint Luke’s Hospital, Cleveland, Ohio 44104.
This section is suitable for reporting cases, results of therapeutic trials, and descriptions of new procedures or instruments. Reports should be limited to seven hundred words and two references. Use of an illustration or table requires a proportionate reduction in total words.
Table I. T and B cell determinations at birth and at six weeks in four erythroblastotic infants whose mothers were treated with promethazine hydrochloride
The effect of promethazine hydrochloride on fetaJ and maternal lymphocytes JOHN
P.
GUSDON,
JR.,
HERBST,
M.D.
GLENN
A.
M.A.
Department of Medicine Carolina
of Obstetrics and Gynecoloa, of Wahe Forest University,
At birth Bowman Gray School Winston-Salem, North
T 7 * Mean
has been involved in the study of promethazine hydrochloride and its use in erythroblastosis for many years. Our interest in this drug was originally aroused by the report by Bierme-Alie-Enjalbert’ of a possible amelioration of the effects of erythroblastosis in 1 I patients treated with that drug during pregnancy. The possibility that its ability to ameliorate this immunologically mediated disease by an immunosuppressive capability was studied by us in patients and in the laboratory. Our most recent publication reviews our prior work and documents the in vitro inhibition, by promethazine hydrochloride, of the ability of macrophages of the newborn infant to form rosettes with anti-D opsonized red blood cells.’ We have previously shown that fetal red blood cell destruction in erythroblastosis caused by the Rh antigen was not accomplished directly by the antibody and that the anti-D was not complement-fixing. In addition, we have also documented the ability of promethazine hydrochloride to inhibit lymphocyte-mediated functions in vivo in animals. The first paper we published concerning the effectiveness of promethazine hydrochloride in the treatment of erythroblastosis involved only 15 patients. A subsequent paper involving more than 40 patients is in preparation. However, we have maintained a concern regarding the effects which promethazine hydroTHIS
LABORATORY
Reprint requests: School of Medicine Salem, North Carolina
Dr. John of Wake 27103.
Six weeks
l1
B
T
B
6 ?c 1.5
41 * 5.1
14 k 4.8
? 1 standard
deviation.
chloride might have on the mother and child. The action of promethazine hydrochloride in ameliorating the effects of erythroblatosis is probably not solely due to its immunosuppressive activities. However, the documented immunosuppressive activity of this drug engenders some concern over the long-term effects of administration of such a drug to both the mother and fetus. Therefore, we have studied some aspects of immunologic function in both the mother and baby at the time of birth and later. We have studied T and B lymphocytes in pregnancies complicated by erythroblastosis. All studies were conducted on patients at the North Carolina Baptist Hospital, Winston-Salem, North Carolina. Lymphocyte preparations and T and B cell determinations were performed according to methods outlined at a World Health Organization workshop.3 A modification 01 those methods was used only where indicated. The results of T and B cell determinations done on the cord blood of erythroblastototic infants are presented in Table I. Using the standard methods of lymphocyte separation, we found an apparently significant suppression of T and B cells at birth in these infants. However, in the process of gradient separation of the lymphocytes from the cord blood samples, we noticed that the cell layer which collected at the Ficollhypaque interface was red. This was in distinct contrast to the color of lymphocyte preparations made from
P. Gusdon, Bowman Gray Forest University, Winston-
730
Communications
blood collected from their respective mothers at the time of delivery. Those preparations were always white. We studied these cells carefully and found a significant contamination of the lymphocytes by nucleated red ceils. When blood from these children was again studied six to eight weeks later with the same methods, we found that the T and B cells had apparently returned to normal. Smears had been made for differential analysis from the cord blood of those erythroblastotic children. We found a range of 89 to 120 nucleated red cells per 100 white cells in those infants. By six weeks of age, the reticulocyte count in these inf-ants had returned to normal levels of 4 per cent or less. coincident with the elevation to apparently normal levels of the T and B cells. It was not until the fifth erythroblastotic infant that we finally found an effective technique by which we could remove the nucleated red cells from the lymphocyte preparations without negativeIy affecting the lymphocytes. We found that the simple modification of adding 2 ml, of distilled water to the lymphocyte pellet, dispersing the cells for twenty seconds by pipetting, and then immediatelv adding a salt solution of sufficient strength to biing the molarity of the solution to O.l.?hi resulted in lysis of the nucleated red cells only, with retained functional lymphocytes. Cord blood was obtained at the time of delivery of this erythroblastotic infant, and the lymphocytes from the Ficollhypdque gradient interface were separated into two halves. Half of these cells were processed in the usual fashion, and half were water lysed. T and B cell determinations were then accomplished in the standard fashion on both samples. The sampIe that was studied without removal of. nucleated red blood cells was read as being 6 per cent T cells and 4 per cent B cells while t.he other aliquot gave 41 per cent T cells and 15 per cent B cells. All treated mothers who were studied (five) were fomld to have normal adult levels of both T and B cells (54 ? 6 r’s and 15 ? 3 B’s). There was no apparent effect of the promethazine hydrochloride on maternal lymphocyte numbers. 0ur first impression that the low levels of T and B cells found in the erythroblastotic newborn infant might be clue to the promethazine hydrochloride treatment was erroneous. When contaminating nucleated red cells were removed from the lymphocyte preparation, the levels of T and B cells in the patients studied so far appeared to be normal. In a recvnt paper by Rubinstein and associates,’ the authors have taken d situation with two variables. the disease pj-ocess erythrobiastosis and the treatment promethazine hydrochloride. and, we believe, erroneously attributed the low levels of T and B lymphocytes to the treatment modality.4 It appears that the disease process and the infants’ response to it rather than the treatment are really at fault. However, we are continuing to study the patients we have treated.
in brief
731
REFERENCES
Bierme- Alie-Enjalbert, S.: Les formes graveh %I utero” de la maladie hemohtique perinatale par incompatabilite rhesus, Thesis, Toulouse, 1967, Centrt: Regional de Transfusion Sanguine et &Hematologie. Gusdon. 1. P.. Ir.. Caudle. M. R.. Herbst. G. A.. and IannuzLi, N. P.: Phagocytosis and erythroblas~osis. I. Modification of the neonatal response by promethaxine hydrochloride, AM. J. OBSTET. GYNECOL. 1% 224, 1976. Aiuti, F., et al.: IULS report of a WHO sponsored workshop on identification, enumeration and isolation OFB and T lymphocytes from human peripheral blc>od, Clin. Immunol. Immunopathol. 3: 584, 1975. Rubinstein A., eidelman, A. I., Melamed, J., Gartner, L M., Kandall, S.. and Schulman, H.: Possible effect uf maternal promethazine therapy on neonatal in1m~~nolo+c f\lnclions, J. Pediatr. 89: 136, 1976.
Hormonal evaluation of premature rnenarche produced by a follicular cyst RALPH G. WlELAND, M.D RAFAEL BENDEZG, M.D. M.dRVlN C, HALLBERG. %l.S. PETER TANG, M.D. KENNETH WEBSTER, M.D.
ov A RK A N follicular cysts have been reported as a cause of precocious pseudopuberty.’ The autonomicity of the tumor has been a matter of debate, mainly because of persistence or recurrence of the precocity after puncture of the cyst and in very rare instances after removal~ Sensitive assay techniques for gonadotropins and steroid hormones now make it possible to delineate the hormonal profile of these individuals. We have had the opportunity to study a case of precocious pseudopuberty associated with a follicular cyst. Serial determinations of serum gonadotropins, estradiol, and testosterone h the peripheral venous blood and of estradiol in the ovarian vein and cystic fluid for the first time confirm the impression of autonomous estrogen production. The patient, a 4-2/12-year-old girl, was admitted because of moderate vaginal bleeding of 1 week’s duration. Two days before admission the mother had noticed slight enlargement of the nipples. The patient was the product of an 8 month twin pregnancy. She and her twin brother had previously normal development. On examination slight nipple enlargement was noted. No raf( au lait spots were observed. The labia majwa were prominent. The clitoris was norn~al. The rugae Supported in part by a General Research %pport Grant from the Xational Institutes of Heal& Reprint requests: Dr. Ralph G, Wieland. Saint Luke’s Hospital, Cleveland, Ohio 44104.
