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Serum bacterial opsonins and polymorphonuclear leukocytes in mothers and newborns
Volume 74 Number 5
DR. COEN. The cell pellet was analyzed on a hemocytometer, and as best we could determine these were neutrophils rather than eosinophils. The number of eosinophils was v e r y low in the differential count. De. KENNY, Pittsburgh. I wonder if you studied hexose monophosphate shunt activity in the white blood cells of the infants under 12 hours of age with and without antibiotics to see if shunt activity was affected by the antibiotic. DR. COEN. No, we did not. DR. TAYLOR, Pittsburgh. Is there a possibility of a humoral activator or inhibitor? Have you studied babies' white blood cells incubated with adult plasma or adult cells incubated with babies' plasma ? DR. COEN. Yes, we have and this seems to be a cellular rather than a plasma phenomenon. DR. BAUBLIS, Ann Arbor. Pretreatment of cells with steroids can stabilize the lysosome, and I guess if you have shown degranulation, it may be that the lysosomes are still rupturing, I wonder whether an interval of pretreatment or a period during which the lysosomcs of the cells are exposed to levels of steroids higher than in the adult might be incriminated here ? DR. COEN. We haven't looked into that phase of it, and I would not care to speculate on it. DR. FOllMAN, Madison. I wonder if you could tell me again what the findings were with the C 14 production. DR. COEN. The ratio of C1402 production of active/resting leukocytes obtained from the older infants ranged up to the level of 2 or 3, whereas the infants that were less than 12 hours of age had levels around 0.8. DR, FORMAN. On your second slide, when you were comparing your control subjects with antibiotics and the newborn infants with antibiotics, you use a log graph. I think the difference between the babies with antibiotics and the normal control subjects with antibiotics figures ought to be about 0.1 per cent difference in total killing of the initial counts. Do you feet this is a reproducible enough system to make this statement on a 0.1 per cent difference ? DR. COEN. There is a tenfold difference. DR. FORMAN. But it is only 0.1 per cent of the initial bacterial count. This is a biological system, and I wonder if this very small difference is significant in a biological system when there are a lot of judgments that have to be made in trying to differentiate white blood cells and measuring bacteria by Coleman spectrophotometry. DR. COEN. Since the antibiotics destroy all extraeellular organisms, the results are based entirely on the number of viable intracellular organisms. Although this number is small in relation to the initial bacterial count, the differences between experimental and control cells are nevertheless significant and reproducible. DR. CHERNIGK,Winnipeg. How specific is the system for Staphylococcus aureus? Do you see differences with E. coli for instance? DR. CogN. We have not as yet studied E. coli.
Abstracts
8 17
8. Serum bacterial opsonlns and polymorphonuclear leukocytes in mothers and newborns J. H. Dossett and P. G. Quie, Minneapolis, Minn. The bactericidal capacity of whole blood, polymorphonuclear leukocyte function, and serum opsonic titers were measured in 60 normal newborn infants a n d their mothers. Fresh whole blood of mothers killed 98 per cent of inoculated E. coli in 2 hours; blood of newborn infants killed 30 to 93 per cent. This difference was not noted with Staphylococcus aureus or group B streptococcus. Separated PMN's from 18 mothers and newborn infants incubated with pooled adult serum as opsonin demonstrated normal phagocytosis and killing. Serum opsonie titers were determined on fresh whole sera in an in vitro phagocytic system. Geometric mean opsonic titers for E. coli were 46.7 in mothers and 4.3 in newborn infants. There was no difference between mothers' and newborn infants' opsonic titers for S. aureus or group B streptococcus. Heat inactivation (56 ~ C., 30 rain.) abolished opsonic activity in ~ of the mothers' sera and decreased the titer to 1:5 in the others. Heated sera of newborn infants had no opsonic activity. Maternal 19S and 7S fractions had no opsonic activity for E. coll. Addition of a complement source to the system enhanced the opsonic potential of the 19S and 7S fractions. In the presence of complement, 19S fractions were fonnd to be 50 to I00 times more effective as opsonins for E. coli than 7S. Deficient bactericidal capacity of blood of newborn infants and the low opsonic titer of serum of newborn infants for E. coli may be related to low levels of 19S antibodies at birth. Moreover, it appears that complement is necessary to manifest the opsonic potential of 19S antibody. DISCUSSION DR. BAUBLIS, A n n Arbor. I think the paper raises several questions very basic to immunology: whether natural antibodies or probodies exist and what their role together with complement is in producing opsonization and bactericidal effects. The results you showed suggest that cord serum has about ~0 of the bactericidal capacity of the maternal serum. It is interesting that this is similar to IgM values. The question of the natural antibody arises because the bulk of this macroglobulin is actually produced by the fetus. So, the question is: Is this an IgM response to some antigen, or is this perhaps the p h a n t o m - - t h e natural antibody or probody ? DR. DOSSETT. Drs. Michael (J. Exper Med. 118: 619, 1963) and Gitlin (Pediatrics 31: 197, 1963) have studied this rather extensively. They found that the extracellular bactericidal factors, for bacteria susceptible to these factors, were also deficient in newborn infants. They also have located these so-called natural antibodies to the
8 1 8 Abstracts
macroglobulin and 193 fraction; for gram-negative bacteria, that is. DR. NAGI-IMAN, Columbus. Do you find any difference between sexes? DR. DOSSETT. We have not tested them. DR. KAUDER, Cincinnati. Since Dr. Coen's data indicated a rapidly changing difference in the intracellular function of cells with age, at what age did you sample the blood in your patients? DR. DOSSETT. All of our babies were tested after birth. These determinations were done on babies between 1 and 3 days of age, and most were done in the first 2 days of life. DR. REINER, Columbus. Would it be out of place to straighten out the misconception in the previous paper about the 0.1 per cent? DR. DOSSETT. I would be glad to speak of it. DR. REINER. It was 900 intracellular organisms, not 10 killed out of 1,000: This is a log scale. DR. DOSSETT. Maybe I can demonstrate what Dr. Forman was talking about. Dr. Coen started with 1 million organisms (106). To lower this one log tO l0 s is 90 per cent killing, and to lower it 2 logs is 99 per cent, and lowering it another one goes even beyond this. We are dealing with very small differences down here. The percentage relative to the beginning inoculum is 0.1 per cent. DR. KAUDER. May I comment on the same point? In the system Dr. Coen used, he was focusing attention on the intracellular organisms by the addition of antibiotics. This represents a very small percentage of the total number of organisms listed. So, if one looks only at the percent difference as represented by intracellular versus total number of organisms, this tends to obscure the intracellular difference that occurs. There is still a tenfold difference in what is occurring inside the cell in terms of killing. I wonder what the coefficient of variation of the count is. DR. DOSSETT. I would say 5 per cent.
9. Rhinovirus injections in nursery school children Mare Beem, Chicago0 Ill. The occurrence of virus infection and acute respiratory illness was determined in 3- to 5-yearold children attending a day care nursery school. Over a 41 week period between November 5, 1962, and August 16, 1963, a group ranging in number from 16 to 24 provided 5,639 child days of observation. Three hundred and twenty-one respiratory illnesses were observed (5.7 per 100 child days, 21.8 per child year). The significance of this exceptionally high rate of illness will be discussed. Seventy-three virus-shedding infections were identified (rhino -~ 46, RS /12, para-influenza ~--- 2, influenza = 2, adeno ~ 5, entero = 4, herpes = 2). The 46 rhinoviruses exceeded the total of all other virus infections identified. They occurred in a near perennial pattern and were significantly related to illness. They were caused by a total of 14 different serotypes. Although 3 types spread extensively through the group and 4 others infected more than one child, with 7
The Journal o[ Pediatrics May 1969
infection appeared limited to a single child. These observations will be discussed in relation to the relative importance of rhinoviruses in acute respiratory illnesses of children and the polytypic epidemiology of these viruses. DISCUSSION DR. STARR, Cleveland. I was intetested in the difference in severity between summer and winter respiratory infection. Is it possible that this might be explained by parental attitudes about sending a child to school? I frequently" observe that mothers tend not to send a child to school during the winter, whereas with an illness of the same severity they would send the child to school during the summer. DR. BEgN. It is a possibility, and something we could not control for completely, but we did notice this seasonal difference when we compared the incidence of illnesses that lasted 3 or more days and did not require absence from school. DR. BAUBLIS, Ann Arbor. What was the frequency with which you isolated the rhinovirus, particularly from well children? In other words, do you have any idea how frequently infection is followed by disease ? DR. BEEN. We were doing a longitudinal study, culturing each child once a week, with additional culturing at times. As discussed, there were significantly more days of illness in the week following than that preceding the isolation of virus. The rhinovirus isolation rate for weeks during which there was cultural evidence of rhinovirus in the school was 11 per cent for specimens taken from well children and 26 per cent for those from children with respiratory illness. DR. BAUBLIS. What is the duration of shedding ? DR. BEEN. As long as 2 weeks. DR. 1V[OFFET, Chicago. Were these nasal or throat swabbings? How often have you found these acid-labeled viruses in rectal swabbings? Did you check the teachers at all, and was polio virus isolated ? DR. BEEN. The polio was a vaccine strain. The teachers opted out of regular culturing. The cultures were primarily nasal. The children didn't tolerate repeated pharyngeal cultures. We have done many rectal swabs, with negative results, on children from whom we have recovered rhinovirus from the respiratory tract. DR. 1V[OFFET. What was the difference between throat and nasal cultures for frequency of isolation ? DR. BEEN. I don't know it from this study, but other studies indicate a higher isolation rate for nasal specimens.
10. Methotrexate liver toxicity H. Sharp, M. Nesbit, J. White, and W. Krivit, Minneapolis, Minn. be
The liver toxicity of methotrexate continues to grossly underestimated. Ten children were
DR. COEN. The cell pellet was analyzed on a hemocytometer, and as best we could determine these were neutrophils rather than eosinophils. The number of eosinophils was v e r y low in the differential count. De. KENNY, Pittsburgh. I wonder if you studied hexose monophosphate shunt activity in the white blood cells of the infants under 12 hours of age with and without antibiotics to see if shunt activity was affected by the antibiotic. DR. COEN. No, we did not. DR. TAYLOR, Pittsburgh. Is there a possibility of a humoral activator or inhibitor? Have you studied babies' white blood cells incubated with adult plasma or adult cells incubated with babies' plasma ? DR. COEN. Yes, we have and this seems to be a cellular rather than a plasma phenomenon. DR. BAUBLIS, Ann Arbor. Pretreatment of cells with steroids can stabilize the lysosome, and I guess if you have shown degranulation, it may be that the lysosomes are still rupturing, I wonder whether an interval of pretreatment or a period during which the lysosomcs of the cells are exposed to levels of steroids higher than in the adult might be incriminated here ? DR. COEN. We haven't looked into that phase of it, and I would not care to speculate on it. DR. FOllMAN, Madison. I wonder if you could tell me again what the findings were with the C 14 production. DR. COEN. The ratio of C1402 production of active/resting leukocytes obtained from the older infants ranged up to the level of 2 or 3, whereas the infants that were less than 12 hours of age had levels around 0.8. DR, FORMAN. On your second slide, when you were comparing your control subjects with antibiotics and the newborn infants with antibiotics, you use a log graph. I think the difference between the babies with antibiotics and the normal control subjects with antibiotics figures ought to be about 0.1 per cent difference in total killing of the initial counts. Do you feet this is a reproducible enough system to make this statement on a 0.1 per cent difference ? DR. COEN. There is a tenfold difference. DR. FORMAN. But it is only 0.1 per cent of the initial bacterial count. This is a biological system, and I wonder if this very small difference is significant in a biological system when there are a lot of judgments that have to be made in trying to differentiate white blood cells and measuring bacteria by Coleman spectrophotometry. DR. COEN. Since the antibiotics destroy all extraeellular organisms, the results are based entirely on the number of viable intracellular organisms. Although this number is small in relation to the initial bacterial count, the differences between experimental and control cells are nevertheless significant and reproducible. DR. CHERNIGK,Winnipeg. How specific is the system for Staphylococcus aureus? Do you see differences with E. coli for instance? DR. CogN. We have not as yet studied E. coli.
Abstracts
8 17
8. Serum bacterial opsonlns and polymorphonuclear leukocytes in mothers and newborns J. H. Dossett and P. G. Quie, Minneapolis, Minn. The bactericidal capacity of whole blood, polymorphonuclear leukocyte function, and serum opsonic titers were measured in 60 normal newborn infants a n d their mothers. Fresh whole blood of mothers killed 98 per cent of inoculated E. coli in 2 hours; blood of newborn infants killed 30 to 93 per cent. This difference was not noted with Staphylococcus aureus or group B streptococcus. Separated PMN's from 18 mothers and newborn infants incubated with pooled adult serum as opsonin demonstrated normal phagocytosis and killing. Serum opsonie titers were determined on fresh whole sera in an in vitro phagocytic system. Geometric mean opsonic titers for E. coli were 46.7 in mothers and 4.3 in newborn infants. There was no difference between mothers' and newborn infants' opsonic titers for S. aureus or group B streptococcus. Heat inactivation (56 ~ C., 30 rain.) abolished opsonic activity in ~ of the mothers' sera and decreased the titer to 1:5 in the others. Heated sera of newborn infants had no opsonic activity. Maternal 19S and 7S fractions had no opsonic activity for E. coll. Addition of a complement source to the system enhanced the opsonic potential of the 19S and 7S fractions. In the presence of complement, 19S fractions were fonnd to be 50 to I00 times more effective as opsonins for E. coli than 7S. Deficient bactericidal capacity of blood of newborn infants and the low opsonic titer of serum of newborn infants for E. coli may be related to low levels of 19S antibodies at birth. Moreover, it appears that complement is necessary to manifest the opsonic potential of 19S antibody. DISCUSSION DR. BAUBLIS, A n n Arbor. I think the paper raises several questions very basic to immunology: whether natural antibodies or probodies exist and what their role together with complement is in producing opsonization and bactericidal effects. The results you showed suggest that cord serum has about ~0 of the bactericidal capacity of the maternal serum. It is interesting that this is similar to IgM values. The question of the natural antibody arises because the bulk of this macroglobulin is actually produced by the fetus. So, the question is: Is this an IgM response to some antigen, or is this perhaps the p h a n t o m - - t h e natural antibody or probody ? DR. DOSSETT. Drs. Michael (J. Exper Med. 118: 619, 1963) and Gitlin (Pediatrics 31: 197, 1963) have studied this rather extensively. They found that the extracellular bactericidal factors, for bacteria susceptible to these factors, were also deficient in newborn infants. They also have located these so-called natural antibodies to the
8 1 8 Abstracts
macroglobulin and 193 fraction; for gram-negative bacteria, that is. DR. NAGI-IMAN, Columbus. Do you find any difference between sexes? DR. DOSSETT. We have not tested them. DR. KAUDER, Cincinnati. Since Dr. Coen's data indicated a rapidly changing difference in the intracellular function of cells with age, at what age did you sample the blood in your patients? DR. DOSSETT. All of our babies were tested after birth. These determinations were done on babies between 1 and 3 days of age, and most were done in the first 2 days of life. DR. REINER, Columbus. Would it be out of place to straighten out the misconception in the previous paper about the 0.1 per cent? DR. DOSSETT. I would be glad to speak of it. DR. REINER. It was 900 intracellular organisms, not 10 killed out of 1,000: This is a log scale. DR. DOSSETT. Maybe I can demonstrate what Dr. Forman was talking about. Dr. Coen started with 1 million organisms (106). To lower this one log tO l0 s is 90 per cent killing, and to lower it 2 logs is 99 per cent, and lowering it another one goes even beyond this. We are dealing with very small differences down here. The percentage relative to the beginning inoculum is 0.1 per cent. DR. KAUDER. May I comment on the same point? In the system Dr. Coen used, he was focusing attention on the intracellular organisms by the addition of antibiotics. This represents a very small percentage of the total number of organisms listed. So, if one looks only at the percent difference as represented by intracellular versus total number of organisms, this tends to obscure the intracellular difference that occurs. There is still a tenfold difference in what is occurring inside the cell in terms of killing. I wonder what the coefficient of variation of the count is. DR. DOSSETT. I would say 5 per cent.
9. Rhinovirus injections in nursery school children Mare Beem, Chicago0 Ill. The occurrence of virus infection and acute respiratory illness was determined in 3- to 5-yearold children attending a day care nursery school. Over a 41 week period between November 5, 1962, and August 16, 1963, a group ranging in number from 16 to 24 provided 5,639 child days of observation. Three hundred and twenty-one respiratory illnesses were observed (5.7 per 100 child days, 21.8 per child year). The significance of this exceptionally high rate of illness will be discussed. Seventy-three virus-shedding infections were identified (rhino -~ 46, RS /12, para-influenza ~--- 2, influenza = 2, adeno ~ 5, entero = 4, herpes = 2). The 46 rhinoviruses exceeded the total of all other virus infections identified. They occurred in a near perennial pattern and were significantly related to illness. They were caused by a total of 14 different serotypes. Although 3 types spread extensively through the group and 4 others infected more than one child, with 7
The Journal o[ Pediatrics May 1969
infection appeared limited to a single child. These observations will be discussed in relation to the relative importance of rhinoviruses in acute respiratory illnesses of children and the polytypic epidemiology of these viruses. DISCUSSION DR. STARR, Cleveland. I was intetested in the difference in severity between summer and winter respiratory infection. Is it possible that this might be explained by parental attitudes about sending a child to school? I frequently" observe that mothers tend not to send a child to school during the winter, whereas with an illness of the same severity they would send the child to school during the summer. DR. BEgN. It is a possibility, and something we could not control for completely, but we did notice this seasonal difference when we compared the incidence of illnesses that lasted 3 or more days and did not require absence from school. DR. BAUBLIS, Ann Arbor. What was the frequency with which you isolated the rhinovirus, particularly from well children? In other words, do you have any idea how frequently infection is followed by disease ? DR. BEEN. We were doing a longitudinal study, culturing each child once a week, with additional culturing at times. As discussed, there were significantly more days of illness in the week following than that preceding the isolation of virus. The rhinovirus isolation rate for weeks during which there was cultural evidence of rhinovirus in the school was 11 per cent for specimens taken from well children and 26 per cent for those from children with respiratory illness. DR. BAUBLIS. What is the duration of shedding ? DR. BEEN. As long as 2 weeks. DR. 1V[OFFET, Chicago. Were these nasal or throat swabbings? How often have you found these acid-labeled viruses in rectal swabbings? Did you check the teachers at all, and was polio virus isolated ? DR. BEEN. The polio was a vaccine strain. The teachers opted out of regular culturing. The cultures were primarily nasal. The children didn't tolerate repeated pharyngeal cultures. We have done many rectal swabs, with negative results, on children from whom we have recovered rhinovirus from the respiratory tract. DR. 1V[OFFET. What was the difference between throat and nasal cultures for frequency of isolation ? DR. BEEN. I don't know it from this study, but other studies indicate a higher isolation rate for nasal specimens.
10. Methotrexate liver toxicity H. Sharp, M. Nesbit, J. White, and W. Krivit, Minneapolis, Minn. be
The liver toxicity of methotrexate continues to grossly underestimated. Ten children were