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CHANGES INDUCED BY TUBERCULIN IN LEUCOCYTE CULTURES
1091 DEATHS NOT ASSOCIATED WITH POSTOPERATIVE HEMORRHAGE (25 CASES)
Errors of Surgical or Nursing Care (4 cases) Case 12.-A boy, aged 6 years, was restless after operation. He was given 5 minims of nepenthe, which was repeated three and a half hours later. The restlessness was now controlled and the child settled down to sleep. Three-quarters of an hour later he was found dead in bed. At necropsy no abnormality
Attributable
was
to
found. ATTRIBUTABLE TO ERRORS
OF ANESTHESIA
(21 CASES) During Induction (4 cases), during Operation (9 cases), or during Recovery (8 cases) Case 13.-A boy, aged 8 years, was given ethyl chloride and ether on a mask. After twenty minutes, and before the operation had been started, there was sudden cardiorespiratory arrest. With artificial respiration and cardiac massage the heart-beat returned, but the child died six hours later without having regained consciousness. The operation was not
performed. Case 14.-A boy, aged 5 years, was anaathetised with nitrous oxide and ether through a Boyle-Davis gag. When the operation was started, anaesthesia was found to be too light and 100 mg. of thiopentone was given. Later the boy was given another 50 mg. of thiopentone. Respiration stopped, and the child became cyanosed. The gag was removed, an endotracheal tube was introduced, and the lungs were inflated with oxygen. The expiratory valve was tightly shut, and widespread surgical emphysema immediately followed. The patient died. Case 15.-A girl, aged 6 years, was returned to the ward five minutes after the operation had been completed. A few minutes later respiration became obstructed and ceased. The anaesthetist was sent for, but resuscitation was only partly successful, and the child died twelve hours later without having regained consciousness. Comment
The suggestions contained in this report are offered with considerable reserve. Most of the data were obtained long after the events, and in many cases were extremely Nevertheless, certain pointers emerged that scanty. could be helpful. The mortality, though not to be accepted with complacency, is not catastrophic: out of 200,000 patients a year who undergo the operation in this country, about 20 lose their lives through it. A common source of error seems to be undue delay in giving effective treatment for the control of hxmorrhage. To give nepenthe, or other opiate, in the hope that the patient will settle down and stop bleeding, is not only disappointingly ineffective but also highly dangerous. Even without hxmorrhage and in a normal clinical dose, nepenthe, when given on top of an anxsthetic and other depressant drugs, can cause death from central depression and obstruction of the airway. In the presence of hxmorrhage there is the added danger of blood being aspirated. The restlessness for which the nepenthe is given, if not a sequel of barbiturate premedication (which is best avoided in tonsillectomy cases) may be a sign of hxmorrhage, which the nepenthe may help to conceal. In a small child the loss of less than a pint of blood may be fatal, and more blood may have been swallowed than is seen on the pillow. Propping up the patient to arrest hxmorrhage is hazardous. But this continues to be taught. The only effective treatment is to secure haemostasis under general anaesthesia, and, if necessary, to give blood. The longer surgical interference is postponed, the more difficult and hazardous it becomes. A wise precaution is to make preparations for giving blood and get an intravenous infusion going as soon as haemorrhage is confirmed.
Anaesthesia for removal of the tonsils and adenoids is by no means as safe and simple as is commonly supposed; and re-anaesthesia for the control of haemorrhage may call for the highest skill and experience. There is also considerable risk during recovery from anaesthesia, and recovery-room facilities are essential for this work. On-the-spot inquiry immediately after a fatality is necessary in order to elucidate its cause. Attention should be directed particularly to the preoperative and postoperative medication of the patient and its timing, the details of the anaesthesia, the position in which the patient was nursed after operation, the’ measures taken to treat any postoperative haemorrhage, and the general suitability of the hospital and its staff for undertaking work of this kind. I am grateful to my colleague, Dr. J. G. Bourne, for helpful advice and criticism. I wish also to thank the surgeons, pathologists, and anxsthetists for the case-records, and the Association of Anxsthetists of Great Britain and Ireland for financial assistance.
CHANGES INDUCED BY TUBERCULIN IN LEUCOCYTE CULTURES D. C. COWLING Melb., F.R.A.C.P., M.C.P.A. E. DAVIDSON D. QUAGLINO M.D. Turin, Ph.D. Cantab. M.B. Cape Town, M.R.C.P. M.D.
From the
Department of Medicine, University of Cambridge
CYTOCHEMICAL observations on short-term leucocyte cultures after phytohasmagglutin stimulation were reported by Quaglino et al. (1962). Following the reports of Pearmain et al. (1963) and Elves et al. (1963) that tuberculin can induce transformation of lymphocytes to blast cells with mitotic activity, we have studied this phenomenon with autoradiographic and cytochemical techniques and have examined the cultured cells for modified activity by injecting them intradermally with
tuberculin. Methods cytochemical methods were those previously reported (Quaglino et al. 1962). Autoradiographic slides were prepared using Kodak AR 10 plates. 1-0 ml. of cell suspension was incubated for one hour with 0.1 ml. of tritiated thymidine diluted to an activity of 5 (.LC per ml. Films were developed after contact for 6 days and counterstained by conventional methods. The autoradiographic preparations were made after culturing cells for 3 and 7 days. Preliminary leucocyte cultures were carried out on blood from two Mantoux-positive subjects to ascertain the optimal dilution of tuberculin for stimulating cells. Eight cultures were set up, four from healthy Mantoux-positive subjects and four from Mantoux-negative subjects. In each case three cultures were prepared, one with 0-2 ml. of phytohwmagglutinin M (Difco) (PHA), one with Old Tuberculin in a final dilution of 1/4000, and the third with medium T.c. 199 only. To assess the changes in the activity of the cells after culture, intradermal injections of 0-1 ml. of the culture (as set out in the results) were performed on the cell donors in a latin-square pattern with suitable controls. These were read at 48 and 72 hours, and the mean diameter of the induration produced in the duplicate tests was recorded. In designing the intradermal tests we attempted to exclude the non-specific absorption of tuberculin by leucocytes demonstrated by Favour (1949). Cultures and
Results
Cytological Observations Table i shows the extent of blastic transformation in cultures of two Mantoux-positive subjects, using various dilutions of Old Tuberculin. During all cultures except those with very dilute tuberculin, polymorphs virtually disappeared at 3 days
1092 TABLE I-PERCENTAGE OF BLAST CELLS IN CULTURES WITH VARIOUS DILUTIONS OF OLD TUBERCULIN
and lymphocytes showed considerable smearing in the most concentrated tuberculin cultures. Tables n and III show the main morphological, cytochemical, and autoradiographic features. 1. Cells from Mantoux-positive and Mantoux-negative subjects cultured with medium T.c. 199 alone showed no increase in periodic-acid/Schiff (P.A.S.) positivity in the lymphocytes and very little enhancement of succinic and lactic dehydrogenase activity. The only relevant morphological finding after 7 days was the presence of variable numbers of mononuclear histiocytic cells, which cytochemically were characterised by a diffuse P.A.s.-positive tinge and in some cases by fine and
nucleoli less prominent. The percentage of the blast cells, however, was increased. 3. When cells from Mantoux-positive subjects were incubated with tuberculin, no prominent cytochemical or morphological changes occurred until the 3rd day, at which time from 1 to 5 % of cells showed nuclear and cytoplasmic characteristics similar to PHA-induced blast cells. At the same time coarse granular P.A.S. positivity was seen in a small proportion of nucleolated cells, which were undergoing blastic transformation (fig. 1). These cells, as well as the fully developed blast cells, were Sudan-black-negative and showed strong lactic and, to a lesser degree, succinic dehydrogenase activity. TABLE III-FINDINGS IN LEUCOCYTE CULTURES FROM MANTOUXPOSITIVE SUBJECTS (CASES 1-3) AND MANTOUX-NEGATIVE SUBJECTS .
(CASES 5-7)
-
moderately
coarse
sudanophilic granules. Thymidine
was
small proportion of cells, usually 0-5-1%, which morphologically resembled the blast cells seen in cultures with PHA. 2. Cultures with PHA from both Mantoux-positive and Mantoux-negative subjects always showed, after 18-24 hours, a number of nucleolated and basophilic cells containing coarse P.A.S. positivity, increasing the initial P.A.S. score of the lymphocytes. Both succinic and lactic dehydrogenases were greatly increased in the lymphoblasts and prolymphocytes. After 3 days, a variable but high proportion of blast cells showed an intense degree of thymidine incorporation. The percentage of cells with thymidine uptake was strikingly small after 7 days and the blast cells appeared less basophilic and the
present in
a
TABLE II-FINDINGS IN LEUCOCYTE CULTURES FROM MANTOUX-POSITIVE AND MANTOUX-NEGATIVE SUBJECTS SUBJECTS
(CASES 1-4)
(CASES 5-8)
The blast cells and the cells showing thymidine uptake are expressed as percentage of the total nucleated cells present in the culture.
a
days blast cells
were more numerous, but their proin contrast to the more extensive exceeded 20%, portion blastic transformation of the lymphocytic population in PHA cultures. Another point of difference from PHA cultures was the continued presence of P.A.S. positivity in the nucleolated cells undergoing blastic transformation. Only a few histiocytic cells were seen at the 7th day. Thymidine incorporation appeared in a small number of cells (0-5-4%) on the 3rd day and was present in a greater proportion after 7 days (3-5%) (fig. 2). 4. No appreciable morphological, cytochemical, or autoradiographic changes were seen when tuberculin was added to cultures of Mantoux-negative subjects, except the occasional histiocytic cell and a 0-5-1% thymidine uptake, features common to the T.c. 199 control culture.
After 7
never
The blast cells and P.A.s.-positive cells (+ + +) are expressed percentage of the total nucleated cells present in the culture.
as
a
Results of Intradermal Tests The results are tabulated diagrammatically (table iv). a. A 72-hour cell-rich culture incubated with tuberculin was injected into two subjects, cell-free plasma in T.c. 199 medium with tuberculin acting as a control. By 48 hours a clear difference was present, the fluid containing the cultured cells giving a diminished response. Fig. 3 illustrates a test at 96 hours with a fading lesion at the sites of cell injection and considerable induration still present in the controls. A similar result was obtained in one subject after 7 days’ incubation. In some injections containing cells, the immediate reaction appeared greater than the control. b. In one subject, a 72-hour cell-rich culture incubated with tuberculin was injected, the control being a 2-hour cell-rich culture with tuberculin. The response was diminished at the site of injection of the 72-hour culture.
1093
Autoradiographic studies with thymidine indicate that with phytohxmagglutinin (PHA) nuclear protein synthesis is maximal at 3 days. While the number of blast cells continues to increase up to 7 days, the thymidine uptake in PHA cultures at this stage is diminished and nucleoli and basophilia in the blast cells appear less prominent. With tuberculin as the stimulating agent, transformation proceeds slowly, and in some cultures P.A.S. positivity continues to increase up to 7 days, in parallel with the thymidine uptake. In the specimens from tuberculin cultures, examined at 3 and 7 days, mitotic activity is less than 0-5% of all nucleolated cells. Our present experiments suggest that the changes which take place in tuberculin cultures differ from the changes Fig. I-Two P.A.S.-negative lymphocytes and a prolymphocyte showing coarse (+++) P.A.S. positivity. (P.A.S. periodicacid/Schiff.)
TABLE IV-RESULTS OF INTRADERMAL TESTS
=
c. In one subject a 7-day cell-rich culture incubated with tuberculin was injected. 2 hours before injection an equivalent number of fresh leucocytes was added to the control plasma incubated with tuberculin and T.c. 199 medium for 7 days. The intradermal test from the 7-day cell-culture showed a diminished response. This was repeated in another subject using a 4-day cell-rich culture with tuberculin, giving similar results. d. The cell-free supernatants from 4-day cell-cultures with tuberculin were injected into two subjects. In the one control the cells were in contact with the plasma during the first 2 hours of the incubation period, and in the second control the cells were added to the control 2 hours before injection. Prior to injection the culture bottles were centrifuged at 3000 r.p.m. for 15 minutes and the cell-free supernatants injected. No decrease in the reaction was noted at sites injected with the supernatant fluid from 4-day cell-cultures. e. Leucocytes from a Mantoux-negative subject were incubated with tuberculin in T.c. 199 medium for 2 hours with plasma and tuberculin in T.c. 199 medium as the control. These were injected into a Mantoux-positive subject and gave identical reactions. Discussion
Reports indicate that phytohxmagglutinin stimulates the majority of lymphocytes to blastic transformation with some mitotic activity. This change is associated with maximal metabolic activity, as assessed by periodic-acid/ Schiff (P.A.S.) staining in relation to carbohydrate metabolism, at 24-48 hours (Quaglino et al. 1962).
Fig.2-A group of lymphocytes and tive cells show varying degrees
Areas of induration, the mean of two reaction sites, are expressed in centimetres. Tuberculin 1/4000 was added to cultures and controls at the commencement of the time stated, and cultures and controls injected and read together.
in
cultures both in the rate and extent of blastic transformation, suggesting that PHA is a more potent stimulus to which the majority of lymphocytes can react or that its mode of action differs qualitatively from that of tuberculin. It may in fact be a growth-stimulating factor, as has been suggested by Grasbeck (1963), by virtue of its surface attachment to all lymphocytes. With regard to the intradermal tests, various reactions PHA
3-Tests at 96 hours, series (a). A are control sites with induration still present. B are sites of cell-rich culture injections
Fig. two blast cells. The two
of
thymidine uptake.
primi-
showing erythema only.
1094
between tuberculin, serum, and cells have been described. Tuberculin neutralisation by normal serum and serum from patients with sarcoid is discussed by the British Medical Journal (1958). Leucocytes from patients (and laboratory animals) with active tuberculosis may be lysed by tuberculin, the reaction being abolished by inactivation of the serum with heat (Fremont Smith and Favour
1948). Leucocytes from healthy subjects (positive or negative to Mantoux tests) are not lysed. The leucocytes from normal subjects will absorb tuberculin when incubated for 30 minutes at 37 °C, as demonstrated by the lysis test on leucocytes from tuberculous subjects (Favour 1949). The present studies have excluded non-specific absorption of tuberculin by leucocytes as the cause of the diminished reaction from leucocyte cultures with tuberculin. From the results of our intradermal testing on a limited scale we conclude that the cells derived from cultures, when reinjected into the donor, reduce the intensity of the Mantoux reaction after 48 hours whereas the supernatant cell-free medium does not do so. It appears that culture of lymphocytes in the presence of tuberculin activates the cells. Lawrence (1960) has described the transfer of delayed hypersensitivity by leucocytes, or extracts of leucocytes, from Mantouxpositive donors to Mantoux-negative volunteers. For a tuberculin reaction to develop in these negative subjects, the tissue must be sensitised; but they would hardly have time to develop a cell line of lymphocytes specifically directed to act in the tuberculin reaction. If indeed the cells from cultures with tuberculin, when injected, perform an immunological function in reducing the intensity of the tuberculin reaction, they may correspond to the mononuclear cells which normally invade the reaction site at 24-48 hours (Boughton and Spector 1963). As tuberculin is apparently not inactivated in vitro by the growth of lymphocytes, these cells probably play their part by suppressing the reaction of the tissue itself to tuberculin. By introducing stimulated lymphocytes initially at the site of injection, as compared with their arrival in the normal sequence of events at 24-48 hours, the timing of events at the site of injection of cells cultured with tuberculin may be foreshortened, thus producing a diminished reaction at 72 hours. We have not yet determined whether lymphocytes activated by other antigens will show a similar effect.
Summary 1. Blastic transformation of
less more in cultures with and tuberculin frequently slowly than in cultures with phytohasmagglutinin. This suggests a possible difference in the mechanism of action between these two agents. At 7 days, in cultures with tuberculin, up to 20% of lymphocytes showed blastic transformation, whereas in cultures with phytohmmagglutinin the proportion was up to 92%. Morphologically and cytochemically the blast cells produced by tuberculin are similar to those produced by phytohsemagglutinin. 2. A wide range of dilutions of tuberculin induce this change. The proportion of blast cells on culture tails off at high and low dilutions of tuberculin. 3. The cells produced in culture-possibly lymphoblasts-seem to diminish the Mantoux reaction at 72 hours. It is yet to be determined whether this is a specific effect of lymphocytes stimulated by tuberculin. We are grateful to Dr. F. G. J. Hayhoe for advice in preparing this paper, and to Mr. R. J. Flemans for technical assistance.
lymphocytes
.
References at foot of next column
occurs
ACHRESTIC DIABETES I. MAGYAR M.D. Budapest DIRECTOR AND PROFESSOR
D. LEHOCZKY M.D.
Budapest
LECTURER
DEPARTMENT OF
MEDICINE,
I. MÁRTON M.D.
Budapest
LECTURER
POSTGRADUATE MEDICAL
SCHOOL,
BUDAPEST, HUNGARY
INSULIN resistance has been reported in patients in whom the insulin-like activity of the plasma is normal or increased (Presland and Todd 1956, Field et al. 1959, Leonards and Martin 1959, Tyler and Beigelman 1960, Field et al. 1961, Shipp et al. 1961). The supposition in these cases is that the tissues are unable to use the sugar even in the presence of insulin. In one of our cases (Magyar 1961), a 28-year-old woman who had had diabetes for 7 years, the blood-sugar was not lowered after the intravenous injection of 100 units of insulin. A daily dose of 16,000 units was needed to end the comatose state of the patient, and she had to have an average daily dose of 300-1000 units of insulin to be kept alive. On eight occasions of determining insulin by the rat-diaphragm method, we found that normal or greater than normal insulin-like activity was manifest. We concluded that the basic disturbance of the patient’s metabolism was peripheral: the tissues were incapable of utilising the sugar for some unknown reason despite the presence of biologically active insulin at their disposal. This, and similar cases, we call " achrestic diabetes". A piece of abdominal muscle was removed by biopsy from the patient. This was tested for sugar consumption in plasma from a healthy subject with a known quantity of glucose, in plasma an hour after administering glucose, and also in the plasma of the patient.
In our previous examinations we found that tissue from the rectus muscle of non-diabetic patients obtained at various abdominal operations can be used for determining insulin activity in the same way as rat-diaphragm. SUGAR CONSUMPTION OF MUSCLE-TISSUE
In plasma from a healthy fasting subject in which we raised the sugar content to 300 mg. per 100 ml. the glucose consumed for 10 mg. of dried tissue after 90 minutes’ incubation was 0-208 in an average of 11 determinations with rat-diaphragm, and 0-230 in an average of 26 with human muscle-tissue. The result in each case was determined by the sugar consumption of two pieces of muscle or two rathalfdiaphragms (Vallance-Owen 1956). The difference was less than ±5%. The smallest sugar intake was 0-180 mg. and the largest was 0-280 mg. for 10 mg. of dried muscle. According to the type of diabetes, the DR. COWLING AND OTHERS: REFERENCES
Boughton, B., Spector, W. G. (1963) J. Path. Bact. 85, 371. British Medical Journal (1958), i, 1403. Elves, M. W., Roath, S., Israëls, M. C. G. (1963) Lancet, i, 806. Favour, C. B. (1949) Proc. Soc. exp. Biol. N.Y. 70, 369. Fremont Smith, P., Favour, C. B. (1948) ibid. 67, 502. Gräsbeck, R., Nordman, C., de la Chapelle, A. (1963) Lancet, ii, 385. Lawrence, S. H. (1960) in Ciba Foundation Symposium on Cellular Aspects of Immunity (edited by G. E. W. Wolstenholme and M. O’Connor); p. 243. London. Pearmain, G., Lycette, R. R., Fitzgerald, P. H. (1963) Lancet, i, 637. Quaglino, D., Hayhoe, F. G. J., Flemans, R.J. (1962) Nature, Lond. 196, 338.
Errors of Surgical or Nursing Care (4 cases) Case 12.-A boy, aged 6 years, was restless after operation. He was given 5 minims of nepenthe, which was repeated three and a half hours later. The restlessness was now controlled and the child settled down to sleep. Three-quarters of an hour later he was found dead in bed. At necropsy no abnormality
Attributable
was
to
found. ATTRIBUTABLE TO ERRORS
OF ANESTHESIA
(21 CASES) During Induction (4 cases), during Operation (9 cases), or during Recovery (8 cases) Case 13.-A boy, aged 8 years, was given ethyl chloride and ether on a mask. After twenty minutes, and before the operation had been started, there was sudden cardiorespiratory arrest. With artificial respiration and cardiac massage the heart-beat returned, but the child died six hours later without having regained consciousness. The operation was not
performed. Case 14.-A boy, aged 5 years, was anaathetised with nitrous oxide and ether through a Boyle-Davis gag. When the operation was started, anaesthesia was found to be too light and 100 mg. of thiopentone was given. Later the boy was given another 50 mg. of thiopentone. Respiration stopped, and the child became cyanosed. The gag was removed, an endotracheal tube was introduced, and the lungs were inflated with oxygen. The expiratory valve was tightly shut, and widespread surgical emphysema immediately followed. The patient died. Case 15.-A girl, aged 6 years, was returned to the ward five minutes after the operation had been completed. A few minutes later respiration became obstructed and ceased. The anaesthetist was sent for, but resuscitation was only partly successful, and the child died twelve hours later without having regained consciousness. Comment
The suggestions contained in this report are offered with considerable reserve. Most of the data were obtained long after the events, and in many cases were extremely Nevertheless, certain pointers emerged that scanty. could be helpful. The mortality, though not to be accepted with complacency, is not catastrophic: out of 200,000 patients a year who undergo the operation in this country, about 20 lose their lives through it. A common source of error seems to be undue delay in giving effective treatment for the control of hxmorrhage. To give nepenthe, or other opiate, in the hope that the patient will settle down and stop bleeding, is not only disappointingly ineffective but also highly dangerous. Even without hxmorrhage and in a normal clinical dose, nepenthe, when given on top of an anxsthetic and other depressant drugs, can cause death from central depression and obstruction of the airway. In the presence of hxmorrhage there is the added danger of blood being aspirated. The restlessness for which the nepenthe is given, if not a sequel of barbiturate premedication (which is best avoided in tonsillectomy cases) may be a sign of hxmorrhage, which the nepenthe may help to conceal. In a small child the loss of less than a pint of blood may be fatal, and more blood may have been swallowed than is seen on the pillow. Propping up the patient to arrest hxmorrhage is hazardous. But this continues to be taught. The only effective treatment is to secure haemostasis under general anaesthesia, and, if necessary, to give blood. The longer surgical interference is postponed, the more difficult and hazardous it becomes. A wise precaution is to make preparations for giving blood and get an intravenous infusion going as soon as haemorrhage is confirmed.
Anaesthesia for removal of the tonsils and adenoids is by no means as safe and simple as is commonly supposed; and re-anaesthesia for the control of haemorrhage may call for the highest skill and experience. There is also considerable risk during recovery from anaesthesia, and recovery-room facilities are essential for this work. On-the-spot inquiry immediately after a fatality is necessary in order to elucidate its cause. Attention should be directed particularly to the preoperative and postoperative medication of the patient and its timing, the details of the anaesthesia, the position in which the patient was nursed after operation, the’ measures taken to treat any postoperative haemorrhage, and the general suitability of the hospital and its staff for undertaking work of this kind. I am grateful to my colleague, Dr. J. G. Bourne, for helpful advice and criticism. I wish also to thank the surgeons, pathologists, and anxsthetists for the case-records, and the Association of Anxsthetists of Great Britain and Ireland for financial assistance.
CHANGES INDUCED BY TUBERCULIN IN LEUCOCYTE CULTURES D. C. COWLING Melb., F.R.A.C.P., M.C.P.A. E. DAVIDSON D. QUAGLINO M.D. Turin, Ph.D. Cantab. M.B. Cape Town, M.R.C.P. M.D.
From the
Department of Medicine, University of Cambridge
CYTOCHEMICAL observations on short-term leucocyte cultures after phytohasmagglutin stimulation were reported by Quaglino et al. (1962). Following the reports of Pearmain et al. (1963) and Elves et al. (1963) that tuberculin can induce transformation of lymphocytes to blast cells with mitotic activity, we have studied this phenomenon with autoradiographic and cytochemical techniques and have examined the cultured cells for modified activity by injecting them intradermally with
tuberculin. Methods cytochemical methods were those previously reported (Quaglino et al. 1962). Autoradiographic slides were prepared using Kodak AR 10 plates. 1-0 ml. of cell suspension was incubated for one hour with 0.1 ml. of tritiated thymidine diluted to an activity of 5 (.LC per ml. Films were developed after contact for 6 days and counterstained by conventional methods. The autoradiographic preparations were made after culturing cells for 3 and 7 days. Preliminary leucocyte cultures were carried out on blood from two Mantoux-positive subjects to ascertain the optimal dilution of tuberculin for stimulating cells. Eight cultures were set up, four from healthy Mantoux-positive subjects and four from Mantoux-negative subjects. In each case three cultures were prepared, one with 0-2 ml. of phytohwmagglutinin M (Difco) (PHA), one with Old Tuberculin in a final dilution of 1/4000, and the third with medium T.c. 199 only. To assess the changes in the activity of the cells after culture, intradermal injections of 0-1 ml. of the culture (as set out in the results) were performed on the cell donors in a latin-square pattern with suitable controls. These were read at 48 and 72 hours, and the mean diameter of the induration produced in the duplicate tests was recorded. In designing the intradermal tests we attempted to exclude the non-specific absorption of tuberculin by leucocytes demonstrated by Favour (1949). Cultures and
Results
Cytological Observations Table i shows the extent of blastic transformation in cultures of two Mantoux-positive subjects, using various dilutions of Old Tuberculin. During all cultures except those with very dilute tuberculin, polymorphs virtually disappeared at 3 days
1092 TABLE I-PERCENTAGE OF BLAST CELLS IN CULTURES WITH VARIOUS DILUTIONS OF OLD TUBERCULIN
and lymphocytes showed considerable smearing in the most concentrated tuberculin cultures. Tables n and III show the main morphological, cytochemical, and autoradiographic features. 1. Cells from Mantoux-positive and Mantoux-negative subjects cultured with medium T.c. 199 alone showed no increase in periodic-acid/Schiff (P.A.S.) positivity in the lymphocytes and very little enhancement of succinic and lactic dehydrogenase activity. The only relevant morphological finding after 7 days was the presence of variable numbers of mononuclear histiocytic cells, which cytochemically were characterised by a diffuse P.A.s.-positive tinge and in some cases by fine and
nucleoli less prominent. The percentage of the blast cells, however, was increased. 3. When cells from Mantoux-positive subjects were incubated with tuberculin, no prominent cytochemical or morphological changes occurred until the 3rd day, at which time from 1 to 5 % of cells showed nuclear and cytoplasmic characteristics similar to PHA-induced blast cells. At the same time coarse granular P.A.S. positivity was seen in a small proportion of nucleolated cells, which were undergoing blastic transformation (fig. 1). These cells, as well as the fully developed blast cells, were Sudan-black-negative and showed strong lactic and, to a lesser degree, succinic dehydrogenase activity. TABLE III-FINDINGS IN LEUCOCYTE CULTURES FROM MANTOUXPOSITIVE SUBJECTS (CASES 1-3) AND MANTOUX-NEGATIVE SUBJECTS .
(CASES 5-7)
-
moderately
coarse
sudanophilic granules. Thymidine
was
small proportion of cells, usually 0-5-1%, which morphologically resembled the blast cells seen in cultures with PHA. 2. Cultures with PHA from both Mantoux-positive and Mantoux-negative subjects always showed, after 18-24 hours, a number of nucleolated and basophilic cells containing coarse P.A.S. positivity, increasing the initial P.A.S. score of the lymphocytes. Both succinic and lactic dehydrogenases were greatly increased in the lymphoblasts and prolymphocytes. After 3 days, a variable but high proportion of blast cells showed an intense degree of thymidine incorporation. The percentage of cells with thymidine uptake was strikingly small after 7 days and the blast cells appeared less basophilic and the
present in
a
TABLE II-FINDINGS IN LEUCOCYTE CULTURES FROM MANTOUX-POSITIVE AND MANTOUX-NEGATIVE SUBJECTS SUBJECTS
(CASES 1-4)
(CASES 5-8)
The blast cells and the cells showing thymidine uptake are expressed as percentage of the total nucleated cells present in the culture.
a
days blast cells
were more numerous, but their proin contrast to the more extensive exceeded 20%, portion blastic transformation of the lymphocytic population in PHA cultures. Another point of difference from PHA cultures was the continued presence of P.A.S. positivity in the nucleolated cells undergoing blastic transformation. Only a few histiocytic cells were seen at the 7th day. Thymidine incorporation appeared in a small number of cells (0-5-4%) on the 3rd day and was present in a greater proportion after 7 days (3-5%) (fig. 2). 4. No appreciable morphological, cytochemical, or autoradiographic changes were seen when tuberculin was added to cultures of Mantoux-negative subjects, except the occasional histiocytic cell and a 0-5-1% thymidine uptake, features common to the T.c. 199 control culture.
After 7
never
The blast cells and P.A.s.-positive cells (+ + +) are expressed percentage of the total nucleated cells present in the culture.
as
a
Results of Intradermal Tests The results are tabulated diagrammatically (table iv). a. A 72-hour cell-rich culture incubated with tuberculin was injected into two subjects, cell-free plasma in T.c. 199 medium with tuberculin acting as a control. By 48 hours a clear difference was present, the fluid containing the cultured cells giving a diminished response. Fig. 3 illustrates a test at 96 hours with a fading lesion at the sites of cell injection and considerable induration still present in the controls. A similar result was obtained in one subject after 7 days’ incubation. In some injections containing cells, the immediate reaction appeared greater than the control. b. In one subject, a 72-hour cell-rich culture incubated with tuberculin was injected, the control being a 2-hour cell-rich culture with tuberculin. The response was diminished at the site of injection of the 72-hour culture.
1093
Autoradiographic studies with thymidine indicate that with phytohxmagglutinin (PHA) nuclear protein synthesis is maximal at 3 days. While the number of blast cells continues to increase up to 7 days, the thymidine uptake in PHA cultures at this stage is diminished and nucleoli and basophilia in the blast cells appear less prominent. With tuberculin as the stimulating agent, transformation proceeds slowly, and in some cultures P.A.S. positivity continues to increase up to 7 days, in parallel with the thymidine uptake. In the specimens from tuberculin cultures, examined at 3 and 7 days, mitotic activity is less than 0-5% of all nucleolated cells. Our present experiments suggest that the changes which take place in tuberculin cultures differ from the changes Fig. I-Two P.A.S.-negative lymphocytes and a prolymphocyte showing coarse (+++) P.A.S. positivity. (P.A.S. periodicacid/Schiff.)
TABLE IV-RESULTS OF INTRADERMAL TESTS
=
c. In one subject a 7-day cell-rich culture incubated with tuberculin was injected. 2 hours before injection an equivalent number of fresh leucocytes was added to the control plasma incubated with tuberculin and T.c. 199 medium for 7 days. The intradermal test from the 7-day cell-culture showed a diminished response. This was repeated in another subject using a 4-day cell-rich culture with tuberculin, giving similar results. d. The cell-free supernatants from 4-day cell-cultures with tuberculin were injected into two subjects. In the one control the cells were in contact with the plasma during the first 2 hours of the incubation period, and in the second control the cells were added to the control 2 hours before injection. Prior to injection the culture bottles were centrifuged at 3000 r.p.m. for 15 minutes and the cell-free supernatants injected. No decrease in the reaction was noted at sites injected with the supernatant fluid from 4-day cell-cultures. e. Leucocytes from a Mantoux-negative subject were incubated with tuberculin in T.c. 199 medium for 2 hours with plasma and tuberculin in T.c. 199 medium as the control. These were injected into a Mantoux-positive subject and gave identical reactions. Discussion
Reports indicate that phytohxmagglutinin stimulates the majority of lymphocytes to blastic transformation with some mitotic activity. This change is associated with maximal metabolic activity, as assessed by periodic-acid/ Schiff (P.A.S.) staining in relation to carbohydrate metabolism, at 24-48 hours (Quaglino et al. 1962).
Fig.2-A group of lymphocytes and tive cells show varying degrees
Areas of induration, the mean of two reaction sites, are expressed in centimetres. Tuberculin 1/4000 was added to cultures and controls at the commencement of the time stated, and cultures and controls injected and read together.
in
cultures both in the rate and extent of blastic transformation, suggesting that PHA is a more potent stimulus to which the majority of lymphocytes can react or that its mode of action differs qualitatively from that of tuberculin. It may in fact be a growth-stimulating factor, as has been suggested by Grasbeck (1963), by virtue of its surface attachment to all lymphocytes. With regard to the intradermal tests, various reactions PHA
3-Tests at 96 hours, series (a). A are control sites with induration still present. B are sites of cell-rich culture injections
Fig. two blast cells. The two
of
thymidine uptake.
primi-
showing erythema only.
1094
between tuberculin, serum, and cells have been described. Tuberculin neutralisation by normal serum and serum from patients with sarcoid is discussed by the British Medical Journal (1958). Leucocytes from patients (and laboratory animals) with active tuberculosis may be lysed by tuberculin, the reaction being abolished by inactivation of the serum with heat (Fremont Smith and Favour
1948). Leucocytes from healthy subjects (positive or negative to Mantoux tests) are not lysed. The leucocytes from normal subjects will absorb tuberculin when incubated for 30 minutes at 37 °C, as demonstrated by the lysis test on leucocytes from tuberculous subjects (Favour 1949). The present studies have excluded non-specific absorption of tuberculin by leucocytes as the cause of the diminished reaction from leucocyte cultures with tuberculin. From the results of our intradermal testing on a limited scale we conclude that the cells derived from cultures, when reinjected into the donor, reduce the intensity of the Mantoux reaction after 48 hours whereas the supernatant cell-free medium does not do so. It appears that culture of lymphocytes in the presence of tuberculin activates the cells. Lawrence (1960) has described the transfer of delayed hypersensitivity by leucocytes, or extracts of leucocytes, from Mantouxpositive donors to Mantoux-negative volunteers. For a tuberculin reaction to develop in these negative subjects, the tissue must be sensitised; but they would hardly have time to develop a cell line of lymphocytes specifically directed to act in the tuberculin reaction. If indeed the cells from cultures with tuberculin, when injected, perform an immunological function in reducing the intensity of the tuberculin reaction, they may correspond to the mononuclear cells which normally invade the reaction site at 24-48 hours (Boughton and Spector 1963). As tuberculin is apparently not inactivated in vitro by the growth of lymphocytes, these cells probably play their part by suppressing the reaction of the tissue itself to tuberculin. By introducing stimulated lymphocytes initially at the site of injection, as compared with their arrival in the normal sequence of events at 24-48 hours, the timing of events at the site of injection of cells cultured with tuberculin may be foreshortened, thus producing a diminished reaction at 72 hours. We have not yet determined whether lymphocytes activated by other antigens will show a similar effect.
Summary 1. Blastic transformation of
less more in cultures with and tuberculin frequently slowly than in cultures with phytohasmagglutinin. This suggests a possible difference in the mechanism of action between these two agents. At 7 days, in cultures with tuberculin, up to 20% of lymphocytes showed blastic transformation, whereas in cultures with phytohmmagglutinin the proportion was up to 92%. Morphologically and cytochemically the blast cells produced by tuberculin are similar to those produced by phytohsemagglutinin. 2. A wide range of dilutions of tuberculin induce this change. The proportion of blast cells on culture tails off at high and low dilutions of tuberculin. 3. The cells produced in culture-possibly lymphoblasts-seem to diminish the Mantoux reaction at 72 hours. It is yet to be determined whether this is a specific effect of lymphocytes stimulated by tuberculin. We are grateful to Dr. F. G. J. Hayhoe for advice in preparing this paper, and to Mr. R. J. Flemans for technical assistance.
lymphocytes
.
References at foot of next column
occurs
ACHRESTIC DIABETES I. MAGYAR M.D. Budapest DIRECTOR AND PROFESSOR
D. LEHOCZKY M.D.
Budapest
LECTURER
DEPARTMENT OF
MEDICINE,
I. MÁRTON M.D.
Budapest
LECTURER
POSTGRADUATE MEDICAL
SCHOOL,
BUDAPEST, HUNGARY
INSULIN resistance has been reported in patients in whom the insulin-like activity of the plasma is normal or increased (Presland and Todd 1956, Field et al. 1959, Leonards and Martin 1959, Tyler and Beigelman 1960, Field et al. 1961, Shipp et al. 1961). The supposition in these cases is that the tissues are unable to use the sugar even in the presence of insulin. In one of our cases (Magyar 1961), a 28-year-old woman who had had diabetes for 7 years, the blood-sugar was not lowered after the intravenous injection of 100 units of insulin. A daily dose of 16,000 units was needed to end the comatose state of the patient, and she had to have an average daily dose of 300-1000 units of insulin to be kept alive. On eight occasions of determining insulin by the rat-diaphragm method, we found that normal or greater than normal insulin-like activity was manifest. We concluded that the basic disturbance of the patient’s metabolism was peripheral: the tissues were incapable of utilising the sugar for some unknown reason despite the presence of biologically active insulin at their disposal. This, and similar cases, we call " achrestic diabetes". A piece of abdominal muscle was removed by biopsy from the patient. This was tested for sugar consumption in plasma from a healthy subject with a known quantity of glucose, in plasma an hour after administering glucose, and also in the plasma of the patient.
In our previous examinations we found that tissue from the rectus muscle of non-diabetic patients obtained at various abdominal operations can be used for determining insulin activity in the same way as rat-diaphragm. SUGAR CONSUMPTION OF MUSCLE-TISSUE
In plasma from a healthy fasting subject in which we raised the sugar content to 300 mg. per 100 ml. the glucose consumed for 10 mg. of dried tissue after 90 minutes’ incubation was 0-208 in an average of 11 determinations with rat-diaphragm, and 0-230 in an average of 26 with human muscle-tissue. The result in each case was determined by the sugar consumption of two pieces of muscle or two rathalfdiaphragms (Vallance-Owen 1956). The difference was less than ±5%. The smallest sugar intake was 0-180 mg. and the largest was 0-280 mg. for 10 mg. of dried muscle. According to the type of diabetes, the DR. COWLING AND OTHERS: REFERENCES
Boughton, B., Spector, W. G. (1963) J. Path. Bact. 85, 371. British Medical Journal (1958), i, 1403. Elves, M. W., Roath, S., Israëls, M. C. G. (1963) Lancet, i, 806. Favour, C. B. (1949) Proc. Soc. exp. Biol. N.Y. 70, 369. Fremont Smith, P., Favour, C. B. (1948) ibid. 67, 502. Gräsbeck, R., Nordman, C., de la Chapelle, A. (1963) Lancet, ii, 385. Lawrence, S. H. (1960) in Ciba Foundation Symposium on Cellular Aspects of Immunity (edited by G. E. W. Wolstenholme and M. O’Connor); p. 243. London. Pearmain, G., Lycette, R. R., Fitzgerald, P. H. (1963) Lancet, i, 637. Quaglino, D., Hayhoe, F. G. J., Flemans, R.J. (1962) Nature, Lond. 196, 338.