- Email: [email protected]
IgE LEVELS IN INTERSTITIAL NEPHRITIS
1254 the start of the infusion when the rate had reached 4.8 jug. per minute. The infusion was switched off and over the next 45 minutes th contractions settled to pressures of between 40 and 50 mm. Hg. This patient was not given any further stimulation and she progressed to a spontaneous delivery. There were two abnormal fetal heart traces but both were associated with normal labour traces. One trace showed late decelerations associated with a baseline tachycardia, but at this time the mother was hyperventilating because she was practising psychoprophylaxis. The fetal pH was 7.45 and the maternal venous pH was 7.58. The other trace showed a persistent baseline bradycardia of 110 beats per minute, but fetal-blood samples repeated on four occasions throughout labour all showed normal pH values.
Discussion When oxytocin is used to induce labour, it has been shown that the contractile response of the uterus is more important than the specific dose of oxytocic used, and that automatic oxytocin titration utilising the Cardiff infusion pump is a highly efficient method of administering intravenous oxytocin.4 It is logical to use prostaglandins in the same way. The most worrying aspect of the induction of labour with this highly potent substance is the fear that there may be episodes of hypertonus. All of the reported instances of hypertonus associated with prostaglandin infusion have occurred at high initial infusion-rates,5 or after rapid increases in the infusion-rate,6,7 or when relatively high infusion-rates were achieved before the membranes were ruptured.8 Dewhurst9 suggested that infusion-rates should be increased at intervals of more than 30 minutes. Beazley and Gillespie 10 in their series increased their infusion-rates at hourly intervals; the highest infusion-rate they reached to induce labour was 6.7 jug. of prostaglandin E2 per minute (equivalent to 67 µg. of p.G.F2a) but this was only achieved after 5 hours of infusion. The advantages of the Cardiff pump lie in the accuracy with which small concentrations of the drug can be infused in very small volumes of fluid, and the smoothness of the increase of the infusion-rate to the dose required to initiate labour. The automatic safety cut-outs in the
of hypertonic uterine activity are a comfeature. The results of this preliminary surpelling labour induced in this way is likely indicate that vey to be both safe and efficient.
Preliminary Communication IgE LEVELS IN INTERSTITIAL NEPHRITIS
Requests for reprints should be addressed
to
J. R. N.
REFERENCES 1. Karim, S., Trussell, R., Patel, R., Hillier, K. Br. med. J. 1965, iv, 621. 2. Naismith, W., Barr, W. J. Obstet. Gynœc. Br. Commonw. 1973, 80, 531. 3. Johnson, A., Hyatt, D., Newton, J. R., Phillips, L. ibid. (in the press). 4. Francis, J., Turnbull, A., Thomas, F. ibid. 1970, 70, 594. 5. Karim, S. M. M., Filshie, G. M. Lancet, 1970, i, 157. 6. Anderson, G., Covdero, L., Hobbins, J., Speroff, L. Ann. N.Y. Acad. Sci. 1970, 180, 499. 7. Vakhariya, V., Sherman, A. Am. J. Obstet. Gynec. 1972, 113, 2, 212. 8. Gillespie, A., Dewhurst, C., Beazley, J. Br. med. J. 1971, ii, 222. 9. Dewhurst, C. Am. J. Obstet. Gynec. 1972, 113, 2, 221. 10. Beazley, J. M., Gillespie, A. Lancet, 1971, i, 152.
V. E. POLLAK
Division, Veterans Administration Hospital, and Division of Nephrology, Department of Medicine, University of Cincinnati Medical Center,
Renal
Cincinnati, Ohio I. L. BERNSTEIN Division of Immunology, Department of Medicine, University of Cincinnati Medical Center WELLINGTON JAO Department of Pathology, Michael Reese Hospital Medical Center, Chicago, Illinois, U.S.A.
Abnormal levels of
serum IgE
and were
Summary found in 3 of 5 patients developing
nephritis, probably induced by drug of sequential serum samples Measurement therapy. in a single patient showed that the rise of IgE paralleled the onset of interstitial nephritis. interstitial
INTRODUCTION
RAISED levels of serum-IgE have been found in a number of atopic disorders 1-3 and in parasitic infestations.4-6 We have detected increased levels in patients developing interstitial nephritis, probably induced by
drug therapy. PATIENTS AND METHODS
Five patients with the clinicopathological features of interstitial nephritis were studied. Renal tissue obtained by percutaneous renal biopsy was processed for light and electron microscopy 7; semiquantitative analysis of histological features was then done. Serum-IgE concentrations were measured by radioimmunosorbent, using a ’Phadebas ’ IgE test (Pharmacia Laboratories, Piscataway, New Jersey). Normal values in our laboratory were 20-1040 ng. per ml. (geometric mean ±2 s.D.). IgE levels of ten other patients with acute renal failure were also measured. Serum IgG, IgA, IgM and /3iC globulin levels were estimated by radial immunodiffusion.8 RESULTS
event
We thank Mr W. West, of Pye Dynamics, for the loan of the Cardiff mark in infusion system and Dr W. Ashton, of May and Baker, for the r.c.F.2a and a grant to support this project.
M. R. FIRST
B. S. OOI A. T. PESCE
Clinical Data The patients
were four males and one female, ranging in age from 17 to 39. None of them had a past history of atopic disorders, or any evidence of parasitic infestation. The underlying disease process three varied, patients having some infective process antibiotic treatment and the other two being requiring treated for the nephrotic syndrome. A skin rash developed in one patient, while eosinophilia occurred in three patients at about the onset of interstitial nephritis. Renal function deteriorated strikingly in two patients, while the remaining three had only mild but significant rises in serum-creatinine. The drug history was complex because multiple antibiotic combinations in fairly high doses had been given to four patients; of importance is the fact that penicillin or a penicillin analogue was one of the drugs administered. In the fifth patient, only chlorthali-
1255 LABORATORY FINDINGS
IN
FIVE PATIENTS NEPHRITIS
WITH
INTERSTITIAL
—————,————————.————————————————————.———————,———————
Renal Histology
Light microscopy.-Except for the two patients with the nephrotic syndrome who had focal glomerular sclerosis, there were no glomerular lesions. By contrast, striking and disproportionate tubular and interstitial changes were evident. Tubulitis and peritubular inflammation characterised by aggregation of inflammatory cells in close contact with tubular basemembrane were also noted. Distal tubules were severely affected than proximal tubules. The interstitium was infiltrated with abundant numbers of acute and chronic inflammatory cells; a distinctive lesion was the presence of significant numbers of ment
more
Normal values
(±2 s.D.): IgG 6 0-15mg. per ml., IgA 06-29 mg. ml., IgM 0-5-2-0 mg. per ml., IgE 20-1010 ng. per ml., (i1Cglobulin 90-200 mg. per 100 ml.
per
N.D. = not done.
eosinophils.
done, 50 mg. daily, had been administered for three months before onset of the nephritis. Complete recovery of excellent renal function occurred in all patients without evidence of under-
lying glomerular disease. Laboratory Results
laboratory results are shown in the table and in figs. 1 and 2. Serum-IgE levels were abnormally high in three of five patients. In patient 1 the serumIgE fell to 650 ng. per ml. in the convalescent phase of his illness. The relation between IgE levels and the clinical course of the illness in one patient is shown in fig. 2. Baseline levels of 320 ng. per ml. The
rose to
1760 ng. per ml. at about the time when
interstitial
nephritiswas diagnosed.
Unfortunately,
available in the convalescent period. IgE levels of all ten patients with other forms of renal disease were within normal limits. /31C-globulin levels measured in four patients were within normal limits. no sera were
Fig. 2-Sequential IgE levels (on log scale) of patient 5, developing interstitial nephritis on the 22nd day.
Electron-microscopy.-Utrastructural observations confirmed the pathological process visible by light microscopy. In addition, lymphocytes were insinuating between epithelial cells or between
seen
base-
membrane and tubular cells. Of interest were the small amounts of electron-dense material occasionally identified in the’ basement membranes of tubules.
ment
DISCUSSION
Fig. 1--Comparison
of serum-IgE levels (on log scale) between patients with interstitial nephritis and patients with other forms of rapidly progressive renal failure.
Normal IgE levels ± 2 s.D., 20-1010 ng. per ml.
The induction of interstitial nephritis by drug therapy is well documented." The clinical picture, biopsy findings, and recovery ,after discontinuation of drug therapy in our patients accord with the reported experience. The pathogenesis of this lesion is obscure. The occurrence of skin rashes and eosinophilia with the renal lesion suggests that there is an allergic basis for this disorder. The occurrence of raised IgE levels in a number of disorders with peripheral-blood eosinophilia led us to investigate the role of IgE in this disease. In addition, raised IgE concentrations have been recorded in patients with drug reactions while being treated with gold, and the occurrence of a positive Prausnitz-Kustner reaction for the sensitising drug in a patient with renal failure induced by phenazone.11 Three of five patients in this series had raised concentrations of In one patient the rise in IgE paralleled serum IgE. the onset of renal disease. Of the two patients with normal IgE levels, one showed a significant drop from There is a high-normal level during convalescence. no obvious explanation for the normal level in the
1256 It is relevant to note that a lag period of one year reported between infestation by hookworm and a rise in IgE level in a single patient 1 None of the patients had a common underlying pathological process which is known definitely to cause an increase in serum-IgE. We postulate that drug hypersensitivity in these patients evoked the formation of sufficient reaginic antibody to cause raised levels of circulating total IgE.
other.
was
Whether allergen-reaginic antibody complexes are involved in the pathogenesis of these renal lesions is not known. However, the presence of electrondense material in some tubular basement membranes and the pronounced interstitial eosinophilic infiltrate are consistent with a reagin-mediated immune injury. Further studies to characterise the role of IgE in this pathological process are warranted. In any case, measurements of serum-IgE may be a useful diagnostic tool in the differentiation of interstitial nephritis from others giving rise to the syndrome of rapidly progressive renal failure. This work was supported in part by National Institutes of Health research grant AM 17196 and U.S. Public Health Service training grant AM 05509.
INCREASED UPTAKE OF 35S-HEPARIN BY LYMPHOCYTES FROM PATIENTS WITH CYSTIC FIBROSIS
Requests for reprints should be addressed to B. S. 0., Veterans Administration Hospital, 3200 Vine Street, Cincinnati, Ohio 45220, U.S.A. REFERENCES
Berg, T., Johansson, S. G. O. Int. Archs Allergy appl. Immun. 1969, 36, 219. Johansson, S. G. O., Bennich, H. in Gamma Globulins, Structure and Control of Biosynthesis (edited by J. Killander); p. 193. Stockholm, 1967. Juhlin, L., Johansson, S. G. O., Bennich, H., Högman, C. F., Thyresson, N. Archs Derm. 1969, 100, 12. Johansson, S. G. O., Melbin, T., Vahlqvist, B. Lancet, 1968, i, 1118. Hogarth-Scott, R. S., Johansson, S. G. O., Bennich, R. Clin. exp. Immun. 1969, 5, 619. Rosenberg, E. B., Whalen, G. E., Bennich, H., Johansson, S. G. O. New Engl. J. Med. 1971, 283, 1148. Pirani, C. L., Pollak, V. E., Schwartz, F. D. Nephron, 1964, 1, 230. Mancini, G., Vaerman, J. P., Carbonara, A. O., Heremans, J. F. in Proceedings of the 11th Colloquium on Protides of the Biological Fluids (edited by H. Peeters), p. 370. Amsterdam, 1964. Simenhoff, M. L., Guild, W. R., Dammin, G. J. Amer. J. Med. 1968, 44, 618. Baldwin, D. S., Levine, B. B., McCluskey, R. T., Gallo, G. R. New Engl. J. Med. 1968, 279, 1245. Lyons, H., Pinn, V. W., Cortell, S., Cohen, J. J., Harrington, J. T. ibid. 1973, 288, 124. Davis, P., Ezeoke, A., Munro, J., Hobbs, J. R., Hughes, G. R. V. Br. med. J. 1973, iii, 676. Joaquin, O., Botella, J. Lancet, 1973, ii, 1673. Ball, P. A. J., Bartlett, A. Trans. R. Soc. trop. Med. Hyg. 1969, 63, 362.
1. 2.
3. 4. 5.
6. 7. 8.
9. 10. 11. 12.
13. 14.
have
leucocytes,6 of increased cytes.
prompted us to report our findings heparin uptake in cultures of c.F. lymphoMETHOD
JOANNE A. ROBERTSON BARBARA CHERNICK
DAVID
J. SEGAL
ERNEST E. MCCOY
Department of Pediatrics, University of Alberta, Edmonton, Alberta, Canada Previous studies on cystic fibrosis (C.F.) have shown increased mucopolysaccharide synthesis in C.F. cells. The uptake of 35S-heparin sulphonate into purified lymphocytes was examined after 3 days’ culture in medium 199 supplemented with 20% autochthonous plasma. Fourteen C.F. homozygotes, seven siblings, and nine parents all showed significantly greater uptake (P< 0.001) of 35S-heparin than cultures from twenty-three normal controls. There was partial overlap in heparin-uptake values between C.F. patients and their families. This procedure thus differentiates C.F. families from the normal population, but does not differentiate C.F. homozygotes from heterozygotes. It is hoped that either variations in culture methods or a combination of this procedure with other measurements of C.F., such as ciliary dyskinesis or cellular metachromasia, will permit unequivocal identification of C.F. carriers.
Sum ary
INTRODUCTION
IN 1971 an altered uptake of 35S-heparin by leucocytes in culture from patients with cystic fibrosis (C.F.) was described 1 In the light of previous reports of an increased accumulation of sulphated mucopolysaccharides in C.F. leucocytes2 and fibroblasts,3,4 we have also examined 35S-heparin uptake by c.F. cells. Although our results are preliminary, other reports of the effect of heparin on the C.F. ciliary dyskinesis factorand increased sulphate incorporation into C.F.
10 ml. of
(E.D.T.A.) in
blood
venous
was
collected into edetic acid
containers, and the lymphocytes were isolated on a’Ficoll-Hypaque’ discontinuous gradient. The cells (>85% pure) were washed twice with M199 medium (Gibco) and resuspended in medium 199 at 2-5 X 106 cells per ml. 0-2 ml. autochthonous plasma and 0-8 ml. HEPES buffered M199 containing 1 /Ci per ml. [N-sulphonate-35S]-heparin (specific activity 14-26 mCi per g.; Amersham Searle) were added to 0-2 ml. of cell suspension. Two to five cultures were set up for each donor, and were incubated for 72 hours at 37°C. Cultures were harvested by centrifugation (500 g for 5 minutes), the medium was removed by suction, and the cells were resuspended in 2 ml. isotonic saline solution and collected on glass fibre filters. After two rinses with 2 ml. saline solution, the filters were washed with 3 ml. ethanol, dried in a warm oven, and counted in a Packard ’Tricarb’,., using a toluene-based scintillation fluid. Standard decay tables were used to correct raw values for isotope decay in the batch of heparin used. Multiple cultures from the same donor rarely varied more than 15 %. Different donor groups were compared by hierarchical analysis of variance performed on the common logarithms of the raw counts to
vacuum
stabilise variances. RESULTS
The data show that the uptake of S-heparin by from c.F. patients and their families was
lymphocytes
Applying
an
UPTAKE OF
8’S-HEPARIN BY LYMPHOCYTES
F test
the group
to
0001; Av D, P < 0.001; B ficantC v D, not significant. p<
v
means:
C,
not
A
v
B,
r<
significant; B
0001; A v C, D, not signi-
v
Discussion When oxytocin is used to induce labour, it has been shown that the contractile response of the uterus is more important than the specific dose of oxytocic used, and that automatic oxytocin titration utilising the Cardiff infusion pump is a highly efficient method of administering intravenous oxytocin.4 It is logical to use prostaglandins in the same way. The most worrying aspect of the induction of labour with this highly potent substance is the fear that there may be episodes of hypertonus. All of the reported instances of hypertonus associated with prostaglandin infusion have occurred at high initial infusion-rates,5 or after rapid increases in the infusion-rate,6,7 or when relatively high infusion-rates were achieved before the membranes were ruptured.8 Dewhurst9 suggested that infusion-rates should be increased at intervals of more than 30 minutes. Beazley and Gillespie 10 in their series increased their infusion-rates at hourly intervals; the highest infusion-rate they reached to induce labour was 6.7 jug. of prostaglandin E2 per minute (equivalent to 67 µg. of p.G.F2a) but this was only achieved after 5 hours of infusion. The advantages of the Cardiff pump lie in the accuracy with which small concentrations of the drug can be infused in very small volumes of fluid, and the smoothness of the increase of the infusion-rate to the dose required to initiate labour. The automatic safety cut-outs in the
of hypertonic uterine activity are a comfeature. The results of this preliminary surpelling labour induced in this way is likely indicate that vey to be both safe and efficient.
Preliminary Communication IgE LEVELS IN INTERSTITIAL NEPHRITIS
Requests for reprints should be addressed
to
J. R. N.
REFERENCES 1. Karim, S., Trussell, R., Patel, R., Hillier, K. Br. med. J. 1965, iv, 621. 2. Naismith, W., Barr, W. J. Obstet. Gynœc. Br. Commonw. 1973, 80, 531. 3. Johnson, A., Hyatt, D., Newton, J. R., Phillips, L. ibid. (in the press). 4. Francis, J., Turnbull, A., Thomas, F. ibid. 1970, 70, 594. 5. Karim, S. M. M., Filshie, G. M. Lancet, 1970, i, 157. 6. Anderson, G., Covdero, L., Hobbins, J., Speroff, L. Ann. N.Y. Acad. Sci. 1970, 180, 499. 7. Vakhariya, V., Sherman, A. Am. J. Obstet. Gynec. 1972, 113, 2, 212. 8. Gillespie, A., Dewhurst, C., Beazley, J. Br. med. J. 1971, ii, 222. 9. Dewhurst, C. Am. J. Obstet. Gynec. 1972, 113, 2, 221. 10. Beazley, J. M., Gillespie, A. Lancet, 1971, i, 152.
V. E. POLLAK
Division, Veterans Administration Hospital, and Division of Nephrology, Department of Medicine, University of Cincinnati Medical Center,
Renal
Cincinnati, Ohio I. L. BERNSTEIN Division of Immunology, Department of Medicine, University of Cincinnati Medical Center WELLINGTON JAO Department of Pathology, Michael Reese Hospital Medical Center, Chicago, Illinois, U.S.A.
Abnormal levels of
serum IgE
and were
Summary found in 3 of 5 patients developing
nephritis, probably induced by drug of sequential serum samples Measurement therapy. in a single patient showed that the rise of IgE paralleled the onset of interstitial nephritis. interstitial
INTRODUCTION
RAISED levels of serum-IgE have been found in a number of atopic disorders 1-3 and in parasitic infestations.4-6 We have detected increased levels in patients developing interstitial nephritis, probably induced by
drug therapy. PATIENTS AND METHODS
Five patients with the clinicopathological features of interstitial nephritis were studied. Renal tissue obtained by percutaneous renal biopsy was processed for light and electron microscopy 7; semiquantitative analysis of histological features was then done. Serum-IgE concentrations were measured by radioimmunosorbent, using a ’Phadebas ’ IgE test (Pharmacia Laboratories, Piscataway, New Jersey). Normal values in our laboratory were 20-1040 ng. per ml. (geometric mean ±2 s.D.). IgE levels of ten other patients with acute renal failure were also measured. Serum IgG, IgA, IgM and /3iC globulin levels were estimated by radial immunodiffusion.8 RESULTS
event
We thank Mr W. West, of Pye Dynamics, for the loan of the Cardiff mark in infusion system and Dr W. Ashton, of May and Baker, for the r.c.F.2a and a grant to support this project.
M. R. FIRST
B. S. OOI A. T. PESCE
Clinical Data The patients
were four males and one female, ranging in age from 17 to 39. None of them had a past history of atopic disorders, or any evidence of parasitic infestation. The underlying disease process three varied, patients having some infective process antibiotic treatment and the other two being requiring treated for the nephrotic syndrome. A skin rash developed in one patient, while eosinophilia occurred in three patients at about the onset of interstitial nephritis. Renal function deteriorated strikingly in two patients, while the remaining three had only mild but significant rises in serum-creatinine. The drug history was complex because multiple antibiotic combinations in fairly high doses had been given to four patients; of importance is the fact that penicillin or a penicillin analogue was one of the drugs administered. In the fifth patient, only chlorthali-
1255 LABORATORY FINDINGS
IN
FIVE PATIENTS NEPHRITIS
WITH
INTERSTITIAL
—————,————————.————————————————————.———————,———————
Renal Histology
Light microscopy.-Except for the two patients with the nephrotic syndrome who had focal glomerular sclerosis, there were no glomerular lesions. By contrast, striking and disproportionate tubular and interstitial changes were evident. Tubulitis and peritubular inflammation characterised by aggregation of inflammatory cells in close contact with tubular basemembrane were also noted. Distal tubules were severely affected than proximal tubules. The interstitium was infiltrated with abundant numbers of acute and chronic inflammatory cells; a distinctive lesion was the presence of significant numbers of ment
more
Normal values
(±2 s.D.): IgG 6 0-15mg. per ml., IgA 06-29 mg. ml., IgM 0-5-2-0 mg. per ml., IgE 20-1010 ng. per ml., (i1Cglobulin 90-200 mg. per 100 ml.
per
N.D. = not done.
eosinophils.
done, 50 mg. daily, had been administered for three months before onset of the nephritis. Complete recovery of excellent renal function occurred in all patients without evidence of under-
lying glomerular disease. Laboratory Results
laboratory results are shown in the table and in figs. 1 and 2. Serum-IgE levels were abnormally high in three of five patients. In patient 1 the serumIgE fell to 650 ng. per ml. in the convalescent phase of his illness. The relation between IgE levels and the clinical course of the illness in one patient is shown in fig. 2. Baseline levels of 320 ng. per ml. The
rose to
1760 ng. per ml. at about the time when
interstitial
nephritiswas diagnosed.
Unfortunately,
available in the convalescent period. IgE levels of all ten patients with other forms of renal disease were within normal limits. /31C-globulin levels measured in four patients were within normal limits. no sera were
Fig. 2-Sequential IgE levels (on log scale) of patient 5, developing interstitial nephritis on the 22nd day.
Electron-microscopy.-Utrastructural observations confirmed the pathological process visible by light microscopy. In addition, lymphocytes were insinuating between epithelial cells or between
seen
base-
membrane and tubular cells. Of interest were the small amounts of electron-dense material occasionally identified in the’ basement membranes of tubules.
ment
DISCUSSION
Fig. 1--Comparison
of serum-IgE levels (on log scale) between patients with interstitial nephritis and patients with other forms of rapidly progressive renal failure.
Normal IgE levels ± 2 s.D., 20-1010 ng. per ml.
The induction of interstitial nephritis by drug therapy is well documented." The clinical picture, biopsy findings, and recovery ,after discontinuation of drug therapy in our patients accord with the reported experience. The pathogenesis of this lesion is obscure. The occurrence of skin rashes and eosinophilia with the renal lesion suggests that there is an allergic basis for this disorder. The occurrence of raised IgE levels in a number of disorders with peripheral-blood eosinophilia led us to investigate the role of IgE in this disease. In addition, raised IgE concentrations have been recorded in patients with drug reactions while being treated with gold, and the occurrence of a positive Prausnitz-Kustner reaction for the sensitising drug in a patient with renal failure induced by phenazone.11 Three of five patients in this series had raised concentrations of In one patient the rise in IgE paralleled serum IgE. the onset of renal disease. Of the two patients with normal IgE levels, one showed a significant drop from There is a high-normal level during convalescence. no obvious explanation for the normal level in the
1256 It is relevant to note that a lag period of one year reported between infestation by hookworm and a rise in IgE level in a single patient 1 None of the patients had a common underlying pathological process which is known definitely to cause an increase in serum-IgE. We postulate that drug hypersensitivity in these patients evoked the formation of sufficient reaginic antibody to cause raised levels of circulating total IgE.
other.
was
Whether allergen-reaginic antibody complexes are involved in the pathogenesis of these renal lesions is not known. However, the presence of electrondense material in some tubular basement membranes and the pronounced interstitial eosinophilic infiltrate are consistent with a reagin-mediated immune injury. Further studies to characterise the role of IgE in this pathological process are warranted. In any case, measurements of serum-IgE may be a useful diagnostic tool in the differentiation of interstitial nephritis from others giving rise to the syndrome of rapidly progressive renal failure. This work was supported in part by National Institutes of Health research grant AM 17196 and U.S. Public Health Service training grant AM 05509.
INCREASED UPTAKE OF 35S-HEPARIN BY LYMPHOCYTES FROM PATIENTS WITH CYSTIC FIBROSIS
Requests for reprints should be addressed to B. S. 0., Veterans Administration Hospital, 3200 Vine Street, Cincinnati, Ohio 45220, U.S.A. REFERENCES
Berg, T., Johansson, S. G. O. Int. Archs Allergy appl. Immun. 1969, 36, 219. Johansson, S. G. O., Bennich, H. in Gamma Globulins, Structure and Control of Biosynthesis (edited by J. Killander); p. 193. Stockholm, 1967. Juhlin, L., Johansson, S. G. O., Bennich, H., Högman, C. F., Thyresson, N. Archs Derm. 1969, 100, 12. Johansson, S. G. O., Melbin, T., Vahlqvist, B. Lancet, 1968, i, 1118. Hogarth-Scott, R. S., Johansson, S. G. O., Bennich, R. Clin. exp. Immun. 1969, 5, 619. Rosenberg, E. B., Whalen, G. E., Bennich, H., Johansson, S. G. O. New Engl. J. Med. 1971, 283, 1148. Pirani, C. L., Pollak, V. E., Schwartz, F. D. Nephron, 1964, 1, 230. Mancini, G., Vaerman, J. P., Carbonara, A. O., Heremans, J. F. in Proceedings of the 11th Colloquium on Protides of the Biological Fluids (edited by H. Peeters), p. 370. Amsterdam, 1964. Simenhoff, M. L., Guild, W. R., Dammin, G. J. Amer. J. Med. 1968, 44, 618. Baldwin, D. S., Levine, B. B., McCluskey, R. T., Gallo, G. R. New Engl. J. Med. 1968, 279, 1245. Lyons, H., Pinn, V. W., Cortell, S., Cohen, J. J., Harrington, J. T. ibid. 1973, 288, 124. Davis, P., Ezeoke, A., Munro, J., Hobbs, J. R., Hughes, G. R. V. Br. med. J. 1973, iii, 676. Joaquin, O., Botella, J. Lancet, 1973, ii, 1673. Ball, P. A. J., Bartlett, A. Trans. R. Soc. trop. Med. Hyg. 1969, 63, 362.
1. 2.
3. 4. 5.
6. 7. 8.
9. 10. 11. 12.
13. 14.
have
leucocytes,6 of increased cytes.
prompted us to report our findings heparin uptake in cultures of c.F. lymphoMETHOD
JOANNE A. ROBERTSON BARBARA CHERNICK
DAVID
J. SEGAL
ERNEST E. MCCOY
Department of Pediatrics, University of Alberta, Edmonton, Alberta, Canada Previous studies on cystic fibrosis (C.F.) have shown increased mucopolysaccharide synthesis in C.F. cells. The uptake of 35S-heparin sulphonate into purified lymphocytes was examined after 3 days’ culture in medium 199 supplemented with 20% autochthonous plasma. Fourteen C.F. homozygotes, seven siblings, and nine parents all showed significantly greater uptake (P< 0.001) of 35S-heparin than cultures from twenty-three normal controls. There was partial overlap in heparin-uptake values between C.F. patients and their families. This procedure thus differentiates C.F. families from the normal population, but does not differentiate C.F. homozygotes from heterozygotes. It is hoped that either variations in culture methods or a combination of this procedure with other measurements of C.F., such as ciliary dyskinesis or cellular metachromasia, will permit unequivocal identification of C.F. carriers.
Sum ary
INTRODUCTION
IN 1971 an altered uptake of 35S-heparin by leucocytes in culture from patients with cystic fibrosis (C.F.) was described 1 In the light of previous reports of an increased accumulation of sulphated mucopolysaccharides in C.F. leucocytes2 and fibroblasts,3,4 we have also examined 35S-heparin uptake by c.F. cells. Although our results are preliminary, other reports of the effect of heparin on the C.F. ciliary dyskinesis factorand increased sulphate incorporation into C.F.
10 ml. of
(E.D.T.A.) in
blood
venous
was
collected into edetic acid
containers, and the lymphocytes were isolated on a’Ficoll-Hypaque’ discontinuous gradient. The cells (>85% pure) were washed twice with M199 medium (Gibco) and resuspended in medium 199 at 2-5 X 106 cells per ml. 0-2 ml. autochthonous plasma and 0-8 ml. HEPES buffered M199 containing 1 /Ci per ml. [N-sulphonate-35S]-heparin (specific activity 14-26 mCi per g.; Amersham Searle) were added to 0-2 ml. of cell suspension. Two to five cultures were set up for each donor, and were incubated for 72 hours at 37°C. Cultures were harvested by centrifugation (500 g for 5 minutes), the medium was removed by suction, and the cells were resuspended in 2 ml. isotonic saline solution and collected on glass fibre filters. After two rinses with 2 ml. saline solution, the filters were washed with 3 ml. ethanol, dried in a warm oven, and counted in a Packard ’Tricarb’,., using a toluene-based scintillation fluid. Standard decay tables were used to correct raw values for isotope decay in the batch of heparin used. Multiple cultures from the same donor rarely varied more than 15 %. Different donor groups were compared by hierarchical analysis of variance performed on the common logarithms of the raw counts to
vacuum
stabilise variances. RESULTS
The data show that the uptake of S-heparin by from c.F. patients and their families was
lymphocytes
Applying
an
UPTAKE OF
8’S-HEPARIN BY LYMPHOCYTES
F test
the group
to
0001; Av D, P < 0.001; B ficantC v D, not significant. p<
v
means:
C,
not
A
v
B,
r<
significant; B
0001; A v C, D, not signi-
v