48 Our initial studies, confirmed in part by Lieberman2 and Hann et al.,5 seem to make tenable the hypothesis that c.F. may have a complement, or complement-mediated, xtiology. Our hypothesis6 that the c.F. ciliary dyskinesia factor (c.D.F.) is possibly a C3a-IgG complex is further strengthened by Danes et al.,7 who show that mostly IgG1 (and IgG;) bind the C.D.F. small-molecular-weight substance, and by Molenaar et al. who demonstrate that C3a links to IgG1 and suggest that the activity of C3a might be evolved on the surface of an immune complex. In addition, Wilson and Jahn 9 reported that cytolysis of Colpidium by c.F. serum may be complement-mediated. We are supporting the hypothesis that C.F. is either a
complement-caused
or
complement-mediated disorder,
in which a C3a-like molecule (C3a, bradykinin 10) either alone or by complexing with IgG gives rise to the physiological and pathological state of c.F. Division of Medical Genetics, Department of Pediatrics, Mount Sinai School of Medicine, City University of New York, New York, New York 10029, U.S.A.
JAMES H. CONOVER ELAINE J. CONOD.
RESISTANT PSEUDOMONADS IN A NEUROSURGICAL UNIT
SIR,-During the past decade gentamicin has been widely used in the treatment of severe infections caused by gram-negative bacilli, particularly Pseudomonas ceruginosa. Its usefulness in treating pseudomonas infections increased with the emergence of carbenicillin-resistant strains,l1 but pseudomonads resistant to gentamicin also began to appear. In the intensive-therapy ward of a nerosurgical unit 2 patients have recently been clinically infected with gentamicin-resistant Ps. aeruginosa. The consumption of gentamicin and carbenicillin in this neurosurgical unit with 56 beds is given in table I. Patient A, who was admitted after a subarachnoid haemorrhage, required ventilation, bladder catheterisation and peritoneal dialysis. Three days after admission his sputum became purulent and klebsiella was isolated; it was also present on two subsequent occasions. Gentamicin was given, to a total of 80 mg. per day, according to the results of the serum-gentamicin assays, since his blood-urea was consistently high (around 140 mg. per .100 ml.) A gentamicin-sensitive strain of Ps. ceruginosa was isolated on July 1, from a urethral swab and a catheter specimen of urine. It continued to appear in his urine, together with leuco-
6. 7.
8.
Conover, J., Conod, E., Hirschhorn, K. Life Sci. 1974, 14, 253. Danes, B. S., Litwin, S., Hutteroth, T., Cleve, H., Bearn, A. G. J. exp. Med. 1973, 137, 1538. Molenaar, J., Muller, M., Engelfriet, C., Pondman, K., J. Immun.
1974, 112, 1444. Wilson, G., Jahn, T. Life Sci. 1974, 15, 551. Conover, J., Beratis, N., Conod, E., Hathaway, P., Hirschhorn, K. Clin. Res. 1973, 21, 531. 11. Lowbury, E. J. L., Kidson, A., Lilly, H. A., Ayliffe, G. A. J., Jones, R. J. Lancet, 1969, ii, 448. 12. Darrell, J. H., Waterworth, P. M. Br. med. J. 1967, ii, 535. 9. 10.
TABLE
I--CONSUMPTION OF INJECTABLE GENTAMICIN AND CARBENICILLIN IN A NEUROSURGICAL UNIT (56 BEDS) -
cytes, and 22 days after its initial isolation was found to be moderately resistant to gentamicin by the disc diffusion method. Gentamicin was stopped, but the gentamicin-resistant pseudomonas persisted in his urine. The patient later died from recurrent subarachnoid haemorrhage. All the isolates of pseudomonas were untypable by the pyocine production method, lysed by typing phage 188/1alone and belonged to serological type 10. Table n includes the minimum inhibitory concentrations (M.iC.) and minimum bactericidal concentrations (M.B.c.) of gentamicin against these isolates; during July the M.I.c. increased 8-fold and the M.B.c. more than 32-fold, but the organism isolated in August was less resistant to gentamicin than the one isolated on July 23. Patient B, admitted with an infected Spitz-Holter valve and pneumococcal meningitis, required bladder catheterisation; subsequent examination of his urine showed leucocytes and a pseudomonas moderately resistant to gentamicin and highly resistant to carbenicillin. This strain was untypable by the pyocine production and phage-typing methods but belonged to serological type 11. Table n includes the M.l.c.s and M.B.C.S of gentamicin and carbenicillin against the pseudomonas. Administration of penicillin and sulphadimidine eradicated the pneumococci and his urinary infection was successfully treated with polymyxin-B sodium methane sulphonate.
Emergence of resistance to gentamicin during therapy has been reported 12,14,15; it probably results from selection by the antibiotic either of bacteria with R-factor-determined ability to inactivate gentamicin enzymatically or of mutants, whose cell walls have become impermeable to gentamicin or which possess an altered ribosome.16 The big difference between the M.i.c. and the M.B.C. of gentamicin for the pseudomonas isolated from patient A on July 23, could be explained by the outgrowth of mutants; the reduced sensitivity to tobramycin suggests an alteration in permeability of the pseudomonas. The necessarily low dose of gentamicin given to this patient probably encouraged the development of resistance 1. 2; resistance decreased after gentamicin therapy was discontinued (table 11). thank the Cross-Infection Reference
Laboratory,
Colindale, for typing the strains of pseudomonas.
We wish
Mr A. E.
to
13. Garrod, L. P., Waterworth, P. M. J. clin. Path. 1969, 22, 534. 14. Waterworth, P. M. ibid. 1972, 25, 979. 15. Hamilton-Miller, J. M. T., Reynolds, A. V., Brumfitt, W. Lancet, 16.
1974, ii, 527. Davies, J. J. infect. Dis. 1971, 124, suppl.
7.
TABLE II-ACTIVITIES OF FOUR ANTIBIOTICS AGAINST PSEUDOMONADS ISOLATED FROM PATIENT A AND PATIENT B
(a) Minimum inhibitory concentrations (µg. per mL). (b) Minimum bactericidal concentrations (µg. per mL). All M.I.C.S. were measured using nutrient broth (pH 7-2 Mg - -
0-4
mg./100 ml.)and
a
1,100 dilution of an overnight broth culture of each organism.
49
Richardson and Mr D. Uttley kindly allowed details of the 2 patients. Public Health Laboratory, St. George’s Hospital,
us to
report clinical
D. V. SEAL
Tooting Grove,
J. E. M. STRANGEWAYS.
London SW17.
ASSAY OF THYMOSIN IN BLOOD
SIR The observation that removal of the thymus gland in adult myasthenia gravis patients, as well as in normal adult guineapigs, results in a rapid loss of the ability of blood lymphocytes to recognise antigen1 suggests a method of estimating the level of the factor in the blood (thymosin) which " opsonises lymphocytes and is necessary for recognition of antigens.1,2 The disappearance of this factor-from the blood begins within 1 hour of removal of the gland and is complete by 24 hours. If lymphocytes from a thymectomised sensitised adult guineapig are used as test cells, then the titre of serum just able to restore recognitional ability may be measured. In this way the level of circulating thymosin can be estimated. Measurements are made by the macrophage electrophoretic mobility (M.E.M.) test.3,4 Guineapigs which had been sensitised to P.P.D. by intradermal injection of 10 µg. P.P.D. (of Mycobacterium tuberculosis) in Freund’s complete adjuvant (0-1 ml.) 10 days previously were thymectomised. Their blood cells before operation were highly sensitised to P.P.D.; 24 hours or more afterwards they failed to respond to this antigen. Addition of 0-1 ml. of increasing dilutions of normal human or guineapig serum restored the reaction to P.P.D. (see accompanying figure). It can be seen that normal guineapig serum is effective in a higher dilution than is normal human serum. Thus, whilst in the blood has an interspecies activity, the thymosin hormone is more effective in its own species. Indeed it may be that autologous thymosin is more effective than homologous, as is found with lymphocyte depressing factor (L.D.F.).5,6L.D.F. is present in normal serum at a dilution of 1/60, but absent at dilutions greater than 1/120. At "
Field, E. J., Bates, D., Shaw, D. A., Griffin, S. G., Shenton, B. K., Smith, J. K. Lancet, 1973, ii, 675. 2. Bach, J.-F., Dardenne, M., Bach, M.-A., Transplant. Proc. 1973, 5, 99. 3. Field, E. J., Caspary, E. A. Lancet, 1970, ii, 1197. 4. Caspary, E. A., Field, E. J. Br. med. J. 1971, i, 612. 5. Field, E. J., Caspary, E. A. Lancet, 1971, ii, 95. 6. Field, E. J., Caspary, E. A. Br. J. Cancer, 1972, 26, 164. 1.
higher dilutions thymosin is still active. At 1/60 any thymosin action is masked by L.D.F. It seems possible that thymosin and L.D.F. may again represent a " brakeaccelerator " mechanism in the control of lymphocyte reactivity. Serum taken from the P.p.D.-sensitised guineapig before it was thymectomised showed a clearly higher titre of thymosin than did normal guineapig serum. It seems that sensitisation is associated with an increased level of thymosin in the blood just as occurs with lymphocyte depressing factor (L.D.F.).5 This method offers a simple and direct means of assay of circulating thymosin (and so assessment of one index of thymic function) in man both in health and disease. It has already been applied to a study of patients thymectomised for myasthenia gravis with special reference to the relation between successful outcome of the operation and completeness of removal of the gland as shown by level of residual thymosin in the blood. such and
The authors would like to express their appreciation for the help of Prof. D. Mucke and Prof. H. Friemel and their staff in the immunology division of the physiological chemistry department of the University of Rostock. B. K. S. was supported by the Newcastle Regional Hospital Board and the work was aided by the M.S. Society and the Multiple Sclerosis Research Fund Ltd.
University of Rostock, Immunological Research Division, Department of Physiological Chemistry, Lenin Allee 70, 25 Rostock, German Democratic Republic, and Institute of Pathology, Newcastle General Hospital, Westgate Road, Newcastle upon Tyne NE4 6BE.
E. J. FIELD B. K. SHENTON.
HETEROGENEITY OF CALCITONIN SIR,-Since the first radioimmunoassay for calcitonin was developed, there has been considerable disagreement concerning basal levels of this hormone in the peripheral blood of man. Some laboratories were unable to demonstrate calcitonin levels in response to calcium infusion. In addition, several investigators have been unable to detect calcitonin in thyroid venous blood, thereby casting doubt upon its physiological relevance in man. We have reported a sensitive and specific radioimmunoassay for calcitonin which is
capable
of
detecting
and
measuring this hormone
in the peripheral blood of most normal persons, and have found that the levels of the hormone change appropriately to perturbations in serum-calcium.l We have also reported that thyroid venous calcitonin levels of persons without disorders of calcium metabolism significantly exceed peripheral values.2 These findings accord with those of and Franchimont.3 We have undertaken an investigation which may elucidate some of the possible causes for the reported discrepancies. Recent studies in our laboratory, using gel-filtration techniques, have demonstrated a heterogeneity of endogenous human calcitonin. For example, using gel filtration on G-75 ’ Sephadex’ at pH 7-5 and pH 2-0, we have been able to separate and identify at least five major immunoreactive calcitonin fractions in the serum of patients with Immunoreactive material was medullary carcinoma. found in the void volume (fraction I), and at apparent molecular weights of 14,000 (fraction II), 5200 (fraction III), 3400 (fraction IV), and 2400 (fraction v). These peaks were completely reproducible on refiltration. The synthetic 32-aminoacid calcitonin monomer gave a single peak on
Heynen
Lymphocytes from P.P.D.-sensitised animal subjected 24 hours previously to thymectomy. Ordinate: response of lymphocytes to P.P.D. (M.E.M. test). 0-1ml. serum added, dilutions as abscissa. Note restoration by serum of ability of lymphocytes to react. This can be used as an assay for
thymosin.
1. 2. 3.
Silva, O. L., Snider, R. H., Becker, K. L. Clin. Chem. 1974, 20, 337. Silva, O. L., Becker, K. L., Doppman, J., et al. Pharmacologist, 1974, 16, 319 (abstract 726). Heynen, G., Franchimont, P. Eur. J. clin. Invest. 1974, 4, 213.
complement-caused
or
complement-mediated disorder,
in which a C3a-like molecule (C3a, bradykinin 10) either alone or by complexing with IgG gives rise to the physiological and pathological state of c.F. Division of Medical Genetics, Department of Pediatrics, Mount Sinai School of Medicine, City University of New York, New York, New York 10029, U.S.A.
JAMES H. CONOVER ELAINE J. CONOD.
RESISTANT PSEUDOMONADS IN A NEUROSURGICAL UNIT
SIR,-During the past decade gentamicin has been widely used in the treatment of severe infections caused by gram-negative bacilli, particularly Pseudomonas ceruginosa. Its usefulness in treating pseudomonas infections increased with the emergence of carbenicillin-resistant strains,l1 but pseudomonads resistant to gentamicin also began to appear. In the intensive-therapy ward of a nerosurgical unit 2 patients have recently been clinically infected with gentamicin-resistant Ps. aeruginosa. The consumption of gentamicin and carbenicillin in this neurosurgical unit with 56 beds is given in table I. Patient A, who was admitted after a subarachnoid haemorrhage, required ventilation, bladder catheterisation and peritoneal dialysis. Three days after admission his sputum became purulent and klebsiella was isolated; it was also present on two subsequent occasions. Gentamicin was given, to a total of 80 mg. per day, according to the results of the serum-gentamicin assays, since his blood-urea was consistently high (around 140 mg. per .100 ml.) A gentamicin-sensitive strain of Ps. ceruginosa was isolated on July 1, from a urethral swab and a catheter specimen of urine. It continued to appear in his urine, together with leuco-
6. 7.
8.
Conover, J., Conod, E., Hirschhorn, K. Life Sci. 1974, 14, 253. Danes, B. S., Litwin, S., Hutteroth, T., Cleve, H., Bearn, A. G. J. exp. Med. 1973, 137, 1538. Molenaar, J., Muller, M., Engelfriet, C., Pondman, K., J. Immun.
1974, 112, 1444. Wilson, G., Jahn, T. Life Sci. 1974, 15, 551. Conover, J., Beratis, N., Conod, E., Hathaway, P., Hirschhorn, K. Clin. Res. 1973, 21, 531. 11. Lowbury, E. J. L., Kidson, A., Lilly, H. A., Ayliffe, G. A. J., Jones, R. J. Lancet, 1969, ii, 448. 12. Darrell, J. H., Waterworth, P. M. Br. med. J. 1967, ii, 535. 9. 10.
TABLE
I--CONSUMPTION OF INJECTABLE GENTAMICIN AND CARBENICILLIN IN A NEUROSURGICAL UNIT (56 BEDS) -
cytes, and 22 days after its initial isolation was found to be moderately resistant to gentamicin by the disc diffusion method. Gentamicin was stopped, but the gentamicin-resistant pseudomonas persisted in his urine. The patient later died from recurrent subarachnoid haemorrhage. All the isolates of pseudomonas were untypable by the pyocine production method, lysed by typing phage 188/1alone and belonged to serological type 10. Table n includes the minimum inhibitory concentrations (M.iC.) and minimum bactericidal concentrations (M.B.c.) of gentamicin against these isolates; during July the M.I.c. increased 8-fold and the M.B.c. more than 32-fold, but the organism isolated in August was less resistant to gentamicin than the one isolated on July 23. Patient B, admitted with an infected Spitz-Holter valve and pneumococcal meningitis, required bladder catheterisation; subsequent examination of his urine showed leucocytes and a pseudomonas moderately resistant to gentamicin and highly resistant to carbenicillin. This strain was untypable by the pyocine production and phage-typing methods but belonged to serological type 11. Table n includes the M.l.c.s and M.B.C.S of gentamicin and carbenicillin against the pseudomonas. Administration of penicillin and sulphadimidine eradicated the pneumococci and his urinary infection was successfully treated with polymyxin-B sodium methane sulphonate.
Emergence of resistance to gentamicin during therapy has been reported 12,14,15; it probably results from selection by the antibiotic either of bacteria with R-factor-determined ability to inactivate gentamicin enzymatically or of mutants, whose cell walls have become impermeable to gentamicin or which possess an altered ribosome.16 The big difference between the M.i.c. and the M.B.C. of gentamicin for the pseudomonas isolated from patient A on July 23, could be explained by the outgrowth of mutants; the reduced sensitivity to tobramycin suggests an alteration in permeability of the pseudomonas. The necessarily low dose of gentamicin given to this patient probably encouraged the development of resistance 1. 2; resistance decreased after gentamicin therapy was discontinued (table 11). thank the Cross-Infection Reference
Laboratory,
Colindale, for typing the strains of pseudomonas.
We wish
Mr A. E.
to
13. Garrod, L. P., Waterworth, P. M. J. clin. Path. 1969, 22, 534. 14. Waterworth, P. M. ibid. 1972, 25, 979. 15. Hamilton-Miller, J. M. T., Reynolds, A. V., Brumfitt, W. Lancet, 16.
1974, ii, 527. Davies, J. J. infect. Dis. 1971, 124, suppl.
7.
TABLE II-ACTIVITIES OF FOUR ANTIBIOTICS AGAINST PSEUDOMONADS ISOLATED FROM PATIENT A AND PATIENT B
(a) Minimum inhibitory concentrations (µg. per mL). (b) Minimum bactericidal concentrations (µg. per mL). All M.I.C.S. were measured using nutrient broth (pH 7-2 Mg - -
0-4
mg./100 ml.)and
a
1,100 dilution of an overnight broth culture of each organism.
49
Richardson and Mr D. Uttley kindly allowed details of the 2 patients. Public Health Laboratory, St. George’s Hospital,
us to
report clinical
D. V. SEAL
Tooting Grove,
J. E. M. STRANGEWAYS.
London SW17.
ASSAY OF THYMOSIN IN BLOOD
SIR The observation that removal of the thymus gland in adult myasthenia gravis patients, as well as in normal adult guineapigs, results in a rapid loss of the ability of blood lymphocytes to recognise antigen1 suggests a method of estimating the level of the factor in the blood (thymosin) which " opsonises lymphocytes and is necessary for recognition of antigens.1,2 The disappearance of this factor-from the blood begins within 1 hour of removal of the gland and is complete by 24 hours. If lymphocytes from a thymectomised sensitised adult guineapig are used as test cells, then the titre of serum just able to restore recognitional ability may be measured. In this way the level of circulating thymosin can be estimated. Measurements are made by the macrophage electrophoretic mobility (M.E.M.) test.3,4 Guineapigs which had been sensitised to P.P.D. by intradermal injection of 10 µg. P.P.D. (of Mycobacterium tuberculosis) in Freund’s complete adjuvant (0-1 ml.) 10 days previously were thymectomised. Their blood cells before operation were highly sensitised to P.P.D.; 24 hours or more afterwards they failed to respond to this antigen. Addition of 0-1 ml. of increasing dilutions of normal human or guineapig serum restored the reaction to P.P.D. (see accompanying figure). It can be seen that normal guineapig serum is effective in a higher dilution than is normal human serum. Thus, whilst in the blood has an interspecies activity, the thymosin hormone is more effective in its own species. Indeed it may be that autologous thymosin is more effective than homologous, as is found with lymphocyte depressing factor (L.D.F.).5,6L.D.F. is present in normal serum at a dilution of 1/60, but absent at dilutions greater than 1/120. At "
Field, E. J., Bates, D., Shaw, D. A., Griffin, S. G., Shenton, B. K., Smith, J. K. Lancet, 1973, ii, 675. 2. Bach, J.-F., Dardenne, M., Bach, M.-A., Transplant. Proc. 1973, 5, 99. 3. Field, E. J., Caspary, E. A. Lancet, 1970, ii, 1197. 4. Caspary, E. A., Field, E. J. Br. med. J. 1971, i, 612. 5. Field, E. J., Caspary, E. A. Lancet, 1971, ii, 95. 6. Field, E. J., Caspary, E. A. Br. J. Cancer, 1972, 26, 164. 1.
higher dilutions thymosin is still active. At 1/60 any thymosin action is masked by L.D.F. It seems possible that thymosin and L.D.F. may again represent a " brakeaccelerator " mechanism in the control of lymphocyte reactivity. Serum taken from the P.p.D.-sensitised guineapig before it was thymectomised showed a clearly higher titre of thymosin than did normal guineapig serum. It seems that sensitisation is associated with an increased level of thymosin in the blood just as occurs with lymphocyte depressing factor (L.D.F.).5 This method offers a simple and direct means of assay of circulating thymosin (and so assessment of one index of thymic function) in man both in health and disease. It has already been applied to a study of patients thymectomised for myasthenia gravis with special reference to the relation between successful outcome of the operation and completeness of removal of the gland as shown by level of residual thymosin in the blood. such and
The authors would like to express their appreciation for the help of Prof. D. Mucke and Prof. H. Friemel and their staff in the immunology division of the physiological chemistry department of the University of Rostock. B. K. S. was supported by the Newcastle Regional Hospital Board and the work was aided by the M.S. Society and the Multiple Sclerosis Research Fund Ltd.
University of Rostock, Immunological Research Division, Department of Physiological Chemistry, Lenin Allee 70, 25 Rostock, German Democratic Republic, and Institute of Pathology, Newcastle General Hospital, Westgate Road, Newcastle upon Tyne NE4 6BE.
E. J. FIELD B. K. SHENTON.
HETEROGENEITY OF CALCITONIN SIR,-Since the first radioimmunoassay for calcitonin was developed, there has been considerable disagreement concerning basal levels of this hormone in the peripheral blood of man. Some laboratories were unable to demonstrate calcitonin levels in response to calcium infusion. In addition, several investigators have been unable to detect calcitonin in thyroid venous blood, thereby casting doubt upon its physiological relevance in man. We have reported a sensitive and specific radioimmunoassay for calcitonin which is
capable
of
detecting
and
measuring this hormone
in the peripheral blood of most normal persons, and have found that the levels of the hormone change appropriately to perturbations in serum-calcium.l We have also reported that thyroid venous calcitonin levels of persons without disorders of calcium metabolism significantly exceed peripheral values.2 These findings accord with those of and Franchimont.3 We have undertaken an investigation which may elucidate some of the possible causes for the reported discrepancies. Recent studies in our laboratory, using gel-filtration techniques, have demonstrated a heterogeneity of endogenous human calcitonin. For example, using gel filtration on G-75 ’ Sephadex’ at pH 7-5 and pH 2-0, we have been able to separate and identify at least five major immunoreactive calcitonin fractions in the serum of patients with Immunoreactive material was medullary carcinoma. found in the void volume (fraction I), and at apparent molecular weights of 14,000 (fraction II), 5200 (fraction III), 3400 (fraction IV), and 2400 (fraction v). These peaks were completely reproducible on refiltration. The synthetic 32-aminoacid calcitonin monomer gave a single peak on
Heynen
Lymphocytes from P.P.D.-sensitised animal subjected 24 hours previously to thymectomy. Ordinate: response of lymphocytes to P.P.D. (M.E.M. test). 0-1ml. serum added, dilutions as abscissa. Note restoration by serum of ability of lymphocytes to react. This can be used as an assay for
thymosin.
1. 2. 3.
Silva, O. L., Snider, R. H., Becker, K. L. Clin. Chem. 1974, 20, 337. Silva, O. L., Becker, K. L., Doppman, J., et al. Pharmacologist, 1974, 16, 319 (abstract 726). Heynen, G., Franchimont, P. Eur. J. clin. Invest. 1974, 4, 213.
