446

McCurdy and co-workers have reported results of studies in two c.L.L. patients heterozygous at the G.-6-P.D. locus.2o 21 Both A and B isoenzyme types were seen in blood-lymphocytes from the first patient, and it was suggested that c.L.L. had a multicellular origin.2O However, the lymphocyte preparation was moderately contaminated with platelets, and it is not known if it consisted predominantly of T or B cells. In contrast, purified B lymphocytes isolated from the second patient showed only one G.-6-P.D., suggesting a unicellular origin of C.L.L.21

Approximately equal

of B and A G.-6-P.D. activities were found in the skin from both of our patients, indicating that they are heterozygous at the G.-6-P.D. locus. In striking contrast to the skin, only a single enzyme type was seen in the B lymphocytes from patient 1, and 95% of the activity in the B-lymphocyte preparation from patient 2 was of one type (A). The simexplanation for the very small amount of type-B enzyme observed in this last patient’s B cells is admixture of the neoplastic B cells with normal cells such as granulocytes or monocytes. The findings of essentially single-enzyme phenotypes in B lymphocytes from our two G.-6-P.D. heterozygotes with C.L.L. and the one reported by McCurdy et al. 21 provide strong confirmation for the assumption, based on Ig studies, that c.L.L. has a clonal origin. Since the Ig system can only be applied to cells that synthesise Ig, it cannot be used to determine if C.L.L. involves a multipotent haemopoietic stem cell. This question is of particular interest since all other chronic proliferative disorders of hxmopoietic cells studied with G.-6-P.D., including chronic myelocytic leukaemia, polycythxmia vera, and myeloid metaplasia, involve stem cells multipotent for granulocytes, monocytes, erythro22 23 Furthermore, in chronic cytes, and platelets.1-3 G.-6-P.D. leuksemia findings suggest that the myelocytic leukaemic stem cell is also multipotent for B lymphocytes.34 In contrast, the results reported here indicate that c.L.L. occurs in a progenitor after the B-cell pathway has diverged from the myeloid and T-lymphocyte pathways. Thus, C.L.L. is the first neoplasm in man so far shown to involve a committed haemopoietic progeniamounts

plest

ABSENCE OF A NEWLY DESCRIBED CYTOCHROME b FROM NEUTROPHILS OF PATIENTS WITH CHRONIC GRANULOMATOUS DISEASE ANTHONY W. SEGAL DAVID WEBSTER

*OWEN T. G. JONES ANTHONY C. ALLISON

Clinical Research Centre, Harrow, and *Department of

Biochemistry, University of Bristol

unique cytochrome b which becomes incorporated into phagocytic vacuoles has been described in human neutrophils. This cytochrome b appears to be situated in the plasma membrane of these cells, and acts as a component of the microbicidal oxidase system. Absence or gross abnormality of this cytochrome b was demonstrated in all four patients with chronic granulomatous disease who were investigated, and reduced concentrations in two mothers

Summary

A

known to be carriers of the disease. Introduction NEUTROPHILIC polymorphonuclear leucocytes (neutrophils) form an important component of the antimicrobial defence system.l These cells must not only phagocytose the invading microbe, but also kill it. A major component of this killing system is dependent upon a burst of oxygen metabolism which is associated with phagocytosis.2 The oxygen is not consumed to provide energy for these cells, which can phagocytose anaerobically,3 but is important in the killing process itself4 In chronic granulomatous disease (C.G.D.) patients are unusually susceptible to bacterial infection as a result of a defect in this oxygen-dependent microbicidal system.s Neutrophils from these patients fail to demonstrate the normal burst of oxygen consumption when they phagocytose particles and are unable to kill certain bacterial particularly those that 5contain catalase and can catabolise hydrogen peroxide. The oxidase enzyme system responsible for the phagocytosis-induced burst of oxygen metabolism has been the

tor. very grateful to Dr Johan Kriel and Dr Clive Sinoff, of the of medicine, Baragwanath Hospital, for sending us materials from these patients for study. We thank Dr Grace Penfold and Dr Ivan A. F. Dukes for haamatological studies and P. Basford, C. Ernst, G. Herner, and C. Whalen for their expert assistance. This research was supported by grants GM 15253 and CA 18029 from the Institute of General Medical Sciences and the National Cancer Institute, National Institutes of Health, Department of Health, Education and Welfare, and by the Medical Research Service of the Veterans Administration. Requests for reprints should be addressed to P. J. F., Medical Service, Veterans Administration Hospital, 4435 Beacon Avenue South, Seattle, Washington 98108, U.S.A.

We

are

department

REFERENCES

Fialkow, P. J., Gartler, S. M., Yoshida, A. Proc. natn. Acad. Sct. U.S.A. 1967, 58, 1468. 2. Fialkow, P. J., Jacobson, R. J., Papayannopoulou, T Am. J. Med. 1977, 63, 1.

125.

3. Fialkow, P. J., Denman, A. M., Singer, J., Jacobson, R. J., Lowenthal, M. N. Proc. V Cold Spring Harbor Conference on Cell Proliferation, Sept. 6-11, 1977 (in the press). 4. Fialkow, P. J., Denman, A. M., Jacobson, R. J., Lowenthal, M. N. J. clin. Invest. (in the press).

5. Wolff, D. A. Methods in Cell Biology; vol. 10, p. 85. New York, 1975. 6. Hudson, L., Hay, F. C. Practical Immunology; p. 201. London, 1976. 7. Bentwich, Z., Douglas, S. D., Skutelsky, E., Kunkel, H. G. J. exp. Med. 8. 9.

1973, 137, 1532. Rosenszajn, L. A., Shohan, D., Kalechman, I. Immunology, 1975, 29, 1641. Galbraith, R. M., Goast, J. M., Fudenberg, H. H. J. exp. Med. 1977, 146,

1821. 10. Wybran, R. J., Cerr, M. C., Fudenberg, H. H.J. clin. Invest. 1972, 51, 2537. 11. Forbes, I. J., Zalewski, P. D. Clin. exp. Immun. 1976, 26, 99. 12. Brown, G., Greaves, M. F. Eur.J. Immun. 1974, 4, 302. 13. Williams, W. J., Beutler, E., Erslev, A. J. Hematology; p. 1393. New York, 1972. 14. Preud’Homme, J. L., Klein, M., Verroust, P., Seligmann, M. Revue eur. Etud. clin. Biol. 1971, 16, 1025. 15. Froland, S. S., Natvig, J. B., Stavem, P. Scand. J. Immun. 1972, 1, 351. 16. Aisenberg, A. C., Bloch, K. J. New Engl.J. Med. 1972, 287, 272. 17. Salsano, F., Froland, S. S., Natvig, J. B., Michaelsen, T. E. Scand. J. Immun. 1974, 3, 841. 18. Schroer, K. R., Briles, D. E., Van Boxel, J. A., Davie, J. M. J. exp. Med.

1974, 140, 1416. 19. Fu, S. M., Winchester, R. J., Feizi, T., Walzer, P. D., Kunkel, H. G. Proc. natn. Acad. Sci. U.S.A. 1974, 71, 4487. 20. McCurdy, P. R. Clin. Res. 1967, 15, 65 (abstr). 21. McCurdy, P. R., Solanki, D., McDermott, R. P. Blood, 1977, suppl. 1, p 1393 (abstr). 22. Jacobson, R. J., Salo, A., Fialkow, P. J. Blood, 1978, 51, 189. 23. Adamson, J. W., Fialkow, P. J., Murphy, S., Prchal, J. F., Steinmann, L. New Engl.J. Med. 1976, 295, 913.

447 and controversy. NADH7 and NADPH"oxidases" have been identified in neutrophils. However, the cell preparations that were used for these experiments were crude homogenates or mixtures of particles, and NADH and NADPH can act as substrates for many dehydrogenase enzymes and can be oxidised by

subject of much investigation

many

compounds including peroxidases.9 Analytical

subcellular fractionation techniques revealed a diaphorase enzyme system in the plasma membrane of human neutrophils and the characteristics of this diaphorase system were shown to be abnormal in neutrophils from patients with C.G.D.11 In some patients the diaphorase system seemed to be present, but could only be demonstrated by the use of an abnormally high concentration OfNADH. Attempts were then made to identify the components of this diaphorase system, and, because cytochromes transfer electrons in the oxidase systems of the endoplasmic reticulum and mitochondria, a search was made for a cytochrome system that could fulfil the oxidase function in neutrophils. A unique cytochrome b has been identified in these cells.12 It was shown to be independent of the cyto-

chrome P450 of the endoplasmic reticulum, and mitochondrial cytochrome oxidase, and to become incorporated into the phagocytic vacuole. Preliminary studies on two patients with C.G.D. showed this cytochrome b to be undetectable in the boy and abnormal in the girl.’2 We have investigated the spectral properties of this cytochromeb in other patients with C.G.D., and in the families of patients with C.G.D. in an attempt to determine the mode of inheritance of the disease in these patients, and, in particular, whether it is possible to identify carriers of the disease.

Subjects

and Methods

Family1 (fig. 1) Both parents and their parents were well and unrelated. The first male child, born in 1961, died at 14 months of age of cervical adenitis and pneumonia. The patient has had suppurative adenitis of the cervical and inguinal regions and a liver abscess in the past but was well at the time of examination. The four female sibs were healthy.

Family 2 (fig. 2) The patient had repeated episodes of cervical adenitis and u

(’[)H)’n

Fig. I-Family Family

tree

1.

’

and dithionite difference spectra of

neutrophil

homo-

genates from members of a family of a patient with chronic granulomatous disease (arrows). Amplitude of spectra has been standardised ,

to a pretem concentration of 4 mg in each cuvette. Absorbence scale marker=0’0056 of full scale. Difference spectrum of pure myeloperoxidase is also shown as a dotted line in this figure.

Fig. 2-Family 2. Legend as for fig.

1.

448

‘

Fig. 3-Family 3. Legend as for fig. 1.

Fig. 4-Family 4. Legend as for fig. 1.

pneumonia but was well at the time of the present study. Other members of the family were well and non-consanguineous. The patient’s father and grandfather could not be traced.

son et

Family 3 (fig. 3) The patient had had repeated episodes of cervical and inguinal adenitis, pneumonia and infected skin lesions. He had a draining inguinal sinus at the time of study. An older brother had repeated pyogenic infections and died at 4tyears of age with were

a liver abscess. The parents and other well and non-consanguineous.

family

members

Family 4 (fig. 4) In this family of East-African Asians the patient’s parents and sister were well and there was no history of consanguinity in the family. The patient had chronic infected eczema of the axillae and inguinal regions and repeated chest infections. She was well at the time of study.

Methods was based upon the diagnosis inability of their neutrophils to kill staphylococci,’3 reduce nitroblue tetrazolium,14 or consume additional oxygen after stimulation with latex particles opsonised with IgG. Neutrophils were purified from 1 dl blood by dextran sedimentation and centrifugation through a gradient of ’Ficoll’/ sodium metrizoate. Residual erythrocytes were removed by hypotonic lysis with 10 ml of water for 45 s. The cells were pelleted, and the hypotonic lysis step was then repeated. The cells were counted, centrifuged at 800g for 10 min, and the pellet was homogenised in (0.34 mol/1) sucrose solution containing (1-0 mmol/1) ethylenediamine tetra-acetate and (5 i.u./ml) heparin at a concentration of 1 x 108 cells/ml in a Dounce homogeniser with 100 strokes of a B pestle.’° Dithionite difference spectra of the homogenate (approximately 8 mg of protein) were determined from 400 to 600 nm by means of a split-

The

of C.G.D. in these patients

beam spectrophotometer.

12

myeloperoxidase was purified by the method of Olsassayed by that of Bretz- and Baggiolini. 11 Protein was assayed as described by Lowry et al. 17 Human

al.15 and

Results Dithionite difference spectra of neutrophil homogenates from normal subjects showed the same features as have been described before 12—i.e., a small peak at 559 nm with a large peak in the position of myeloperoxidase at 474 nm, a lower peak or shoulder at 452 nm, and a sharp peak at 429 nm (fig. 1). The 559 nm peak seems to be a composite one made up of a small sharp peak of the cytochrome b superimposed on a flat broad peak, the identity of which has not yet been established. All four patients with C.G.D. lacked the 429 nm and sharp 559 nm peaks. Patients Q and U had a peak at about 433 nm and the other two had a shoulder in this region. Patients G and M had a peak at 474 nm corresponding to that of myeloperoxidase, whereas this was missing in Q and U. All four had a peak or a shoulder in the region of 452 nm. The absence of the normal myeloperoxidase peak from the spectrum of patients Q and U could be due either to an absence of this enzyme or to a change in its spectrum. The enzyme had previously been assayed and been found to be present in patient U. When peroxidase activity was measured in the homogenates of patients G and Q it was 13.6 and 14.8 milliunits/mg protein compared with 13 2±06 (n=19) milliunits/mg in other members of the families and normal controls studied at the same time. Thus in patient Q the enzyme is present but has a different spectrum,18 possibly because abnormalities of the cytochrome b18 had changed its oxidation state.

449

The two obligate heterozygotes (B and 0) had spectra that resembled mixtures of the normal pattern and that observed in the c.G.D. patients. The spectrum of the mother of the Asian patient also seemed to be midway between the abnormal and the normal and the spectra of the fathers in families 1 and 4 also look abnormal. The spectra of all the relatives of the affected boy in family 2 that we studied appeared normal but we were unable to trace his father; possibly the disease may have developed in this boy as a result of a spontaneous mutation. Discussion These studies confirm the presence of a cytochromeb in human neutrophils, which was undetectable or abnormal in four patients with C.G.D. In C.G.D. the causal biochemical defect and mechanism of genetic transmission is likely to vary from family to family. Although inheritance is thought to be X-linked in boys and autosomal recessive in girls,19 this has not been definitely established because of the absence of a good marker of the disease. Some workers20 believe that the fathers of some affected male patients are abnormal and that in these cases there is an autosomal recessive pattern of inheritance with sex modification. We are unable to draw firm conclusions from our family studies because we do not know the normal variation in the spectral characteristics of neutrophil homogenates. Many normal people will have to be studied to establish this. Nevertheless, two obligate heterozygotes, the mothers in families 1 and 3 who have had two affected sons, had cytochrome b peaks which were detectably smaller than normal. It is surprising that none of the five daughters in these families (four in family 1) seem to be carriers of the disease. In family 2, none of the relatives had a detectably abnormal spectrum. Both parents in family 4 had slightly abnormal spectra with a high peak at 452 nm. These are the only relatives of a female patient that we have studied. This observation would accord with descriptions of an autosomal recessive mode of inheritance in these patients.19 However, both parents are Asian, and we do not know whether neutrophil spectra differ in populations from different continents. The observation that both obligate heterozygotes (subjects B and 0) had abnormally low concentrations of cytochromeb strengthens the case for its involvement in the disease, since both subjects are clinically unaffected. This indicates that the cytochromeb changes are not a manifestation of the C.G.D.

The

direct way in which to link the defect of the cytochrome b to the syndrome of C.G.D. is to propose that it is a component of the oxidase system that transports electrons from the substrate to oxygen. This cytochromeb seems to have spectral properties similar to those of cytochrome o, which by definition is a cytochromeb that binds oxygen and functions as an oxidase.21 Such a cytochrome has not previously been described in higher animals but occurs in bacteria, where the transport of electrons is coupled to a proton pump and A.T.P. synthesis. Although it is generally thought that the oxidase enzyme in neutrophils generates hydrogen peroxide as substrate for a myeloperoxidasemediated microbicidal system,22 it is possible that an important function of the "oxidase system" is to pump protons into the phagocytic vacuole, which is known to become acidic, thereby producing optimum conditions for microbicidal activity. most

The abnormality of this cytochromeb system is probably the molecular basis for the previous observation of an abnormality of a diaphorase enzyme system in the plasma membrane of patients with C.G.D. 11 Although the diaphorase system seemed to be present in these patients, it could only be measured at an abnormally high concentration of the substrate, NADH—a situation that is consistent with an absent or abnormal subunit in an elec-

tron-transporting system. Spectroscopic abnormalities of the cytochrome b of neutrophils have been confirmed in four patients with The identification of this distinct biochemical abnormality should simplify further studies to identify the complete oxidase system, and with further experience it should be possible to clarify the mode of inheritance and carrier status in families with the disease. We thank Dr D. I. K. Evans and Prof. J. Soothill and Prof. M. Greaves for allowing us to study patients under their care and Dr M. Crawfurd for helpful advice. 0. T. G. J. is supported by a grant from C.G.D.

the Science Research Council. Requests for reprints should be addressed to A. W. S., Division of Cell Pathology, Clinical Research Centre, Watford Road, Harrow, Middlesex HA13UJ. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.

Klebanoff, S. J. A. Rev. Med. 1971, 22, 39. Baldridge, C. W., Gerard, R. W. Am. J. Physiol. 1933, 103, 235. Sbarra, A. J., Karnovsky, M. L.J. biol. Chem. 1959, 234, 1355. Mandell, G. L. Infect. Immun. 1974, 9, 337. Holmes, B., Page, A. R.J. clin. Invest. 1967, 46, 1422. Mandell, G. L., Hook, E. W.J. Bact. 1969, 100, 531. Cagan, R. H., Karnovsky, M. L. Nature, 1964, 204, 255. Iyer, G. Y. N., Islam, M. F., Quastel, J. H. ibid. 1961,192, 535. Takanaka, K., O’Brien, P. J. Biochem. Biophys. Res. Commun. 1975, 62,

10. 11. 12. 13.

Segal, A. W., Peters, T. J. Clin. Sci. mol. Med. 1977, 52, 429. Segal, A. W Peters, T.J.Q. Jl Med 1978, 47, 213. Segal, A. W., Jones, O. T. G. Biochem. Soc. Trans. (in the press). Quie, P. G., White, J. G., Holmes, B., Good, R. A.J. clin Invest. 1967, 46,

966.

668. 14. Segal, A. W., Peters, T. J. Clin. Sct. mol. Med. 1975, 49, 591. 15. Olsson, I., Olofsson, T., Odeberg, H. Scand. J. Hœmat. 1972, 16. Bretz, U., Baggiolini, M. J. cell. Biol. 1974, 63, 251. 17. Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R.

9, 483.

J. J. biol. Chem. 1951, 193, 265. 18. Odajima, T., Yamazaki, I. Biochim. biophys Acta. 1972, 284, 360. 19. Babior, B. M. New Engl. J. Med. 1978, 298, 721. 20. Thompson, E. N., Soothill, J. F. Archs Dis Childh, 1970, 45, 24. 21. Lemberg, R., Barrett, J. Cytochromes; p. 226. London, 1973. 22. Klebanoff, S. J. Semin Hæmat. 1975, 12, 117.

"BODY-BRACE" ŒSOPHAGITIS, A COMPLICATION OF KYPHOSCOLIOSIS THERAPY SAMUEL A. KOCOSHIS JOYCE D. GRYBOSKI BIRGIR GUDJONSSON JOHN H. SEASHORE JAMES C. DRENNAN Departments of Pediatrics, Pediatric Surgery, and Medicine,

University School of Medicine, and Department of Orthopedic Surgery, Newington Children’s Hospital, Newington, Connecticut, U.S.A.

Yale

Summary

Oesophagitis developed

in 4

patients,

3

of whom had scoliosis braces and 1 a body cast after surgery for kyphoscoliosis. Symptoms varied from chronic epigastric pain to gastrointestinal hæmorrhage. Prophylaxis of œsophageal disease in children undergoing correction of scoliosis may prevent severe

œsophagitis.