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SOME IMMUNOLOGICAL STUDIES OF THE NEPHROTIC SYNDROME OF NIGERIAN CHILDREN
629
the foetus whose delivery subsequently occurs past term can synthesise oestrogens almost as rapidly as the foetus who delivers before term, thus tending to obliterate the trends shown at 34 weeks. If the foetus does play a role in initiating labour, it is not surprising that there was no correlation between maternal pregnanediol excretion and gestation at the onset of labour, for placental progesterone production seems to be almost independent of the foetal circulation (Cassmer 1959). Macnaughton (1967) found the results of maternal pregnanediol excretion too variable to be of much help in assessing the growth and development of the foetus in utero.
Although in the
foetal lamb an intact pituitary-adrenal axis seems essential for the initiation of labour in the pregnant sheep (Liggins et al. 1966), in human pregnancy at mid-term Schwers et al. (1965) have shown that the foetal liver plays a more vital role in the metabolism of cestrogens and hence possibly in the control of the onset of labour than does the adrenal gland. However, as gestation advances, the foetal adrenal may assume a more important role in oestrogen metabolism than it has in midpregnancy. In support of this, Frandsen and Stakemann (1964) found a low excretion of oestrogens in late pregnancy in women pregnant with an anencephalic fcetus in whom the foetal zone of the adrenal was missing or grossly hypoplastic; oestriol excretion was depressed more than twice that of cestrone or oestradiol. In one of their patients where the adrenals of the anencephalic foetus were normal, maternal urinary oestrogen excretion was normal for gestation. Although in individual patients the correlation between oestriol and oestrone was not statistically significant, cases with a high oestriol excretion at the 34th week usually had a low urinary oestrone and an early onset of labour. In cases with a high oestrone output, however, urinary oestriol could be either low or high. The correlation between cestriol and oestrone excretion in these cases may have been upset by synthesis of some cestriol from oestrone in the maternal liver or by differences in the maternal renal excretion of oestrogens. The final answer to the role of the foetus in the initiation of labour in human pregnancy will not be given until further knowledge is acquired of the synthesis and metabolism of steroids by the foetus itself in the latter part of gestation, and of the effects of these metabolites in regulat-
ing myometrial contractility. This work was carried out during the tenure of a grant from the Medical Research Council to A. C. T. All urinary steroid estimations were carried out by Mrs. Sandra Ritchie in the clinical research unit of the Aberdeen Maternity Hospital. We are grateful to Dr. Arnold Klopper for helpful criticism and to Dr. H. G. Lovel, of the M.R.C. Epidemiological Research Unit, Cardiff, for advice on statistical aspects.
Requests for reprints should be addressed to A. C. T., Department of Obstetrics and Gynaecology, Maternity Hospital, Glossop Terrace, Cardiff. REFERENCES
Brown, J. B. (1955) Biochem. J. 60, 185. Cassmer, O. (1959) Acta endocr., Copenh. suppl. 45. Diczfalusy, E., Cassmer, O., Alonso, C., De Miquel, M. (1961) ibid. 38, 31. Frandsen, V. A., Stakemann, G. (1964) ibid. 47, 265. Kirschner, M. A., Wiqvist, N., Diczfalusy, E. (1966) ibid. 53, 584. Klopper, A. (1963) Proc. Ass. clin. Biochem. 2, 163. Billewicz, W. J. (1963) J. Obstet. Gynœc. Br. Commonw. 70, 1024. Michie, E. A., Brown, J. B. (1955) J. Endocr. 12, 209. Turnbull, A. C., Anderson, A. B. M. (1966) J. Obstet. Gynœc. Br. Commonw. 73, 90. Wilson, G. R. (1962) ibid. 69, 28. Liggins, G. C., Holm, L. W., Kennedy, P. C. (1966) J. Reprod. Fert. 12, 419. Macnaughton, M. C. (1967) Am. J. Obstet. Gynec. 97, 998. Palmer, R., Eriksson, G., Wiqvist, N., Diczfalusy, E. (1966) Acta endocr., Copenh. 52, 598. Schwers, J., Eriksson, G., Diczfalusy, E. (1965) ibid. 49, 65. —
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SOME IMMUNOLOGICAL STUDIES OF THE NEPHROTIC SYNDROME OF NIGERIAN CHILDREN
J. F. SOOTHILL M.A., M.B. Cantab., M.R.C.P., M.C.Path. HEAD, DEPARTMENT OF IMMUNOLOGY, UNIVERSITY OF LONDON INSTITUTE OF CHILD HEALTH,
W.C.1
R. G. HENDRICKSE M.D. Cape Town, M.R.C.P.E. PROFESSOR OF
PÆDIATRICS,
UNIVERSITY OF
IBADAN, NIGERIA
The
selectivity of the proteinuria differs significantly in childhood nephrotic syndrome in Nigeria from that in Europe. In most of the Nigerian children, the proteinuria is moderately selective, of the " dog’s-leg " type, but a minority of children with highly selective proteinuria exists. Preliminary results suggest that this provides a means of detecting the steroidresponsive minority in this population. Immunoelectrophoresis studies of the complement component &bgr;1C provides evidence that the predominant disease in this population is an immunological one. &bgr;1C was detected in the first peak of ’ G-200 Sephadex ’ gel filtration of the serum of some of these patients, as well as in the middle peak as normally, showing that it is incorporated into a macromolecular form, as would be anticipated, if it were bound to antigen excess soluble antigen-antibody complexes. This is taken as evidence in support of the idea that this is soluble complex glomerulonephritis and, if so, the antigen should be identifiable. The therapeutic implications are discussed. Summary
Introduction
nephrotic syndrome is very common in children in Nigeria (Hendrickse and Gilles 1963). Epidemiological THE
evidence indicates that it is related to the Plasmodium malariae infection (Gilles and Hendrickse 1963) which reaches a peak in the fifth year of life. This is also when the peak of incidence of nephrotic syndrome occurs in Nigeria, unlike the earlier incidence of this syndrome in
Europe. Contrary to experience elsewhere, in Nigeria steroid therapy is only occasionally successful and many patients are made worse or die of complications of the treatment (Hendrickse 1966). While there can be little doubt that the high incidence of the nephrotic syndrome in Nigerian children is associated with a predominant local cause, namely quartan malaria, there is no reason to believe that other well-recognised causes of the nephrotic syndrome do not contribute to the total incidence of the syndrome. If this is so, a minority of steroid-sensitive individuals would be anticipated in this population. Because of difficulty in identifying this minority and because steroids are ineffective and often harmful to the majority, some children who would benefit from steroids are not receiving such treatment at present. Immunochemically determined differential protein clearances (Soothill 1962) provide an objective means of separating patients with steroid-sensitive nephrotic syndrome, the predominant type in European children which is associated with minimal renal histological abnormality (Blainey et al. 1960, Cameron and White 1965) from most other causes of the nephrotic syndrome. We have investigated differential protein clearance in Nigerian children with the nephrotic syndrome to evaluate this procedure as a method of identifying steroid-sensitive
patients. N2
630 between them.
They gave comparable results in the subsequent
analyses. Results
Fig. I-Malarial nephrosis showing increase of periodic-acid/Schiff positive intercapillary material and endothelial cells, and obliteration of some capillary loops.
One of the ways of producing glomerulonephritis experimentally is by administering soluble antigen-excess, antigen-antibody complexes or inducing their production in vivo (Dixon 1963). Most Nigerian children with the nephrotic syndrome have a form of glomerulonephritis (fig. 1) (Edington and Mainwaning 1966) similar to this experimental lesion. We describe here preliminary studies of serum complement in these children, which throw light upon the possible role of soluble antigen-antibody complex in the xtiology of their disease, and discuss possible therapeutic implications of these findings. This work has been reported in abstract previously (Soothill and Hendrickse 1966).
The clearances of four proteins expressed as a percentage of the clearance of albumin, are plotted in fig. 2 against the molecular weight of each protein, in a manner similar to that used by Blainey, Brewer, Hardwicke, and Soothill (1960). Also on this plot are mean values of similar clearance studies of forty-eight European children with the nephrotic syndrome. The majority of the Nigerian children were much less selective for IgG than were the European ones, though there was very little «2-macroglobulin cleared by either group; the Nigerian children thus represent a fairly homogeneous group of the type of proteinuria which has been called " dog’s-leg ". Clearly, the IgG/albumin clearance ratio provides the ideal separation of these two groups. Values for this ratio for the Nigerian children are shown in the table. They differ significantly (p < 0-001) from similar data for European children (fig. 3). The values for the two known steroid responders, much lower than for most of the other Nigerian children, were plotted in the figure, but were not included in the statistical analysis. Values of antigenically estimated serum complement component &bgr;IC are shown in the table. Satisfactory control data on a similar population are not available, but most of the values are probably normal, though the two patients with the shortest duration of disease may have a rather low
Patients and Methods
nephrosis clinic in the Department of Paediatrics, University College Hospital, Ibadan, were selected randomly for study. Two patients, known to have responded to steroids previously but who were in relapse at the time, were also investigated. All patients had oedema, heavy proteinuria, and low serum-albumin. The blood-urea was normal or only slightly raised (highest value 65 mg. per 100 ml.). Five children attending the children’s emergency room for febrile illness, who had no proteinuria, were studied as controls for the complement observations. Immunochemically determined differential protein clearances were performed by the technique of Soothill (1962). Immunoelectrophoresis of the complement component Pic was performed as described by Soothill (1967a). Serum complement (31c was estimated by the double-diffusion technique described by Ellis and Gell (1958), as modified for inclusion in the differential protein-clearance study. Results were expressed as a percentage of a reference serum from a healthy adult male (R.N.S.) (Soothill 1962). Complement component Pic in a complexed macromolecular form was detected by’G-200 Sephadex ’ gel filtration of 2 ml. of serum on a 45 x 2-5 cm. glass column with ultraviolet monitoring of the eluate. Venous blood was defibrinated and then edetic acid was added, before the serum was separated; the gel filtration was done within 3 hours of collection of the blood. The fractions containing the first half of the first peak were pooled, concentrated tenfold by dialysis against sucrose, and analysed for the &bgr;IC antigen by the double-diffusion precipitin technique in agar. Two rabbit antisera against pic were used, one kindly provided by Dr. D. S. Rowe, and one from Baxter Laboratories. Both gave only a single precipitin line with whole human serum on immunoelectrophoresis and geldiffusion analysis, and a reaction of identity was obtained Attenders
at
the
Fig. 2-Clearance of four plasma proteins-siderophilin (S), IgG, complement component Pic. and (x-macroglobulin—expressed as % clearance of albumin, and plotted against the molecular weight of the protein, for sixteen randomly selected Nigerian children with the nephrotic syndrome. Mean values for forty-eight European children with nephrotic
syndrome
are
indicated
by
detected in the urine, the in each case.
x .
arrow
Where the protein concerned was not indicates the lowest detectable value
631 drome (Soothill 1967a), the two children with highly selective steroid-responsive nephrotic syndrome did not show this abnormality. Protein reacting with the anti-Ric antiserum was detected in the first peak of sephadex G-200 chromatography in three out of the five patients with moderately selective proteinuria studied (including a second study of one), but in the febrile children with no renal abnormality and one of the children with highly selective proteinuria (studied twice) it was not.
NEPHROTIC SYNDROME IN NIGERIAN CHILDREN
Discussion clearances separate the steroid-
Differential protein sensitive European nephrotic syndrome patients, whether adults or children, from the steroid insensitive, by their high selectivity (Blainey et al. 1960, Cameron and White 1965). Our results show that most Nigerian children with nephrotic syndrome have proteinuria much less selective for IgG and, usually lc (though there is very much less oc-macroglobulin cleared-what has been informally called "
value. On immunoelectrophoresis against the anti-&bgr;IC antiserum, eight out of the eleven randomly selected nephrotic children showed the abnormality of the Pic component which has been found in various forms of glomerulonephritis (Soothill 1967a). A control plasma from a healthy man was run with every one of these studies and only the single plc arc was obtained. One of the control children also showed this abnormality. This child had a very severe infection by both Falciparum and Plasmodium malaria parasites, and Salmonella septicxmia. Like most European cases of childhood nephrotic syn-
dog’s-leg " proteinuria) than most European nephrotic children. The IgG/albumin clearance ratio provides the ideal and statistically highly significant means of separating them from European children. The two Nigerian children known to be steroid responsive had very low values for this ratio, and it seems likely that clearance studies of these two proteins alone (a very simple test which can be done on finger-prick blood and a random urine sample) will permit identification of the steroid-responsive minority in this population, and so permit their treatment. A prospective study to confirm this separation is under way. The detection by immunoelectrophoresis of the altered form of the complement component &bgr;IC in the serum of most of the Nigerian children with the nephrotic syndrome confirms the view that their disease is an immunological This finding occurs almost invariably in acute one. nephritis, and in some other forms of nephritis (Soothill 1965, 1967a); it probably represents the breakdown products of &bgr;IC, OC2D, and/or &bgr;IA (West et al. 1967). The finding of normal levels of &bgr;IC in most of these sera is not inconsistent with this, since the qualitative abnormality
may be associated with such normal levels in various other
ratio of IgG albumin for forty-eight British children and sixteen Nigerian children with the nephrotic syndrome.
Fig. 3-Clearance
Of the Nigerian children, random attenders at the clinic indicated 8 and the two known steroid responders, in relapse, indicated X.
are are
forms of nephritis (Soothill 1967a). The detection of this abnormality in one of the control children is new, but hardly surprising since it seems likely that this material is normally formed and rapidly eliminated, but that our techniques are not sensitive enough to detect it in health. This child certainly had a profound antigenic stimulus. It is hoped that some time this procedure will be on a quantitative basis. The aetiological role of antigen-excess soluble antigenantibody complex, though established in experimental nephritis including human serum sickness, remains speculative in other human nephritis (Dixon 1963). Such complexes bind the complement component pic- Plc is normally present in the middle peak of G-200 sephadex gel filtration separation of serum-protein, so detection of Pic in the first peak, which contains the large-size proteins, provides evidence of incorporation of it into some complex or aggregate form. Its presence in this form, in the sera of three of the five Nigerian children with the typical form of nephrotic syndrome studied (all those with illness of shortest duration, and in one the observation was repeated) provides supporting evidence for the hypothesis, based on histology and epidemiology, that this form of nephritis might be of the antigen-excess soluble-complex type. We did not detect this material in the serum of five controls,
632
samples from a girl who had nephrotic syndrome of the selective of highly type proteinuria, and children with febrile illnesses who were infected with malaria parasites. Clearly more data is needed for conclusive interpretation, but our present results strongly suggest a separation of these patients from our controls by this parameter. It seems likely that small amounts of soluble complex are formed normally, but these would be eliminated rapidly and would be below the level of detection by our techniques, as one postulates for the presence of the PIA/oe2D abnormality described above. If this material is confirmed to be antigen-excess soluble complex, it should be possible to identify the antigen, permitting the trial of antigen-specific treatment as postulated by Soothill (1967b). But success will depend on whether an autoimmune process is superimposed. The techniques described here would also permit similar investigation of other forms of human nephritis, possibly leading to a new approach to treatment there too. including tests had
on
of
We thank Dr. H. Goodman and Dr. I. Riha, of the World Health Organisation Immunology Section, who supported the visit of one of us (J. F. S.) to Ibadan and in whose laboratories much of the work was done, the Medical Research Council and the Wellcome Trust for support for the study of Nigerian nephrotic syndrome, and the Medical Research Council for support to the Research Group in Immunology at the London University Institute of Child
Health.
Requests for reprints should be addressed to J. F. S., Department Immunology, University of London Institute of Child Health, 30 Guilford Street, London W.C.I.
of
REFERENCES
Blainey, J. D., Brewer, D. B., Hardwicke, J., Soothill, J. F. (1960) Q. Jl Med. 29, 235. Cameron, J. S., White, R. H. R. (1965) Lancet, i, 463. Dixon, F. J. (1963) Harvey Lect. Series 58. Feldman, J. D., Vazquez, J. J. (1961) J. exp. Med. 113, 899. Edington, G. M., Mainwaning, A. R. (1966) in International Academy of Pathology Monographs no. 6 (edited by F. K. Mostofi and D. E. Smith); p. 488. Baltimore. Ellis, H. A., Gell, P. G. H. (1958) Nature, Lond. 181, 1667. Gilles, H. M., Hendrickse, R. G. (1963) Br. med. J. ii, 27. Hendrickse, R. G. (1966) Int. Congr. Nephrol. Abstracts, p. 208. Washington. Gilles, H. M. (1963) E. Afr. med. J. 40, 186. Soothill, J. F. (1962) J. Lab. clin. Med. 59, 859. (1965) Nephron, 2, 63. (1967a) Clin. exp. Immun. 2, 83. (1967b) Proc. R. Soc. Med. (in the press). Hendrickse, R. G. (1966) Int. Congr. Nephrol. Abstracts, p. 276. Washington. West, C. D., Winter, S., Forristal, J., McConville, J. M., Davis, N. C. (1967) J. clin. Invest. 46, 539. —
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I’m for a National Health Service; I don’t want to set up standards of care within it; we can’t afford two health services, so I’m not in favour of extending private practice or offering inducements to contract out. The position today is that the government is the sole giver of the nation’s Health Service, because it is financed almost wholly out of taxation ... Now that we have no prescription charges and a Health Service contribution that is standing still, our direct participation in financing the service is getting smaller every year. I favour reversing this process, and doing it by a system of charge, graduated according to income and based on the PAYE coding system ... The picture of the impoverished sick being burdened with demands for payment simply is not true. I don’t say that charges would be absolutely painless but I do say that they would cause no hardship. Some think that there would be the tendency for those better-off patients for whom Health charges might be substantial to mov into the private sector. In my view it’s not likely to happen on any scale. All charges would be well below the economic cost, and a great deal less than the fees for private treatment."-DOUGLAS HOUGHTON, Listener, ...
two
Sept. 7, 1967,
RESPONSE TO BYPASS ILEOSTOMY IN ULCERATIVE COLITIS AND CROHN’S DISEASE OF THE COLON
two serum
a recent recurrence
p. 304.
RAYMOND M. KIVEL M.D. California ASSISTANT PROFESSOR OF MEDICINE
KEITH B. TAYLOR D.M. Oxon., M.R.C.P. BARNETT PROFESSOR OF MEDICINE
HARRY
OBERHELMAN, JR.
M.D.
Chicago
PROFESSOR OF SURGERY
STANFORD UNIVERSITY SCHOOL OF
CALIFORNIA, Summary
MEDICINE,
PALO
ALTO,
U.S.A.
Ileostomy, excluding intestinal
contents
from the colon, was done in sixteen with chronic inflammatory disease of the large patients intestine. Ten patients had Crohn’s disease, five had ulcerative colitis, and one could not be classified. These designations were based on histological assessment. Clinical recovery followed operation in the Crohn’s disease group; the only relapse to occur was in one of the two patients who had intestinal continuity restored. In the ulcerative colitis group, on the other hand, relapse was universal; four of the five patients required total colectomy. The response to bypass ileostomy suggests that there is a fundamental difference between ulcerative colitis and Crohn’s disease. Introduction
THE inflammatory process described by Crohn et al. (1932), often called regional ileitis, predominantly affects the small bowel. However, involvement of the colon with lesions indistinguishable from those of regional ileitis has been reported (Colp 1934, Crohn and Rosenak 1936, Wells 1952), and Brooke (1959) and Lockhart-Mummery and Morson (1960, 1964) attempted to define a clinical and pathological entity which they have called " Crohn’s disease of the colon ". Although there is still some lack of agreement about terminology, the term Crohn’s disease of the colon does emphasise that the pathological findings
identical with those of the disease in the small bowel. Crohn’s disease of the large bowel may be segmental or involve the whole organ. A small minority of patients with ulcerative colitis respond poorly to standard medical therapy or become dependent on continuous corticosteroid administration. The preliminary results of a diverting ileostomy in such patients (Truelove et al. 1965) were encouraging, and we adopted a similar approach in both ulcerative colitis and Crohn’s disease of the colon. Patients with Crohn’s disease of the colon improved dramatically when a bypass operation was done, in contradistinction to those with
are
ulcerative colitis. Patients and Methods With two exceptions, the colitis patients selected for operation were assessed as requiring colectomy because of: (1) failure of salicylazosulfapyridine (’ Salazopyrin ’) and corticosteroids to to
produce
a
satisfactory remission; (2) complications related
the colitis or to prolonged corticosteroid therapy; or (3) retardation of growth and maturation in adolescence, due to the disease or to therapy. In case 12 surgery was done primarily to remove an enlarging polyp of the transverse colon, which proved to be inflammatory. In case 1, uncertainty about the cause of his severe illness led
the foetus whose delivery subsequently occurs past term can synthesise oestrogens almost as rapidly as the foetus who delivers before term, thus tending to obliterate the trends shown at 34 weeks. If the foetus does play a role in initiating labour, it is not surprising that there was no correlation between maternal pregnanediol excretion and gestation at the onset of labour, for placental progesterone production seems to be almost independent of the foetal circulation (Cassmer 1959). Macnaughton (1967) found the results of maternal pregnanediol excretion too variable to be of much help in assessing the growth and development of the foetus in utero.
Although in the
foetal lamb an intact pituitary-adrenal axis seems essential for the initiation of labour in the pregnant sheep (Liggins et al. 1966), in human pregnancy at mid-term Schwers et al. (1965) have shown that the foetal liver plays a more vital role in the metabolism of cestrogens and hence possibly in the control of the onset of labour than does the adrenal gland. However, as gestation advances, the foetal adrenal may assume a more important role in oestrogen metabolism than it has in midpregnancy. In support of this, Frandsen and Stakemann (1964) found a low excretion of oestrogens in late pregnancy in women pregnant with an anencephalic fcetus in whom the foetal zone of the adrenal was missing or grossly hypoplastic; oestriol excretion was depressed more than twice that of cestrone or oestradiol. In one of their patients where the adrenals of the anencephalic foetus were normal, maternal urinary oestrogen excretion was normal for gestation. Although in individual patients the correlation between oestriol and oestrone was not statistically significant, cases with a high oestriol excretion at the 34th week usually had a low urinary oestrone and an early onset of labour. In cases with a high oestrone output, however, urinary oestriol could be either low or high. The correlation between cestriol and oestrone excretion in these cases may have been upset by synthesis of some cestriol from oestrone in the maternal liver or by differences in the maternal renal excretion of oestrogens. The final answer to the role of the foetus in the initiation of labour in human pregnancy will not be given until further knowledge is acquired of the synthesis and metabolism of steroids by the foetus itself in the latter part of gestation, and of the effects of these metabolites in regulat-
ing myometrial contractility. This work was carried out during the tenure of a grant from the Medical Research Council to A. C. T. All urinary steroid estimations were carried out by Mrs. Sandra Ritchie in the clinical research unit of the Aberdeen Maternity Hospital. We are grateful to Dr. Arnold Klopper for helpful criticism and to Dr. H. G. Lovel, of the M.R.C. Epidemiological Research Unit, Cardiff, for advice on statistical aspects.
Requests for reprints should be addressed to A. C. T., Department of Obstetrics and Gynaecology, Maternity Hospital, Glossop Terrace, Cardiff. REFERENCES
Brown, J. B. (1955) Biochem. J. 60, 185. Cassmer, O. (1959) Acta endocr., Copenh. suppl. 45. Diczfalusy, E., Cassmer, O., Alonso, C., De Miquel, M. (1961) ibid. 38, 31. Frandsen, V. A., Stakemann, G. (1964) ibid. 47, 265. Kirschner, M. A., Wiqvist, N., Diczfalusy, E. (1966) ibid. 53, 584. Klopper, A. (1963) Proc. Ass. clin. Biochem. 2, 163. Billewicz, W. J. (1963) J. Obstet. Gynœc. Br. Commonw. 70, 1024. Michie, E. A., Brown, J. B. (1955) J. Endocr. 12, 209. Turnbull, A. C., Anderson, A. B. M. (1966) J. Obstet. Gynœc. Br. Commonw. 73, 90. Wilson, G. R. (1962) ibid. 69, 28. Liggins, G. C., Holm, L. W., Kennedy, P. C. (1966) J. Reprod. Fert. 12, 419. Macnaughton, M. C. (1967) Am. J. Obstet. Gynec. 97, 998. Palmer, R., Eriksson, G., Wiqvist, N., Diczfalusy, E. (1966) Acta endocr., Copenh. 52, 598. Schwers, J., Eriksson, G., Diczfalusy, E. (1965) ibid. 49, 65. —
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SOME IMMUNOLOGICAL STUDIES OF THE NEPHROTIC SYNDROME OF NIGERIAN CHILDREN
J. F. SOOTHILL M.A., M.B. Cantab., M.R.C.P., M.C.Path. HEAD, DEPARTMENT OF IMMUNOLOGY, UNIVERSITY OF LONDON INSTITUTE OF CHILD HEALTH,
W.C.1
R. G. HENDRICKSE M.D. Cape Town, M.R.C.P.E. PROFESSOR OF
PÆDIATRICS,
UNIVERSITY OF
IBADAN, NIGERIA
The
selectivity of the proteinuria differs significantly in childhood nephrotic syndrome in Nigeria from that in Europe. In most of the Nigerian children, the proteinuria is moderately selective, of the " dog’s-leg " type, but a minority of children with highly selective proteinuria exists. Preliminary results suggest that this provides a means of detecting the steroidresponsive minority in this population. Immunoelectrophoresis studies of the complement component &bgr;1C provides evidence that the predominant disease in this population is an immunological one. &bgr;1C was detected in the first peak of ’ G-200 Sephadex ’ gel filtration of the serum of some of these patients, as well as in the middle peak as normally, showing that it is incorporated into a macromolecular form, as would be anticipated, if it were bound to antigen excess soluble antigen-antibody complexes. This is taken as evidence in support of the idea that this is soluble complex glomerulonephritis and, if so, the antigen should be identifiable. The therapeutic implications are discussed. Summary
Introduction
nephrotic syndrome is very common in children in Nigeria (Hendrickse and Gilles 1963). Epidemiological THE
evidence indicates that it is related to the Plasmodium malariae infection (Gilles and Hendrickse 1963) which reaches a peak in the fifth year of life. This is also when the peak of incidence of nephrotic syndrome occurs in Nigeria, unlike the earlier incidence of this syndrome in
Europe. Contrary to experience elsewhere, in Nigeria steroid therapy is only occasionally successful and many patients are made worse or die of complications of the treatment (Hendrickse 1966). While there can be little doubt that the high incidence of the nephrotic syndrome in Nigerian children is associated with a predominant local cause, namely quartan malaria, there is no reason to believe that other well-recognised causes of the nephrotic syndrome do not contribute to the total incidence of the syndrome. If this is so, a minority of steroid-sensitive individuals would be anticipated in this population. Because of difficulty in identifying this minority and because steroids are ineffective and often harmful to the majority, some children who would benefit from steroids are not receiving such treatment at present. Immunochemically determined differential protein clearances (Soothill 1962) provide an objective means of separating patients with steroid-sensitive nephrotic syndrome, the predominant type in European children which is associated with minimal renal histological abnormality (Blainey et al. 1960, Cameron and White 1965) from most other causes of the nephrotic syndrome. We have investigated differential protein clearance in Nigerian children with the nephrotic syndrome to evaluate this procedure as a method of identifying steroid-sensitive
patients. N2
630 between them.
They gave comparable results in the subsequent
analyses. Results
Fig. I-Malarial nephrosis showing increase of periodic-acid/Schiff positive intercapillary material and endothelial cells, and obliteration of some capillary loops.
One of the ways of producing glomerulonephritis experimentally is by administering soluble antigen-excess, antigen-antibody complexes or inducing their production in vivo (Dixon 1963). Most Nigerian children with the nephrotic syndrome have a form of glomerulonephritis (fig. 1) (Edington and Mainwaning 1966) similar to this experimental lesion. We describe here preliminary studies of serum complement in these children, which throw light upon the possible role of soluble antigen-antibody complex in the xtiology of their disease, and discuss possible therapeutic implications of these findings. This work has been reported in abstract previously (Soothill and Hendrickse 1966).
The clearances of four proteins expressed as a percentage of the clearance of albumin, are plotted in fig. 2 against the molecular weight of each protein, in a manner similar to that used by Blainey, Brewer, Hardwicke, and Soothill (1960). Also on this plot are mean values of similar clearance studies of forty-eight European children with the nephrotic syndrome. The majority of the Nigerian children were much less selective for IgG than were the European ones, though there was very little «2-macroglobulin cleared by either group; the Nigerian children thus represent a fairly homogeneous group of the type of proteinuria which has been called " dog’s-leg ". Clearly, the IgG/albumin clearance ratio provides the ideal separation of these two groups. Values for this ratio for the Nigerian children are shown in the table. They differ significantly (p < 0-001) from similar data for European children (fig. 3). The values for the two known steroid responders, much lower than for most of the other Nigerian children, were plotted in the figure, but were not included in the statistical analysis. Values of antigenically estimated serum complement component &bgr;IC are shown in the table. Satisfactory control data on a similar population are not available, but most of the values are probably normal, though the two patients with the shortest duration of disease may have a rather low
Patients and Methods
nephrosis clinic in the Department of Paediatrics, University College Hospital, Ibadan, were selected randomly for study. Two patients, known to have responded to steroids previously but who were in relapse at the time, were also investigated. All patients had oedema, heavy proteinuria, and low serum-albumin. The blood-urea was normal or only slightly raised (highest value 65 mg. per 100 ml.). Five children attending the children’s emergency room for febrile illness, who had no proteinuria, were studied as controls for the complement observations. Immunochemically determined differential protein clearances were performed by the technique of Soothill (1962). Immunoelectrophoresis of the complement component Pic was performed as described by Soothill (1967a). Serum complement (31c was estimated by the double-diffusion technique described by Ellis and Gell (1958), as modified for inclusion in the differential protein-clearance study. Results were expressed as a percentage of a reference serum from a healthy adult male (R.N.S.) (Soothill 1962). Complement component Pic in a complexed macromolecular form was detected by’G-200 Sephadex ’ gel filtration of 2 ml. of serum on a 45 x 2-5 cm. glass column with ultraviolet monitoring of the eluate. Venous blood was defibrinated and then edetic acid was added, before the serum was separated; the gel filtration was done within 3 hours of collection of the blood. The fractions containing the first half of the first peak were pooled, concentrated tenfold by dialysis against sucrose, and analysed for the &bgr;IC antigen by the double-diffusion precipitin technique in agar. Two rabbit antisera against pic were used, one kindly provided by Dr. D. S. Rowe, and one from Baxter Laboratories. Both gave only a single precipitin line with whole human serum on immunoelectrophoresis and geldiffusion analysis, and a reaction of identity was obtained Attenders
at
the
Fig. 2-Clearance of four plasma proteins-siderophilin (S), IgG, complement component Pic. and (x-macroglobulin—expressed as % clearance of albumin, and plotted against the molecular weight of the protein, for sixteen randomly selected Nigerian children with the nephrotic syndrome. Mean values for forty-eight European children with nephrotic
syndrome
are
indicated
by
detected in the urine, the in each case.
x .
arrow
Where the protein concerned was not indicates the lowest detectable value
631 drome (Soothill 1967a), the two children with highly selective steroid-responsive nephrotic syndrome did not show this abnormality. Protein reacting with the anti-Ric antiserum was detected in the first peak of sephadex G-200 chromatography in three out of the five patients with moderately selective proteinuria studied (including a second study of one), but in the febrile children with no renal abnormality and one of the children with highly selective proteinuria (studied twice) it was not.
NEPHROTIC SYNDROME IN NIGERIAN CHILDREN
Discussion clearances separate the steroid-
Differential protein sensitive European nephrotic syndrome patients, whether adults or children, from the steroid insensitive, by their high selectivity (Blainey et al. 1960, Cameron and White 1965). Our results show that most Nigerian children with nephrotic syndrome have proteinuria much less selective for IgG and, usually lc (though there is very much less oc-macroglobulin cleared-what has been informally called "
value. On immunoelectrophoresis against the anti-&bgr;IC antiserum, eight out of the eleven randomly selected nephrotic children showed the abnormality of the Pic component which has been found in various forms of glomerulonephritis (Soothill 1967a). A control plasma from a healthy man was run with every one of these studies and only the single plc arc was obtained. One of the control children also showed this abnormality. This child had a very severe infection by both Falciparum and Plasmodium malaria parasites, and Salmonella septicxmia. Like most European cases of childhood nephrotic syn-
dog’s-leg " proteinuria) than most European nephrotic children. The IgG/albumin clearance ratio provides the ideal and statistically highly significant means of separating them from European children. The two Nigerian children known to be steroid responsive had very low values for this ratio, and it seems likely that clearance studies of these two proteins alone (a very simple test which can be done on finger-prick blood and a random urine sample) will permit identification of the steroid-responsive minority in this population, and so permit their treatment. A prospective study to confirm this separation is under way. The detection by immunoelectrophoresis of the altered form of the complement component &bgr;IC in the serum of most of the Nigerian children with the nephrotic syndrome confirms the view that their disease is an immunological This finding occurs almost invariably in acute one. nephritis, and in some other forms of nephritis (Soothill 1965, 1967a); it probably represents the breakdown products of &bgr;IC, OC2D, and/or &bgr;IA (West et al. 1967). The finding of normal levels of &bgr;IC in most of these sera is not inconsistent with this, since the qualitative abnormality
may be associated with such normal levels in various other
ratio of IgG albumin for forty-eight British children and sixteen Nigerian children with the nephrotic syndrome.
Fig. 3-Clearance
Of the Nigerian children, random attenders at the clinic indicated 8 and the two known steroid responders, in relapse, indicated X.
are are
forms of nephritis (Soothill 1967a). The detection of this abnormality in one of the control children is new, but hardly surprising since it seems likely that this material is normally formed and rapidly eliminated, but that our techniques are not sensitive enough to detect it in health. This child certainly had a profound antigenic stimulus. It is hoped that some time this procedure will be on a quantitative basis. The aetiological role of antigen-excess soluble antigenantibody complex, though established in experimental nephritis including human serum sickness, remains speculative in other human nephritis (Dixon 1963). Such complexes bind the complement component pic- Plc is normally present in the middle peak of G-200 sephadex gel filtration separation of serum-protein, so detection of Pic in the first peak, which contains the large-size proteins, provides evidence of incorporation of it into some complex or aggregate form. Its presence in this form, in the sera of three of the five Nigerian children with the typical form of nephrotic syndrome studied (all those with illness of shortest duration, and in one the observation was repeated) provides supporting evidence for the hypothesis, based on histology and epidemiology, that this form of nephritis might be of the antigen-excess soluble-complex type. We did not detect this material in the serum of five controls,
632
samples from a girl who had nephrotic syndrome of the selective of highly type proteinuria, and children with febrile illnesses who were infected with malaria parasites. Clearly more data is needed for conclusive interpretation, but our present results strongly suggest a separation of these patients from our controls by this parameter. It seems likely that small amounts of soluble complex are formed normally, but these would be eliminated rapidly and would be below the level of detection by our techniques, as one postulates for the presence of the PIA/oe2D abnormality described above. If this material is confirmed to be antigen-excess soluble complex, it should be possible to identify the antigen, permitting the trial of antigen-specific treatment as postulated by Soothill (1967b). But success will depend on whether an autoimmune process is superimposed. The techniques described here would also permit similar investigation of other forms of human nephritis, possibly leading to a new approach to treatment there too. including tests had
on
of
We thank Dr. H. Goodman and Dr. I. Riha, of the World Health Organisation Immunology Section, who supported the visit of one of us (J. F. S.) to Ibadan and in whose laboratories much of the work was done, the Medical Research Council and the Wellcome Trust for support for the study of Nigerian nephrotic syndrome, and the Medical Research Council for support to the Research Group in Immunology at the London University Institute of Child
Health.
Requests for reprints should be addressed to J. F. S., Department Immunology, University of London Institute of Child Health, 30 Guilford Street, London W.C.I.
of
REFERENCES
Blainey, J. D., Brewer, D. B., Hardwicke, J., Soothill, J. F. (1960) Q. Jl Med. 29, 235. Cameron, J. S., White, R. H. R. (1965) Lancet, i, 463. Dixon, F. J. (1963) Harvey Lect. Series 58. Feldman, J. D., Vazquez, J. J. (1961) J. exp. Med. 113, 899. Edington, G. M., Mainwaning, A. R. (1966) in International Academy of Pathology Monographs no. 6 (edited by F. K. Mostofi and D. E. Smith); p. 488. Baltimore. Ellis, H. A., Gell, P. G. H. (1958) Nature, Lond. 181, 1667. Gilles, H. M., Hendrickse, R. G. (1963) Br. med. J. ii, 27. Hendrickse, R. G. (1966) Int. Congr. Nephrol. Abstracts, p. 208. Washington. Gilles, H. M. (1963) E. Afr. med. J. 40, 186. Soothill, J. F. (1962) J. Lab. clin. Med. 59, 859. (1965) Nephron, 2, 63. (1967a) Clin. exp. Immun. 2, 83. (1967b) Proc. R. Soc. Med. (in the press). Hendrickse, R. G. (1966) Int. Congr. Nephrol. Abstracts, p. 276. Washington. West, C. D., Winter, S., Forristal, J., McConville, J. M., Davis, N. C. (1967) J. clin. Invest. 46, 539. —
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I’m for a National Health Service; I don’t want to set up standards of care within it; we can’t afford two health services, so I’m not in favour of extending private practice or offering inducements to contract out. The position today is that the government is the sole giver of the nation’s Health Service, because it is financed almost wholly out of taxation ... Now that we have no prescription charges and a Health Service contribution that is standing still, our direct participation in financing the service is getting smaller every year. I favour reversing this process, and doing it by a system of charge, graduated according to income and based on the PAYE coding system ... The picture of the impoverished sick being burdened with demands for payment simply is not true. I don’t say that charges would be absolutely painless but I do say that they would cause no hardship. Some think that there would be the tendency for those better-off patients for whom Health charges might be substantial to mov into the private sector. In my view it’s not likely to happen on any scale. All charges would be well below the economic cost, and a great deal less than the fees for private treatment."-DOUGLAS HOUGHTON, Listener, ...
two
Sept. 7, 1967,
RESPONSE TO BYPASS ILEOSTOMY IN ULCERATIVE COLITIS AND CROHN’S DISEASE OF THE COLON
two serum
a recent recurrence
p. 304.
RAYMOND M. KIVEL M.D. California ASSISTANT PROFESSOR OF MEDICINE
KEITH B. TAYLOR D.M. Oxon., M.R.C.P. BARNETT PROFESSOR OF MEDICINE
HARRY
OBERHELMAN, JR.
M.D.
Chicago
PROFESSOR OF SURGERY
STANFORD UNIVERSITY SCHOOL OF
CALIFORNIA, Summary
MEDICINE,
PALO
ALTO,
U.S.A.
Ileostomy, excluding intestinal
contents
from the colon, was done in sixteen with chronic inflammatory disease of the large patients intestine. Ten patients had Crohn’s disease, five had ulcerative colitis, and one could not be classified. These designations were based on histological assessment. Clinical recovery followed operation in the Crohn’s disease group; the only relapse to occur was in one of the two patients who had intestinal continuity restored. In the ulcerative colitis group, on the other hand, relapse was universal; four of the five patients required total colectomy. The response to bypass ileostomy suggests that there is a fundamental difference between ulcerative colitis and Crohn’s disease. Introduction
THE inflammatory process described by Crohn et al. (1932), often called regional ileitis, predominantly affects the small bowel. However, involvement of the colon with lesions indistinguishable from those of regional ileitis has been reported (Colp 1934, Crohn and Rosenak 1936, Wells 1952), and Brooke (1959) and Lockhart-Mummery and Morson (1960, 1964) attempted to define a clinical and pathological entity which they have called " Crohn’s disease of the colon ". Although there is still some lack of agreement about terminology, the term Crohn’s disease of the colon does emphasise that the pathological findings
identical with those of the disease in the small bowel. Crohn’s disease of the large bowel may be segmental or involve the whole organ. A small minority of patients with ulcerative colitis respond poorly to standard medical therapy or become dependent on continuous corticosteroid administration. The preliminary results of a diverting ileostomy in such patients (Truelove et al. 1965) were encouraging, and we adopted a similar approach in both ulcerative colitis and Crohn’s disease of the colon. Patients with Crohn’s disease of the colon improved dramatically when a bypass operation was done, in contradistinction to those with
are
ulcerative colitis. Patients and Methods With two exceptions, the colitis patients selected for operation were assessed as requiring colectomy because of: (1) failure of salicylazosulfapyridine (’ Salazopyrin ’) and corticosteroids to to
produce
a
satisfactory remission; (2) complications related
the colitis or to prolonged corticosteroid therapy; or (3) retardation of growth and maturation in adolescence, due to the disease or to therapy. In case 12 surgery was done primarily to remove an enlarging polyp of the transverse colon, which proved to be inflammatory. In case 1, uncertainty about the cause of his severe illness led