1060
glands in the newborn’6 or to their high dietary phosphate when given cow’s milk,17 seemed to be especially precipitated in the Asian babies by their mother’s low vitamin-D intake"—a factor which sometimes operated in negro and Puerto Rican mothers and babies.19 Vitamin-D status in patients is now best assessed20 by plasma assay of its main circulating metabolite 25-hydroxyvitamin-D (23-o.H.D.), so we have used this method to reinvestigate the problem of vitamin-D requirements in pregnancy. A non-pregnant non-lactating woman requires 70-100 LV. (2-5 pLg) of vitamin D daily.21 The W.H.O. and the Nutrition Board, U.S.A., 22 recommend an intake of 400 i.u. (10 jig) during pregnancy and lactation. An average English diet provides about 100 i.u. of vitamin D so is just sufficient to maintain health under ordinary circumstances.23 The intake of the Asian vegetarian is, however, much lower24 and they are less prone to augment this by exposing their skin to the sun. Clearly if pregnancy increases the vitamin-D requirement they would all be in danger of developing osteomalacia. Our results (figs 1 and 2, tables i and n) show that there was no significant fall in plasma, 23-o.H.D. levels during pregnancy even in the group of Asian vegetarians whose stores must have been minimal on account of their known diets and whose plasma levels were correspondingly a little lower than in the non-vegetarians. We submit that these results argue against the idea that pregnancy increases vitamin-D requirement, and therefore predisposes to osteomalacia. Furthermore, there was nothing to suggest that the fetus suffered in any particular way. The cord bloods showed good transfer of 25-o.H.D. from mother to baby. This latter was to be expected in view of the similar results already obtained by Hillman and Haddad25 in the human and by Haddad et al. 26 in rats. We thank especially Mr R. G. Law and his department for allowing us to use his patients for this study. We are most grateful to the dietitians for
their
diet surveys and
to
Miss Susan Heels for the para-
thyroid-hormone assays. REFERENCES 1. Maxwell, J. P., Miles, L. M. J. Obstet. Gynæc. 1925, 32, 433. 2. Maxwell, J. P. Proc. R. Soc. Med. 1935, 28, 265. 3. Ford, J. A., Davidson, D. C., McIntosh, W. B., Fyfe, W. M., Dunnigan, M. G. Br. med. J. 1973, iii, 211. 4. Davidson, S. Passmore, R., Brock, J. F., Truswell, A. S. Human Nutrition and Dietetics. Edinburgh, 1975. 5. Round, J. M. Br. med. J. 1973, iii, 137. 6. Berry, E. M., Gupta, M. M., Turner, S., Brown, R. ibid. 1973, iv, 640. 7. Haddad, J. G., Chyu, K. J. J. clin. Endocr. 1971, 33, 992. 8. Anson, R. C., Rowe, D. J. F. J. clin. Path., 1970. 23, 499. 9. Yalow, R. S., Berson, S. A. Nature, 1966, 212, 357. 10. Swan, C. H. J., Cooke, W. T. Lancet, 1971, ii, 456. 11. Ford, J. A., Colhoun, E. M., McIntosh, W. B., Dunnigan, M. G. Br. med. J. 1972, ii, 677. 12. Holmes, A. M., Enoch, B. A., Taylor, J. L., Jones, M. E. Q. Jl. Med. 1973,
42, 125. 13. Preece, M. A., Ford, J. A., McIntosh, W. B., Dunnigan, M. G., Tomlinson, S., O’Riordan, J. L. H. Lancet, 1973, i, 907. 14. Felton, D. J. C., Stone, W. D. Br. med. J. 1966, 1521. 15. Vaishnava, H. P., Rizvi, S. N. A. ibid, i, 112. 16. Fairney, A. Jackson, D., Clayton, B. E. Archs. Dis. Childh. 1973, 48, 419. 17. Pincus, J. B., Gittleman, I. F., Marius, N., Bachra, B. Am. J. Dis. Child. 1958, 96, 16. 18. Watney, P. J. M., Chance, G. W., Scott, P., Thompson, J. M. Br. med. J. 1971, ii, 432. 19. Rosen, J. F., Roginsky, G., Nathenson, G., Finberg, L. Am. J. Dis. Child.
1974, 127, 220. 20. Haddad, J. G., Stamp, T. C. B. Am. J. Med. 1974, 57, 57. 21. Tech. Rep. Ser. Wld. Hlth Org. 1970, no. 452, p. 34. 22. Pitkin, R. M., Kaminetzky, H. A., Newton, M. Pritchard, J. A. Obstet. Gynec. 1972, 40, 773. 23. Dent, C. E., Smith, R. Q.Jl. Med. 1969, 38, 195. 24. Lumb, G. A., Mawer, E. B., Stanbury, S. W. Am. J. Med. 1971, 50, 421. 25. Hillman, L. S., Haddad, J. G. J. Pediat. 1974, 84, 742. 26. Haddad, J. G., Boisseau, V., Avioli, L. V. J. Lab. clin. Med. 1971, 77, 908.
INTESTINAL BIOPSY IN THE DIAGNOSIS OF COW’S MILK PROTEIN INTOLERANCE WITHOUT ACUTE SYMPTOMS MARGOT SHINER C. G. D. BROOK
JANET BALLARD S. HERMAN
Medical Research Council Gastroenterology Unit and Department of Pædiatrics, Central Middlesex Hospital, London NW10 4 infants, suspected of cow’s milk protein intolerance, were placed on an elimination diet and then challenged with cow’s milk. None reacted clinically, yet in 2 of the 4 patients jejunal biopsy revealed clear histological, ultrastructural, and immunological changes. It is suggested that these changes are the objective criteria on which the diagnosis of cow’s milk intolerance should be based, and that the clinical evidence derived from milk challenge after an elimination diet may be unreliable.
Summary
Patients and Methods
patients aged 5-11 months were investigated. All presented with failure to thrive and diarrhoea and/or vomiting, and patient 2 had blood in the stools. Only, patient 4 had a family history of atopy, and he had mild eczema and was sensitive to ampicillin. All were investigated to exclude lactose intolerance, enteric infection, and immune deficiency. They subsequently made good progress when changed to soya-bean or hydrolysed casein formula. Patients 2 and 3 had previously been exposed to gluten and this was 4
continued. After 3-12 weeks the infants were admitted for challenge with whole cow’s milk. A baseline jejunal biopsy (0) was taken with a paediatric Crosby capsule after a 12-hour fast, and blood was examined for haemoglobin, serum immunoglobulins, complement (measured as IA) and the R.A.S.T. test for IgE milk-specific antibodies (’Phadebas’). This was followed by the oral administration of a single 30 ml dose of cow’s milk, repeated, in the absence of symptoms, over the next 8 hours to a total of 150 ml. A second jejunal biopsy specimen was taken on the next day after a further 12-hour fast and followed by repeat blood examinations. The biopsy specimens were examined histologically, ultrastructurally, and by immunofluorescent techniques’ by three different observers who were unaware of the clinical details. Additional sources of antisera used since our original paper were IgE (Miles-Yeda Ltd) and C3 complement
(Dakopatts).
Results None of the 4 patients reacted clinically to the mik Haematological data are summarised in table I. Patients 1 and 4 had high initial serum IgE values (90 and 149 I.u.jml respectively) and patient 1 alone had circulating milk specific IgE antibodies. The only changes noted in the serum after the milk challenge were an increase in plA (C3 activator) which was observed in patient 1 at 4 hours and in patient 2 at 5 hours (84 and 90 mg/100 ml respectively). Normal levels had returned after 18 and 23 hours respectively. It is particularly noteworthy that immunoglobulin levels were normal and that no eosinophilia was observed after the challenge. Light microscopy (table 11).- The histology of the pre-challenge biopsies (0) showed a mild partial villous atrophy (N./P.v.A.) in patients 1, 2, and 4. Patient 3 had a normal mucosa. Advanced pathological changes were
1061 TABLE I-SERUM VALUES
N.D.
=
not
determined.
’see text.
TABLE II-SUSPECTED
N Inc. P,C.
COW’S
MILK INTOLERANCE BEFORE AND AFTER MILK CHALLENGE
normal. increased. = plasma cells.
=
P.V.A.
=
B.M./C.T.
noted in the post-challenge biopsies of patients 1 and 2 after 18 and 23 hours respectively. These consisted in shortening and broadening of villi and intense inflammatory-cell infiltration which included polymorphs and mast cells (figs. 1 and 2). The histological appearances did not change in the biopsies of patients 3 and 4,12 and 22 hours respectively after milk challenge. Immunofluorescence of the jejunal mucosa (table II).In patient 1 an increase in IgG, IgE, and C3 complement could be demonstrated extracellularly after challenge. In patient 2, IgM, IgA, and IgE were increased extracellularly and IgE plasma cells were also more numerous. Except for staining of basement membrane and connective tissue with antisera to IgA in patient 3, no other change in the mucosal staining patterns were observed in patients 3 and 4. Electron Microscopy (table tn) Patient 1.-After challenge there were changes affecting the epithelium, subepithelium, and inflammatory cells. The villous epithelial cells showed an increase in lysosomal and other smooth-walled vacuoles, and mitochondrial swelling with partial disruption of the cristas mitochondrales. The microvilli appeared unaffected. There was widening of the connective-tissue spaces below the epithelium, presumably due to oedema. The endothelium of small blood-vessels showed swelling and increased cytoplasmic vacuoles. There was an increase in degranu-
S.V.A.
= =
=
partial villous atrophy. basement membrane and/or connective tissue. subtotal villous atrophy.
lated mast cells, eosinophils, and macrophages, and plasma cells showed dilated cystemae suggesting increased immunoglobulin production. A few polymorphs were also noted. Patient 2.-The mucosal ultrastructure, which was normal before challenge except for some increase in macrophages, showed the most pronounced changes after milk challenge. These were similar to those seen in patient 1, but there were additional cytopathological features: shortening of the villous epithelial microvilli, thickening of the basement membranes of epithelial and endothelial cells, and the appearance of dense, electron-opaque fibres, presumably collagen, in the connective-tissue spaces. Numerous polymorphs and degranulated mast cells were also seen. Patient 3.-The ultrastructure of the jejunal mucosa was entirely normal both before and after challenge. Patient 4.-The noteworthy features present in both pre-challenge and post-challenge mucosae were "inactive" plasma cells in which the cytoplasmic cystemae appeared in parallel fashion with apposed limiting
membranes, and numerous macrophages containing large phagocytic vacuoles partially filled with a dense homogeneous material. We
have
pathological
Discussion previously described the immunochanges occurring after milk challenge in
1062 TABLE III-SUSPECTED
B.M. .
=
basement membrane.
=
degranulated.
COW’S
MILK INTOLERANCE:
ELECTRONMICROSCOPY BEFORE AND AFTER MILK CHALLENGE
Inc. Dec.
the
jejunal mucosa of 2 infants with cow’s milk protein allergy.! In both cases there was a clear clinical reaction within 2 hours of challenge. We have now tested 4 other infants suspected of milk allergy in a similar manner, but none of these showed any evidence of a clinical reaction to the challenge. Our investigations of these infants therefore indicate that cow’s milk protein intolerance may be demonstrated from changes
= =
increased. decreased.
in the jejunal mucosa in the absence of an acute clinical response to cow’s milk challenge. Two recent textbooks of paediatrics2 3 have commented on the lack of rigid criteria for the diagnosis of cow’s milk allergy which would make this condition an acceptable clinical entity. In common with other investigators3-6 we doubt whether the criteria of Goldman et al.7 embrace all patients with this syndrome
Fig. 2-Patient 2: normal villus from jejunal mucosal 3 months after elimination of cow’s milk.
Fig. I-Patient The biopsy E x 200.)
was
taken
immediately before the
milk
biopsy;
challenge. (H
&
2: mucosa, 23 hours after cow’s milk
challenge.
Compared to fig. 1 there is shortening and broadening of the villus, reduced villous epithelial-cell height, elongation of crypts, and intense inflammatory-cell infiltration which includes inter-epithelial lymphocytes and polymorphs. (H & E x 200.)
1063 since at least one of these-the return of symptoms within 48 hours of challenge-has been shown here to be fallible. Anderson and Burke3state that "at present the situation regarding cow’s milk protein intolerance somewhat resembles the diagnosis of cceliac disease it is hoped before intestinal biopsy was introduced that a reliable diagnostic method applicable to ’cow’s milk allergy’ will soon emerge and remove the confusion." We would suggest that the method of detecting milk allergy described in this paper, especially in the absence of clinical reactivity to milk challenge, provides the objective criterion required. We consider routine light microscopy of the pre and post challenge jejunal biopsies to be the most helpful for diagnosis. Our 4 patients had similar clinical presentations and did not react to repeated small milk challenges, at least for the next 48 hours. Patients 1 and 2, however, showed morphological and immunological changes in the jejunal mucosa, as well as evidence of serumcomplement activation after milk challenge. Our preliminary conclusions are that with these techniques we were able to detect those infants who, in the absence of symptoms, show local (mucosal) reactions which can only be attributed to cow’s milk. It is conceivable that, if this insult to the tissue is not detected, chronic malabsorption with villous atrophy will result.4 The time relation between milk challenge and subsequent jejunal biopsy is important. Kuitunen and his colleagues8 carried out their post-challenge biopsies at the onset of symptoms, which took from a few hours to several weeks to develop. We have chosen to repeat the biopsy within 24 hours of challenge so as to minimise the effect on the mucosa of other factors, such as the introduction of different food antigens or intercurrent infections. We have demonstrated that within this short period of time, when only cow’s milk has been added to the elimination diet, severe mucosal damage may occur. Walker-Smith has shown that this damage may persist for 48 hours after a single milk ...
challenge.5 Our previous reportl showed that when biopsy was repeated as early as 6 hours after milk challenge, local immunological changes (increased in IgM, IgA, and IgE plasma cells without C3 complement increase) preceded the pathological changes. At 11 hours both pathological and immunological changes occurred, the latter including increased C3 complement staining. These findings are almost. identical to those which occurred in patients 1 and 2, 18 and 23 hours after milk challenge. The time relation of these changes suggests an early reaginic reaction within the jejunal mucosa (increased mucosal IgE staining and mast-cell degranulation) followed by an Arthus reaction (oedema, endothelial reaction, thickening of basement membranes, appearance of collagen fibres, and polymorph infiltration). We suggest that these two types of local reactions are a consequence of antigenic permeability of the affected mucosa and that they should be sought in the diagnosis of cow’s milk protein intolerance. REFERENCES
1. Shiner, M., Ballard, J., Smith, M. E. Lancet, 1975, i, 136. 2. Silverman, A., Roy, C. C., Cozzetto, F. J. (editors) Pediatric Gastro-enterology;
3.
p. 182. St.
Clinical
Louis, 1971. Anderson, C. M., Burke, V. (editors) Pædiatric Gastro-enterology; p. 225. Oxford, 1975.
Kuitunen, P., Visakorpi, J. K., Savilahti, E., Pelkonen, P. Archs Dis. Childh. 1975, 50, 351. 5. Walker-Smith, J. ibid. p. 347. 6. Freier, S. Clin. Allergy 1973, 3 suppl. p. 597. 7. Goldman, A. S., Anderson, D. W., Sellers, W. A., Saperstein, S., Kniker, W. T., Halpern, S. R. Pediatrics, Springfield, 1963, 32, 425. 8. Kuitunen, P., Rapola, J., Savilahti, E., Visakorpi, J. K. Acta pædiat. scand. 1973, 62, 585. 4.
Addendum The 4 patients have now been followed up for 3 to 6 months after their milk challenge. Patients 1 and 2 are doing well on a replacement diet of soya-bean milk formula for cow’s milk. Patient 3 has been on cow’s milk for 3 months and is thriving despite episodes of recurrent diarrhoea; repeat jejunal biopsy showed a microscopically normal mucosa. Patient 4 has been on a diet excluding cow’s milk or any other milk formula but is still not thriving and continues to have diarrhoea.
DEPRESSED MONOCYTE CHEMOTAXIS DURING ACUTE INFLUENZA INFECTION RALPH SNYDERMAN EUGENIE S. KLEINERMAN CHARLES A. DANIELS Division of Rheumatic and Genetic Disease, Department of Medicine, and Departments of Immunology and Pathology, Duke University Medical Center, Durham, North Carolina 27710, U.S.A.
Summary
The
chemotactic
responsiveness of patients with serologiinfection has been quantified in acute influenza had a significant
monocytes from proven influenza vitro. Individuals with
cally
(P
glands in the newborn’6 or to their high dietary phosphate when given cow’s milk,17 seemed to be especially precipitated in the Asian babies by their mother’s low vitamin-D intake"—a factor which sometimes operated in negro and Puerto Rican mothers and babies.19 Vitamin-D status in patients is now best assessed20 by plasma assay of its main circulating metabolite 25-hydroxyvitamin-D (23-o.H.D.), so we have used this method to reinvestigate the problem of vitamin-D requirements in pregnancy. A non-pregnant non-lactating woman requires 70-100 LV. (2-5 pLg) of vitamin D daily.21 The W.H.O. and the Nutrition Board, U.S.A., 22 recommend an intake of 400 i.u. (10 jig) during pregnancy and lactation. An average English diet provides about 100 i.u. of vitamin D so is just sufficient to maintain health under ordinary circumstances.23 The intake of the Asian vegetarian is, however, much lower24 and they are less prone to augment this by exposing their skin to the sun. Clearly if pregnancy increases the vitamin-D requirement they would all be in danger of developing osteomalacia. Our results (figs 1 and 2, tables i and n) show that there was no significant fall in plasma, 23-o.H.D. levels during pregnancy even in the group of Asian vegetarians whose stores must have been minimal on account of their known diets and whose plasma levels were correspondingly a little lower than in the non-vegetarians. We submit that these results argue against the idea that pregnancy increases vitamin-D requirement, and therefore predisposes to osteomalacia. Furthermore, there was nothing to suggest that the fetus suffered in any particular way. The cord bloods showed good transfer of 25-o.H.D. from mother to baby. This latter was to be expected in view of the similar results already obtained by Hillman and Haddad25 in the human and by Haddad et al. 26 in rats. We thank especially Mr R. G. Law and his department for allowing us to use his patients for this study. We are most grateful to the dietitians for
their
diet surveys and
to
Miss Susan Heels for the para-
thyroid-hormone assays. REFERENCES 1. Maxwell, J. P., Miles, L. M. J. Obstet. Gynæc. 1925, 32, 433. 2. Maxwell, J. P. Proc. R. Soc. Med. 1935, 28, 265. 3. Ford, J. A., Davidson, D. C., McIntosh, W. B., Fyfe, W. M., Dunnigan, M. G. Br. med. J. 1973, iii, 211. 4. Davidson, S. Passmore, R., Brock, J. F., Truswell, A. S. Human Nutrition and Dietetics. Edinburgh, 1975. 5. Round, J. M. Br. med. J. 1973, iii, 137. 6. Berry, E. M., Gupta, M. M., Turner, S., Brown, R. ibid. 1973, iv, 640. 7. Haddad, J. G., Chyu, K. J. J. clin. Endocr. 1971, 33, 992. 8. Anson, R. C., Rowe, D. J. F. J. clin. Path., 1970. 23, 499. 9. Yalow, R. S., Berson, S. A. Nature, 1966, 212, 357. 10. Swan, C. H. J., Cooke, W. T. Lancet, 1971, ii, 456. 11. Ford, J. A., Colhoun, E. M., McIntosh, W. B., Dunnigan, M. G. Br. med. J. 1972, ii, 677. 12. Holmes, A. M., Enoch, B. A., Taylor, J. L., Jones, M. E. Q. Jl. Med. 1973,
42, 125. 13. Preece, M. A., Ford, J. A., McIntosh, W. B., Dunnigan, M. G., Tomlinson, S., O’Riordan, J. L. H. Lancet, 1973, i, 907. 14. Felton, D. J. C., Stone, W. D. Br. med. J. 1966, 1521. 15. Vaishnava, H. P., Rizvi, S. N. A. ibid, i, 112. 16. Fairney, A. Jackson, D., Clayton, B. E. Archs. Dis. Childh. 1973, 48, 419. 17. Pincus, J. B., Gittleman, I. F., Marius, N., Bachra, B. Am. J. Dis. Child. 1958, 96, 16. 18. Watney, P. J. M., Chance, G. W., Scott, P., Thompson, J. M. Br. med. J. 1971, ii, 432. 19. Rosen, J. F., Roginsky, G., Nathenson, G., Finberg, L. Am. J. Dis. Child.
1974, 127, 220. 20. Haddad, J. G., Stamp, T. C. B. Am. J. Med. 1974, 57, 57. 21. Tech. Rep. Ser. Wld. Hlth Org. 1970, no. 452, p. 34. 22. Pitkin, R. M., Kaminetzky, H. A., Newton, M. Pritchard, J. A. Obstet. Gynec. 1972, 40, 773. 23. Dent, C. E., Smith, R. Q.Jl. Med. 1969, 38, 195. 24. Lumb, G. A., Mawer, E. B., Stanbury, S. W. Am. J. Med. 1971, 50, 421. 25. Hillman, L. S., Haddad, J. G. J. Pediat. 1974, 84, 742. 26. Haddad, J. G., Boisseau, V., Avioli, L. V. J. Lab. clin. Med. 1971, 77, 908.
INTESTINAL BIOPSY IN THE DIAGNOSIS OF COW’S MILK PROTEIN INTOLERANCE WITHOUT ACUTE SYMPTOMS MARGOT SHINER C. G. D. BROOK
JANET BALLARD S. HERMAN
Medical Research Council Gastroenterology Unit and Department of Pædiatrics, Central Middlesex Hospital, London NW10 4 infants, suspected of cow’s milk protein intolerance, were placed on an elimination diet and then challenged with cow’s milk. None reacted clinically, yet in 2 of the 4 patients jejunal biopsy revealed clear histological, ultrastructural, and immunological changes. It is suggested that these changes are the objective criteria on which the diagnosis of cow’s milk intolerance should be based, and that the clinical evidence derived from milk challenge after an elimination diet may be unreliable.
Summary
Patients and Methods
patients aged 5-11 months were investigated. All presented with failure to thrive and diarrhoea and/or vomiting, and patient 2 had blood in the stools. Only, patient 4 had a family history of atopy, and he had mild eczema and was sensitive to ampicillin. All were investigated to exclude lactose intolerance, enteric infection, and immune deficiency. They subsequently made good progress when changed to soya-bean or hydrolysed casein formula. Patients 2 and 3 had previously been exposed to gluten and this was 4
continued. After 3-12 weeks the infants were admitted for challenge with whole cow’s milk. A baseline jejunal biopsy (0) was taken with a paediatric Crosby capsule after a 12-hour fast, and blood was examined for haemoglobin, serum immunoglobulins, complement (measured as IA) and the R.A.S.T. test for IgE milk-specific antibodies (’Phadebas’). This was followed by the oral administration of a single 30 ml dose of cow’s milk, repeated, in the absence of symptoms, over the next 8 hours to a total of 150 ml. A second jejunal biopsy specimen was taken on the next day after a further 12-hour fast and followed by repeat blood examinations. The biopsy specimens were examined histologically, ultrastructurally, and by immunofluorescent techniques’ by three different observers who were unaware of the clinical details. Additional sources of antisera used since our original paper were IgE (Miles-Yeda Ltd) and C3 complement
(Dakopatts).
Results None of the 4 patients reacted clinically to the mik Haematological data are summarised in table I. Patients 1 and 4 had high initial serum IgE values (90 and 149 I.u.jml respectively) and patient 1 alone had circulating milk specific IgE antibodies. The only changes noted in the serum after the milk challenge were an increase in plA (C3 activator) which was observed in patient 1 at 4 hours and in patient 2 at 5 hours (84 and 90 mg/100 ml respectively). Normal levels had returned after 18 and 23 hours respectively. It is particularly noteworthy that immunoglobulin levels were normal and that no eosinophilia was observed after the challenge. Light microscopy (table 11).- The histology of the pre-challenge biopsies (0) showed a mild partial villous atrophy (N./P.v.A.) in patients 1, 2, and 4. Patient 3 had a normal mucosa. Advanced pathological changes were
1061 TABLE I-SERUM VALUES
N.D.
=
not
determined.
’see text.
TABLE II-SUSPECTED
N Inc. P,C.
COW’S
MILK INTOLERANCE BEFORE AND AFTER MILK CHALLENGE
normal. increased. = plasma cells.
=
P.V.A.
=
B.M./C.T.
noted in the post-challenge biopsies of patients 1 and 2 after 18 and 23 hours respectively. These consisted in shortening and broadening of villi and intense inflammatory-cell infiltration which included polymorphs and mast cells (figs. 1 and 2). The histological appearances did not change in the biopsies of patients 3 and 4,12 and 22 hours respectively after milk challenge. Immunofluorescence of the jejunal mucosa (table II).In patient 1 an increase in IgG, IgE, and C3 complement could be demonstrated extracellularly after challenge. In patient 2, IgM, IgA, and IgE were increased extracellularly and IgE plasma cells were also more numerous. Except for staining of basement membrane and connective tissue with antisera to IgA in patient 3, no other change in the mucosal staining patterns were observed in patients 3 and 4. Electron Microscopy (table tn) Patient 1.-After challenge there were changes affecting the epithelium, subepithelium, and inflammatory cells. The villous epithelial cells showed an increase in lysosomal and other smooth-walled vacuoles, and mitochondrial swelling with partial disruption of the cristas mitochondrales. The microvilli appeared unaffected. There was widening of the connective-tissue spaces below the epithelium, presumably due to oedema. The endothelium of small blood-vessels showed swelling and increased cytoplasmic vacuoles. There was an increase in degranu-
S.V.A.
= =
=
partial villous atrophy. basement membrane and/or connective tissue. subtotal villous atrophy.
lated mast cells, eosinophils, and macrophages, and plasma cells showed dilated cystemae suggesting increased immunoglobulin production. A few polymorphs were also noted. Patient 2.-The mucosal ultrastructure, which was normal before challenge except for some increase in macrophages, showed the most pronounced changes after milk challenge. These were similar to those seen in patient 1, but there were additional cytopathological features: shortening of the villous epithelial microvilli, thickening of the basement membranes of epithelial and endothelial cells, and the appearance of dense, electron-opaque fibres, presumably collagen, in the connective-tissue spaces. Numerous polymorphs and degranulated mast cells were also seen. Patient 3.-The ultrastructure of the jejunal mucosa was entirely normal both before and after challenge. Patient 4.-The noteworthy features present in both pre-challenge and post-challenge mucosae were "inactive" plasma cells in which the cytoplasmic cystemae appeared in parallel fashion with apposed limiting
membranes, and numerous macrophages containing large phagocytic vacuoles partially filled with a dense homogeneous material. We
have
pathological
Discussion previously described the immunochanges occurring after milk challenge in
1062 TABLE III-SUSPECTED
B.M. .
=
basement membrane.
=
degranulated.
COW’S
MILK INTOLERANCE:
ELECTRONMICROSCOPY BEFORE AND AFTER MILK CHALLENGE
Inc. Dec.
the
jejunal mucosa of 2 infants with cow’s milk protein allergy.! In both cases there was a clear clinical reaction within 2 hours of challenge. We have now tested 4 other infants suspected of milk allergy in a similar manner, but none of these showed any evidence of a clinical reaction to the challenge. Our investigations of these infants therefore indicate that cow’s milk protein intolerance may be demonstrated from changes
= =
increased. decreased.
in the jejunal mucosa in the absence of an acute clinical response to cow’s milk challenge. Two recent textbooks of paediatrics2 3 have commented on the lack of rigid criteria for the diagnosis of cow’s milk allergy which would make this condition an acceptable clinical entity. In common with other investigators3-6 we doubt whether the criteria of Goldman et al.7 embrace all patients with this syndrome
Fig. 2-Patient 2: normal villus from jejunal mucosal 3 months after elimination of cow’s milk.
Fig. I-Patient The biopsy E x 200.)
was
taken
immediately before the
milk
biopsy;
challenge. (H
&
2: mucosa, 23 hours after cow’s milk
challenge.
Compared to fig. 1 there is shortening and broadening of the villus, reduced villous epithelial-cell height, elongation of crypts, and intense inflammatory-cell infiltration which includes inter-epithelial lymphocytes and polymorphs. (H & E x 200.)
1063 since at least one of these-the return of symptoms within 48 hours of challenge-has been shown here to be fallible. Anderson and Burke3state that "at present the situation regarding cow’s milk protein intolerance somewhat resembles the diagnosis of cceliac disease it is hoped before intestinal biopsy was introduced that a reliable diagnostic method applicable to ’cow’s milk allergy’ will soon emerge and remove the confusion." We would suggest that the method of detecting milk allergy described in this paper, especially in the absence of clinical reactivity to milk challenge, provides the objective criterion required. We consider routine light microscopy of the pre and post challenge jejunal biopsies to be the most helpful for diagnosis. Our 4 patients had similar clinical presentations and did not react to repeated small milk challenges, at least for the next 48 hours. Patients 1 and 2, however, showed morphological and immunological changes in the jejunal mucosa, as well as evidence of serumcomplement activation after milk challenge. Our preliminary conclusions are that with these techniques we were able to detect those infants who, in the absence of symptoms, show local (mucosal) reactions which can only be attributed to cow’s milk. It is conceivable that, if this insult to the tissue is not detected, chronic malabsorption with villous atrophy will result.4 The time relation between milk challenge and subsequent jejunal biopsy is important. Kuitunen and his colleagues8 carried out their post-challenge biopsies at the onset of symptoms, which took from a few hours to several weeks to develop. We have chosen to repeat the biopsy within 24 hours of challenge so as to minimise the effect on the mucosa of other factors, such as the introduction of different food antigens or intercurrent infections. We have demonstrated that within this short period of time, when only cow’s milk has been added to the elimination diet, severe mucosal damage may occur. Walker-Smith has shown that this damage may persist for 48 hours after a single milk ...
challenge.5 Our previous reportl showed that when biopsy was repeated as early as 6 hours after milk challenge, local immunological changes (increased in IgM, IgA, and IgE plasma cells without C3 complement increase) preceded the pathological changes. At 11 hours both pathological and immunological changes occurred, the latter including increased C3 complement staining. These findings are almost. identical to those which occurred in patients 1 and 2, 18 and 23 hours after milk challenge. The time relation of these changes suggests an early reaginic reaction within the jejunal mucosa (increased mucosal IgE staining and mast-cell degranulation) followed by an Arthus reaction (oedema, endothelial reaction, thickening of basement membranes, appearance of collagen fibres, and polymorph infiltration). We suggest that these two types of local reactions are a consequence of antigenic permeability of the affected mucosa and that they should be sought in the diagnosis of cow’s milk protein intolerance. REFERENCES
1. Shiner, M., Ballard, J., Smith, M. E. Lancet, 1975, i, 136. 2. Silverman, A., Roy, C. C., Cozzetto, F. J. (editors) Pediatric Gastro-enterology;
3.
p. 182. St.
Clinical
Louis, 1971. Anderson, C. M., Burke, V. (editors) Pædiatric Gastro-enterology; p. 225. Oxford, 1975.
Kuitunen, P., Visakorpi, J. K., Savilahti, E., Pelkonen, P. Archs Dis. Childh. 1975, 50, 351. 5. Walker-Smith, J. ibid. p. 347. 6. Freier, S. Clin. Allergy 1973, 3 suppl. p. 597. 7. Goldman, A. S., Anderson, D. W., Sellers, W. A., Saperstein, S., Kniker, W. T., Halpern, S. R. Pediatrics, Springfield, 1963, 32, 425. 8. Kuitunen, P., Rapola, J., Savilahti, E., Visakorpi, J. K. Acta pædiat. scand. 1973, 62, 585. 4.
Addendum The 4 patients have now been followed up for 3 to 6 months after their milk challenge. Patients 1 and 2 are doing well on a replacement diet of soya-bean milk formula for cow’s milk. Patient 3 has been on cow’s milk for 3 months and is thriving despite episodes of recurrent diarrhoea; repeat jejunal biopsy showed a microscopically normal mucosa. Patient 4 has been on a diet excluding cow’s milk or any other milk formula but is still not thriving and continues to have diarrhoea.
DEPRESSED MONOCYTE CHEMOTAXIS DURING ACUTE INFLUENZA INFECTION RALPH SNYDERMAN EUGENIE S. KLEINERMAN CHARLES A. DANIELS Division of Rheumatic and Genetic Disease, Department of Medicine, and Departments of Immunology and Pathology, Duke University Medical Center, Durham, North Carolina 27710, U.S.A.
Summary
The
chemotactic
responsiveness of patients with serologiinfection has been quantified in acute influenza had a significant
monocytes from proven influenza vitro. Individuals with
cally
(P
