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EVIDENCE OF CYTOTOXICITY IN ULCERATIVE COLITIS FROM IMMUNOFLUORESCENT STAINING OF THE RECTAL MUCOSA
1014 a myocardial antigenic factor in some cases commilk protein, in some cases common to egg white protein, and in some cases common to both. This seems untenable.
postulate mon to
We believe that the relationship with food antigenicity is one of cause and effect. The possession of antibody to cow’s milk protein and egg white measured soon after infarction seems to be highly predictive of death. We think that these antibodies are of considerable significance and have a causal relationship through an immunological mechanism. An immunological mechanism in atherosclerotic cardiovascular disease could operate in several ways, either directly through the formation of toxic complexes,3 through disturbance of complement" or indirectly by action on platelets or fibrinogen. The almost ubiquitous occurrence of atherosclerosis means that if it has an immunological basis, antigens must be operative in all or most of us. Food provides such a source throughout our lives.s We have examined only three food antigens, and two seem to be implicated. Both the milk and egg antigen used were derived from heated foodstuff, and there is evidence that heating of a protein modifies its antigenicity. Processed foods could, therefore, constitute an almost unlimited source of possible antigens. The antigens we have studied were crude mixtures of proteins, and so further work on other possible antigens, preferably purified single proteins, required.
is
now
We thank all
hospital staff who have cooperated with us. The Carmarthen was supported by an M.R.C. project grant. Requests for reprints should be addressed to D. F. D.
work
at
REFERENCES 1. 2.
3. 4. 5. 6. 7. 8. 9. 10.
Keys, A. et al. Circulation, 1972, 45, 815. Davies, D. F., Davies, J. R., Richards, M. A. J. Atheroscler. Res. 1969, 9, 103. Davies, D. F. ibid. 1969, 10, 253. Davies, D. F. Am. Heart J. 1971, 81, 289. Poston, R. N., Davies, D. F. J. Atheroscler. Res. 1974, 19, 353. Annan, W., Isherwood, D. M. J. med. Lab. Technol. 1969, 26, 202. Rees, B. W. G. ibid. 1973, 30, 167. Siegel, S. Nonparametric Statistics for the Behavioural Sciences. New York, 1956. Ehrenfield, E. N., Gery, I., Davies, A. M. Lancet, 1961, i, 1138. Geertinger, P., Sørensen, H. J. Atheroscler. Res. 1973, 18, 65.
EVIDENCE OF CYTOTOXICITY IN ULCERATIVE COLITIS FROM IMMUNOFLUORESCENT STAINING OF THE RECTAL MUCOSA
JANET BALLARD
MARGOT SHINER
Medical Research Council Gastroenterology Unit, Central Middlesex Hospital, Park Royal, London NW10 7NS
rectal mucosal biopsy from 11 patients with specimens ulcerative colitis and five biopsy specimens from 5 control patients were studied by tissue immunofluorescence. In all patients with ulcerative colitis, the number of IgG-containing plasma-cells was greatly increased, being highest in those with the most active disease. There was also intense staining
Sum ary
Seventeen
of the connective tissue of the rectal mucosa with IgG antisera and a similar staining pattern was seen with the C3 component of complement in these patients. IgA plasma-cells were increased in ten out of seventeen biopsy specimens, and IgM plasma-cells were increased in six out of seventeen specimens. This great increase of IgG plasma-cells may be due to stimulation by bacterial antigens, especially by the strictly anaerobic bacteria, since IgG from the rectal mucosa in ulcerative colitis is known to react strongly with these organisms. The finding of complement deposition in the rectal mucosa suggests that a cytotoxic reaction is occurring and may account for the pathological features, such as ulceration, seen in ulcerative colitis. Introduction
NEITHER the cause nor the mechanism involved in ulcerative colitis has been established despite extensive investigations.1 Immunological reactions are thought to be important, but their exact nature remains unknown. An antibody, or antibodies, has been found in the rectal mucosa of patients with ulcerative colitis which reacted with certain of the obligate anaerobic faecal bacterial We have examined the type and distribution of immunoglobulins in the rectal mucosa in ulcerative colitis and the evidence of cytotoxic reactions involving complement. Reports on tissue immunoglobulins in ulcerative colitis conflict. Gelzayd et al.1 reported that both IgG and IgA plasma-cells were reduced in the rectal mucosa of mild or moderately active colitis, whereas Søltoft et a1.4 found the opposite. To our knowledge, there have been no reports on mucosal complement in ulcerative colitis. Materials and Methods
patients with ulcerative colitis were studied; 4 were investigated on more than one occasion. The clinical activity of the colitis, the radiological and histological findings, and the treatment at the time of study are given in the accompanying table. Patient 5 was studied three times (a, b, c) during his stay in hospital, and again fifteen months later (d). Patient 6 was studied before (a) and sixteen months after (b) colectomy and ileostomy, when the rectum was left with a view to eventual ileorectal anastomosis. Patient 9 was investigated during an acute relapse (a) and again during a moderately active phase (b). Patient 11 had proctocolitis which, at investigation, was restricted to the lower rectum. Biopsy specimens were taken 8 cm. (a) and 17 cm. (b) from the anus. The control group consisted of 5 patients with diarrhcea or constipation in whom the diagnosis of ulcerative colitis or Crohn’s disease had been excluded. Rectal biopsies were taken via a sigmoidoscope and cut in half; one portion was kept for routine histology and the other was snap-frozen in liquid nitrogen. The latter was stored at -80°C for study by immunofluorescence. The direct immunofluorescence technique5 was used with fluorescein-isothiocyanate-conjugated antisera to IgG, IgM, IgA, and C3 complement (Wellcome Reagents and Hyland Laboratories). The sections were studied in random order on a Leitz ’Orthoplan’ microscope. Between 2 and 5 slides of each type were studied without prior knowledge of the antiserum used, and the number of plasma-cells seen was graded on a scale from 1 to 6, the upper limits of normal being 1 for IgG, 2 for IgM, and 4 for IgA. The staining of the connective tissue seen with 11
1015 was graded as absent (0), positive (+), strongly positive (+ +), a normal result being 0.
C3 antiserum or
Results In the 5 control patients, the cell immunoglobulin was IgA
predominant plasma(grade 4) (see table). IgG-containing plasma-cells (fig. 1) were seen only occasionally (grade 0 or 1), with IgM cells being slightly more numerous (grade 2). In the normal rectum, no staining was seen for the C3 component of complement. A fine outline of the connective tissue was always seen with IgG antiserum and has also been reported in the normal jejunum.s In contrast, large numbers of IgG plasma-cells were seen in the rectal mucosae from all patients with active ulcerative colitis (grades 3 to 5) as well as intense staining of the connective tissue and basement membrane (fig. 2). In six biopsy specimens, IgG plasma-
cells outnumbered IgA cells, although these were often also increased. Connective-tissue staining was also seen in eight of the seventeen biopsy specimens tested with IgA antiserum. In the cases with histologically inactive colitis (patients 10 and llb), the number of IgG cells remained slightly increased (grade 2). In six of seventeen biopsy specimens,
IgM plasma-cells grade 3).
were
moderately increased (to
In nine of the seventeen specimens, fluorescence of the connective tissue was strongly positive (+ +) with antiserum to the C3 component of complement (fig. 3). In four biopsy specimens staining was positive (+) and in four it was absent. Of the latter four, the rectal mucosa showed only moderate activity in 2 patients (5d and 9b) and inactivity in the other 2 (10 and llb). Where crypt abscesses were seen, the material in
DATA ON PATIENTS WITH ULCERATIVE COLITIS AND CONTROLS
= not done. N.R. = not repeated. A.C.C. = active, chronic colitis (defined as acute and chronic inflammations mucus depletion of epithelium M.A.C. =moderately active colitis (acute and chronic inflammation only). C.I.C. = chronic, inactive colitis (chronic innammation, often with thin mucosa and crypt distortion).
N.D.
and crypt abscesses).
1016
Fig. 1-Rectal
mucosa
from
a
control patient stained with IgG
antisera.
Few stained plasma-cells are seen (small arrows), and the basement membrane and connective tissue are finely outlined (large arrows). E == epithelium. ( x 225.)
Fig. 3-Rectal mucosa from patient lla, showing C3-complement deposition in connective tissue and basement membrane. No stained cells are seen. E= epithelium. ( x 225.)
We chose our method of grading beexperience of the variable distribution of plasma-cells throughout the mucosa, and it has proved entirely consistent. Certain bacteria in the fasces of patients with ulcerative colitis possessed antigen(s) which reacted in vitro with antibody in rectal mucosal homogenates from such patients.2 These bacteria were obligate anaerobes and the reaction involved mainly IgG, and to a lesser extent IgA. Complement was not taken up by this reaction, as shown by negative staining for C3. The IgG involved was not circulating immunoglobulin because the result was negative when the patient’s serum was tested against the fscal
superfluous. cause
of
our
anaerobes. emerge from our study: (1) The increase in IgG plasma-cells in the rectal mucosa suggests that the bacterial antigen stimulates local, and not systemic, IgG production. This is noteworthy because IgA is normally the major mucosal immunoglobulin and IgG is the major serum-immunoglobulin. (2) The intense staining of connective tissue with C3 complement and also with IgG and IgA antisera indicates that there has been a reaction between these immunoglobulins and antigen (?bacterial) in this region and that
Several
Fig. 2-Rectal mucosa from patient lla stained for IgG. Stained plasma-cells in the lamina propria are greatly increased in number. Connective tissue and basement membrane are also stained. A small, fluorescent abscess (A) is seen in the lumen of a crypt. E=epithelium. (x 225.)
the crypt lumen fluoresced brightly with IgG, and sometimes IgA, antisera, but rarely with IgM and only once with C3 antisera. In most biopsy specimens from patients with ulcerative colitis, the number of macrophages, polymorphs, and eosinophils were usually increased and easily identified by their red autofluorescence. The extent of infiltration with plasma-cells did not seem to be affected by treatment. The immunofluorescence results of repeat biopsies in patients 5, 6, and 9 showed some relation to the patients’ clinical condition, in that the IgG grading and/or C3 staining were less when the disease was less active. The correlation between sigmoidoscopical and histological appearances and immunofluorescence grading in biopsy specimens a and b from
patient
11
was
good. Discussion
The present study has demonstrated an increase in rectal mucosal plasma-cells producing IgG, and also IgA, in ulcerative colitis. This increase in IgG plasma-cells was so great as to make quantitation
points
complement was taken up. (3) This complement-binding complex would be cytotoxic and produce the pathological features of colitis (i.e., vascular congestion, inflammation, ulceration, and abscesses). (4) The complement-requiring reaction of IgG with antigen found in the mucosa may well be the same reaction that occurs in vitro between bacteria and IgG from the mucosal homogenate, except that there is no free complement to complete the reaction in vitro. The mucosal complement would be bound in complexes with IgG and antigen but mucosal IgG liberated from plasma-cells during homogenisation of the tissue would be free to react with faecal anaerobes in vitro. This possibility is being tested.
(5) The increase in mucosal IgA plasma-cells and connective-tissue staining with IgA antiserum in ulcerative colitis suggests that this immunoglobulin is also involved in immune-complex formation. If this complex binds complement, it could do so by invoking the alternate pathway. We noted that the material in the crypt abscesses
1017 with antiserum to IgG, and to a lesser extent with IgA, but not with complement. Hence, there could be a free exchange of antigen and antibody across the mucosa, but the formation of cytotoxic complexes may be confined to the mucosa. The present study attempted to relate bacterial antigens to mucosal pathology in ulcerative colitis. It does not exclude the possibility that there are other sources of antigen leading to stimulation of local IgG antibody, with the formation of complexes which bind complement. Such antigen could be a common mucosal antigen related to Escherichia coli 014 8 or to reticulin 9 or to any combination of anaerobes and aerobes. Many of the points we raise can, and should, be tested, to give a fuller understanding of the local (mucosal) immunological processes and their relation to the pathology of ulcerative colitis.
stained
brightly
Requests for reprints should be addressed
to
J.
B.
REFERENCES 1. 2.
3. 4.
5. 6. 7. 8. 9.
Kraft, S. C., Kirsner, J. B. Gastroenterology, 1971, 60, 922. Monteiro, E. Fossey, J., Shiner, M., Drasar, B. S., Allison, A. C. Lancet, 1971, i, 249. Gelzayd, E. A., Kraft, S. C., Fitch, F. W., Kirsner, J. B. Gastroenterology, 1968, 54, 341. Søltoft, J., Binder, V., Gudmand-Höyer, E. Scand. J. Gastroent. 1973, 8, 4. Shiner, R. J., Ballard, J. Gut, 1973, 14, 778. Shiner M. Ballard, J. Lancet, 1972, i, 796. Lachman, P. J. in Defence and Recognition (edited by R. R. Porter); p. 361. London, 1973. Perlman, P., Hammarstrom, S., Lagercrantz, R., Campbell, D. Proc. Soc. exp. Biol. Med. 1967, 125, 975. Seah, P. P., Fry, L., Holborow, E. J., Rossiter, M. A., Doe, W. F., Magalhes, A. F., Hoffbrand, A. V. Gut, 1973, 14, 311.
THE VALUE OF PATCH AND OTHER SKIN TESTS IN DRUG ERUPTIONS*
R. H. FELIX
J. S. COMAISH Department of Dermatology, Royal Victoria Infirmary, Newcastle upon Tyne NE1 4LP Four
patients in whom severe adverse to systemically administered had drugs developed positive in-vivo skin tests. Summary reactions
These case-reports illustrate that under some circumstances suitably conducted and controlled in-vivo skin-testing can be of diagnostic value. Introduction
SKIN-TESTING, especially patch testing, is an established method of diagnosing contact dermatitis, whether caused by occupational factors or topically applied medicaments. The status of skin-testing in the diagnosis of reactions resulting from systemically taken drugs is more debatable because of the imprecise descriptions of types of skin eruptions observed, which is often linked to inadequate data on controls (if any), and the concentrations and techniques used in the skin tests themselves. But what better alternatives are available? Many would agree with the opinion that, apart from clinical challenge, there is no consistently reliable in-vivo or in-vitro test to confirm * Based on a paper read at a meeting of the Scottish Dermatological Society in Edinburgh, May 25, 1973.
the cause of an adverse systemic reaction.1 This means that any investigation affording even fragmentary information should be exploited. The following case-reports illustrate some of the circumstances in which suitably conducted and controlled in-vivo skin-testing can be of diagnostic value. a
drug
as
Case-reports Patient 1 was a 61-year-old woman with a 2-month history of an -itchy eruption on her face, which later spread to the rest of the body. She had previously had a myocardial infarct and was taking digoxin, frusemide, and On examination she had a generalised practolol. eczematous eruption and she was admitted to hospital. Abnormal findings on investigation were an erythrocyte sedimentation-rate (E.S.R.) of 17 mm. per hour, total serum-protein of 7-8 g. per 100 ml., with an increase in y-globulin, and a positive antinuclear factor. Her rash was suspected to be drug-induced and the digoxin, frusemide, and practolol were stopped. The skin cleared slowly after treatment with a topical corticosteroid, and she was discharged on digoxin, frusemide, and ’Slow-K’ (potassium supplement). Since then her rash has not recurred, although the antinuclear factor remained positive for 16 months. In view of this finding the possibility of systemic lupus erythematosus had to be considered as an underlying diagnosis. Because of the beneficial effect of practolol on her cardiac condition, it was decided to challenge the patient with this drug. She was asked to take a 100 mg. tablet every 12 hours and report immediately if she experienced any reaction. Within 12 hours of taking the second tablet some erythema of the face and forearms developed, and after the third tablet there was swelling of the face with erythema over the right shoulder. She took no further tablets and within 3 days the rash had disappeared. There were no adverse systemic effects. She was later patch tested with practolol, using the pure powder made up in yellow soft paraffin at a concentration of 50% and 10% and the injectable form (2 mg. per ml.) made up in propylene glycol at concentrations of 50% and 10%. No reaction was seen after 48 hours (see accompanying table). Intradermal tests were then performed in which 0-05 ml. of practolol (10 µg. per ml.) was injected into the forearm. Within 20 minutes a weal and flare developed which persisted for 11 hours. Results were negative in two controls. The intradermal tests were repeated and punch biopsies were performed at 20 minutes and at 5 hours. Histology showed cedema of the dermis with lymphocytes close to the dermoepidermal junction at 20 minutes and a perivascular infiltration within the dermis at
5 hours
(fig. 1). RESULTS OF SKIN-TESTING
postulate mon to
We believe that the relationship with food antigenicity is one of cause and effect. The possession of antibody to cow’s milk protein and egg white measured soon after infarction seems to be highly predictive of death. We think that these antibodies are of considerable significance and have a causal relationship through an immunological mechanism. An immunological mechanism in atherosclerotic cardiovascular disease could operate in several ways, either directly through the formation of toxic complexes,3 through disturbance of complement" or indirectly by action on platelets or fibrinogen. The almost ubiquitous occurrence of atherosclerosis means that if it has an immunological basis, antigens must be operative in all or most of us. Food provides such a source throughout our lives.s We have examined only three food antigens, and two seem to be implicated. Both the milk and egg antigen used were derived from heated foodstuff, and there is evidence that heating of a protein modifies its antigenicity. Processed foods could, therefore, constitute an almost unlimited source of possible antigens. The antigens we have studied were crude mixtures of proteins, and so further work on other possible antigens, preferably purified single proteins, required.
is
now
We thank all
hospital staff who have cooperated with us. The Carmarthen was supported by an M.R.C. project grant. Requests for reprints should be addressed to D. F. D.
work
at
REFERENCES 1. 2.
3. 4. 5. 6. 7. 8. 9. 10.
Keys, A. et al. Circulation, 1972, 45, 815. Davies, D. F., Davies, J. R., Richards, M. A. J. Atheroscler. Res. 1969, 9, 103. Davies, D. F. ibid. 1969, 10, 253. Davies, D. F. Am. Heart J. 1971, 81, 289. Poston, R. N., Davies, D. F. J. Atheroscler. Res. 1974, 19, 353. Annan, W., Isherwood, D. M. J. med. Lab. Technol. 1969, 26, 202. Rees, B. W. G. ibid. 1973, 30, 167. Siegel, S. Nonparametric Statistics for the Behavioural Sciences. New York, 1956. Ehrenfield, E. N., Gery, I., Davies, A. M. Lancet, 1961, i, 1138. Geertinger, P., Sørensen, H. J. Atheroscler. Res. 1973, 18, 65.
EVIDENCE OF CYTOTOXICITY IN ULCERATIVE COLITIS FROM IMMUNOFLUORESCENT STAINING OF THE RECTAL MUCOSA
JANET BALLARD
MARGOT SHINER
Medical Research Council Gastroenterology Unit, Central Middlesex Hospital, Park Royal, London NW10 7NS
rectal mucosal biopsy from 11 patients with specimens ulcerative colitis and five biopsy specimens from 5 control patients were studied by tissue immunofluorescence. In all patients with ulcerative colitis, the number of IgG-containing plasma-cells was greatly increased, being highest in those with the most active disease. There was also intense staining
Sum ary
Seventeen
of the connective tissue of the rectal mucosa with IgG antisera and a similar staining pattern was seen with the C3 component of complement in these patients. IgA plasma-cells were increased in ten out of seventeen biopsy specimens, and IgM plasma-cells were increased in six out of seventeen specimens. This great increase of IgG plasma-cells may be due to stimulation by bacterial antigens, especially by the strictly anaerobic bacteria, since IgG from the rectal mucosa in ulcerative colitis is known to react strongly with these organisms. The finding of complement deposition in the rectal mucosa suggests that a cytotoxic reaction is occurring and may account for the pathological features, such as ulceration, seen in ulcerative colitis. Introduction
NEITHER the cause nor the mechanism involved in ulcerative colitis has been established despite extensive investigations.1 Immunological reactions are thought to be important, but their exact nature remains unknown. An antibody, or antibodies, has been found in the rectal mucosa of patients with ulcerative colitis which reacted with certain of the obligate anaerobic faecal bacterial We have examined the type and distribution of immunoglobulins in the rectal mucosa in ulcerative colitis and the evidence of cytotoxic reactions involving complement. Reports on tissue immunoglobulins in ulcerative colitis conflict. Gelzayd et al.1 reported that both IgG and IgA plasma-cells were reduced in the rectal mucosa of mild or moderately active colitis, whereas Søltoft et a1.4 found the opposite. To our knowledge, there have been no reports on mucosal complement in ulcerative colitis. Materials and Methods
patients with ulcerative colitis were studied; 4 were investigated on more than one occasion. The clinical activity of the colitis, the radiological and histological findings, and the treatment at the time of study are given in the accompanying table. Patient 5 was studied three times (a, b, c) during his stay in hospital, and again fifteen months later (d). Patient 6 was studied before (a) and sixteen months after (b) colectomy and ileostomy, when the rectum was left with a view to eventual ileorectal anastomosis. Patient 9 was investigated during an acute relapse (a) and again during a moderately active phase (b). Patient 11 had proctocolitis which, at investigation, was restricted to the lower rectum. Biopsy specimens were taken 8 cm. (a) and 17 cm. (b) from the anus. The control group consisted of 5 patients with diarrhcea or constipation in whom the diagnosis of ulcerative colitis or Crohn’s disease had been excluded. Rectal biopsies were taken via a sigmoidoscope and cut in half; one portion was kept for routine histology and the other was snap-frozen in liquid nitrogen. The latter was stored at -80°C for study by immunofluorescence. The direct immunofluorescence technique5 was used with fluorescein-isothiocyanate-conjugated antisera to IgG, IgM, IgA, and C3 complement (Wellcome Reagents and Hyland Laboratories). The sections were studied in random order on a Leitz ’Orthoplan’ microscope. Between 2 and 5 slides of each type were studied without prior knowledge of the antiserum used, and the number of plasma-cells seen was graded on a scale from 1 to 6, the upper limits of normal being 1 for IgG, 2 for IgM, and 4 for IgA. The staining of the connective tissue seen with 11
1015 was graded as absent (0), positive (+), strongly positive (+ +), a normal result being 0.
C3 antiserum or
Results In the 5 control patients, the cell immunoglobulin was IgA
predominant plasma(grade 4) (see table). IgG-containing plasma-cells (fig. 1) were seen only occasionally (grade 0 or 1), with IgM cells being slightly more numerous (grade 2). In the normal rectum, no staining was seen for the C3 component of complement. A fine outline of the connective tissue was always seen with IgG antiserum and has also been reported in the normal jejunum.s In contrast, large numbers of IgG plasma-cells were seen in the rectal mucosae from all patients with active ulcerative colitis (grades 3 to 5) as well as intense staining of the connective tissue and basement membrane (fig. 2). In six biopsy specimens, IgG plasma-
cells outnumbered IgA cells, although these were often also increased. Connective-tissue staining was also seen in eight of the seventeen biopsy specimens tested with IgA antiserum. In the cases with histologically inactive colitis (patients 10 and llb), the number of IgG cells remained slightly increased (grade 2). In six of seventeen biopsy specimens,
IgM plasma-cells grade 3).
were
moderately increased (to
In nine of the seventeen specimens, fluorescence of the connective tissue was strongly positive (+ +) with antiserum to the C3 component of complement (fig. 3). In four biopsy specimens staining was positive (+) and in four it was absent. Of the latter four, the rectal mucosa showed only moderate activity in 2 patients (5d and 9b) and inactivity in the other 2 (10 and llb). Where crypt abscesses were seen, the material in
DATA ON PATIENTS WITH ULCERATIVE COLITIS AND CONTROLS
= not done. N.R. = not repeated. A.C.C. = active, chronic colitis (defined as acute and chronic inflammations mucus depletion of epithelium M.A.C. =moderately active colitis (acute and chronic inflammation only). C.I.C. = chronic, inactive colitis (chronic innammation, often with thin mucosa and crypt distortion).
N.D.
and crypt abscesses).
1016
Fig. 1-Rectal
mucosa
from
a
control patient stained with IgG
antisera.
Few stained plasma-cells are seen (small arrows), and the basement membrane and connective tissue are finely outlined (large arrows). E == epithelium. ( x 225.)
Fig. 3-Rectal mucosa from patient lla, showing C3-complement deposition in connective tissue and basement membrane. No stained cells are seen. E= epithelium. ( x 225.)
We chose our method of grading beexperience of the variable distribution of plasma-cells throughout the mucosa, and it has proved entirely consistent. Certain bacteria in the fasces of patients with ulcerative colitis possessed antigen(s) which reacted in vitro with antibody in rectal mucosal homogenates from such patients.2 These bacteria were obligate anaerobes and the reaction involved mainly IgG, and to a lesser extent IgA. Complement was not taken up by this reaction, as shown by negative staining for C3. The IgG involved was not circulating immunoglobulin because the result was negative when the patient’s serum was tested against the fscal
superfluous. cause
of
our
anaerobes. emerge from our study: (1) The increase in IgG plasma-cells in the rectal mucosa suggests that the bacterial antigen stimulates local, and not systemic, IgG production. This is noteworthy because IgA is normally the major mucosal immunoglobulin and IgG is the major serum-immunoglobulin. (2) The intense staining of connective tissue with C3 complement and also with IgG and IgA antisera indicates that there has been a reaction between these immunoglobulins and antigen (?bacterial) in this region and that
Several
Fig. 2-Rectal mucosa from patient lla stained for IgG. Stained plasma-cells in the lamina propria are greatly increased in number. Connective tissue and basement membrane are also stained. A small, fluorescent abscess (A) is seen in the lumen of a crypt. E=epithelium. (x 225.)
the crypt lumen fluoresced brightly with IgG, and sometimes IgA, antisera, but rarely with IgM and only once with C3 antisera. In most biopsy specimens from patients with ulcerative colitis, the number of macrophages, polymorphs, and eosinophils were usually increased and easily identified by their red autofluorescence. The extent of infiltration with plasma-cells did not seem to be affected by treatment. The immunofluorescence results of repeat biopsies in patients 5, 6, and 9 showed some relation to the patients’ clinical condition, in that the IgG grading and/or C3 staining were less when the disease was less active. The correlation between sigmoidoscopical and histological appearances and immunofluorescence grading in biopsy specimens a and b from
patient
11
was
good. Discussion
The present study has demonstrated an increase in rectal mucosal plasma-cells producing IgG, and also IgA, in ulcerative colitis. This increase in IgG plasma-cells was so great as to make quantitation
points
complement was taken up. (3) This complement-binding complex would be cytotoxic and produce the pathological features of colitis (i.e., vascular congestion, inflammation, ulceration, and abscesses). (4) The complement-requiring reaction of IgG with antigen found in the mucosa may well be the same reaction that occurs in vitro between bacteria and IgG from the mucosal homogenate, except that there is no free complement to complete the reaction in vitro. The mucosal complement would be bound in complexes with IgG and antigen but mucosal IgG liberated from plasma-cells during homogenisation of the tissue would be free to react with faecal anaerobes in vitro. This possibility is being tested.
(5) The increase in mucosal IgA plasma-cells and connective-tissue staining with IgA antiserum in ulcerative colitis suggests that this immunoglobulin is also involved in immune-complex formation. If this complex binds complement, it could do so by invoking the alternate pathway. We noted that the material in the crypt abscesses
1017 with antiserum to IgG, and to a lesser extent with IgA, but not with complement. Hence, there could be a free exchange of antigen and antibody across the mucosa, but the formation of cytotoxic complexes may be confined to the mucosa. The present study attempted to relate bacterial antigens to mucosal pathology in ulcerative colitis. It does not exclude the possibility that there are other sources of antigen leading to stimulation of local IgG antibody, with the formation of complexes which bind complement. Such antigen could be a common mucosal antigen related to Escherichia coli 014 8 or to reticulin 9 or to any combination of anaerobes and aerobes. Many of the points we raise can, and should, be tested, to give a fuller understanding of the local (mucosal) immunological processes and their relation to the pathology of ulcerative colitis.
stained
brightly
Requests for reprints should be addressed
to
J.
B.
REFERENCES 1. 2.
3. 4.
5. 6. 7. 8. 9.
Kraft, S. C., Kirsner, J. B. Gastroenterology, 1971, 60, 922. Monteiro, E. Fossey, J., Shiner, M., Drasar, B. S., Allison, A. C. Lancet, 1971, i, 249. Gelzayd, E. A., Kraft, S. C., Fitch, F. W., Kirsner, J. B. Gastroenterology, 1968, 54, 341. Søltoft, J., Binder, V., Gudmand-Höyer, E. Scand. J. Gastroent. 1973, 8, 4. Shiner, R. J., Ballard, J. Gut, 1973, 14, 778. Shiner M. Ballard, J. Lancet, 1972, i, 796. Lachman, P. J. in Defence and Recognition (edited by R. R. Porter); p. 361. London, 1973. Perlman, P., Hammarstrom, S., Lagercrantz, R., Campbell, D. Proc. Soc. exp. Biol. Med. 1967, 125, 975. Seah, P. P., Fry, L., Holborow, E. J., Rossiter, M. A., Doe, W. F., Magalhes, A. F., Hoffbrand, A. V. Gut, 1973, 14, 311.
THE VALUE OF PATCH AND OTHER SKIN TESTS IN DRUG ERUPTIONS*
R. H. FELIX
J. S. COMAISH Department of Dermatology, Royal Victoria Infirmary, Newcastle upon Tyne NE1 4LP Four
patients in whom severe adverse to systemically administered had drugs developed positive in-vivo skin tests. Summary reactions
These case-reports illustrate that under some circumstances suitably conducted and controlled in-vivo skin-testing can be of diagnostic value. Introduction
SKIN-TESTING, especially patch testing, is an established method of diagnosing contact dermatitis, whether caused by occupational factors or topically applied medicaments. The status of skin-testing in the diagnosis of reactions resulting from systemically taken drugs is more debatable because of the imprecise descriptions of types of skin eruptions observed, which is often linked to inadequate data on controls (if any), and the concentrations and techniques used in the skin tests themselves. But what better alternatives are available? Many would agree with the opinion that, apart from clinical challenge, there is no consistently reliable in-vivo or in-vitro test to confirm * Based on a paper read at a meeting of the Scottish Dermatological Society in Edinburgh, May 25, 1973.
the cause of an adverse systemic reaction.1 This means that any investigation affording even fragmentary information should be exploited. The following case-reports illustrate some of the circumstances in which suitably conducted and controlled in-vivo skin-testing can be of diagnostic value. a
drug
as
Case-reports Patient 1 was a 61-year-old woman with a 2-month history of an -itchy eruption on her face, which later spread to the rest of the body. She had previously had a myocardial infarct and was taking digoxin, frusemide, and On examination she had a generalised practolol. eczematous eruption and she was admitted to hospital. Abnormal findings on investigation were an erythrocyte sedimentation-rate (E.S.R.) of 17 mm. per hour, total serum-protein of 7-8 g. per 100 ml., with an increase in y-globulin, and a positive antinuclear factor. Her rash was suspected to be drug-induced and the digoxin, frusemide, and practolol were stopped. The skin cleared slowly after treatment with a topical corticosteroid, and she was discharged on digoxin, frusemide, and ’Slow-K’ (potassium supplement). Since then her rash has not recurred, although the antinuclear factor remained positive for 16 months. In view of this finding the possibility of systemic lupus erythematosus had to be considered as an underlying diagnosis. Because of the beneficial effect of practolol on her cardiac condition, it was decided to challenge the patient with this drug. She was asked to take a 100 mg. tablet every 12 hours and report immediately if she experienced any reaction. Within 12 hours of taking the second tablet some erythema of the face and forearms developed, and after the third tablet there was swelling of the face with erythema over the right shoulder. She took no further tablets and within 3 days the rash had disappeared. There were no adverse systemic effects. She was later patch tested with practolol, using the pure powder made up in yellow soft paraffin at a concentration of 50% and 10% and the injectable form (2 mg. per ml.) made up in propylene glycol at concentrations of 50% and 10%. No reaction was seen after 48 hours (see accompanying table). Intradermal tests were then performed in which 0-05 ml. of practolol (10 µg. per ml.) was injected into the forearm. Within 20 minutes a weal and flare developed which persisted for 11 hours. Results were negative in two controls. The intradermal tests were repeated and punch biopsies were performed at 20 minutes and at 5 hours. Histology showed cedema of the dermis with lymphocytes close to the dermoepidermal junction at 20 minutes and a perivascular infiltration within the dermis at
5 hours
(fig. 1). RESULTS OF SKIN-TESTING