.01F;11”1atilkot.4 (cid:9)
`
`N4
`
`. .
`
`July 1991 Vol. 67 No. 789
`
`(cid:9) (cid:9)
`
`Postgraduate
`.1 ournal
`M (cid:9)
`
`WI PW,W7t1
`1991
`NO.7t14 (cid:9)
`sE0: p.41660000
`,11
`L: PO:T(7 RAJUATb MEDICAL
`JOURNAL
`
`248345?
`
`
`
`POSTGRADUATE MED II
`
`1971 VOLUME 67 ISSUE
`SISAC
`
`pF
`CZArt,
`
`111111111111111111111111
`0072-5417 3(1791..67:789-0
`Ref:WI P0957H
`3042230/01
`
`MIL
`
`Reviews in Medicine —
`Medical genetics
`
`Published for
`The Fellowship of Postgraduate Medicine
`
`1
`
`1
`
`Ex. 1038 - Page 1
`
`

`

`The Postgraduate Medical Journal is published
`monthly on behalf of the Fellowship of
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`Subject USCdpyright Laws
`
`Ex. 1038 - Page 2
`
`

`

`Postgrad Med J (1991) 67, 666 — 671 (cid:9)
`
`The Fellowship of Postgraduate Medicine, 1991
`
`Hypothesis
`
`A new hypothesis for the aetiology of Crohn's disease —
`evidence from lipid metabolism and intestinal tuberculosis
`
`W.E.W. Roediger
`
`University Department of Surgery, The Queen Elizabeth Hospital, Woodville, S.A. 5011, Australia
`
`The stimulus for the immune response in Crohn's disease is unknown. In each of 19 cases of
`Summary: (cid:9)
`Crohn's disease evaluated by electron microscopy, epithelial cells of the ileum contained phagolysosomes
`with lamellar layers of lipid. These structures, now termed R or reactant bodies, are the proffered antigenic
`stimulus. They are proposed to be an amalgam of lipid (cholesterol esters, or phospholipids) and bacterial
`fragments (mycoplasma, mycobacteria or streptococci), which in combination are hypothesized to
`produce a powerful immunological response analogous to the adjuvant effect. For disease expression to
`occur, lipids and specific bacterial populations are needed in the bowel lumen. These factors may account
`for the success of elemental diets that are low in fat in the treatment of Crohn's disease and for the regional
`distribution of disease along the intestinal tract.
`
`Introduction
`
`Over the past three decades the incidence of recent study almost half of a group of patients with
`Crohn's ileo-colitis has increased in Western cavitating pulmonary tuberculosis were shown to
`societies. Search for an aetiological agent has have intestinal involvement, affecting either the
`devolved upon atypical mycobacteria' or cell-wall ileo-caecum or ascending colon.' The association
`deficient organisms, but consistent observations of tuberculosis with the ileo-caecal region is similar
`have not been made. Because an inflammatory to CD where 90% of cases will eventually have a
`response is usually present in Crohn's disease (CD) disease focus.' The similarity of distribution begs
`an antigenic agent should be detectable in most, if the question whether the two diseases have com-
`not all, cases. A place for such an agent can be parable pathogenetic mechanisms.
`constructed from electron microscopic data and
`separate observations in clinical medicine,
`immunology, biochemistry and in microbiology. (cid:9)
`
`The antigenicity of M. tuberculosis
`
`M. tuberculosis induces a strong cellular and lesser
`Intestinal involvement with tuberculosis and Crohn's humoral immune response. The cellular and
`disease (cid:9)
`associated delayed-type hypersensitivity were
`shown, by Freund et al. in 1937, to be enhanced if
`A similarity between intestinal tuberculosis and mycobacteria were administered with paraffin oil.'
`Crohn's disease was mentioned in the classical Subsequent experiments showed that myco-
`bacterial cell walls' or chemically defined fractions
`descriptions of regional ileitis by Dalziel in 1913'
`and Crohn et al. in 1932.3 Caseation and detectable of mycobacteria9 with certain lipids and emulsify-
`acid-fast bacilli distinguish tuberculosis from CD. ing agents strongly enhanced the immune response
`Analysis of a series of patients with intestinal or elicited pulmonary granulomas in experimental
`tuberculosis reported in 1985 showed that 64 of 72 animals. Why the combination of oils with
`cases (89%) had disease involving the ileocaecal mycobacteria or mycobacterial fragments pro-
`region, amongst which were 20 cases of generalized duces striking antigenicity has not been explained.
`peritonitis but none of miliary spread.' In a more
`
`Correspondence: Associate Professor W.E.W. Roediger,
`M.B., B.Ch., M.Sc., D.Phil.(Oxon.), F.C.S.(S.A.),
`F.R.A.C.S.
`Accepted: 5 February 1991
`
`The observation that lipids emulsified with Tween
`80 together with bacterial fragments promote a
`powerful antigenic response is known as the
`
`Lipids, antigenicity and bacterial assimilation
`
`This material was co-pi ed
`at the NLM and may be
`Subject US Copyright Laws
`
`Ex. 1038 - Page 3
`
`(cid:9)
`

`

`AETIOLOGY OF CROHN'S DISEASE
`
`667
`
``adjuvant effect'." Barclay et al." emphasize that
`any factor which interferes with the association of
`oil and mycobacterial fragments reduces or
`eliminates the immunogenicity. Coating of bacteria
`to promote phagocytosis or prolonged retention of
`bacteria in tissue has been suggested for the
`adjuvant effect. Other concepts also seem possible.
`All bacterial cell walls lack cholesterol' and only
`mycoplasmas are known to require cholesterol or
`phospholipid for growth.' Mycoplasmas take up
`cholesterol/phospholipids from human low density
`lipoproteins by a simple exchange process," so
`altering fluidity of bacterial membranes" which in
`turn may promote uptake of bacteria by epithelial
`cells or immune cells. Thus the presence of surface
`receptors for lipids'." in ileal epithelial cells may
`promote endocytosis of lipid-associated bacterial
`fragments. Some mycobacteria require exogenous
`lipid for chain elongation of intrinsic fatty acids
`and growth." The presence and absorption of lipid
`in the distal ileum may enhance mycobacterial
`growth and hence the frequent manifestation of
`intestinal tuberculosis in the ileum.
`
`literature's-20 and two further cases of Crohn's
`disease were personally observed (Table I). None of
`6 control cases"' and neither of 2 cases of
`radiation ileitis, now observed, contained these
`structures. They have not been observed in
`enterocytes of coeliac disease' or experimental
`Yersinia infection.' The lipid-lysosomal bodies
`appear in the apical, basal and lateral portions of
`enterocytes, a feature which suggests that these
`bodies undergo transport across epithelial cells —
`transcytosis — a well known property of lyso-
`somes.' These bodies were also found with the
`electron microscope in macrophages" but aleady
`40 years ago lipid bodies were observed by light
`microscopy in activated macrophages of Crohn's
`disease.'
`Nine electron microscopic studies of colonic
`epithelial cells (Table 11)2" in ulcerative colitis
`comprising 159 cases, revealed 4 instances' where
`
`Table I Electron microscopy of ileal epithelial cells in
`active Crohn's disease with relevance to phagolysosomes
`(R bodies)
`
`Lamellar-lipid lysosomal bodies in Crohn's disease
`
`Crohn's
`cases
`
`Control*
`cases
`
`Ileal epithelial cell
`characteristics in CD
`
`1
`
`3
`
`Small dense vesicles and
`lysosomes
`Lysosomal inclusions
`and myelin-like
`figures
`2 (colon) Lysosomes with mem-
`brane bound vesicles
`R bodies
`
`2t
`
`Year
`
`1970
`
`198119
`
`198429
`
`19901
`
`Many ileal epithelial cells involved with CD display
`lysosomes or phagolysomomes containing lipid in
`lamellar layers showing a whorl-like pattern similar
`to myelin (Figure 1). The lipid nature of these
`bodies was inferred from the characteristic lamella-
`tion of lipids' and their affinity on electron micro-
`scopy for osmium tetroxide, a characteristic of
`lipids. These structures were found in 19 out of 19
`cases of CD: 17 cases were reported in the
`
`.1 4
`
`• Voo•
`
`x.
`
`•
`
`3
`
`8
`
`6
`
`2
`
`* No R bodies observed; eradiation ileitis; tcurrent cases.
`
`Figure 1
`Ilea! enterocyte showing phagolysosomes (R bodies — arrows) from a case of active Crohn's disease. Original
`magnification x 43,000.
`
`This material was copied
`at the NLAA and may be
`Subject US Copyright Laws
`
`Ex. 1038 - Page 4
`
`

`

`668 (cid:9) W.E.W. ROEDIGER
`
`Table H Electron microscopy (EM) of colonic epithelial
`cells in active and quiescent ulcerative colitis (UC) with
`relevance to phagolysosomes (R bodies)
`
`No. of cases Colonic epithelial cells
`
`1
`7
`
`27
`9
`16
`14
`37
`33
`15
`
`No R bodies
`R bodies but in shigellosis
`and Crohn's disease (one
`case each)
`No R bodies
`R bodies in one case of UC*
`No R bodies
`No R bodies
`No R bodies
`No R bodies
`No R bodies
`
`Year
`196426
`1960
`
`196628
`1967'
`1972'
`19753'
`197532
`198933
`1989'
`
`*Other EM features suggest a case of Crohn's colitis.
`
`myelin bodies were found and these appear to be
`cases of Crohn's colitis and shigellosis rather than
`ulcerative colitis. Healthy colonic epithelial cells
`revealed no lipid lysosomal bodies.
`
`A new hypothesis for the aetiology of Crohn's
`disease (R bodies)
`
`The hypothesis now proposed is that the lamellar
`lipid lysosomal bodies, together with bacterial
`fragments, are the antigenic stimulus and underly-
`ing cause of CD (Figure 2). The lysosomally
`derived bodies are termed R (reactant) bodies to
`
`distinguish them from non-reactant, non-tissue
`damaging lysosomes or phagolysosomes. The
`lamellar lipid layering in R bodies may vary in
`character but the configuration of thin lamellae is
`suggestive of cholesterol esters' while the
`configuration illustrated in Figure I is suggestive of
`phospholipids. The current proposal implies that
`the combination of a non-antigenic lipid, with
`bacterial fragments or bacterial cell wall together
`become strongly antigenic. Such an antigenic
`stimulus may be of long duration (6 — 12 months) as
`shown with dermal injection of mycobacteria in
`oily suspensions.'
`Cholesterol esters are taken up selectively by a
`variety of cells where both lysosomes and extra
`lysosomal sites' participate in their hydrolysis and
`further metabolism. Macrophages in particular
`take up cholesterol esters where they are also
`normally hydrolysed.' Hydrolysis appears im-
`paired in Crohn's disease judged by the appearance
`of lipid lamellar bodies in macrophages' of active
`disease but not in health.
`The conditions prevalent in the distal ileum for
`the causation of CD — cholesterol absorption,
`presence of emulsifying agents (bile) and specific
`bacteria — are analogous to the conditions required
`for inducing tuberculosis in experimental animals.
`These conditions are the presence of a lipid, an
`emulsifying agent (Tween) and mycobacteria. Such
`analogous sets of requisites may account for why
`both tuberculosis and Crohn's disease are so
`frequently found in the ileo-caecal region.
`
`Intestinal
`lumen
`
`Cholesterol — esters, phospholipid
`
`Mycoplasma, mycobacteria, streptococci
`
`(Non-antigenic)
`
`(Weakly antigenic)
`
`I
`
`Bacteria/bacterial fragments
`
`Ilea)/colonic
`epithelial cells
`
`Lamina propria
`and muscle layers
`
`Phagolysosomes
`(R bodies)
`(Strongly antigenic)
`
`?Cell
`damage
`
`Endocytosis
`
`Transcytosis
`
`UPTAKE OF Ft BODIES BY MACROPHAGES
`
`IMMUNE ACTIVATION AND TISSUE
`RESPONSE OBSERVED IN ACTIVE DISEASE
`
`Figure 2 Proposed events in the causation of Crohn's disease.
`
`This material was copi ed
`attheNLM and may be
`Subject US Copyright Laws
`
`Ex. 1038 - Page 5
`
`

`

`AETIOLOGY OF CROHN'S DISEASE
`
`669
`
`Clinical and experimental evidence for the Experiments with animals provide further support
`hypothesis (cid:9)
`for the hypothesis:
`
`Besides the biochemical and electron microscopic
`data a number of clinical observations uphold the
`present contentions.
`
`1. The content of lipids in the intestinal lumen
`should determine the formation of R bodies
`and the subsequent disease process. Total
`parenteral nutrition, which curtails lipid and
`other luminal nutrients, produces remission in
`66-89% of cases of active Crohn's disease."
`More specific evidence that removal of lipid
`induces remission was obtained in three pro-
`spectively controlled trials with elemental diets
`for active CD." They showed that elemental
`diets were as effective as prednisolone in induc-
`ing remission of disease and even reversed the
`radiological lesions of Crohn's disease.' All
`three diets' reflected a drastic reduction in
`lipid intake from the 20-30% of a normal diet
`to 0.66% of the total nutritive value. Most of the
`lipids in elemental diets were vegetable oils.
`These oils when used with mycobacteria do not
`promote antigenicity as seen with mineral or
`other oils."
`2. The hypothesis suggests that CD should occur
`in those regions of the bowel that have a good
`capacity for absorption of fatty acids and bile
`and where bacteria are plentiful. The distal
`ileum and colon fulfil these criteria and corre-
`spond to the predilective sites of disease.
`3. Crohn's disease in Africa rarely occurs in black
`population groups compared with those of
`European descent." The fat intake of blacks is
`very significantly less than that observed in
`whites' and is reflected in differences in
`manifestation of other bowel diseases in the
`black populations.
`4. Guthy of Germany" put forward the hypothesis
`that the lipid content of Western diets may
`cause CD. He then showed that a high lipid diet
`in pigs produced intestinal inflammation when
`intestinal transit was slowed by reversed ileal
`loops.'
`5. Because R bodies contain no viable bacteria,
`bacterial growth from tissue with Crohn's
`disease would not be expected. This is in keeping
`with the general microbiological observations
`of the disease.
`
`1. Suspensions of bacterial cell fragments (Strep-
`tococci group A or D) injected into the ileal wall
`of rats produced chronic inflammation and
`granulomas in 46% of animals," while injection
`of live or irradiated B C G (Pasteur) into bowel
`of guinea pigs gave a good model of
`granulomatous bowel disease," observations
`which, to some degree, are supportive of the
`hypothesis.
`2. R bodies of CD closely resemble structures
`found in macrophages of chronic arthritis pro-
`duced by injecting fragments of streptococci
`into joints. No bacteria but morphological
`arrangements similar to R bodies were found in
`inflamed joints after 4 months.'
`
`Conclusions
`
`Evidence from several disciplines has led to pro-
`posals that antigens causing CD are a combination
`of luminal lipids, emulsifiers (bile) and bacterial
`fragments which together become powerfully anti-
`genic when normally each individual substance is
`either non-antigenic or only weakly antigenic. The
`agents are amalgamated into phagolysosomes,
`now termed R (reactant) bodies, which, after trans-
`cytosis, activate immune cells in the lamina pro-
`pria. Intestinal tuberculosis is analogous to CD but
`viable bacteria are taken up where absorption of
`bile and cholesterol is optimal. The analogy
`explains the siting of both diseases in the ileo-
`colonic region. Clinical and dietary evidence sug-
`gests that luminal lipids play a role in the causation
`of CD but the precise lipid and precise bacterial
`fragments that are involved remain to be defined. A
`case can be made for fragments of mcoplasma or
`atypical mycobacteria based on their metabolic
`needs for exogenous lipids. These bacterial
`fragments would ideally be detected by DNA
`hybridization techniques' in Crohn's disease tis-
`sue. The new hypothesis has strong implications for
`the treatment of CD. Modification of specific lipid
`intake and elimination of luminal mycoplasma/
`atypical mycobacteria with specific therapy may be
`future strategies for the treatment of Crohn's
`disease.
`
`References
`
`I . Burnham, W.R. Le role des mycobacteries dans la maladie de
`Crohn. Gastroenterol Clin Biol 1989, 13: 1033-1035.
`2. Dalziel, T.K. Chronic interstitial enteritis. Br Med J 1913, 21:
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`
`3. Crohn, B.B., Ginzburg, L. & Oppenheimer, G.D. Regional
`ileitis: a pathologic and clinical entity. JAMA 1932, 99:
`1323-1329.
`
`This material was copied
`at the NUM and may be
`Subject LISCopyright Lams
`
`Ex. 1038 - Page 6
`
`

`

`670 (cid:9) W.E.W ROEDIGER
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`

`

`(cid:9) (cid:9) WOOLF, A.D. Osteoporosis — an opporunity to
`
`Postgraduate Medical Journal
`July 1991
`Volume 67, No. 789
`
`Contents
`
`601
`
`LEADING ARTICLE
`
`prevent being missed
`
`606 (cid:9)
`
`REVIEW ARTICLE
`JONES, S.C. & AXON, A.T.R. Bleeding peptic
`ulcer — endoscopic and pharmacological manage-
`ment
`
`REVIEWS IN MEDICINE
`613 SUPER, M. Medical genetics
`
`638 (cid:9)
`
`PAPERS
`632 (cid:9) ABRAMSON, M.J., BARNEY!', A.J., LITTLE-
`JOHN, G.O., SMITH, M.M. & HALL, S. Lung
`function abnormalities and decline of spirometry
`in scleroderma: an overrated danger?
`PARKER, S.G. Transient hyperphosphatasaemia
`in association with acute infection in adults
`643 LONG, C.A., MARIN, P., BAYER, A.J.,
`SHETTY, H.G.M. & PATHY, M.S.J. Hyperna-
`traemia in an adult in-patient population
`646 VIJAN, S.G., MANNING, G. & MILLAR-
`CRAIG, M.W. How reliable is the electrocardio-
`gram in detecting left ventricular hypertrophy in
`hypertension?
`LIM, C.W., BENNIE, M.J. & LIM, R. Bradyarr-
`hythmias in acute myocardial infarction: should
`thrombolysis lower the decision threshold for
`temporary pacing?
`652 RAO, R.A.C., HEGDE, B.M., BHAT, E.K.,
`VIDYAVATHI, U. & RAO, R.R. Lipid profile
`studies in long term thiazide treated hypertensives
`LI KAM WA, T.C., LAWSON, M., JACKSON,
`S.H.D., HITOGLOU-MAKEDOU, A. &
`TURNER, P. Interaction of ketoprofen and
`frusemide in man
`CARR, B., HAWLEY, K. & CHANNER, K.S.
`Cardioversion of atrial fibrillation of recent onset
`with fiecainide
`
`649 (cid:9)
`
`655 (cid:9)
`
`659 (cid:9)
`
`V.6
`
`r>c:
`
`666 (cid:9)
`
`HYPOTHESIS
`ROEDIGER, W.E.W. A new hypothesis for the
`aetiology of Crohn's disease — evidence from lipid
`metabolism and intestinal tuberculosis
`
`672 (cid:9)
`
`MISSED DIAGNOSIS
`ROGIERS, Ph., VERSCHAKELEN, J., KNOCK-
`AERT, D. & VANNESTE, S. Occult tuberculous
`postpneumonectomy space empyema four years
`after lung resection
`675 CANTWELL, B.M.J., RICHARDSON, P.G.G.
`& CAMPBELL, S.J. Gynaecomastia and extra-
`gonadal symptoms leading to diagnosis delay of
`germ cell tumours in young men
`
`678 (cid:9)
`
`CLINICAL TOXICOLOGY
`STIFF, G., ROBINSON, D., CUGNONI, H.L.,
`TOUQUET, R. & DALTON, A.M. Massive
`chloroquine overdose — a survivor
`
`CLINICAL REPORTS
`680 GAINES, P., CHAN, J.C.N. & COCKRAM,
`C.S. Histiocytosis X involving the thyroid and
`hypothalamus
`683 SAUNDERS, M.P. A solitary jejunal vascular
`abnormality: a source of massive rectal bleeding
`LOH, A. & JONES, P.A. Evisceration and other
`complications of abdominal drains
`689 CUMMINS, D., WEBB, G., SHAH, N. &
`DAVIES, S.C. Delayed haemolytic tranfusions
`reactions in patients with sickle cell disease
`692 (cid:9) GIBNEY, E.J. Diverticulum of a Meckel's diver-
`ticulum containing a stone
`
`687 (cid:9)
`
`695 (cid:9)
`
`EUROPEAN MEDICAL RESEARCH GROUP
`ABSTRACTS
`
`663 (cid:9)
`
`MEDICINE IN THE ELDERLY
`ELIZABETH, J.E. & GREEN, G.J. Permanent
`pacemakers in nonagenarians
`
`696 (cid:9)
`
`LETTERS TO THE EDITOR
`
`704 (cid:9)
`
`POSTGRADUATE DIARY
`
`This matarial Was copied
`
`Ex. 1038 - Page 9
`
`(cid:9)
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