UNIDENTIFIED CURVED BACILLI ON GASTRIC
`EPITHELIUM IN ACTIVE CHRONIC GASTRITIS
`SIR,-Gastric microbiology has been sadly neglected. Half the
`patients coming to gastroscopy and biopsy show bacterial
`colonisation of their stomachs, a colonisation remarkable for the
`constancy of both the bacteria involved and the associated
`histological changes. During the past three years I have observed
`small curved and S-shaped bacilli in 135 gastric biopsy specimens.
`The bacteria were closely associated with the surface epithelium,
`both within and between the gastric pits. Distribution was
`continuous, patchy,’ or focal. They were difficult to see with
`haematoxylin and eosin stain, but stained well by the Warthin-
`Starry silver method (figure).
`I have classified gastric biopsy findings according to the type of
`inflammation, regardless of other features, as "no inflammation",
`"chronic gastritis" (CG), or "active chronic gastritis" (ACG). CG
`shows more small round cells than normal while ACG is
`characterised by an increase in polymorphonuclear neutrophil
`leucocytes, besides the features of CG. It was unusual to find no
`inflammation. CG usually showed superficial oedema of the
`mucosa. The leucocytes in ACG were usually focal and superficial,
`in and near the surface epithelium. In many cases they only
`infiltrated the necks of occasional gastric glands. The superficial
`epithelium was often irregular, with reduced mucinogenesis and a
`cobblestone surface.
`When there was no inflammation bacteria were rare. Bacteria
`were often found in CG, but were rarely numerous. The curved
`bacilli were almost always present in ACG, often in large numbers
`and often growing between the cells of the surface epithelium
`(figure). The constant morphology of these bacteria and their
`intimate relationship with the mucosal architecture contrasted with
`the heterogeneous bacteria often seen in the surface debris. There
`was normally a layer of mucous secretion on the surface of the
`mucosa. When this layer was intact, the debris was spread over it,
`while the curved bacilli were on the epithelium beneath, closely
`spread over the surface (figure).
`The curved bacilli and the associated histological changes may be
`present in any part of the stomach, but they were seen most
`consistently in the gastric antrum. Inflammation, with no bacteria,
`occurred in mucosa near focal lesions such as carcinoma or peptic
`ulcer. In such cases, the leucocytes were spread through the full
`thickness of the nearby mucosa, in contrast- to the superficial
`infiltration associated with the bacteria. Both the bacteria and the
`typical histological changes were commonly found in mucosa
`unaffected by the focal lesion.
`The extraordinary features of these bacteria are that they are
`almost unknown to clinicians and pathologists alike, that they are
`closely associated with granulocyte infiltration, and that they are
`present in about half of our routine gastric biopsy specimens in
`numbers large enough to see on routine histology. The only other
`organism I have found actively growing in the stomach is Candida,
`sometimes seen in the floor of peptic ulcers. These bacteria were not
`mentioned in two major studies of gastrointestinal microbiology 1,2
`possibly because of their unusual atmospheric requirements and
`slow growth in culture (described by Dr B. Marshall in the
`accompanying letter). They were mentioned in passing by Fung et
`al.3
`How the bacteria survive is uncertain. There is a pH gradient
`from acid in the gastric lumen to near neutral in the mucosal vessels.
`The bacteria grow in close contact with the epithelium, presumably
`near the neutral end of this gradient, and are protected by the
`overlying mucus.
`The identification and clinical significance of this bacterium
`remain uncertain. By light microscopy it resembles Campylobacter
`jejuni but cannot be classified by reference to Bergey’s Manual of
`
`1. Gray JDA, Shiner M. Influence of gastric pH on gastric and jejunal flora Gut 1967; 8:
`574-81.
`2. Drasar BS, Shiner M, McLeod GM. Studies on the intestinal flora I I: The bacterial flora
`of the gastrointestinal tract in healthy and achlorhydric persons. Gastroenterology
`1969; 56: 71-79.
`3. Fung WP, Papadimitriou JM, Matz LR. Endoscopic, histological and ultrastructural
`correlations in chronic gastritis. Am J Gastroenterol 1979; 71: 269-79.
`
`1273
`
`Curved bacilli on gastric epithelium.
`Section is cut at acute angle to show bacteria on surface, forming network
`between epithelial cells. (Warthm-Starry silver stain; bar= 10 m.)
`Determinative Bacteriology. The stomach must not be viewed as a
`sterile organ with no permanent flora. Bacteria in numbers
`sufficient to see by light microscopy are closely associated with an
`active form of gastritis, a cause of considerable morbidity (dyspeptic
`disease). These organisms should be recognised and their
`significance investigated.
`Department of Pathology,
`Royal Perth Hospital,
`Perth, Western Australia 6001
`
`J. ROBIN WARREN
`
`‘
`
`SIR,-The above description of S-shaped spiral bacteria in the
`gastric antrum, by my colleague Dr J. R. Warren, raises the
`following questions: why have they not been seen before; are they
`pathogens or merely commensals in a damaged mucosa, and are they
`campylobacters?
`In 1938 Doenges’ found "spirochaetes" in 43% of 242 stomachs
`at necropsy but drew no conclusions because autolysis had rendered
`most of the specimens unsuitable for pathological diagnosis.
`Freedburg and Barron2 studied 35 partial gastrectomy specimens
`and found "spirochaetes" in 37%, after a long search. They
`concluded that the bacteria colonised the tissue near benign or
`malignant ulcers as non-pathogenic opportunists. When Palmer3 3
`examined 1140 gastric suction biopsy specimens he did not use
`silver stains, so, not surprisingly, he found "no structure which
`could reasonably be considered to be of a spirochaetal nature". He
`concluded that the gastric "spirochaetes" were oral contaminants
`which multiplied only in post mortem specimens or close to ulcers.
`Since that time, the spiral bacteria have rarely been mentioned,
`except as curiosities,4 and the subject was not reopened with the
`
`1. Doenges JL Spirochaetes in the gastric glands of Macacus rhesus and humans without
`definite history of related disease. Proc Soc Exp Med Biol 1938, 38: 536-38
`2. Freedburg AS, Barron LE. The presence of spirochaetes in human gastric mucosa. Am
`J Dig Dis 1940, 7: 443-45.
`3 Palmer ED Investigation of the gastric spirochaetes of the human? Gastroenterology
`1954; 27: 218-20
`4 Ito S. Anatomic structure of the gastric mucosa. In: Heidel US, Cody CF, eds.
`Handbook of physiology, section 6. Alimentary canal, vol II· Secretion
`Washington, DC: American Physiological Society, 1967 705-41.
`
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`1274
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`Fig 1-Thin-section micrograph showing spiral bacteria on surface of a mucous cell in gastric biopsy specimen. (Bar= = 1 m.)
`
`advent of gastroscopic biopsy. Silver staining is not routine for
`mucosal biopsy specimens, and the bacteria have been overlooked.
`In other mammals spiral gastric bacteria are well known and are
`thought to be commensals (eg, Doenges 1 found them in all offorty-
`three monkeys). They usually have more than two spirals and
`inhabit the acid-secreting gastric fundus. In cats they even occupy
`the canaliculi of the oxyntic cells, suggesting tolerance to acid. The
`animal bacteria do not cause any inflammatory response, and no
`illness has ever been associated with them.
`Investigation of gastric bacteria in man has been hampered by the
`false assumption that the bacteria were the same as those in animals
`and would therefore be acid-tolerant inhabitants of the fundus.
`Warren’s bacteria are, however, shorter, with only one or two
`spirals and resemble campylobacters rather than spirochaetes. They
`live beneath the mucus of the gastric antrum well away from the
`
`acid-secreting cells.
`We have cultured the bacteria from antral biopsy specimens,
`They are
`isolation
`Campylobacter
`techniques.
`using
`microaerophilic and grow on moist chocolate agar at 37°C, showing
`up in 3-4 days as a faint transparent layer. They are about 0’ /-1m in
`diameter and 2’ 5 m in length, appearing as short spirals with one
`or two wavelengths (fig 1). The bacteria have smooth coats with up
`to five sheathed flagellae arising from one end (fig 2). In some cells,
`including dividing forms, flagellae may be seen at both ends and in
`negative stain preparations they have bulbous tips, presumably an
`artefact. 7
`These bacteria do not fit any known species either
`morphologically or biochemically. Similar sheathed flagellae have
`been described in vibrios but micro-aerophilic vibrios have now
`
`5. Lockard VG, Boler RK. Ultrastructure of a spiraled micro-organism in the gastric
`mucosa of dogs. Am J Vet Res 1970; 31: 1453-62.
`6. Vial JD, Orrego H. Electron microscope observations on the fine structure of parietal
`cells. J Biophys Biochem Cytol 1960; 7: 367-72
`
`7. Glauert AM, Kerridge D, Horne RW. The fine structure and mode of attachment of the
`sheathed flagellum of Vibrio metchnikovii. J Cell Biol 1963; 18: 327-36.
`8 Shewan JM, Veron M. Genus I vibrio. In: Buchanan RE, Gibbons NE, eds. Bergey’s
`manual of determinative microbiology, 8th ed. Baltimore: Williams & Wilkins,
`1974·341.
`
`Fig 2-Negative stain micrograph of dividing bacterium from broth culture.
`Multiple polar flagellae have terminal bulbs, (2% phosphotungstate, pH 6.8; bar = 1 m.) Inset: detail showing sheathbed flagellum and basal disc associated II nl
`plasma membrane. (3% ammonium molybdate, pH 6 - 5; bar= 100 nm.)
`
`Page 2 Dr. Reddy's Exh. 1048
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`been transferred to the family Spirillaceae genus Campylobacter.1
`Campylobacters however, have "a single polar flagellum at one or
`both ends of the cell" and the campylobacter flagellum is
`unsheathed.9 Warren’s bacteria may be of the genus Spirillum.
`The pathogenicity of these bacteria remains unproven but their
`association with polymorphonuclear infiltration in the human
`antrum is highly suspicious. If these bacteria are truly associated
`with antral gastritis, as described by Warren, they may have a part to
`play in other poorly understood, gastritis associated diseases (ie,
`peptic ulcer and gastric cancer).
`I thank Miss Helen Royce for microbiological assistance, Dr J. A. Armstrong
`for electronmicroscopy, and Dr Warren for permission to use fig 1.
`Department of Gastroenterology,
`Royal Perth Hospital,
`Perth, Western Australia 6001
`
`BARRY MARSHALL
`
`VASODILATOR PROSTANOIDS AND ACTH-
`DEPENDENT HYPERTENSION
`SIR,-Dr Axelrod (April 23, p 904) proposes that the permissive
`effect of glucocorticoids on vascular tone is mediated via inhibition
`of prostacyclin production and that this may contribute to the
`hypertension of Cushing’s syndrome. We became interested in this
`possibility following the suggestion by Rascher at all that
`glucocorticoids may produce hypertension as a result of inhibition
`of phospholipase Az and a subsequent reduction in "vasodilator"
`prostaglandin synthesis. The demonstration by Weeks and Sutter2
`prostacyclin (epoprostenol) infusion attenuated the
`that
`development of DOCA (desoxycortone) induced hypertension in
`the rat was also relevant. We have reviewed the evidence for such a
`hypothesis in relation to steroid and corticotropin (ACTH)
`dependent hypertension.3 Our own studies have been concerned
`with the mechanism of ACTH induced hypertension in sheep, a
`form of experimental hypertension and features of glucocorticoid
`and mineralocorticoid excess but in which these two classes of
`adrenocortical steroid activity do not appear to account for more
`than about half of the hypertension.3 On the basis of detailed
`experiments in conscious sheep we concluded that although
`"vasodilator" prostanoids such as prostacyclin appear to modulate
`the ACTH induced rises in blood pressure they did not play a
`primary role in the development of the hypertension.
`Although in sheep,4 as in other species, indomethacin enhances
`vasoconstrictor responses to angiotensin II, ACTH treatment does
`not alter pressor responsiveness to either angiotensin II,
`noradrenaline, or arginine-vasopressin.5-7 Also, indomethacin
`(3 mg/kg daily for 3 days) had no effect on blood pressure in
`normotensive sheep.ó Further, pretreatment of sheep for 24 h with
`prostacyclin at a dose which lowered total peripheral resistance but
`not blood pressure did not alter the blood pressure response to
`
`9 Pead PJ. Electron microscopy of Campylobacter jejuni. J Med Microbiol 1979; 12:
`383-85.
`1. Rascher W, Dietz R, Schomig A, et al. Modulation of sympathetic vascular tone by
`prostaglandins in corticosterone-induced hypertension in rats. Clin Sci 1979; 57:
`235s-37s
`2. Weeks JR, Sutter DM. An antihypertensive effect of prostacyclin. New York: Raven
`Press, 1979. 253-57.
`3. Scoggms BA, Coghlan JP, Denton DA, Mason RT, Whitworth JA. A review of
`mechanisms involved in the production of steroid induced hypertension with
`particular reference to ACTH dependent hypertension. In: Mantero F, Biglieri EG,
`Edwards CRW, eds. Endocrinology of hypertension. London: Academic Press,
`1982: 41-67.
`4. Beilby DS, Coghlan JP, Denton DA, et al. In vivo-modification of angiotensin II
`pressor responsiveness in sheep by indomethacin. Clin Exp Pharmacol Physiol 1981;
`8: 33-37.
`5. McDougall JG, Barnes AM, Coghlan JP, et al. The effect of corticotrophin (ACTH)
`administration on the pressor action of angiotensin II, noradrenaline and tyramine
`in sheep. Clin Exp Pharmacol Physiol 1978; 5: 449-55.
`6. Mason RT, Coghlan JP, Denton DA. Do prostaglandins play a role in modulating the
`haemodynamic effects of ACTH administration? Proc Endocrinol Soc Aust 1981; 24:
`7
`7 Coghlan JP, Denton DA, Graham WF, et al. Effect of ACTH administration on the
`haemodynamic response to arginine-vasopressin in sheep. Clin Expt Pharmacol
`Physiol 1980; 7: 559-62.
`
`1275
`
`ACTH.6 This suggests to us that the proposal by Axelrod that
`ACTH-dependent hypertension is in any way caused by inhibition
`or prostaglandin synthesis is questionable.
`Our evidence that prostaglandins may modulate the severity of
`ACTH dependent hypertension is based on three series of
`experiments. The first showed that although indomethacin infusion
`for 60 min, at a dose which blocks the vasodepressor effect of
`arachidonic acid, has no effect on blood pressure in normotensive
`sheep, it produced a further increase in mean arterial pressure of 26
`mm Hg in sheep with ACTH-induced hypertension.6 This rise in
`blood pressure was entirely due to a rise in total peripheral
`resistance. In the second series of experiments we showed that in
`animals pretreated with indomethacin for three days the rise in
`blood pressure in response to ACTH was significantly greater. 6
`Finally we found that although graded doses of prostacyclin,
`infused for 10 min, produced similar falls in blood pressure in
`normotensive and ACTH hypertensive sheep, the fall in total
`peripheral resistance is much greater in the ACTH treated
`animals.8 We speculated that plasma levels of vasodilator
`prostanoids such as prostacyclin may rise in response to ACTH
`administration. However, measurement of plasma 6-keto-PGF,,
`(considered by some to reflect prostacyclin production) by Dr
`Murray Mitchell (Dallas, USA) showed a small but significant
`decrease with ACTH treatment. 3
`Our studies in sheep suggest a modulating rather than causal role
`for vasodilator prostanoids in ACTH-dependent hypertension.
`B. A. SCOGGINS
`J. A. WHITWORTH
`J. P. COGHLAN
`D. A. DENTON
`R. T. MASON
`
`Howard Florey Institute of Experimental
`Physiology and Medicine
`and Department of Nephrology,
`Royal Melbourne Hospital,
`Parkville, Victoria 3052, Australia
`
`EPOPROSTENOL (PROSTACYCLIN) DECREASES
`PLATELET DEPOSITION ON VASCULAR
`PROSTHETIC GRAFTS
`SIR,-Prostacyclin (PGI2) is an important regulator of platelet
`deposition on vascular surfaces. When a prosthetic vascular graft is’
`inserted, a few weeks are required before the formation ofPGI2 by
`the pseudovascular wall cells reaches the same level of activity of
`tissue in the vicinityt° because of the slow increase in prostacyclin
`synthetase 11 in the invading cells. Hence platelet deposition on the
`graft surface may be a significant factor in limiting graft survival12
`and causing early occlusion. PGI2 can decrease platelet deposition
`on vascular surfaces,13 so we wondered if platelet deposition on
`prosthetic grafts would be affected by a short term infusion of
`epoprostenol.
`We examined nine male and two female patients aged 53-66 years
`between 48 and 72 h after surgery. Autologous platelet labelling was
`carried out with 100 Ci 11 ’*In-oxine sulphate. 14 Platelet labelling
`efficiency amounted to 92±2%, and recovery 2 h after re-injection of
`autologous labelled platelets was 76±4%. 6 h after re-injection of
`autologous labelled platelets gamma-camera imaging studies were
`done. Epoprostenol (prostacyclin) 5 ng/kg/min was then infused for
`24 h. Gamma-camera imaging was repeated (see figure) during and
`after prostacyclin infusion. Regions of interest (ROI) were
`
`8. Mason RT, Allen KJF, Coghlan JP, Denton, et al. ACTH hypertension modifies the
`haemodynamic effects of prostacyclin infusions in sheep. Clin Exp Pharmacol
`Physiol 1980; 7: 469-72.
`9. Moncada S, Vane JR. Unstable metabolites of arachidonic acid and their role in
`hemostasis and thrombosis. Br Med Bull 1978, 34: 129-36.
`10.Sinzinger H, Silberbauer K, Winter M. Implanted vascular prostheses generate
`prostacyclin Lancet 1978; ii: 840-41.
`11. Eldor A, Falcone D, Hajjar DP, Mimck CR, Weksler BB. Recovery of prostacyclin
`production by deendothelialized rabbit aorta. J Clin Invest 1981; 67: 735-41
`12. Harker LA, Slichter SJ, Sauvaage LR. Platelet consumption by arterial prostheses: The
`effect of endothelialization and pharmacological inhibition of platelet function. Ann
`Surg 1977; 186: 594-600.
`13 Moncada S, Higgs EA, Vane JR. Human arterial and venous tissue generates
`prostacyclin (prostaglandin) a potent inhibitor of platelet aggregation Lancet 1977;
`i 18-21
`14. Sinzinger H, Schwarz M, Leithner Ch, Hofer R. Labelling of autologous human
`platelets with indium-111-oxine sulphate for monitoring of human kidney
`transplants. Nucl Med Biol (Paris) 1982; 2752-55.
`
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