- Email: [email protected]
IMMUNOLOGICAL DIFFERENTIATION OF PATHOGENIC AND NON-PATHOGENIC ISOLATES OF ENTAMOEBA HISTOLYTICA
561
flow, and to an increase in myocardial oxygen consumption. Cyclizine is useful if avoidance, or prompt reversal, of the
hypotensive effects of opioids is required,3 because of this ability to raise venous and arterial pressures-although it has been suggested that cyclizine has anti-analgesic and excitatory actions.4 From this study, we would suggest that cyclizine should not be used routinely as an antiemetic in acute myocardial infarction or severe heart failure. We thank the night sisters of the Coronary Care Unit in East Birmingham Hospital, who helped in the observations reported here.
Preliminary Communication IMMUNOLOGICAL DIFFERENTIATION OF PATHOGENIC AND NON-PATHOGENIC ISOLATES OF ENTAMOEBA HISTOLYTICA W. D. STRACHAN W. M. SPICE
J. P.
P. L. CHIODINI A. H. MOODY ACKERS
Department of Medical Protozoology, London School of Hygiene and Tropical Medicine, London WC1E 7HT; and Hospital for Tropical Diseases, London NW1 0PE Entamoeba histolytica can act as a harmless commensal organism in the lumen of the large intestine, or can cause invasive amoebiasis. Some workers have suggested that there are two distinct subspecies of this organism, and that only one of these is associated with invasive disease. Present isoenzyme tests to identify the subspecies take several days to analyse: we report a technique that uses immunofluorescence with monoclonal antibodies, takes two days to perform, and may, therefore, assist in the clinical management of patients infected with this organism.
Summary
INTRODUCTION
Entamoeba histolytica causes up to 500 million new infections every year across the world,1 but usually lives as a harmless commensal confined to the lumen of the large intestine. In a small proportion of cases, dissemination occurs and causes invasive amoebiasis-typically invasive intestinal disease, or amoebic liver abscess. Some workers believe that all E histolytica amoebae are potentially capable of invasion, but are usually restrained by host-mediated defence mechanisms. Others feel that two kinds (or even two species) are involved---one of which is always potentially invasive, and the other incapable of causing disease. (This argument should not be confused with the disagreement over the pathogenic potential of the "small race" of E histolytica, which is now universally accepted as a different and non-pathogenic species, E hartmanni.) The argument for all being potential pathogens, restrained by the host’s immune system, was supported by exacerbations which seemed to occur during pregnancy2 and after treatment with corticosteroids.3 In 1982, however, Martinez-Palomo showed differences in agglutinability by the lectin concanavalin A in E histolytica isolates obtained from patients with invasive amoebiasis and from symptomless cyst-passers.4 Sargeaunt and Williams have since shown that band patterns obtained after starch-gel electrophoresis with the enzymes hexokinase and phosphoglucomutase
Correspondence should be addressed to L. B. T., Division of Cardiology, Hospital, Lake Shore Drive at 31st Street, Chicago, Illinois
Michael Reese 60616, USA.
REFERENCES 1. Kerr F, Donald KW Analgesia m myocardial infarction Br Heart J 1974; 36: 117-21 2 Gott PH Cyclizine toxicity-intentional drug abuse of a proprietary antihistamine. N
Engl J Med 1968; 279: 596 G, Gershon S, Gray R, Shaw FH, McCance I, Bruce DW Treatment of certain effects of morphine. Br Med J 1958; i: 675-78. 4. Nicholl RM, Moore J, Dundee JW. Alterations m response to somatic pain associated with anaesthesia. XI two non-phenothiazine anti-emetics: cyclizine and trimethobenzamide Br J Anaesth 1962; 34: 287-89.
3. Christie
correlate very closely with clinical fmdings.5.6 E histolytica isolates from patients with invasive disease possess two advanced hexokinase bands (fast), but in isolates from symptomless cyst-passers the two bands are less advanced (slow). Similar results have been obtained by other groups using different electrophoretic mediae and have led to debate about the need to treat patients shedding cysts of a non-invasive zymodeme.lO.11 A non-treatment policy is already practised in populations where the carriage of invasive zymodemes is extremely rare-for example male homosexuals in London.12.13 This policy has’ been questioned by Mirelman and co-workers, who have found that it is possible to change the isoenzyme pattern and the virulence of non-invasive isolates of the parasite, by manipulations of the bacterial flora which accompany most cultures of E histolytica.’,14 The epidemiological results presented over the years by Sargeaunt and his co-workers, however, leave little doubt that the outcome of an infection with E histolytica usually correlates with the zymodeme of the infecting organism. This has important clinical implications, especially if an accurate test could rapidly determine the zymodeme. We report a method, based on immunofluorescence with monoclonal antibodies, that may partly meet this need. S METHODS
All faecal samples found by routine examination at the Hospital for Tropical Diseases to contain cysts of E histolytica were stored at 4° C, for not more than six days, before being inoculated into Robinson’s medium (but with half the starch of the original formula),15 and kept at 37° C. Subcultures were made until sufficient growth was obtained for isoenzyme analysis-usually within seven days. Several old isolates, of known invasiveness, were re-established in the same medium. When enough organisms (about 105) were present, the amoebae from each bottle were harvested by centrifugation, washed, resuspended in 20 ul of lysis buffer (20 mmol/1 2-amino-2 (hydroxymethyl)-1,3-propandiol, 150 mmol/1 NaCl, 1 mrnol/1 phenylmethylsulphonylfluoride, 1 mmol/1 iodoacetate, and 10 fig/mi N-p-Tosyl-L-lysine chloromethylketone, pH 8-0), and lysed by freezing and thawing three times. Centrifugation (12 000 g for 1 min) isolated the lysate used for isoenzyme assay. Only hexokinase was examined, and lysates were run in agarose mini-gels submerged in 01 mmol/1 borate buffer (pH 8-0)’ at 50 V, for 90 min. Enzyme bands were displayed by the method used by Sargeaunt and Williams6,16 except that no overlay was used, and development was performed in a tank. Two isolates of known invasiveness were used as controls, so that distinction between fast bands and slow bands was clear. The monoclonal antibodies 22.3 and 22.5 were members of a large group prepared by standard procedures (Strachan, manuscript in preparation) from BALB/c mice immunised with bacteria-free cultures of the laboratory-adapted strain of E histolytica NIH 200, and supplied in the form of ascitic fluids obtained from pristane-primed mice. NIH 200 (ATCC 30458) was first isolated in 1949, and has since been adapted to axenic (bacteria
562 COMPARISON OF CLINICAL FINDINGS, HEXOKINASE MOBILITY, AND IMMUNOFLUORESCENT STAINING WITH MONOCLONAL ANTIBODIES
RESULTS
22.3 AND 22.5, FOR VARIOUS STRAINS OF ENTAMOEBA HISTOLYTICA
)!!!
M-.h TFAT
52 faecal samples containing cysts of E histolytica were obtained from June to August, 1987. 25 successful cultures were established, although one never grew well enough for isoenzyme analysis and is not included. The results are shown in the table, together with those for 6 cryopreserved isolates and 4 invasive, laboratory-adapted, axenic strains. The patterns of staining produced by the two monoclonal antibodies were not entirely similar: 22.3 produced a granular internal fluorescence, while 22.5 produced an internal vacuolar staining pattern. The two monoclonal antibodies 22.3 and 22.5 appear to bind exclusively to isolates of E histolytica with fast hexokinase bands. Such isolates are regarded as being capable of invasion, and their disease-causing potential was confirmed by the clinical symptoms and serum antibody titres, when available. Immunofluorescence results with non-invasive (slow band) strains of E histolytica were all negative at a dilution of 1/9 (log3 titre = 2), while invasive organisms were positive to log3 titres 8-12. DISCUSSION
The first four strains are from established axenic invasive isolates; the next six strains are from cryopreserved clinical isolates; and the remaining strains
from fresh clinical isolates. All clinical samples contained E histolytzca (cysts unless otherwise stated), and in addition: E h + rbc haematophagous trophozoites; G] Gwrdta lambha; E c Entamoeba coli; I b lodamoeba buetschlzz; E hart Entamoeba hartmannz; E nana Endolimax nana ; Z = zymodeme ; Ax Axemc; are
=
=
=
=
These results have the potential to provide a relatively cheap and rapid method for determining the invasive potential of a clinical isolate of E histolytica. The antibodies do not react directly with E histolytica cysts, and preliminary culture is still required, but sufficient organisms for our technique can be obtained within two days, rather than the seven days needed for preparing the lysate for isoenzyme analysis. The reason for the correlation between zymodeme and invasiveness is still not understood, and these antibodies provide another tool for studying this problem. However, general application of these findings will largely depend on resolution of the controversy about treatment or nontreatment of symptomless cyst-passers.
=
=
Dys = dysentery; Sf= symptom free; diarr = diarrhoea; O = occasional; Int = intermittent; Rec = recurrent; liver + = enlarged liver; IFAT reciprocal slide immunofluoresence titres with fixed NIH 200 organisms as antigen; CAP precipitin reaction on cellulose acetate membrane; ND not done; Mab IFAT titres = monoclonal antibody immunofluorescence titres (log, dilutions); Neg = negative.
We thank the Wellcome Trust and the World Health financial support.
Organisation
for
=
=
=
Correspondence should be addressed to J. P. A., Department of Medical Protozoology, London School of Hygiene and Tropical Medicine, Keppel St, London WC1E 7HT. REFERENCES
free) culture-but it retains a pathogenic zymodeme (Z 11). The reaction of the monoclonal antibodies with the amoebae was assessed by immunofluorescence. Samples of the bottom starch layer of 2-day cultures in Robinson’s medium were removed and gently mixed. Drops of suspension were added to the wells of multispot polytetrafluoroethene-coated slides (Henley) and air dried. Slides were fixed in methanol (5 min), and then stored with desiccant at - 22° C. Tripling dilutions of ascitic fluid were applied to wells, and the slides were incubated in a humidified atmosphere at room temperature for 30 min, then washed twice in phosphatebuffered isotonic saline (PBS) at pH 72. Fluorescein isothiocyanate-conjugated rabbit anti-mouse immunoglobulin (Amersham) was applied, diluted 1:50 in PBS with 1/10 000 Evans blue, and the slides were incubated for a further 30 min. Finally, the slides were washed twice with PBS, and mounted in 50% glycerol ’Citifluor’ in PBS. Wells were examined with a Leitz microscope with incident-light fluorescence, an HB50 high pressure mercury vapour lamp, a TK 510 dichroic mirror, and a 2 x KP 490 (exciting) and a K 515 (suppressing) filter. End-point dilutions were recorded as the highest dilution which produced recognisable specific fluorescence. All cultures were confirmed as E histolytica by morphology, and by staining with the monoclonal antibody 24.2 which is species specific. Immunofluorescence results were read before the hexokinase gels were assessed; subsequently clinical details and the results, when available, of amoebic serology were obtained.
1. Walsh JA Problems
in recognition and diagnosis of amebiasis: estimation of the global magnitude of morbidity and mortality. Rev Inf Dis 1986; 8: 228-38. 2. Abioye AA. Fatal amoebic colitis in pregnancy and puerpenum a new clinicopathological entity. J Trop Med Hyg 1973; 76: 97-100. 3. Kanani SR, Knight R. Relapsing amoebic colitis of 12 years standing exacerbated by corticosteroids. Br Med J 1969; ii: 613-14. 4. Martinez-Palomo A, Gonzales-Robles A, de la Torre M. Selective agglutination of pathogenic strains of Entamoeba histolytica induced by Con A Nature 1973; 245:
186-87.
5. Sargeaunt PG, non-invasive
6
Williams JE, Grene JD. The differentiation of invasive and Entamoeba histolytica by isoenzyme electrophoresis. Trans R Soc
Trop Med Hyg 1978, 72: 519-21 Sargeaunt PG, Williams JE. Electrophoretic isoenzyme patterns of the pathogenic and non-pathogenic intestinal amoebae of man. Trans R Soc Trop Med Hyg 1979; 73:
225-27. 7. Mirelman D, Bracha
8
R, Chayen A, Aust-Kettis A, Diamond LS. Entamoeba histolytica: effect of growth conditions and bacterial associates on isoenzyme patterns and virulence. Exp Parasitol 1986; 62: 142-48 Mathews HM, Moss DM, Healy GR, Visvesvara GS. Polyacrylamide gel electrophoresis of isoenzymes from Entamoeba species. J Clin Microbiol 1983; 17:
1009-12. 9 Moss DM, Mathews HM. A fast
electrophoreuc isoenzyme technique for the and non-invasive Entamoeba histolytica and "E histolytica-like" organisms. J Protozool 1987; 34: 253-55 Editorial Is that amoeba harmful or not? Lancet 1985, i: 732-34. Editorial On the three stages of amoebic research Lancet 1986; ii. 1133-34 Goldmeier D, Sargeaunt PG, Pnce AB, et al Is Entamoeba histolytica in homosexual men a pathogen? Lancet 1986; i: 641-44. Allason-Jones E, Mindel A, Sargeaunt P, Williams P Entamoeba histolytica as a commensal intestinal parasite in homosexual men. N Engl J Med 1986; 315: identification of
10 11. 12. 13.
353-56
invasive
563
Reviews of Books Magnetic Resonance Imaging of the Knee J. H. Mink, M. A. Reicher, and J. V. Cruess. New York: Raven Press. 1987.
Pp 178.$95.50. ISBN 0-881673323.
To "explore" or worse still to remove a knee cartilage without first undertaking an arthrogram and/or an arthroscopy is today to invite criticism. In his monograph on arthrography Wolfe conceded that excellent arthroscopy was ultimately more rewarding than excellent arthrography. The fact remains that arthroscopy involves a day bed and an anaesthetic. Moreover, there is a tendency in some quarters to use arthroscopy as a screening test for almost any vague symptom between mid thigh and mid calf. Now magnetic resonance imaging has arrived to change all this. In a spectacular monograph, Mink and his colleagues explain the basic physics of MRI and apply it to the anatomy in superb detail. They then offer an elegant depiction of the wide range of synovial, meniscal, bony, ligamentous, and soft-tissue pathology that MRI can reveal. No other technique provides such a range of information so we can expect MRI, ultimately, to replace diagnostic arthroscopy. As a pointer to the future of investigating joint pathology this book is destined to be a classic. Department of Orthopaedic Surgery, Hope Hospital, Salford M6 8HD
J. NOBLE
The Causes of Crime New Biological Approaches.-Edited by Samoff A. Mednick, Terrie E. Moffitt, and Susan Stack. Cambridge: Cambridge University Press. 1987. Pp 364. 35. ISBN 0-521304024.
IT is slightly offputting to find that this multi-author book is based on the proceedings of a conference held in 1982. However, it becomes clear to the reader that whoever were the delinquent authors the rest have taken the opportunity to update their contributions. Therefore this volume represents the nearest thing available to a state-of-the-art review of the biological basis of criminality. As such it is bound to give offence in some quarters. The law-abiding world seems inevitably to divide into those who see criminal behaviour as inherent and constitutional and those who see the law-breaker as the hapless victim of his (or her) life circumstances. However, the articles included in this text provide persuasive arguments in favour of a determined occupation of a middle ground. A chapter by Mednick and co-workers reviews genetic factors, focusing particularly on adoption data, which
14. Mirelman D, Bracha R, Wexler A, Chayen A. Changes in isoenzyme patterns of a cloned culture of nonpathogenic Entamoeba histolytica during axenization Inf Immun 1986, 54: 827-32. 15. Robmson GL. The laboratory diagnosis of human parasitic amoebae Trans R Soc Trop Med Hyg 1968, 62: 285-94. 16. Farri TA, Sargeaunt PG, Warhurst DC, Williams JE, Bhojnani R. Electrophoretic studies of the hexokmase of Entamoeba histolytica groups I to IV. Trans R Soc Trop Med Hyg 1980; 74: 672-73. 17 Sargeaunt PG, Williams JE, Neal RA. A comparative study of Entamoeba histolytica (NIH 200, HK9 etc), "E. Histolytica like" and other morphologically identical amoeba using isoenzyme electrophoresis Trans R Soc Trop Med Hyg 1980; 74: 469-74.
provide impressive support for a genetic contribution to chronic offending but clearly also point to important environmental influences. Cloninger and Gottesman perform a masterly synthesis of family, twin, and adoption studies which strongly supports this general conclusion. There are informative reviews of studies of the psychophysiological substrate of antisocial behaviour, among which Venables’ article on autonomic nervous system factors is particularly valuable. Although there’ is some disparity in research findings, there is overall support for the idea that criminality is associated with low levels of tonic activity and low electrodermal responsivity. The idea that criminality is associated with "inability to learn from experience" is explored by Hare and Connelly and the possible role of factors such as disconnection between the "semantic and affective components of language" are interesting, if rather speculative. The tone of the book is not one of undiluted enthusiasm for the biological approach to antisocial behaviour. Cloninger strikes a cautious note in reviewing pharmacological approaches, calling for a more careful attempt at the classification of those subtypes of abnormal behaviour that might benefit from drug treatments. O’Callaghan and Carroll are even more outspoken in their condemnation of psychosurgical methods, which they see as "socially pernicious and ultimately ineffective". Despite its long gestation, this volume has emerged agreeably formed and without tell-tale signs of postmaturity. It is a useful, balanced, and scholarly contribution to a controversial and frequently messy field. Department of Psychological Medicine, University of Wales College of Medicine, Heath Park, Cardiff
PETER MCGUFFIN
Hypoxia, Polycythemia, and Chronic Mountain Sickness Robert M. Winslow and Carlos Monge C. Baltimore: Johns Hopkins University Press. 1988. Pp 255. 33.35. ISBN 0-801834481.
ALTHOUGH chronic mountain sickness (CMS) may seem rather esoteric topic for the average clinician, its study does throw light on many aspects of hypoxia and polycythaemia. Analogies with chronic obstructive lung disease and polycythaemia rubra vera are many and hence make the subjects of interest to a wider audience than high altitude specialists. This book is devoted to a wide ranging discussion of CMS. It does not set out to explore, in any depth, the mechanisms of the hypoxic stimulus to a
erythropoiesis. authors are experts. Robert Winslow is a haematologist who has taken part in several research expeditions to the Peruvian Andes where he collaborated with Carlos Monge. He was also a member of the 1981 American Medical Research Expedition to Everest where he conducted sophisticated haematological studies in a tented laboratory in the Western Cwm. Carlos Monge C is the son of Carlos Monge M, the Peruvian physician who first described CMS and after whom the disease is known. Monge C has worked on CMS for most of his professional life. There are chapters on every aspect of CMS including its history, comparative physiology, red cell mass and its control, effects on circulation, ventilation, renal function, exercise, and the effects of blood letting. The
flow, and to an increase in myocardial oxygen consumption. Cyclizine is useful if avoidance, or prompt reversal, of the
hypotensive effects of opioids is required,3 because of this ability to raise venous and arterial pressures-although it has been suggested that cyclizine has anti-analgesic and excitatory actions.4 From this study, we would suggest that cyclizine should not be used routinely as an antiemetic in acute myocardial infarction or severe heart failure. We thank the night sisters of the Coronary Care Unit in East Birmingham Hospital, who helped in the observations reported here.
Preliminary Communication IMMUNOLOGICAL DIFFERENTIATION OF PATHOGENIC AND NON-PATHOGENIC ISOLATES OF ENTAMOEBA HISTOLYTICA W. D. STRACHAN W. M. SPICE
J. P.
P. L. CHIODINI A. H. MOODY ACKERS
Department of Medical Protozoology, London School of Hygiene and Tropical Medicine, London WC1E 7HT; and Hospital for Tropical Diseases, London NW1 0PE Entamoeba histolytica can act as a harmless commensal organism in the lumen of the large intestine, or can cause invasive amoebiasis. Some workers have suggested that there are two distinct subspecies of this organism, and that only one of these is associated with invasive disease. Present isoenzyme tests to identify the subspecies take several days to analyse: we report a technique that uses immunofluorescence with monoclonal antibodies, takes two days to perform, and may, therefore, assist in the clinical management of patients infected with this organism.
Summary
INTRODUCTION
Entamoeba histolytica causes up to 500 million new infections every year across the world,1 but usually lives as a harmless commensal confined to the lumen of the large intestine. In a small proportion of cases, dissemination occurs and causes invasive amoebiasis-typically invasive intestinal disease, or amoebic liver abscess. Some workers believe that all E histolytica amoebae are potentially capable of invasion, but are usually restrained by host-mediated defence mechanisms. Others feel that two kinds (or even two species) are involved---one of which is always potentially invasive, and the other incapable of causing disease. (This argument should not be confused with the disagreement over the pathogenic potential of the "small race" of E histolytica, which is now universally accepted as a different and non-pathogenic species, E hartmanni.) The argument for all being potential pathogens, restrained by the host’s immune system, was supported by exacerbations which seemed to occur during pregnancy2 and after treatment with corticosteroids.3 In 1982, however, Martinez-Palomo showed differences in agglutinability by the lectin concanavalin A in E histolytica isolates obtained from patients with invasive amoebiasis and from symptomless cyst-passers.4 Sargeaunt and Williams have since shown that band patterns obtained after starch-gel electrophoresis with the enzymes hexokinase and phosphoglucomutase
Correspondence should be addressed to L. B. T., Division of Cardiology, Hospital, Lake Shore Drive at 31st Street, Chicago, Illinois
Michael Reese 60616, USA.
REFERENCES 1. Kerr F, Donald KW Analgesia m myocardial infarction Br Heart J 1974; 36: 117-21 2 Gott PH Cyclizine toxicity-intentional drug abuse of a proprietary antihistamine. N
Engl J Med 1968; 279: 596 G, Gershon S, Gray R, Shaw FH, McCance I, Bruce DW Treatment of certain effects of morphine. Br Med J 1958; i: 675-78. 4. Nicholl RM, Moore J, Dundee JW. Alterations m response to somatic pain associated with anaesthesia. XI two non-phenothiazine anti-emetics: cyclizine and trimethobenzamide Br J Anaesth 1962; 34: 287-89.
3. Christie
correlate very closely with clinical fmdings.5.6 E histolytica isolates from patients with invasive disease possess two advanced hexokinase bands (fast), but in isolates from symptomless cyst-passers the two bands are less advanced (slow). Similar results have been obtained by other groups using different electrophoretic mediae and have led to debate about the need to treat patients shedding cysts of a non-invasive zymodeme.lO.11 A non-treatment policy is already practised in populations where the carriage of invasive zymodemes is extremely rare-for example male homosexuals in London.12.13 This policy has’ been questioned by Mirelman and co-workers, who have found that it is possible to change the isoenzyme pattern and the virulence of non-invasive isolates of the parasite, by manipulations of the bacterial flora which accompany most cultures of E histolytica.’,14 The epidemiological results presented over the years by Sargeaunt and his co-workers, however, leave little doubt that the outcome of an infection with E histolytica usually correlates with the zymodeme of the infecting organism. This has important clinical implications, especially if an accurate test could rapidly determine the zymodeme. We report a method, based on immunofluorescence with monoclonal antibodies, that may partly meet this need. S METHODS
All faecal samples found by routine examination at the Hospital for Tropical Diseases to contain cysts of E histolytica were stored at 4° C, for not more than six days, before being inoculated into Robinson’s medium (but with half the starch of the original formula),15 and kept at 37° C. Subcultures were made until sufficient growth was obtained for isoenzyme analysis-usually within seven days. Several old isolates, of known invasiveness, were re-established in the same medium. When enough organisms (about 105) were present, the amoebae from each bottle were harvested by centrifugation, washed, resuspended in 20 ul of lysis buffer (20 mmol/1 2-amino-2 (hydroxymethyl)-1,3-propandiol, 150 mmol/1 NaCl, 1 mrnol/1 phenylmethylsulphonylfluoride, 1 mmol/1 iodoacetate, and 10 fig/mi N-p-Tosyl-L-lysine chloromethylketone, pH 8-0), and lysed by freezing and thawing three times. Centrifugation (12 000 g for 1 min) isolated the lysate used for isoenzyme assay. Only hexokinase was examined, and lysates were run in agarose mini-gels submerged in 01 mmol/1 borate buffer (pH 8-0)’ at 50 V, for 90 min. Enzyme bands were displayed by the method used by Sargeaunt and Williams6,16 except that no overlay was used, and development was performed in a tank. Two isolates of known invasiveness were used as controls, so that distinction between fast bands and slow bands was clear. The monoclonal antibodies 22.3 and 22.5 were members of a large group prepared by standard procedures (Strachan, manuscript in preparation) from BALB/c mice immunised with bacteria-free cultures of the laboratory-adapted strain of E histolytica NIH 200, and supplied in the form of ascitic fluids obtained from pristane-primed mice. NIH 200 (ATCC 30458) was first isolated in 1949, and has since been adapted to axenic (bacteria
562 COMPARISON OF CLINICAL FINDINGS, HEXOKINASE MOBILITY, AND IMMUNOFLUORESCENT STAINING WITH MONOCLONAL ANTIBODIES
RESULTS
22.3 AND 22.5, FOR VARIOUS STRAINS OF ENTAMOEBA HISTOLYTICA
)!!!
M-.h TFAT
52 faecal samples containing cysts of E histolytica were obtained from June to August, 1987. 25 successful cultures were established, although one never grew well enough for isoenzyme analysis and is not included. The results are shown in the table, together with those for 6 cryopreserved isolates and 4 invasive, laboratory-adapted, axenic strains. The patterns of staining produced by the two monoclonal antibodies were not entirely similar: 22.3 produced a granular internal fluorescence, while 22.5 produced an internal vacuolar staining pattern. The two monoclonal antibodies 22.3 and 22.5 appear to bind exclusively to isolates of E histolytica with fast hexokinase bands. Such isolates are regarded as being capable of invasion, and their disease-causing potential was confirmed by the clinical symptoms and serum antibody titres, when available. Immunofluorescence results with non-invasive (slow band) strains of E histolytica were all negative at a dilution of 1/9 (log3 titre = 2), while invasive organisms were positive to log3 titres 8-12. DISCUSSION
The first four strains are from established axenic invasive isolates; the next six strains are from cryopreserved clinical isolates; and the remaining strains
from fresh clinical isolates. All clinical samples contained E histolytzca (cysts unless otherwise stated), and in addition: E h + rbc haematophagous trophozoites; G] Gwrdta lambha; E c Entamoeba coli; I b lodamoeba buetschlzz; E hart Entamoeba hartmannz; E nana Endolimax nana ; Z = zymodeme ; Ax Axemc; are
=
=
=
=
These results have the potential to provide a relatively cheap and rapid method for determining the invasive potential of a clinical isolate of E histolytica. The antibodies do not react directly with E histolytica cysts, and preliminary culture is still required, but sufficient organisms for our technique can be obtained within two days, rather than the seven days needed for preparing the lysate for isoenzyme analysis. The reason for the correlation between zymodeme and invasiveness is still not understood, and these antibodies provide another tool for studying this problem. However, general application of these findings will largely depend on resolution of the controversy about treatment or nontreatment of symptomless cyst-passers.
=
=
Dys = dysentery; Sf= symptom free; diarr = diarrhoea; O = occasional; Int = intermittent; Rec = recurrent; liver + = enlarged liver; IFAT reciprocal slide immunofluoresence titres with fixed NIH 200 organisms as antigen; CAP precipitin reaction on cellulose acetate membrane; ND not done; Mab IFAT titres = monoclonal antibody immunofluorescence titres (log, dilutions); Neg = negative.
We thank the Wellcome Trust and the World Health financial support.
Organisation
for
=
=
=
Correspondence should be addressed to J. P. A., Department of Medical Protozoology, London School of Hygiene and Tropical Medicine, Keppel St, London WC1E 7HT. REFERENCES
free) culture-but it retains a pathogenic zymodeme (Z 11). The reaction of the monoclonal antibodies with the amoebae was assessed by immunofluorescence. Samples of the bottom starch layer of 2-day cultures in Robinson’s medium were removed and gently mixed. Drops of suspension were added to the wells of multispot polytetrafluoroethene-coated slides (Henley) and air dried. Slides were fixed in methanol (5 min), and then stored with desiccant at - 22° C. Tripling dilutions of ascitic fluid were applied to wells, and the slides were incubated in a humidified atmosphere at room temperature for 30 min, then washed twice in phosphatebuffered isotonic saline (PBS) at pH 72. Fluorescein isothiocyanate-conjugated rabbit anti-mouse immunoglobulin (Amersham) was applied, diluted 1:50 in PBS with 1/10 000 Evans blue, and the slides were incubated for a further 30 min. Finally, the slides were washed twice with PBS, and mounted in 50% glycerol ’Citifluor’ in PBS. Wells were examined with a Leitz microscope with incident-light fluorescence, an HB50 high pressure mercury vapour lamp, a TK 510 dichroic mirror, and a 2 x KP 490 (exciting) and a K 515 (suppressing) filter. End-point dilutions were recorded as the highest dilution which produced recognisable specific fluorescence. All cultures were confirmed as E histolytica by morphology, and by staining with the monoclonal antibody 24.2 which is species specific. Immunofluorescence results were read before the hexokinase gels were assessed; subsequently clinical details and the results, when available, of amoebic serology were obtained.
1. Walsh JA Problems
in recognition and diagnosis of amebiasis: estimation of the global magnitude of morbidity and mortality. Rev Inf Dis 1986; 8: 228-38. 2. Abioye AA. Fatal amoebic colitis in pregnancy and puerpenum a new clinicopathological entity. J Trop Med Hyg 1973; 76: 97-100. 3. Kanani SR, Knight R. Relapsing amoebic colitis of 12 years standing exacerbated by corticosteroids. Br Med J 1969; ii: 613-14. 4. Martinez-Palomo A, Gonzales-Robles A, de la Torre M. Selective agglutination of pathogenic strains of Entamoeba histolytica induced by Con A Nature 1973; 245:
186-87.
5. Sargeaunt PG, non-invasive
6
Williams JE, Grene JD. The differentiation of invasive and Entamoeba histolytica by isoenzyme electrophoresis. Trans R Soc
Trop Med Hyg 1978, 72: 519-21 Sargeaunt PG, Williams JE. Electrophoretic isoenzyme patterns of the pathogenic and non-pathogenic intestinal amoebae of man. Trans R Soc Trop Med Hyg 1979; 73:
225-27. 7. Mirelman D, Bracha
8
R, Chayen A, Aust-Kettis A, Diamond LS. Entamoeba histolytica: effect of growth conditions and bacterial associates on isoenzyme patterns and virulence. Exp Parasitol 1986; 62: 142-48 Mathews HM, Moss DM, Healy GR, Visvesvara GS. Polyacrylamide gel electrophoresis of isoenzymes from Entamoeba species. J Clin Microbiol 1983; 17:
1009-12. 9 Moss DM, Mathews HM. A fast
electrophoreuc isoenzyme technique for the and non-invasive Entamoeba histolytica and "E histolytica-like" organisms. J Protozool 1987; 34: 253-55 Editorial Is that amoeba harmful or not? Lancet 1985, i: 732-34. Editorial On the three stages of amoebic research Lancet 1986; ii. 1133-34 Goldmeier D, Sargeaunt PG, Pnce AB, et al Is Entamoeba histolytica in homosexual men a pathogen? Lancet 1986; i: 641-44. Allason-Jones E, Mindel A, Sargeaunt P, Williams P Entamoeba histolytica as a commensal intestinal parasite in homosexual men. N Engl J Med 1986; 315: identification of
10 11. 12. 13.
353-56
invasive
563
Reviews of Books Magnetic Resonance Imaging of the Knee J. H. Mink, M. A. Reicher, and J. V. Cruess. New York: Raven Press. 1987.
Pp 178.$95.50. ISBN 0-881673323.
To "explore" or worse still to remove a knee cartilage without first undertaking an arthrogram and/or an arthroscopy is today to invite criticism. In his monograph on arthrography Wolfe conceded that excellent arthroscopy was ultimately more rewarding than excellent arthrography. The fact remains that arthroscopy involves a day bed and an anaesthetic. Moreover, there is a tendency in some quarters to use arthroscopy as a screening test for almost any vague symptom between mid thigh and mid calf. Now magnetic resonance imaging has arrived to change all this. In a spectacular monograph, Mink and his colleagues explain the basic physics of MRI and apply it to the anatomy in superb detail. They then offer an elegant depiction of the wide range of synovial, meniscal, bony, ligamentous, and soft-tissue pathology that MRI can reveal. No other technique provides such a range of information so we can expect MRI, ultimately, to replace diagnostic arthroscopy. As a pointer to the future of investigating joint pathology this book is destined to be a classic. Department of Orthopaedic Surgery, Hope Hospital, Salford M6 8HD
J. NOBLE
The Causes of Crime New Biological Approaches.-Edited by Samoff A. Mednick, Terrie E. Moffitt, and Susan Stack. Cambridge: Cambridge University Press. 1987. Pp 364. 35. ISBN 0-521304024.
IT is slightly offputting to find that this multi-author book is based on the proceedings of a conference held in 1982. However, it becomes clear to the reader that whoever were the delinquent authors the rest have taken the opportunity to update their contributions. Therefore this volume represents the nearest thing available to a state-of-the-art review of the biological basis of criminality. As such it is bound to give offence in some quarters. The law-abiding world seems inevitably to divide into those who see criminal behaviour as inherent and constitutional and those who see the law-breaker as the hapless victim of his (or her) life circumstances. However, the articles included in this text provide persuasive arguments in favour of a determined occupation of a middle ground. A chapter by Mednick and co-workers reviews genetic factors, focusing particularly on adoption data, which
14. Mirelman D, Bracha R, Wexler A, Chayen A. Changes in isoenzyme patterns of a cloned culture of nonpathogenic Entamoeba histolytica during axenization Inf Immun 1986, 54: 827-32. 15. Robmson GL. The laboratory diagnosis of human parasitic amoebae Trans R Soc Trop Med Hyg 1968, 62: 285-94. 16. Farri TA, Sargeaunt PG, Warhurst DC, Williams JE, Bhojnani R. Electrophoretic studies of the hexokmase of Entamoeba histolytica groups I to IV. Trans R Soc Trop Med Hyg 1980; 74: 672-73. 17 Sargeaunt PG, Williams JE, Neal RA. A comparative study of Entamoeba histolytica (NIH 200, HK9 etc), "E. Histolytica like" and other morphologically identical amoeba using isoenzyme electrophoresis Trans R Soc Trop Med Hyg 1980; 74: 469-74.
provide impressive support for a genetic contribution to chronic offending but clearly also point to important environmental influences. Cloninger and Gottesman perform a masterly synthesis of family, twin, and adoption studies which strongly supports this general conclusion. There are informative reviews of studies of the psychophysiological substrate of antisocial behaviour, among which Venables’ article on autonomic nervous system factors is particularly valuable. Although there’ is some disparity in research findings, there is overall support for the idea that criminality is associated with low levels of tonic activity and low electrodermal responsivity. The idea that criminality is associated with "inability to learn from experience" is explored by Hare and Connelly and the possible role of factors such as disconnection between the "semantic and affective components of language" are interesting, if rather speculative. The tone of the book is not one of undiluted enthusiasm for the biological approach to antisocial behaviour. Cloninger strikes a cautious note in reviewing pharmacological approaches, calling for a more careful attempt at the classification of those subtypes of abnormal behaviour that might benefit from drug treatments. O’Callaghan and Carroll are even more outspoken in their condemnation of psychosurgical methods, which they see as "socially pernicious and ultimately ineffective". Despite its long gestation, this volume has emerged agreeably formed and without tell-tale signs of postmaturity. It is a useful, balanced, and scholarly contribution to a controversial and frequently messy field. Department of Psychological Medicine, University of Wales College of Medicine, Heath Park, Cardiff
PETER MCGUFFIN
Hypoxia, Polycythemia, and Chronic Mountain Sickness Robert M. Winslow and Carlos Monge C. Baltimore: Johns Hopkins University Press. 1988. Pp 255. 33.35. ISBN 0-801834481.
ALTHOUGH chronic mountain sickness (CMS) may seem rather esoteric topic for the average clinician, its study does throw light on many aspects of hypoxia and polycythaemia. Analogies with chronic obstructive lung disease and polycythaemia rubra vera are many and hence make the subjects of interest to a wider audience than high altitude specialists. This book is devoted to a wide ranging discussion of CMS. It does not set out to explore, in any depth, the mechanisms of the hypoxic stimulus to a
erythropoiesis. authors are experts. Robert Winslow is a haematologist who has taken part in several research expeditions to the Peruvian Andes where he collaborated with Carlos Monge. He was also a member of the 1981 American Medical Research Expedition to Everest where he conducted sophisticated haematological studies in a tented laboratory in the Western Cwm. Carlos Monge C is the son of Carlos Monge M, the Peruvian physician who first described CMS and after whom the disease is known. Monge C has worked on CMS for most of his professional life. There are chapters on every aspect of CMS including its history, comparative physiology, red cell mass and its control, effects on circulation, ventilation, renal function, exercise, and the effects of blood letting. The