group and two patients died among the patients treated with quinine.1 In addition 19 patients with cerebral malaria (12 with unrousable coma) were treated with artemether, and only 1 died.2 From the same hospital treatment with artemether and mefloquine combination or with quinine has been reported in 30 patient pairs with complicated falciparum malaria (including 6 pairs with over 10% of parasite-infected red blood cells).3 All patients treated with artemether/mefloquine survived. 11 with cerebral malaria were also so treated, and all survived.4 The parasite and fever clearance times in patients treated with artemether are shorter than in those receiving quinine. It is noteworthy that in the Burmese studies the best results were obtained with artemether/mefloquine combination. This prevented the recrudescences that are very common among patients treated with artemether alone. In addition, Chawira et al-I have reported the potentiative effect of artemisinin and mefloquine in rodent malaria, and Li et al6 have shown favourable results with mefloquine and artemisinin in patients with chloroquine-resistant falciparum malaria. Although the benefits of exchange blood transfusion are uncertain even in hospitals with good facilities, the inception of clinical trials with qinghaosu and its derivatives outside China and Burma may well have a large impact on mortality from severe and complicated falciparum malaria. Clinical Research Unit, Department of Medical Research,
Yangon (Rangoon), Union of
Institute of Medicine (1),
PE THAN MYINT
Department of Medical Parasitology, London School of Hygiene & Tropical Medicine, London WC1, UK
D. C. WARHURST
Myint PT, Shwe T. A controlled clinical trial of artemether (qinghaosu) versus quinine in complicated and severe falciparum malaria. Trans R Soc Trop Med Hyg 1987; 81: 559-61. 2. Myint PT, Shwe T, Soe L, Htut Y, Myint W. Clinical study of the treatment of cerebral malaria with artemether [qinghaosu derivative]. Trans R Soc Trop Med Hyg 1989; 83: 72. 3. Shwe T, Myint PT, Htut Y, Myint W, Soe L. The effect of mefloquine artemether compared with quinine on patients with complicated falciparum malaria. Trans R Soc Trop Med Hyg 1988; 82: 665-66. 4. Shwe T, Myint PT, Myint W, Htut Y, Soe L, Thwe M. Clinical studies on treatment of cerebral malaria with artemether and mefloquine. Trans R Soc Trop Med Hyg 1989; 83: 489. 5. Chawira AN, Warhurst DC, Robinson BL, Peters W. The effects of combinations of qinghaosu [artemisinin] with standard antimalarial drugs in the suppressive treatment of malaria in mice. Trans R Soc Trop Med Hyg 1987; 81: 554-58. 6. Li GQ, Guo ZB, Jin H, Wang ZC, Jian HX, Li ZY. Clinical studies on treatment of cerebral malaria with qinghaosu and its derivatives. J Tradit Chin Med 1982; 2: 1.
Superoxide dismutase and Parkinson’s disease neurons mainly affected in Parkinson’s the melanised neurons of the substantia nigra pars compacta.’ Although the cause of nigral cell death remains unknown, oxygen free radicals have been implicated as a potential cytotoxic mechanism.2 Biochemical pathways in the synthesis of neuromelanin, including auto-oxidation of dopamine, lead to the production of toxic free radicals.3 Moreover, in Parkinson’s disease the level of lipid peroxidation is increased in the substantia nigra of post-mortem brains.4 These observations suggest that nigral tisue is continually exposed to neurotoxic oxygen species and that alterations in the production and/or removal of oxygen radicals might play a part in dopaminergic cell death. Cytosolic superoxide dismutase (CuZn SOD; E.C.220.127.116.11) is one of the enzymes that protects against oxygen toxicity by catalysing the dismutation of superoxide anions (02’-) to oxygen and hydrogen peroxide (H202). An increased activity of the cytosolic’ and particulatebfractions of SOD has been seen in the substantia nigra ofparkinsonian patients. In human hippocampus7 CuZn SOD gene expression is not uniform among all cell types but is preferential in pyramidal and granular neurons. This finding has led to a search for specific characteristics of this defence system in the human substantia nigra. We have investigated CuZn SOD gene expression at the cellular level in post-mortem substantia nigra by in-situ hybridisation with a 3sS-labelled cDNA probe homologous to human CuZn SOD mRNA.7 Five brains from subjects with no evidence of psychiatric or neurological disease (mean age 83  years; mean post-mortem delay 8 [ 1] hours) were examined. Within 2 hours after necropsy the caudal part of the ventral mesencephalon was dissected and fixed by three-day immersion at 4°C in 4% (weight/volume) paraformaldehyde and 15% (weight/volume) saturated picric acid. Cryostat tissue sections (15 Nrn) were mounted on gelatine-coated slides and processed for in-situ hybridisation. Autoradiograms were generated from sections dipped in NTB2 emulsion (Kodak) (exposure 20 days at 4°C).’’ The distribution of positively hybridised cells was recorded with a computerised plotting system (Biocom,
France). Emulsion-coated sections were heavily labelled (appearing as an accumulation of silver grains). The grains overlay cell somata and were densely packed over the cytoplasm of the melanised neurons and more sparse over the neuromelanin. The labelled cells were densely distributed in substantia nigra pars compacta (figure)
Clinical diversity of sickle-cell anaemia SiR,—Or Christakis and colleagues (March 17, p 637) compare clinical and laboratory findings of their 30 patients who have sickle-cell anaemia with those of 310 Jamaican patients and find several differences. Despite the absence of concomitant alphathalassaemia, mean cell haemoglobin concentration was lower and haemolysis was less in Greek patients. Clinical diversity of sickle-cell anaemia could be genetically determined and/or related to factors of cellular modulation.’ If the haplotypes of these Greek patients had been provided Christakis and colleagues’ fmdings could have been compared with those for such patients in neighbouring countries. Haemolysis is also not severe in patients of Eti-Turk extraction2 and they generally have haplotype 19.3 Department of Paediatrics and Haematology, Hacettepe Children’s Hospital,
Plots of melanised neurons containing CuZn SOD mRNA in substantia nigra at the caudal level.
RP. Clinical diversity of sickle cell anemia. genetic and cellular modulation of disease severity. Am J Hematol 1983; 14: 405-16. 2. Ozsoylu S, Altinoz N. Sickle-cell anaemia in Turkey: evaluation of 97 cases (with parents’ findings). Scand J Haematol 1977; 19: 85-92. 3. Alouch JR, Kilinç Y, Aksoy M, et al. Sickle cell anaemia among Eti-Turks: haematological, clinical and genetic observations. Br J Haematol 1986; 64: 45-55.
Neurons were densely concentrated in pars compacta, topography of each cell type was obtained with computer-assisted microscope system (Biocom) in which each cell was plotted only once Bar 5 mm, CP, cerebral peduncle, RN, red nucleus; ML, median lemniscus; SN, substantia nigra.
Ankara 06100, Turkey 1.
Steinberg MH, Hebbel
whereas some were also present in the ventral tegmental area. No labelled cells were seen in the cerebral peduncle and superior cerebellar peduncle. Among positively hybridised cells (n 805), 95% (763) were the large neuromelanin-containing neurons. These data indicate that, within substantia nigra pars compacta, CuZn SOD gene is preferentially and highly expressed in the neuromelanin-pigmented neurons, a subset of cells vulnerable to degenerative processes in Parkinson’s disease.’ This observation suggests that within these cells biochemical pathways leading to generation of0*- radicals are especially active, thus needing a high CuZn SOD content to facilitate removal of these radicals. Alternatively, a high cellular CuZn SOD activity, by promoting H202 production, might also contribute to the vulnerability of these =
Laboratory of Genetics Biochemistry, CNRS URA 1335,
Hôpital Necker-Enfants Malades, 75015 Paris, France
Laboratory of Experimental Medicine, INSERM U 289, Hôpital de la Salpétrière, Paris
M. LAFON F. JAVOY-AGID E. HIRSCH
Laboratory of Genetics Biochemistry, Hôpital Necker-Enfants Malades
A. NICOLE P. M. SINET
Laboratory of Experimental Medicine, Hôpital de la Salpétrière
E, Graybiel AM, Agid Y. Melanized dopaminergic neurons are differentially susceptible to degeneration in Parkinson’s disease. Nature 1988; 334: 345-17. 2. Calne DB, Langston JW. Aetiology of Parkinson disease. Lancet 1983; ii. 1457-59. 3 Graham DG. Oxidative pathways for catecholamines m the genesis of neuromelanin and cytotoxic quinones. Mol Pharmacol 1978; 14: 633-34. 4. Dexter DT, Carter C, Agid F, et al. Lipid peroxidation as a cause of nigral cell death in Parkinson’s disease. Lancet 1986; ii. 639-40. 5. Martilla RJ, Lorentz H, Rinne UK. Oxygen toxicity protecting enzymes in Parkinson’s disease. J Neurol Sci 1988; 86: 321-31 6. Saggu H, Cooksey J, Dexter D, et al. A selective increase in particulate superoxide dismutase activity in parkinsonian substantia nigra J Neurochem 1989; 53: 692-97. 7. Ceballos I, Javoy-Agid F, Hirsch E, et al. Localization of copper zinc superoxide dismutase mRNA in human hippocampus by in situ hybridization Neurosci Lett 1989; 105: 41-46.
incubated at 30°C for 48 h. In parallel 1 ml of skin homogenate and a cavity swab were added to 20 ml of Oxoid tryptose phosphate broth and incubated at 5°C for 8 weeks before plating. With carcasses obtained on three occasions from the same slaughterhouse, a most probable number technique was used to determine levels of naturally occurring L monocytogenes. The method involved "hot" enrichment2 and plating on the Oxford medium, with incubation as above. Carcasses were examined by taking skin samples. Representative isolates were subjected to confirmatory tests.216 freshly processed carcasses were examined for naturally occurring L monocytogenes. 10 were positive, with counts of 0 36 to 24/cm2 (mean 4-3), which would have had little effect on the levels of artificial inocula. Inoculated carcasses were examined at 0, 7, 14, and 21 days when held at 5°C and 0, 3, 5, and 7 days for birds kept at 10’C, but in the following table results for all storage periods are combined: was
Proportion positive for L monocytogenes after storage at.
5°C 102 Time Before storage After storage
(CFU/cm2) (CFU/cm2) (CFU/cm2) (CFU /CM2) 0/3 0/3 1/33 0/3 3/9 1/9 0/9 4/9
Immediately after irradiation, only 1 of 12 carcasses was positive for L morwcytogenes and no further positives were found until day 14 at 5°C and day 5 at 10°C. On completion of each storage period, the carcasses remained unspoiled but 8/36 (22%) yielded L monocytogenes, mainly from the larger inoculum. At the lower inoculation level, which is more in keeping with natural contamination, only 1 of 18 carcasses showed any surviving listeria. At both 5°C and 10°C, counts on non-irradiated birds increased 100-fold after 7 and 3 days, respectively, but spoilage was becoming evident at 10°C and no further tests were made on birds held at this temperature. Spoilage of non-irradiated birds kept at 5°C was detectable after 7 days, by which time L monocytogenes had increased 1000-fold. Only very slow growth of any survivors was observed initially at either storage temperature for the irradiated birds; after 7 days at 10°C, counts had reached a mean of almost
growth of Listeria monocytogenes on irradiated poultry carcasses
SIR,-In the UK poultry carcasses are often contaminated with Listeria monocytogenes,1,2 a potential pathogen that can grow under chill conditions. Because irradiation is to be permitted in the UK and has been proposed as a means of eliminating listeria,3 we have investigated the effects of irradiation on the survival of L monocytogenes on poultry carcasses and on the behaviour of the organism during cold storage. Previous studies4-7 suggest that the susceptibility of L monocytogenes to irradiation is comparable with that of the salmonellae, and doses of 25-7-0 kGy should be sufficient to effect elimination. However, for chilled poultry doses above 25 kGy may affect the odour, colour, or flavour of the cooked 8
product. We used a mixture of four strains of L monocytogenes, isolated from poultry, to inoculate freshly processed, air-chilled broiler carcasses obtained from a commercial slaughterhouse. Inocula of about 100 or 10 000 colony-forming units/cm2 from diluted broth cultures, were spread over the surface of the carcass with an alginate swab. The abdominal cavity was also inoculated. The carcasses were then placed in double polythene bags, and those requiring irradiation were taken under refrigeration to a nearby treatment plant, where they were given 25 kGy gamma radiation from a cobalt-60 source. Irradiated and non-irradiated carcasses were stored at 5 or 10°C and individual birds were removed at suitable intervals for sampling. The experiment was done three times on different occasions. Carcasses were sampled by taking five 10 cmz portions of skin which were bulked and homogenised for diluting and plating directly on Oxoid listeria selective agar (Oxford formulation), which
study confirms that L monocytogenes is
common on raw
but shows that numbers are likely to be low poultry immediately after processing and that they will largely be destroyed by gamma irradiation at 25 kGy. Where survivors were found, after irradiation, they either recovered slowly from sublethal injury or multiplied to detectable levels from small numbers of uninjured cells. Because listerias seem to grow well on poultry skin at 5OC, any multiplication before irradiation would progressively reduce the chances of elimination from chilled carcasses, and our results do not support the contention of Huhtanen et aF that 20 kGy is sufficient to destroy 10 000 L monocytogenes on poultry. Listeria may sometimes grow under commercial conditions of transportation to a radiation treatment centre, and potential users of irradiation for chilled poultry should be aware of the possibility. carcasses
Bristol Laboratory, AFRC Institute of Food Research, Langford, Bristol BS18 7DY, UK
G. C. MEAD W. R. HUDSON RADZIAH ARIFFIN
1. Pini PN, Gilbert RJ. The occurrence in the UK of Lisieria species in raw chickens and soft cheeses. Int J Food Microbiol 1988, 6: 317-26. 2 Hudson WR, Mead GC. Listeria contamination at a poultry processing plant Lett Appl Microbiol 1989; 9: 211-14. 3. World Health Organisation. Foodborne listeriosis: report of a WHO informal working group (WHO/EHE/FOS 88 5). Geneva WHO, 1988. 4. Stegeman H. Radiation resistance of Listeria monocytogenes. 10th International Symposium on Listeriosis (Pecs, Hungary, Aug, 22-26, 1988 104 (abstr). 5. Tarjan V. The sensitivity of Listeria moncytogenes to gamma radiation 10th International Symposium on Listeriosis (Pecs, Hungary, Aug, 22-26, 1988) 105
(abstr). 6. Patterson M. Sensitivity of Listeria monocytogenes to irradiation on poultry meat and in phosphate-buffered saline Lett Appl Microbiol 1989, 8: 181-84 7. Huhtanen CN, Jenkins RK, Thayer DW Gamma radiation sensitivity of Listeria monocytogenes.J Food Protect 1989; 52: 610-13 8 Mulder RWAW. Salmonella radicidation of poultry carcasses Doctoral thesis, Spelderholt Institute for Poultry Research, Beekbergen, Netherlands, 1982