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absence of breast-feeding is recognized

as the single most important risk factor for diarrhoea in young children 2, so this activity should be strongly encouraged. The development of multiple barriers (such as environmental interventions)in conjunction with other preventative and curative measures to control infection was seen as an important: multidisciplinary approach for achieving significant reduction in the incidence of these diseases. Probably the most important educational intervention would be to motivate and train the populations affected by these dise.ases, and so enable them to make the necessary changes to effectively prevent the transmission of these pathogens. In addition to public information programmes and school-based health education, training courses on important intestinal proto-

zoa should be encouraged for primary health and day care workers, workers in diagnostic laboratories and water treatment plant operators. More effective diagnoses carried out in clinics and laboratories in the developing world will enhance the targeting of treatment. In addition, provided that accurate statistics are collected, the

view of the importance of intestinal protozoal diseases held by governments and international agencies will be more realistic, and lead to correct targeting of aid and research funds. The aim of all these studies, prevention and control of intestinal protozoan disease, is becoming a possibility. Acknowledgements The WHO/PAHO Informal Consultation on Intestinal Protozoa/Infections was held 21-23

October in Mexico City, Mexico. This

report represents the authors' personal view of the proceedings. The full report of the meeting was published in May as WHO/ CDS/IPI/92.2. Copies of the document are available through the Programme for Intestinal Parasitic Infections, Division of Communicable Diseases, World Health Organization, 121 I Geneva 27, Switzerland, and also from Dr Fernando Beltran in PAHO. References I Anon. (I 990) Health Research."Essential Link to Equity in Development, O x f o r d University Press 2 Anon. (1990) Programme for Control of Diar-

rhoeal D~seases: Interim Programme Report, WHO

David Warhurst is at the PHLS Malaria Reference Laboratory, London School of Hygiene and Tropical Medicine, Keppel Street, London, UK WCIE 7HT and Huw Smith is at the Department of Bacteriology, Stobhill General Hospital, Glasgow, UK G21 3UW.

Euroleish III Workshop R. Maingon, M. Segoviaand D. Le Ray Archena, Spain March 1992 Archena, in the Murcia region of Spain, was host to the Third Furoleish Workshop, which drew together senior and young biochemists, immunologists and

molecular biologists from the Euroleish network (of holders of [-C-STD grants on leishmaniasis) so as to establish new perspectives in current research on Leishmania and to discuss co-operation (North-South, North-North and South-South). H I V and Leishmania Co-infection

Although leishmaniasis is often assumed to be a 'tropical' disease, it is still significant in south Mediterranean areas. In Spain, it has increased to I00 new cases per year as a consequence of the increase in the morbidity of canine leishmaniasis (prevalence of 5%) and the emergence of new risk population groups for human visceral leishmaniasis (VL) (Monserrat Port0s, Universidad de Barcelona, Spain). It is ,estimated that 50% of VL in adults are HIV-Leishmania co-infections and that between I% and 3% of AIDS patients have VL in Southwestern Europe. About 30% of co-infected patients do not show anti-Leishmania antibodies. The real chal~) 1992.ElsevierSciencePublishersLtd. (UK)

lenge is to detect the actual percentage of the population carrying cryptic asymptomatic infections. VL-specific primers for the polymerase chain reaction (PCR) with blood samples have been particularly useful Gorge Alvar, Instituto Carlos III, Madrid, Spain). An alternative method, applied to early diagnosis of VL in Brazil, is based on a chemiluminescent VL-specific DNA probe' (Michael Miles, London School of Hygiene and Tropical Medicine, UK). Parasite detection in urine samples is now possible, using a robust and cheap dipstick-coloured colloid assay2 (Cibdle Dourado, Liverpool School of Tropical Medicine, UK). These methods should improve the sensitivity and specificity of current prevalence surveys; direct agglutination tests have shown that 10% of apparently healthy dogs in Corsica and Greece carry L. infantum (Y. Tselentis, University of Crete, Greece). Reaching Parasite Targets W i t h Old and N e w Drugs Leishmania resembles fungi both in its sterol and in its protein carboxymethylation metabolism. This similarity has been exploited by researchers interested in using known anti-fungal compounds as anti-leishmanial agents. Imidazole and triazole drugs, inhibiting

cytochrome P450-dependent 14-0~methylsterol demethylase, affect parasite growth at a rate of I Ftgml-'. Their anti-leishmanial activity is increased synergistically by binary or ternary combinations of inhibitors acting upon different enzymes in the sterol pathway (Michael Chance, Liverpool School of Tropical Medicine, UK). Another interesting anti-fungal is sinefungin, a nucleoside-5'-adenosylmethionine analogue that inhibits protein-O-methyltransferases, enzymes known to regulate the function of membrane protein transporters (M. Robert-Gdro, Institut de Chimie des Substances Naturelles, Gifsur-Yvette, France). Among the new targets for chemotherapeutic attack are the cysteine proteases 3. Since these enzymes have been conserved throughout evolution, extensive studies are necessary before choosing the most appropriate target enzyme for further rational drug design. Two out of seven different types of cysteine protease in Leishmonia are significantly different from the mammalian cysteine proteases (Graham Coombs, University of Glasgow, UK). Anti-leishmanial therapy is hampered by the location of the parasite within phagolysosomes of the macrophage, which restricts the delivery of many 'old' or 'new' potentially useful drugs. Recently, targeting of allopurinolriboside and methotrexate to the mannose-

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fucose receptor on the surface of L. donovani-infected murine macrophages has been possible by using chemical linkage to a mannose-substituted polyc-lysine carrier molecule (Mark Hommel, Liverpool School of Tropical Medicine, UK). Another problem with the chemotherapy of leishmaniasis is that it frequently leads to resistance. Several molecular mechanisms can be responsible for resistance of Leishmania to the same drug: amplification of the target gene, alteration in the transport of the drug into the parasite and expression of an altered target protein. The first example of an altered target enzyme in drug-resistant Leishmania was reported for the bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) enzyme which catalyses sequential reactions in the synthesis of dTMP. The gene was isolated, using the polymerase chain reaction method, from a strain of L. major resistant to methotrexate and was shown to produce a mutant (Met53--~Arg53) enzyme that binds the inhibitor poorly compared to the wild enzyme (Luis M. Ruiz, Instituto Lopez Neyra de Parasitologia, Granada, Spain).

Host Immunity, Parasite Virulence Factors and Pathogenesis Although various relatively wellcharacterized antigens, such as gp63, heat shock proteins, gp46 and lipopolyglycan (LPG) are potential parasite virulence factors, it is the cytokine concentrations and the profile elicited by these and other antigens, as well as the specific CD4+/CD8 + T-cell subsets that predominate in the host, that are the crucial events in determining the healing or exacerbation of the disease. Studies on the human response in 441 Colombian patients with tegumentary leishmaniasis caused by Leishmania of the viannia subgenus revealed that increased duration of the disease is associated with increased cell-mediated and antibody responses to Leishmania antigens (Nancy Saravia, Fundacion ClDEIM, Call, Colombia). Since asymptomatic infections outnumbered the number of cases, and recurrent infection and disease were common, criteria to define susceptible and resistant human populations affected by this subgenus were proposed by analogy with inbred (eg. CBA and BALB/c) mouse models of leishmaniasis. Nonspecific human immune responses are also common. In fact, crude antigen preparations induced proliferation, in

vitro, and the production of gammainterferon (IFN-y), mainly in CD2 ÷ cells from individuals with no previous exposure to Leishmania, suggesting that epitopes are shared between this parasite and environmental antigens. Individuals cured from kala-azar have expanded T-cell clones (secreting IFN-y) that recognize LPG but not gp63 preparations from L. major (Thor Theander, Rigshospitalet, Copenhagen, Denmark). The availability of Leishmania-specific human T-cell clones that secrete either interleukin 4 (THI) or IFN-y (TH2) opens the possibility of identifying human T-cell subset-specific Leishmania epitopes. The role of gp63 as a specific metalIoproteinase, in the binding and invasion of macrophages and as a heavily glycosylated 'virulence' factor, cannot be overstated4. An inhibitory effect of gp63 on the chemotactic response of neutrophils but not of monocytes, combined with a stimulation of the reactive oxygen production in monocytes, relative to neutrophils, should help the chances of parasite survival inside monocytes (Arsalan Kharazmi, Rigshospitalet, Copenhagen, Denmark). Apart from cleavage/activation of C3, gp63 may cleave specifically CD4 ÷ molecules on human T cells, leaving CD8+-presenting T cells in charge of IFN-y production (Adam Hey, Rigshospitalet, Copenhagen, Denmark). Collective evidence suggests that the correlation between gp63 and parasite virulence may not be simple. Leishmania transfection vectors were constructed with the tunicamydn resistance gene as a selective marker for expression of genes encoding potential virulence factors such as gp63 (Kwang Poo Chang, Chicago Medical School, USA). Leishmania transfection of attenuated strains and deletion of genes in wild type strains should provide a powerful tool for examining the role of putative parasite virulence factors. In contrast, in BALB/c mice, an avirulent L. major cloned line (COl) showed relatively higher levels of gp63 and macrophage attachment than did a virulent line (CORI), although the latter and not COl was able to survive inside macrophages (Miguel Navarro, Universidad de Murcia, Spain). Collective evidence suggests that the correlation between gp63 and parasite virulence may not be simple. Other molecules, such as the cell adhesion molecules (eg. beta integrins), may be more relevant in the invasion of mouse macrophages by L. pifanoi. Parasite killing in this model system appears to be the result of increased

nitric oxide production in activated macrophages. Infected macrophages are in turn activated by IFN-y and TNF-0~ secreted by L. pifanoi-immune T cells (Manuel Fresno, Universidad Autonoma de Madrid, Spain). This is in agreement with various promising vaccination studies in mice using either L. infantum sub-cellular fractions or a defined processed 18kDa antigen secreted by macrophages in response to promastigotes (Loic Monjour, Facult~ de M~decine Piti~-Salpetri~re, Paris, France). Similarly, in mice vaccinated with a gp46-vaccinia recombinant virus, protection was associated with high IFN-y production and TNF-0~ by THI cells (Luis Rivas, Centro de Investigaciones Biologicas, Madrid, Spain).

Genome Plasticity in Field Isolates The variability of chromosome structure and selected polymorphic sequences is impressive and suggestive of a role in increasing the chances for adaptability and survival of Leishmania to changing environments. Amplification and deletion of different sequencespecific K-DNA minicircles occurs spontaneously and under tunicamycin selection of L. amazonensis (Kwang Poo Chang). A L. donovani sequence showing stage-specific restriction fragment length polymorphisms and chromosomal polymorphisms among L. majorcloned lines, differing in their virulence in BALB/c mice, may be a mobile genetic elements (Rhaysa Maingon). The L. major model system shows size polymorphic multicopy minichromosomes related to previously described LD I/CD 1/715 class6. The L. major elements appear, de novo, and may also be lost through multiple transfers during culture, in vitro, or passages in mice, in vivo. DNA sequences derived from one of these elements (M 180) are present in either a 1.5Mb or a 770kb chromosome, suggesting multiple sources and/or minichromosome recombination (Manuel Segovia). The elements from different species of Leishmania share a common transcribed region that could code for functions required for their generation or maintenance. Chi-like sequences, similar to those in prokaryotes, flank the common region in CDI elements of L. mexicana and L. infantum. By analogy to E. coil, these sequences may be used by recombination enzymes for the interconversion between the circular and the linear elements (Raymond Hamers, Vrije Universiteit Brussel, Belgium).

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Chromosome variability is not a curiosity of the laboratory models; it occurs in 22 taxonomic~.lly related L. infantum field isolates from the border region between France and Spain. Strains producing cutaneous disease had high karyotypic homology, in contrast with the extreme polymorphism observed for chromosomes I-5 of other strains from the same Ioc~Llity(Christine Blaineau, Faculte de Medecine, Montpellier, France). The analysis of association frequencies between size variants suggested genetic exchange7. Considerable variation is also observed in both the chromosomal localization of protein-coding genes ancl the number and relative proportion of sequencespecific K-DNA minicircles between L. peruviana or L. brasiliensis individual strains (Jorge Arevalo, Universidad Peruana Cayetano Heredia, Lima, Per~).

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Acknowledgements The Euroleish III Workshop: Biochemistry, Immunology and Molecular Biology was held 10-14 March 1992 in Archena, Spain, and was sponsored by the Third Programme for Life Sciences and Technologies for Developing countries (STD3) of the European Communiites. Additional financial support was provided by the Comunidad Autonoma and the Universidad de Murcia. We are specially indebted to Soren Jepsen (DGXllG4 Division, EEC Headquarters) for his constant interest and support. We thank all participants, session's chairpersons and Isabel Navarro (Universidad de Murcia) for their contributions to the running of the workshop. This report represents our personal view of the proceedings. References

I Howard, M. et al. (1992) Trans. R. Sac. Trap. Mec~ Hyg. 86, 35-36 2 Snowden, K. and Hommel, M. (1991) J. Immunol. Methods 140, 57-65 3 Coombs, G., Robertson, C. and Mottram, J.

Legionella pneumophila mip Gene Potentiates Intracellular Infection of Protozoa and H u m a n Macrophages N.P. Cianciotto and B.S. Fields Proc. Nat/Acad. Sci. USA 89, 5188-5191 Strains of Legionella pneumophila, which are ubiquitous within aquatic environments, represent a major environmental pathogenic threat. When inhaled by humans, L. pneumophila invades and proliferates within alveolar macrophages, resulting in acute bronchopneumonia. What has remained a mystery is how it has evolved the ability to parasitize human phagocytes. It flourishes in aquatic environments as an intracellular parasite of protozoa such as

Acanthamoeba, Echinomoeba and TetrahymencJ. Somehow, the ability of L. pneumophila to adapt to intracellular niches within protozoa has given it the ability to infect mammalian cells. It is known that a 24kDa macrophage infectivity potentiator (Mip) surface protein is necessary for optimal infection of macrophages and, in this paper, Cianciotto and Fields look at Hartmannella amoebae and Tetrahymena ciliates to see if a strain of L.

Hydroxylated Metabolites of the A n t i m a l a r i a l Drug Primaquine J. Vasquez-Vivar and O. AugustoJ. Biol. Chem. 267, 6848-6854 This paper presents data on the characterization of products and intermediates formed from primaquine-hydroxylated metabolites, with the aim of determining their possible involvement in the toxic effects of primaquine. Under physiological conditions, the three metabolites oxidize to form hydrogen p e r o x i d e H 2 0 2 and the corresponding quinone-imine derivatives as the main products. The latter are here characterized by visible, NMR and infrared spectroscopy, and the concomitant formation of drug-derived radicals and hydroxy radicals, and their tentative ~) 1992,ElsevierSciencePublishersLtd, (UK)

structures, are ascertained. The quinone-imines undergo redox cycling upon addition of ferredoxin: NADP ÷ oxidoreductase, forming hydrogen peroxide and hydroxyl radicals, the main route being the metal ioncatalyzed reaction between the drugderived radicals and hydrogen peroxide. Altogether, the results from this S~o Paulo group indicate that H 2 0 2 is the potential toxic product formed from primaquine metabolites. Interestingly, they propose a role for H 2 0 2 in the well-documented toxic effect of primaquine in patients with glucose-6-

4 5 6 7

( 1991) in Biochemical Protozoology (Coombs, G. and North, M., eds), pp 208-220, Taylor & Francis Chang, K.P. and Chaudhuri, G. (1990)Annu. Rev. /Microbial. 44, 499-529 Maingon, R. et al. (1990) in UCLA Symp./Viol. Cell. Biol. (Vol. 130) (Agabian, N and Cerami, T., eds), pp 191-204, Wiley-Liss Beverley, S. (1991)Annu. Rev. MIicrobiol. 45, 417-444 Bastien, P., Blaineau, C. and Pages, M. (1992) Parasitology Today 8, 174-177

Rhaysa Maingon is at the Wolfson Unit of Molecular Genetics, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, UK !_3 50__A. Manuel Segovia is at the Departamento de Microbiologia, Facultad de Medicina, Universidad de Murcia, Campus Espinardo, Murcia, Spain and Dominique Le Ray is at the Laboratoire de Protozoologie, Prince Leopold Institute of Tropical Medicine, Nationalestraat 155, B-2000 Antwerpen, Belgium.

pneumophila that lacks Mip retains the ability to infect these protozoa. After three days, although mip mutants retained the ability to bind to amoebae cell surfaces, there were 1000-fold fewer bacteria in protozoan cultures infected with the Mip- strain than in cocultures infected with an isogenic Mip ÷ strain. Hence, there would appear to be similar genes and mechanisms involved in the infection of human cells and protozoa by L. pneumophila, suggesting that intracellular parasites of mammalian ceils may have evolved from free-living forms that first adapted to growth within lower eukaryotes. •

phosphate dehydrogenase deficiency. Primaquine-hydroxylated metabolites accumulate in the blood of patients, undergo redox cycling and produce H 2 0 2 in excess. The removal of H 2 0 2 from erythrocytes is mediated by three primary pathways, each dependent on a different protein. All routes, however, are NADPH dependent, and if cellular metabolism results in decreased NADPH production, as is the case in glucose-6-phosphate dehydrogenase deficiency, then the H 2 0 2 is not removed. • Outlook was compiled by T. Saklatvala from suggestions by A.E. Bianco, S.L. James, P.H. David and C.P.J. Ash.