[Weil's disease: a severe case with respiratory insufficiency]. / Weilova nemoc: tezký prubeh s respiracní insuficiencí.

In the patient case (man, age 25) with suspected leptospirosis, indication for polymerase chain reaction (PCR) are supposed and procedures suitable for taking biological material are recommended. In the presented case of leptospirosis, serious conditions were accompanied by high fever, chills, hepatorenal failure, meningitis, pneumonia, increased bleeding time and further symptoms are described. Introduction of the molecular biological methods (PCR) enables to determine leptospiroses diagnosis even in the early phase of the disease, when the antibodies are not yet formed. Detection of DNA pathogenic leptospires in the PCR method is completed with serological examination by microagglutination-lysis (MAL) method for determination of the corresponding serovar that is important from therapeutic and epidemiological reasons.

PCR-SSCP comparison of 16S rDNA sequence diversity in soil DNA obtained using different isolation and purification methods.

This study compared different methods of direct DNA extraction and purification from a silt loam soil and investigated the relationship between DNA quantity and sequence diversity. Five extraction methods and four purification techniques were investigated. Quantities of DNA extracted were between 3.4+/-0.55 and 54.3+/-8.18 &mgr;g g(-1) (dry wt) of soil with OD(260)/OD(230) purity ratios between 0.80 and 1.15. Analysis of sequence diversity in all extracts was conducted using PCR-single strand conformation polymorphism (SSCP). Profiles generated using universal 16S rDNA primers (Com1/Com2) were found to be identical when used to amplify 16S rDNA extracted directly from soil. The genus Pseudomonas was targeted in order to reduce profile complexity, which was apparent when using universal 16S rDNA primers, and which hindered direct comparison of sequence diversity. A Pseudomonas culture library and non-cultured Pseudomonas 16S rDNA genes were used to provide a background count of Pseudomonas operational taxonomic units present in the soil. Cloning and sequencing of amplicons generated using a Pseudomonas-specific (Ps-for) and a universal 16S rDNA (Com2) primer, coupled with nested amplification (Com1/Com2 amplification from Ps-for/Ps-rev amplicons), used in conjunction with SSCP, revealed that environmental contaminants co-extracted with DNA, such as humic acid, significantly reduced primer specificity. SSCP was sensitive enough to reveal template bias in different primer sets. PCR-restriction fragment length-SSCP of Pseudomonas 16S rDNA amplified from soil-extracted DNA revealed distinct differences in sequence representation between extraction methods and showed that greater DNA yield is not synonymous with higher sequence diversity. We, therefore, suggest that DNA extractions from soil should be evaluated not only in terms of quantity and purity, but also in terms of the sequence diversity present. SSCP proved to be a valuable tool for the assessment of the methodologies commonly used in PCR-mediated microbial ecology studies.

Biota participating in wastewater treatment in a horizontal flow constructed wetland.

During the period 1996-1997, three constructed wetlands with sub-surface horizontal flow were investigated. All systems are designed to treat municipal sewage from small villages (150, 200 and 300 PE). The survey included microscopical identification of organisms in both wastewater and filtration substrate. The organisms were used as an indication of oxygen conditions (aerobic, anoxic and anaerobic) in the particular microenvironment. Saprobiological terms characterizing different levels of saprobity were employed to characterize inflowing wastewater, filtration bed and outflowing water. The occurrence of organisms was correlated with BOD5 values in particular profiles. It has been found that the biocenosis in the inflowing wastewater differs from those found in the filtration bed and water outflowing from the vegetated beds. The organisms were grouped into those living under anaerobic and anoxic conditions and those living under aerobic conditions. More than 70 species of bacteria, amoebae, ciliates, rotifers, colorless flagellates, cyanobacteria and algae were found and the most important 45 species were figured in a plate together with saprobiological information for each species. Biota of the inflowing water is usually restricted to bacteria, ciliata and colorless flagellata while the organisms found in outflowing water as well as in periphyton growing on outflow structures indicate 2-3 levels better quality.

Dechlorination of polychlorinated biphenyls, dibenzo-p-dioxins and dibenzofurans on fly ash.

Dechlorination of commercial mixtures of polychlorinated biphenyls (PCB) as well as polychlorinated dibenzo-p-dioxins (PCDD) and dibenzofurans (PCDF) on extracted and non-extracted fly ash obtained from municipal waste incinerator (MWI) was studied in closed systems under nitrogen atmosphere at temperatures of 260 degrees C and 340 degrees C. Decomposition results (given as the difference between PCB or PCDD/F molar amounts before and after the experiment (in %) due predominantly to dechlorination reactions) and detoxification data (expressed similarly but related to toxic PCB and PCDD/F congeners only and given in I-TEQ units) are reported. Detoxification of Delor 105/80T at 260 degrees C and 340 degrees C at a loading of 0.65 wt%, was 99.48% and 100%, respectively. The decomposition of Delor 103 at 340 degrees C and for the loading of 0.75 wt%, corresponded to 99.99%. The detoxification capability of PCDD/Fs on extracted and non-extracted fly ash for loading of 130 and 264 ng/0.4 g of fly ash at 340 degrees C made 96 and 98%, respectively.

Managing death. Room to mourn.

More than a quarter of deaths occurred on wards which offered relatives and staff no privacy. In a fifth of cases no information was available on how to register a death. Almost half of the staff had received no training in bereavement. More than half of the staff wanted training or further training.

Bee venom melittin blocks neutrophil O2- production.

Bee venom (BV) is used in folk medicine to treat arthritis. It has antiinflammatory effects in animal models of rheumatic disease. We have studied the effects of BV on human neutrophil production of superoxide (O2-) and hydrogen peroxide, finding potent, nontoxic, dose-dependent production inhibition. Melittin, the major fraction of BV (50-70%) shows high-affinity calmodulin binding (Kd 3 nM). Drugs which bind calmodulin, such as trifluoperazine, inhibit O2- production by human neutrophils. For these reasons we have investigated the effect of melittin and other BV peptides on O2- production by human peripheral blood leukocytes. We show that melittin inhibited O2- production both pre- and poststimulation in contrast to other BV fractions which were without effect. Oxygen radicals and their derivatives from inflammatory cells are implicated in the tissue damage occurring during inflammation. The inhibition is due to a direct effect on cells, and not indicator medium, dismutation, toxic or scavenging effects. We propose that melittin may serve as a prototype small (mol wt 1280), cationic, amphipathic, calmodulin-binding, membrane-active, superoxide-production-inhibiting peptide, providing a model for peptides which could have a role in in vivo regulation of radical production.

T-cell subsets and natural killer activity in Plasmodium falciparum-infected children.

Thirty children acutely infected by Plasmodium falciparum and suffering either benign uncomplicated malaria (17 cases), or cerebral malaria (13 cases), were investigated for T-cell number and subset distribution among peripheral blood mononuclear cells using OKT3, OKT4, and OKT8 monoclonal antibodies, and for natural killer (NK) activity using K562 cells as targets. They were compared to a group of 16 age- and sex-matched healthy Senegalese children. OKT8 cell percentage was found increased in both groups of patients with a decrease of OKT4 cell percentage in cerebral malaria patients only. Both groups thus exhibited a decreased OKT4/OKT8 ratio, which was slightly lower in cerebral malaria cases than in benign cases. NK activity was found elevated in uncomplicated cases of malaria, in contrast to patients suffering cerebral malaria, who exhibited a profound depression of NK activity.

Various strains of BCG vaccine and genetic control of the immune response of mice.

Numerous field trials of BCG vaccine, undertaken to evaluate its effectiveness in the prophylaxis of human tuberculosis, indicated that the levels of protection achieved were not uniform. Most probable factors influencing these results are: partial protection due to atypical mycobacteria, tuberculosis expectancy in different population, nutritional and socio-economic factors, potency of BCG vaccines, and genetic factors controlling the host immune response. Specific and non specific hallmarks of cell-mediated immunity after BCG infection were analysed in various outbred, inbred and recombinant lines of mice, testing the capacity of different preparations and strains of BCG vaccine. Results of such studies did show that the by far most important factor involved the type of immune response related to gene(s) controlling the natural resistance and the expression of C.M.I. Consequences for future mass and individual BCG vaccination as well as the proper integration of BCG vaccination in antituberculous policy are discussed.

Vaccines against mycobacteria and other intracellular multiplying bacteria.

As the prototype of a vaccine against mycobacterial infection, the BCG (bacille bilié Calmette et Guérin) has been used against tuberculosis for more than 60 years. It is the only live attenuated vaccine used on humans in more than 182 countries or territories in the world, and very few changes have been made in its fabrication and distribution, except for the production of lyophilized seed-lots. However, its history is marked with controversies concerning its innocuity and efficacy. While BCG safety is no longer a matter of debate, the question of its effectiveness is still pertinent, and results in several controlled trials have shown great variability (from 0 to 80%). The studies of different variables involved in such results have shown statistical bias, and numerous factors are involved in the highly complex interrelationships between the host, the pathogen and environmental factors. World-wide research is now being conducted under the auspices of the World Health Organisation, in order to gain further knowledge of the immunology of tuberculosis and leprosy. Such results are aimed at understanding variations in BCG efficacy and producing strategies for developing new vaccines and alternative methods for prophylaxis and diagnosis. Concerning human infections due to other facultative intracellular multiplying bacteria, there are relatively few vaccines which are able to give long-lasting and efficient protection. Some controversy remains as to the live attenuated mutant GalE S. typhi Ty21a, and there is hope for the new insoluble phenol extract from Brucella abortis, strain B19. Further research is necessary on the others, for instance, Listeria monocytogenes, Chlamydia trachomatis and Legionella sp.

Bee venom inhibits superoxide production by human neutrophils.

Investigation of the antiinflammatory properties of bee venom demonstrates that it inhibits production of superoxide anion by human neutrophils in a potent, selective, nontoxic, dose-dependent fashion, both pre- and poststimulation by particulate and soluble activators of the neutrophil oxidative metabolism burst. The effect is not due to receptor competition, superoxide dismutase, and/or catalase activity, scavenging, or indicator media effects. These findings may explain the antiinflammatory effects of whole bee venom in experimental systems, its widespread use in folk medicine, and lead to the development of potent, new antiinflammatory substances for therapeutic use in man.

Natural resistance to mycobacteria: antimicrobial activity and reactive oxygen intermediate releasing functions of murine macrophages.

After infection with Mycobacterium lepraemurium or with M. bovis strain BCG (bacillus Calmette-Guérin), splenic macrophages from mice naturally resistant (NR) to these pathogens (C3H and A/J) spontaneously produced H2O2, whereas splenic macrophages from naturally susceptible (NS) mice (C57BL/6 and Swiss) did not. None of them produced superoxide anion O2-. In addition, NR macrophages had higher levels of superoxide dismutase (SOD) than did NS macrophages. In vivo treatments thought to enhance H2O2 metabolism (phorbol myristate acetate, SOD, Zymosan) decreased M. lepraemurium survival, whereas treatment with diethyl-dithiocarbamate, a potent inhibitor of SOD, had the converse effect. These results favour the hypothesis of a link between natural resistance to BCG (and M. lepraemurium) and H2O2 metabolism, with higher producers being naturally resistant.

Preliminary evidence of natural resistance to Mycobacterium bovis (BCG) in lepromatous leprosy.

An assay system has been developed based on radiometric quantification of 3H uracil incorporation into viable BCG in the absence or presence of blood monocytes in cultures from untreated lepromatous (LL) or tuberculoid (TT) leprosy patients. 3H Uracil incorporation into BCG was inhibited when the bacilli were cultivated in the presence of blood-derived macrophages in culture for four days, and that inhibition was always greater with macrophages harvested from LL patients compared to TT patients. The reasons for such an observed difference in humans are discussed according to our knowledge obtained in murine models of mycobacterial infections.

Phenotypic expression of genetically-controlled natural resistance to Mycobacterium bovis (BCG).

Mycobacterium bovis (BCG), when maintained in vitro, readily incorporates [3H]uracil, the RNA precursor. The rate of [3H]uracil incorporation into bacilli is sharply reduced when the BCG is phagocytized by murine adherent resident peritoneal macrophages and subsequently released by the lysis of monolayers. Macrophages derived from mouse strains that are innately resistant to BCG infection in vivo (Bcgr) are able to inhibit the [3H]uracil incorporation into the bacilli in a significantly more effective way than macrophages from BCG-susceptible (Bcgs) strains. This difference is best demonstrated with a low rate of infection (BCG: macrophage ratio between 1:1 and 2:1), and is most pronounced at 4 to 5 days after in vitro infection of macrophage monolayers. In vivo interaction of BCG with peritoneal macrophages in situ results in the same pattern of enhanced inhibition of [3H]uracil incorporation by Bcgr macrophages. The use of Bcg-congenic mouse strains has confirmed that the Chromosome 1 Bcg (Ity, Lsh) locus is regulating the antimycobacterial activity of macrophages. We conclude that the resident macrophage is the cell population that expresses the phenotype of genetically determined resistance to BCG infection.

[Use of purified antigens in the serodiagnosis and epidemiologic studies of human malaria. Importance of the ELISA technic for determining specific IgG and IgM]. / Utilisation d'antigènes purifiés pour le sérodiagnostic et les études épidémilogiques du paludisme humain. Intérêt de la technique ELISA pour la mise en évidence des IgG et des IgM spécifiques.

A purified antigen was isolated from the red blood cells of Saimiri sciureus which was experimentally infected by Plasmodium falciparum. An enzyme immunoassay (ELISA) was carried out thanks to this antigen. The results obtained with the test were evaluated from two series of sera: one coming from an endemic area, the other taken out of it. The results were compared with those obtained with indirect fluorescent antibody test (IFAT) which is considerated as reference test. There is a good correlation between the two tests when anti-IgG conjugate is used for ELISA but there is no correlation with anti-IgM conjugate. One of the advantages of the ELISA, is that it allows a separate evaluation of specific IgG and IgM. Consequently distinction between recent and past infection would be possible. This may be useful for estimating the efficiency of an antimalaric campaign.

Defect in the generation of cytotoxic T cells in lepromatous leprosy.

Cytotoxic T cells are consistently produced in normal individuals after in vitro stimulation by a pool of mitomycin-treated normal lymphocytes. Patients suffering from lepromatous leprosy (LL), presenting with large amounts of Mycobacterium leprae and without a history of erythema nodosum leprosum (ENL) are unable to generate such cytotoxic T cells, while lepromatous patients with ENL which, in the present study were all deprived of M. leprae, react normally.