Genetic heterogeneity in the species Leishmania tropica revealed by different PCR-based methods.

A PCR fingerprinting approach, using single non-specific primers, as well as restriction and single-strand conformation polymorphism (SSCP) analyses of the amplified ribosomal internal transcribed spacer, were used to investigate genetic variability in the species Leishmania tropica. Twenty-nine strains of the 'L. tropica complex' from different Old World geographical areas were studied including 4 from Namibia, and 1 strain of L. killicki. All techniques revealed a high degree of genetic heterogeneity among the strains of L. tropica. The PCR fingerprinting displayed the highest discriminatory power, but can be applied only to cultured parasites. The internal transcribed spacer (ITS) region can be amplified directly from infected clinical samples and analysed subsequently. No strict correlation was discerned between the genetic variants and either the geographical origin of the strains or the clinical manifestations associated with human disease, except for the Namibian strains. Also, genetic variation did not correlate well with characterization by enzyme variant electrophoretic analysis. The strain of L. killicki always clustered together with the strains of L. tropica, suggesting it, probably, should not be considered a separate species of Leishmania. However, the 4 Namibian strains formed a distinct, statistically well-supported group closely related to but different from the other strains of L. tropica.

Application of flow cytometry and microscopical methods to characterize the effect of herbal drugs on Leishmania Spp.

The World Health Organization has identified leishmania sis as a major public health problem, particularly in Africa, Asia, and Latin America. About 1.5 to 2 million people are affected annually by this parasitic infection. As there is no vaccine, there is still a strong need for sufficient drugs. In a preliminary screening, extracts of 50 different plants were evaluated for their possible leishmanicidal activity against the promastigote form of Leishmania mexicana amazonensis. Eighteen extracts showed at least 50% inhibition at 100 microg/ml. The ethanolic extract from Yucca filamentosa L. showed the strongest leishmanicidal activity (100% inhibition at 5 microg/ml). The bioactivity-guided fractionation of this extract led to the isolation of three main components (Yuccasaponins MC 1--3). In further experiments, the effect of Yuccasaponin MC 3 on the promastigote form of L. mexicana amazonensis was quantified and characterized using flow cytometry and specific fluorescent dyes [propidium iodide, Syto 9, and DiBAC(4)(3)]. The data revealed that the membrane of the promastigote is attacked. The effect of Yuccasaponin MC 3 on intracellular forms (amastigote) was also characterized; green fluorescent protein-transfected Leishmania major were used. By this method, an inhibition of intracellular growth of L. major was demonstrated. This paper shows that, together, flow cytometry and microscopy are quick, sensitive, and easily reproducible methods to describe the effects of drugs on parasites.

Proteomics in human disease: cancer, heart and infectious diseases.

In recent years, genomics has increased the understanding of many diseases. Proteomics is a rapidly growing research area that encompasses both genetic and environmental factors. The protein composition represents the functional status of a biological compartment. The five approaches presented here resulted in the detection of disease-associated proteins. Calgranulin B was upregulated in colorectal cancer, and hepatoma-derived aldose reductase-like protein was reexpressed in a rat model during hepatocarcinogenesis. In these two investigations, attention was focused on one protein, obviously differing in amount, directly after two-dimensional electrophoresis (2-DE). Additional methods, such as enzyme activity measurements and immunohistochemistry, confirmed the disease association of the two candidates resulting from 2-DE subtractive analysis. The following three investigations take advantage of the holistic potential of the 2-DE approach. The comparison of 2-DE patterns from dilated cardiomyopathy patients with those of controls revealed 25 statistically significant intensity differences, from which 12 were identified by amino acid analysis, Edman degradation or matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). A human myocardial 2-DE database was constructed, containing 3300 protein spots and 150 identified protein species. The number of identified proteins was limited by the capacity of our group, rather than by the principle of feasibility. Another field where proteomics proves to be a valuable tool in identifying proteins of importance for diagnosis is proteome analysis of pathogenic microorganisms such as Borrelia burgdorferi (Lyme disease) and Toxoplasma gondii (toxoplasmosis). Sera from patients with early or late symptoms of Lyme borreliosis contained antibodies of various classes against about 80 antigens each, containing the already described antigens OspA, B and C, flagellin, p83/100, and p39. Similarly, antibody reactivity to seven different marker antigens of T. gondii allowed differentiation between acute and latent toxoplasmosis, an important diagnostic tool in both pregnancy and immunosuppressed patients.

Toxoplasma gondii infection: analysis of serological response by 2-DE immunoblotting.

Toxoplasma gondii is known to cause a variety of diseases ranging from asymptomatic infections to serious conditions in immunocompromised hosts such as AIDS-patients or transplant recipients. In addition they may cause abortion or fetal abnormalities during pregnancy. Despite the clinical importance, diagnosis, treatment and prevention still remain unsatisfactory. Analysis of the parasitic cell determinants, recognized by specific humoral and cellular immune responses, may have important implications for diagnosis, therapy and vaccination strategies. Two-dimensional electrophoresis (2-DE) was used to resolve and compare protein patterns from Toxoplasma gondii strains RH and BK (mouse virulent strains). Comparison of silver-stained gels showed that 35.2% to 60.3% of the spots had the same position. In a second series of experiments, the reactivity of the spots with human sera was tested. Proteins were transferred to PVDF membranes and were detected with sera from different patient groups. Depending upon the immunoglobulin class (IgG, IgM, IgA or IgE) different epitope patterns were observed. Some of the spots seemed to be recognized in different stages of infection. Sera of two patients with similar serology and comparable stage of infection were compared in order to demonstrate an individual immune response.

Toxoplasma infection and cell free extract of the parasites are able to reverse multidrug resistance of mouse lymphoma and human gastric cancer cells in vitro.

A large number of compounds are known to reduce the ATP-dependent efflux pump activity of multidrug resistant (mdr) tumor cells. Here we report that an infection of cancer cells with T. gondii reduced the multidrug resistance of the tumour cells against cytostatic drugs. Two mouse lymphoma cell lines (Mdr L 5718 and Par 5718) were infected with Toxoplasma gondii in vitro and the reduction of efflux pump activity of the cells was measured. The drug accumulation (Rhodamin-123) was increased in the infected mdr cell lines compared with non- infected mdr-cells, and no effect was shown after infection of the parental cell line. The same effect was also achieved by incubation of Mdr-tumor cells with cell lysate of Toxoplasma gondii. Mdr-1-gene expression was reduced in the infected cell lines 48 hours after infection. Co-cultivation of Toxoplasma gondii with mdr cell lines separated by a microfilter from tumor cells was performed, but this cocultivation did not change the mdr efflux activity. The effect of Toxoplasma gondii infection on the efflux pump activity and mdr-1 gene expression was also examined in the human gastric cancer cells. A sensitization of resistant gastric cancer cells was also achieved by parasite infection. This phenomenon is an evidence that a reduction of resistance in tumor cells can be achieved by a natural parasite infection. It is as yet unclear whether an active infection or another substance of T. gondii is responsible for this phenomenon.

Occurrence of S and F1C/S-related fimbrial determinants and their expression in Escherichia coli strains isolated from extraintestinal infections.

The presence of S and F1C/S-related fimbrial determinants was determined in 462 E. coli strains obtained from different extraintestinal infections and in 162 control isolates of E. coli by using two different DNA probes: an oligonucleotide probe consisting of three oligonucleotides that bind specifically to the S adhesin gene and a polynucleotide probe which is not able to distinguish between S, F1C, and S-related sequences. The expression of S and F1C phenotypes was tested by dot enzyme immunoassay with the corresponding monoclonal antibodies. S fimbriae genotypes were observed more frequently in septic (25%) and urinary (12%) isolates of E. coli than in faecal and water isolates (1%) and often occurred together with O2, O6, O18 and O83 antigens. F1C/S-related fimbrial DNA was detected with a higher frequency in UTI isolates (26%) than in septic (16%) and faecal (10%) isolates and was most frequently associated with O4, O6, and O75 serotypes. Since the production of S and F1C fimbriae was comparatively rare in all clinical and control isolates of E. coli, DNA hybridization assays which allow the sensitive and specific detection of fimbrial determinants even in the absence of their expression are preferable to phenotypic assays.

Genotypic and phenotypic determination of five virulence markers in clinical isolates of Escherichia coli.

The presence of the virulence markers K1 capsule, serum resistance, aerobactin, S and P/PR fimbriae were examined in a total of 395 E. coli strains from different extraintestinal infections and in 81 faecal isolates of healthy volunteers using specific DNA probes and classical phenotypic methods. All markers were more frequently detected when genotypic assays were applied. The simultaneous occurrence of 3-4 virulence determinants was typical for isolates derived from patients with septicaemia or meningitis. Isolates from blood cultures and cerebrospinal fluid were expressing the virulence phenotypes to a greater extent than isolates from urine or faeces. The use of colony hybridization with specific oligonucleotide and polynucleotide probes for the detection of virulence determinants has been proven to be more specific and reliable than phenotypic approaches.

Clonal analysis of Escherichia coli serotype O6 strains from urinary tract infections.

A total of 36 Escherichia coli urinary tract isolates (UTI) of serotype O6, with different combinations of capsule (K) and flagellin (H) antigens, were analysed according to the outer membrane pattern (OMP), serum resistance properties, mannose-resistant hemagglutination using various types of erythrocytes, and also for the genetic presence and the expression of P-fimbriae, S fimbriae/F1C fimbriae, Type 1 fimbriae, aerobactin and hemolysin. Twenty selected strains were further analysed by pulsed field gel electrophoresis (PFGE), elaborating genomic profiles by XbaI cleavage and subsequent Southern hybridization to virulence-associated DNA probes. It could be shown that O6 UTI isolates represent a highly heterogeneous group of strains according to the occurrence and combination of these traits. Relatedness on the genetic and the phenotypic level was found for some of the strains exhibiting the same O:K:H:F serotype. DNA long-range mapping further indicated some interesting features, according to the copy number and the genomic linkage of virulence genes.

Determination of S fimbriae among Escherichia coli strains from extraintestinal infections by colony hybridization and dot enzyme immunoassay.

449 E. coli strains obtained from septic infections (124 isolates), urinary tract infections (246) and water (79) were surveyed for S/F1C fimbrial DNA by DNA hybridization and for expression of S fimbriae by enzyme immunoassay. S/F1C fimbrial DNA was detected with greater frequency in septic (35%) and urinary (43%) isolates than in water isolates and was often associated with the O2, O4, O6, O18, O83, and O156 serogroups. Most O45 strains did not possess such sequences. Only 12% and 28%, respectively, of the septic and urinary strains possessing S/F1C fimbrial DNA expressed S fimbriae.

[Determination of serum- and phagocyte resistance of E. coli using bioluminescence]. / Bestimmung der Serum- und Phagozytoseresistenz von E. coli durch Biolumineszenzmessung.

Resistance to serum and phagocytosis belong to the most important virulence markers of the bacteria. These properties enable the microorganism to have some selective advantage by overcoming of host defence, thus increasing the invasiveness of the bacteria. Determination of these properties make it possible to evaluate better the virulence of the facultative-pathogenic microorganisms and can therefore be used for microbiological diagnosis. The method used until now to test these characteristics are very time consuming. For these reason we have employed bioluminescence to determine the number of viable cells. This method is very suitable for this purpose.

Nosocomial infections due to Serratia marcescens--clinical findings, antibiotic susceptibility patterns and fine typing.

We report on nosocomial infections caused by Serratia marcescens occurring in a neonatal intensive care unit and a children's ward for cardiac intensive care. According to the plasmid pattern analysis, all isolated epidemic strains belonged to one clone. Multi-drug resistance, even to cephalosporins of the third generation and amikacin, was characteristic for all strains. Certain markers of S. marcescens (haemolysin, proteases, siderophores) which are thought to be related to virulence were studied but will require further investigation.

Ureaplasma urealyticum and male infertility: an animal model: II. Morphologic changes of testicular tissue at light microscopic level and electron microscopic findings.

Testicular biopsies were obtained from Wistar rats that had been infected artificially with Ureaplasma urealyticum, serotype 3. Approximately 50% of the biopsy specimens obtained 3 and 6 months after infection showed degeneration of the germinal epithelium, giant cell formation and Leydig cell hyperplasia. Electron microscopic studies revealed striking alterations of Sertoli cells, germ cells, and Leydig cells as well as ureaplasma organisms inside the seminiferous tubules. The changes noted in the ertoli cells were apparent as early as one week after infection.

[The modification of phagocytosis by different K1 capsule preparations of Escherichia coli]. / Beeinflussung der Phagocytose durch verschiedene K1-Kapselpräparationen von Escherichia coli.

The influence of two different types of Escherichia coli K1 capsule preparation on the phagosytosis of an E. coli laboratory strain (LN 28) by polymorphnuclear leucocytes (PMNL) is described. Capsule material of E. coli 0.18 and 0.83 was prepared a) from the culture supernatant and b) from a bacterial pellet fraction. When capsule material from the pellet was added to opsonin, before mixing the laboratory strain with the PMNL, it decreased the rate of phagocytosis, compared with untreated opsonin. The preparation from the supernatant of capsule showed no alteration of phagocytosic rate or capacity. These results are explained by the different chemical compositions of the preparations.

Interactions between Sarcocystis gigantea lectin and toxin-containing fractions in human lymphocyte cultures.

Sarcocystis gigantea extract (SGE) was separated by affinity chromatography into one lectin-containing fraction (SGL) that was mitogenic to mononuclear cells (MNC) and another that lacked this lectin activity. The SGL-depleted Sarcocystis extract (SGTF) contained the so-called Sarcotoxin, inducing only a slight increase in MNC proliferation. Furthermore, preincubation of MNC with SGTF for 60 min suppressed the mitogenic capacity of SGL by 60%-90%. The results presented indicate that SGTF interacts with human MNC differently than SGL, particularly by interfering with the mitogenic lectin. These findings suggest that SGL and SGTF may be involved in different immunomechanisms induced by the parasite.

Ureaplasma urealyticum and male infertility: an animal model. I. Artificial infection, breeding experiments and histological preparation of organs.

Male Wistar rats were infected with Ureaplasma urealyticum, serotype 3 or 7, by injecting broth containing organisms into the urinary bladder following laparatomy. Animals were sacrificed 3, 7 and 21 days after infection. Ureaplasmas were detected in the organs of the genital tract by culture in 43% (serotype 3) and in 60% (serotype 7) of the animals. Type-specific ureaplasma antigen was detected in the genital organs in 44% (serotype 3) and 45% (serotype 7) of the animals. Control animals injected with sterile bouillon were negative for organisms and antigen. Male Wistar rats artificially infected with Ureaplasma urealyticum, serotype 3, were mated 3 and 6 months after infection with ureaplasma free female rats. The mating experiment revealed a smaller mean litter size and a lower birth weight in the offspring of infected males compared with the control animals, but no general influence on the fertility of infected animals.

The role of Ureaplasma urealyticum in urinary tract infections. Demonstration of antibodies against ureaplasmas in the urine.

This study reports the presence of antibodies against Ureaplasma urealyticum in the urine in 17.2% (85) of 493 patients with chronic pyelonephritis. At the same time in 37 of antibody-positive urine samples evidence was found of cultivated Ureaplasma urealyticum, other bacterial germs were isolated infrequently. These culturally and serologically positive urine samples mostly showed pathologic findings of urine sediments. These results led us to believe in a possible involvement of the Ureaplasmas in keeping inflammation processes in the upper urinary tract.