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 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.

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.