Lamella formation and emigration from the water by a laboratory colony of Biomphalaria glabrata (SAY) in flow-through system

Lamella formation and emigration from the water were investigated in juvenile Biomphalaria glabrata reared at two temperatures in aquaria with a constant water flow. Most snails (97.4%) reared at the lower temperature (21- C) formed lamella at the shell aperture and emigrated from the water, whereas only 10.1% did so at 25- C. Eighty percent of emigrations at 21- C occurred within a period of 15 days, 70-85 days after hatching. A comparison of the studies done so far indicates that the phenomenon may be affected by the ageing of snail colonies kept in the laboratory and their geographic origin, rather than the rearing conditions. This hypothesis, however, requires experimental confirmation.

On the current existence of a coccidial line of development in the malaria parasites: a theory

Most opinion favors the origin of the malaria parasites from a coccidial ancestor. It is assumed that whatever the process through which the coccidia differentiated into a Plasmodium this phenomenon very probably occured millions of year ago, and during that differentiation process the original coccidia vanished. Therefore it has never repeated. At the light of some experiments the existence, at the present time, of a coccidial cycle of development in the malaria parasites, is proposed. The conection routes and mechanisms through which the malaria parasite changes to a coccidial life, and the routes in reverse are exposed. Transmission of the malaria-coccidial forms is suggested.

A rapid, reliable method of evaluating growth and viability of intraerythrocytic protozoan hemoparasites using fluorescence flow cytometry

Fluorescence flow cytometry was employed to assess the potential of a vital dye, hydroethiedine, for use in the detection and monitoring of the viability of hemoparasites in infected erythrocytes, using Babesia bovis as a model parasite. The studies demonstrated that hydroethidine is taken up by B. bovis and metabolically converted to the DNA binding fluorochrone, ethidium. Following uptake of the dye, erythrocytes contamine viable parasites were readily distinguished and quantitated. Timed studies with the parasiticidal drug, Ganaseg, showed that it is possible to use the fluorochrome assay to monitor the effects of the drug on the rate of replication and viability of B. bovis in culture. The assay provides a rapid method for evaluation of the in vitro effect of drugs on hemoparasites and for analysis of the effect of various components of the immune response, such as lymphokines, monocyte products, antibodies, and effector cells (T, NK, LAK, ADCC) on the growth and viability of intraerythrocytic parasites.

Establishment and Characterization of a Cell Line from the Mosquito Anopheles albimanus (Diptera: Culicidae)

A new cell line designated LSB-AA695BB, was established from embryos of the mosquito Anopheles albimanus. The primary culture was initiated in April, 1995, and the first passage was made 48 days later. Serial subcultures of the cells have been carried through 90 passages from Abril 1995 to February 1996. The cells were grown at 28°C in MK/VP12 medium, supplemented with 20% fetal bovine serum; the pH tolerance ranged between 6.8 to 7.0. The cells have also been adapted to MM/VP12 medium under the same pH, temperature and serum concentration. The majority of the cells were a fibroblast-type. Isozyme characterization showed a pattern similar to that of An. albimanus pupae and adults but distinct from Ae. taeniorhynchus and Ae. albopictus (C6/36) mosquito cell lines. The culture was shown to be free of mycoplasma, bacteria and fungi. Microsporidia contamination of transovarial transmission was controlled with 6.0 mg/ml of albendazole

Establishment and characterization of a new continuous cell line from Lutzomyia longipalpis (Diptera: Psychodidae) and its susceptibility to infections with arboviruses and Leishmania chagasi

Embryonic tissue explants of the sand fly Lutzomyia longipalpis (Lutz & Neiva 1912) the main vector of Leishmania chagasi (Cunha and Chagas), were used to obtain a continuous cell line (Lulo). The tissues were seeded in MM/VP12 medium and these were incubated at 28ºC. The first subculture was obtained 45 days after explanting and 96 passages have been made to date. Lulo is composed of epithelioid cells, showed a 0.04 generations/hour exponential growth rate and population doubling time at 24.7 h. The cell line isoenzymatic profiles were determined by using PGI, PGM, MPI and 6-PGDH systems, coinciding with patterns obtained from the same species and colony's pupae and adults. The species karyotype characteristics were recognized (2n = 8), in which pair 1 is subtelocentric and pairs 2, 3 and 4 are metacentric. Lulo was free from bacterial, fungal, mycoplasmic and viral infection. Susceptibility to five arbovirus was determined, the same as Lulo interaction with Leishmania promastigotes.

Immunological evaluation of human immunedeficiency virus infected individuals by flow cytometry

Human immunodeficiency virus (HIV) infection heavily compromises the immune system. The decrease of the T cell CD4+ subset along the evolution to acquired immunodeficiency syndrome has been considered as a hallmark of HIV infection. In this paper we review some aspects of the immunopathology of HIV infection and discuss the importance of the flow cytometry for the evaluation of the T lymphocyte subsets in the follow-up of HIV infected children and adults, and for the monitoring of the immune reconstitution upon antiretroviral therapy.

Detection of intracytoplasmic cytokines by flow cytometry

Flow cytometry has been used as a powerful technique for studying cell surface antigen expression as well as intracellular molecules. Its capability of analyzing multiple parameters simultaneously on a single cell has allowed identification and studies of functional cell subsets within heterogeneous populations. In this respect, several techniques have been developed during the past few years to study cytokine-producing cells by flow cytometry in humans and several animal models.

Applications of flow cytometry to hematopoietic stem cell transplantation

Applications of flow cytometry to clinical and experimental hematopoietic stem cell transplantation (HSCT) are discussed in this review covering the following topics: diagnosis and classification of lymphohematologic disorders, quantitation of hematopoietic progenitors in the graft, lymphohematopoietic reconstitution following HSCT and animal models of human HSCT. At the end, the utilization of flow cytometry in clinical HSCT by Brazilian transplant centers is briefly reviewed.

Procedures to characterize and study P2Z/P2X7 purinoceptor: flow cytometry as a promising practical, reliable tool

The expression of P2Z/P2X7 purinoceptor in different cell types is well established. This receptor is a member of the ionotropic P2X receptor family, which is composed by seven cloned receptor subtypes (P2X1 - P2X7). Interestingly, the P2Z/P2X7 has a unique feature of being linked to a non-selective pore which allows the passage of molecules up to 900 Da depending on the cell type. Early studies of P2Z/P2X7 purinoceptor were exclusively based on classical pharmacological studies but the recent tools of molecular biology have enriched the analysis of the receptor expression. The majority of assays and techniques chosen so far to study the expression of P2Z/P2X7 receptor explore directly or indirectly the effects of the opening of P2Z/P2X7 linked pore. In this review we describe the main techniques used to study the expression and functionality of P2Z/P2X7 receptor. Additionally, the increasing need and importance of a multifunctional analysis of P2Z/P2X7 expression based on flow cytometry technology is discussed, as well as the adoption of a more complete analysis of P2Z/P2X7 expression involving different techniques.

Flow cytometry in the study of cell death

In this report we present a concise review concerning the use of flow cytometric methods to characterize and differentiate between two different mechanisms of cell death, apoptosis and necrosis. The applications of these techniques to clinical and basic research are also considered. The following cell features are useful to characterize the mode of cell death: (1) activation of an endonuclease in apoptotic cells results in extraction of the low molecular weight DNA following cell permeabilization, which, in turn, leads to their decreased stainability with DNA-specific fluorochromes. Measurements of DNA content make it possible to identify apoptotic cells and to recognize the cell cycle phase specificity of apoptotic process; (2) plasma membrane integrity, which is lost in necrotic but not in apoptotic cells; (3) the decrease in forward light scatter, paralleled either by no change or an increase in side scatter, represent early changes during apoptosis. The data presented indicate that flow cytometry can be applied to basic research of the molecular and biochemical mechanisms of apoptosis, as well as in the clinical situations, where the ability to monitor early signs of apoptosis in some systems may be predictive for the outcome of some treatment protocols.

The beginning and expansion of flow cytometry in Brazil

The author describes and make comments on the favorable conditions that made possible the creation and organization of the first laboratory of flow cytometry in Brazil and South America installed at the Oswaldo Cruz Institute-Fiocruz, Rio de Janeiro, in November 1988.

Comparison of dengue infection in human mononuclear leukocytes with mosquito C6/36 and mammalian Vero cells using flow cytometry to detect virus antigen

Fluorescent activated cell sorter (FACS) analysis is useful for the detection of cellular surface antigens and intracellular proteins. We used this methodology in order to detect and quantify dengue antigens in highly susceptible cells such as clone C6/36 (Aedes albopictus) and Vero cells (green monkey kidney). Additionally, we analyzed the infection in vitro of human peripheral blood mononuclear leukocytes (PBML). FACS analysis turned out to be a reliable technique to quantify virus growth in traditional cell cultures of C6/36 as well as Vero cells. High rates of infection were achieved with a good statistical correlation between the virus amount used in infection and the percentage of dengue antigen containing cells detected in infected cultures. We also showed that human monocytes (CD14+) are preferred target cells for in vitro dengue infection among PBML. Monocytes were much less susceptible to virus infection than cell lines but they displayed dengue antigens detected by FACS five days after infection. In contrast, lymphocytes showed no differences in their profile for dengue specific immunofluorescence. Without an animal model to reproduce dengue disease, alternative assays have been sought to correlate viral virulence with clinical manifestations and disease severity. Study of in vitro interaction of virus and host cells may highlight this relationship.

Flow cytometry as a tool to identify Mycobacterium tuberculosis interaction with the immune system and drug susceptibility

Flow cytometric analysis is a useful and widely employed tool to identify immunological alterations caused by different microorganisms, including Mycobacterium tuberculosis. However, this tool can be used for several others analysis. We will discuss some applications for flow cytometry to the study of M. tuberculosis, mainly on cell surface antigens, mycobacterial secreted proteins, their interaction with the immune system using inflammatory cells recovered from peripheral blood, alveolar and pleura spaces and the influence of M. tuberculosis on apoptosis, and finally the rapid determination of drug susceptibility. All of these examples highlight the usefulness of flow cytometry in the study of M. tuber-culosis infection.

A new continuous cell line from the mosquito Psorophora confinnis (Diptera: Culicidae) and its susceptibility to infections with some arboviruses

A new cell line, PC-0199-BR, was established from embryonated eggs of the mosquito Psorophora confinnis. To date (September 2000) it has had 62 continuous passages. This is the first report of a cell line of mosquitoes belonging to the genus Psorophora. Cell growth initially was achieved in the MM/VP12 medium, supplemented with 20% fetal bovine serum; however, the subcultures were later adapted to Grace's medium with 10% fetal bovine serum. Cell morphology in the primary cultures was heterogeneous; but later in the established cell line, the predominant cell type was epithelioid. Cultured cells were predominantly diploid (2n=6); however, chromosome abnormalities were observed in a small proportion of the cells in later passages. C and G band patterns were also determined in the karyotype. The cell line isozyme profiles coincided with pupae and adult samples of the species taken from the same colony. A preliminary arbovirus susceptibility study for the cell line was undertaken. No evidence was observed of contamination of the cell line with bacteria, fungi or mycoplasma.

Laboratory diagnosis of Schistosomiasis in areas of low transmission: a review of a line of research

After 57 years of successful control of schistosomiasis in Venezuela, the prevalence and intensity of infection have declined. Approximately 80% of the individuals eliminate less than 100 eggs/g of stools, therefore morbidity is mild and the majority are asymptomatic. The sensitivity of Kato-Katz decreases to approximately 60%. Available serological methods for the detection of circulating antigens only reach a 70% of sensitivity. Tests based on the detection of antibodies by immunoenzymatic assays have been improved. The circumoval precipitine test has shown a high sensitivity (97%), specificity (100%), and correlation with oviposition, being considered the best confirmatory diagnostic test. Additionally to the classical immunoenzymatic assays, the development of the alkaline phosphatase immunoassay, allowed to reach a 100% specificity with an 89% sensitivity. Recently, we have developed a modified ELISA in which the soluble egg antigen is treated with sodium metaperiodate (SMP-ELISA) in order to eliminate the glycosilated epitopes responsible for the false positive reactions. The specificity and sensitivity reaches 97% and 99%, respectively. Synthetic peptides from the excretory-secretory enzymes, cathepsin B (Sm31) legumain (Sm32) and cathepsin D (Sm45), have been synthesized. The combination of two peptides derived from the Sm31 have been evaluated, reaching a sensitivity of 96% when analyzed independently and with a 100% specificity. Antibodies raised in rabbits against peptides derived from the Sm31 and Sm32 are currently evaluated in two different antigen-capture-based assays. The development of a simple, cheap and reliable test that correlates with parasite activity is a major goal.