Análise citológica e cariométrica da ação da colchina sôbre a espermatogênese dos hemípteros

The action of colchicine upon the spermatogenesis of Triatoma infestans, (Hemipt. Heteroptera), has been studied and the different categories of giant spermatids that appear during the treatment have been compared with the nuclear volumes of the whole series of normal spermatogenetic stages. The following facts have been ascertained: 1) 4 hours after the treatment the gonial mitotic metaphases, and the 1st. and 2nd. metaphases of meiosis are stopped. The prophasic stages of meiosis and diakynesis appear to be normal. After 9 days of treatment, all the tetrads are broken in the meiotic metaphases and the cells appear with 44 and 22 chromosomes respectively, scattered in the cytoplasm. 2) At 9 days, practically all spermatogenetic stages have disappeared except for a few cysts of spermatogonia, and practically the whole testicle is full of cysts of spermatozoa and spermatid, with some large zones of necrosis with pycnotic nuclei. The spermatids appear to be of different sizes and the statistical analysis of the nuclear volumes gives a polymodal hystogram with 4 modes, whose volumes are in the ratio of 1:2:4:8. Ripe spermatozoa seem to have a certain volume variability, that has not been possible to analyse quantitatively. All these facts confirm what DOOLEY found in the colchicinized Orthoptera testicle. 3) The caryometric analysis conducted statistically on the normal stages of the spermatogenesis (resting spermatogonia, gonial prophases, leptotene, "confused stage", diakynesis, and spermatid) revealed the following facts: a) Considering the volume of the resting, spermatogonia as 1, their mitotic prophases have a volume of 2. Some rare prophases appear to have a volume of 4 and probably belong to tetraployd spermatogonia normally present in the testicle of Hemiptera. b) The first spermatocyte at the beginning of the auxocitary growth (leptotene) has a volume of 2, which is equal to that of them gonial prophase. It grows further during the "confused stage" and reduplicates, reaching thus the volume of 4. Diakynesis has a rather variable nuclear volume and it is higher than volume 4. This is probably of physico-chemical nature and not a growth increase. c) The spermatid at the beginning of the spermiogenetic process has a volume of 1 which is very constant and homogeneous. 4) These results can be summarized concluding that the meiotic process begins from a spermatogonium at the end of his mitotic interphasic growth (vol. 2) and instead of entering into the mitotic prophase transforms itself into the leptotene spermatocyte. During the diplotene ("confused stage") the volume of the nucleus doubles once more and reaches volume 4. In consequence of the two successive meiotic divisions the spermatid, although having an haploid number of chromosomes, has a nuclear volume of 1, just like the diploid spermatogonium. The interpretation of this strange result probably comes from the existence of the "tertiary split" in the chromosomes of the haploid set, that has been illustrated in the Hemiptera by HUGUES SCHRADER and in Orthoptera by MICKEY and co-workers. The tertiary split indicates that the chromosomes of the haploid set are constituted from almost two chromonemata, and this double constitution corresponds to the double cycle of reduplication that takes place during the spermatogenesis starting from the resting gonia. In Triatoma infestans the tertiary split appears in the chromosomes in the 1st. and 2nd. metaphases and in the diakynesis. In the blocked metaphases at the 9th. day of colchicinization some of the 44 elements scattered in the cytoplasm, show, when properly oriented, the split very clearly. Some new and strange facts revealed by SCHRADER and LEUCHTEMBERGER in Arvelius suggest the possibility of other interpretations of the rhythmic growth in special cases. There appears the necessity of more knowledge about the multiple or simple constitution of the chromosomes in somatic and spermatogonial mitosis.

The ribonucleic acid content of some mammalian erythrocytes

RNA was determined in red blood cells of man and other mammals. Our report is based on 41 determinations. Red blood cells of rat showed the highest values in comparison with the blood cells of guinea pig, rabbit, horse and sheep which showed the lowest values, and man with intermediate ones. The method used was a combination of Schimidt and Thanhauser and Schneider extractions with the final reactions of pentose with the orcinol reagent colorimetrically measured.

Toxoplasmose congênita

We had the opportunity to study 6 cases of the congenital form of toxoplasmosis, found in a series of 1200 necropsies of fetuses and newborn babies, realized at 3 different hospitals in Rio de Janeiro, Brazil. Among the 6 cases, 4 were premature babies liveborn at the 6th-8th gestational month and 2 were stillborn (1 premature and 1 at term). In all those cases, the diagnosis was based in the detection of the parasite in tissues and in one case it was even isolated the Toxoplasma from the necrotic material found in the cranial cavity. This strain of Toxoplasma, pathogenic to pigeons, to guinea pigs and to mice, is preserved by successive transfers in mice. Some facts observed in those cases present an interest not only strictly anatomic but also have certain value for the better acknowlegment of the disease. First, we want to call the attention to the presence of a sudden high fever, during or just before pregnancy in the 4 cases in which the maternal anamnesis was perfectly studied; this fever that was preceded by a normal beginning of pregnancy, had relatively rapid remission, but in 2 cases was immediately followed by uterine bleeding and premature delivery, although the puerperium had been apparently normal. It is known that are normal the subsequent children of the mothers that delivered a baby with toxoplasmosis and that several women have normal babies before the toxoplasmotic one. We believe that the fever observed in our cases could be indicative of the beginning of maternal infection and those are the reasons why we emphasize the need of careful anamnesis, specially in the cases actually diagnosed as inapparent infection. Another fact to notice is that in 5 of our cases the event premature delivery happened always between the 6th and the 8th months of pregnancy, and the only term fetus was delivered in advanced stage of maceration. The above mentioned facts could agree with the opinion of FRENKEL (1949), when he declared that "primary infection of the pregnant mother appears more likely to be the commoner mode of fetal toxoplasmic infection", but they would disagree with WEINMAN (1952) who believes that the transmission of Toxoplasma to the fetus is more frequent through a pregnant woman with chronic disease and who says "that infection contracted during pregnancy may and probably does happen from time to time"...Still in connection with the transmission of toxoplasmosis, we want to note the verification of inflammatory lesions in the placental villi and in the umbilical cord in 3 of the 4 cases in which such organs were examined at the microscope. In the case n. 1, we found several pseudocysts of Toxoplasma in the placenta, and the fibroblasts of Wharton's jelly were particularly rich in isolated forms and in colonies of Toxoplasma; the easy multiplication of the parasite in that tissue calls the attention and even suggests its utilisation for Toxoplasma's cultivation. The confirmation of Toxoplasma in human placenta was made only recently by CRISTEN et al. (1951) and by NEGHME et al. (1952), in Chile; it is not frequent in the literature, what gives some value to our present verification. Another observation was that provided by the case n. 6. This baby, a premature one of the 6th month, was 14 days old and-died with signs of respiratory disease, the causa mortis have been pneumonia. At the necropsy, we found no gross change that suggested toxoplasmosis, except the presence of some small necrotic focuses in the cerebral nervous substance around the ventricles. As a matter of fact, there was no enlargement of spleen or liver and neither leptomeningitis nor hydrocephalus. Such focuses were attributed to possible anoxia and in fact they are extremely similar to anoxial softenings, even when they are examined at the microscope; its structure composed of a central necrotic zone, surrounded by proliferated neuroglia and by a variable deposit of calcium salts, closely simulated the anoxial softenings, when the microscopical examination is based in the common histological preparations (hematoxilin-eosin, etc.). But when we examine preparations by the Giemsa or by the periodic acid-Schiff methods, we will note the presence of Toxoplasma, with its typical aspect or a little changed by degeneration. When we describe this observation, we wish to evidence the need of the search of Toxoplasma and closed parasites, in the cases of supposed pure anoxial softenings of nervous substance, in children. The frequency with which the congenital toxoplasmosis was anatomically verified should be emphasized, although the disease had not been clinically suspected, and it should be borne in mind that the second case of toxoplasmosis reported in the world was observed in Brazil by MAGARINOS TORRES; this case was the first to be described of the generalized congenital form of the infection, i. e. with myocardial lesions and parasites in skeletal muscles and skin.

Features of reproduction in laboratory-reared Aedes fluviatilis (Lutz, 1904) (Diptera: Culicidae)

Observations were made on 100 female Aedes fluviatilis (Lutz) maintained individually from the first blood meal onwards and allowed one blood meal during each oviposition cycle, 5% dextrose solution been supplied throughout life. The average length of live was 49.9 ± 17.8 days; the logarithm of the mortality rate increased proportionately to physiological age. The insects took an average of 7.3 ± 3.2 blood meals and produced a mean of 7.9 ± 3.7 clutches of eggs. There was a progressive decrease, proportional to advancing physiological age, in the mean numbers of eggs laid in successive oviposition cycles, in the intervals between blood feeding oviposition, and in the numbers of larvae that hatched. Delayed oviposition, transient sterility and a total loss of fertility were also recorded.

Studies on Trypanosoma rangeli Tejera 1920: VI. Developmental pattern in the haemolymph of Rhodnius prolixus

The morphological sequence of Trypanosoma rangeli development in the body cavity of Rhodnius prolixus is described. The metacyclic trypanosome is the product of successive division and transformation during the intra and extracellular development in the haemocoele. The significance of the early invasion of T. rangeli into the haemolymph is discussed. The epidemiological importance of the developmental pattern of T. rangeli in the vectors haemolymph and the host-response to the parasite are considered.

The cell biology of mosquito vitellogenesis

Insect vitellogenesis involves coordinated activities of the fat body and oocytes. We have studied these activities at the cellular level in the mosquito. During each vitellogenic cycle, the fat body undergoes three successive stages: 1) proliferation of biosynthetic organelles, 2) vitellogenin synthesis, 3) termination of vitellogenin synthesis and degradation of biosynthetic organelles by lysosomes. Analysis with monoclonal antibodies and radiolabelling demonstrated that the mosquito yolk protein consists of two subunits (200-kDa and 65-kDa). Both subunits are glycosylated, their carbohydrate moieties are composed of high-mannose oligosaccharides. The yolk protein subunits are derived from a single 220 kDa precursor detected by an in vitro translation. Oocytes become competent to internalize proteins as a result of juvenile hormone-mediated biogenesis of endocytotic organelles. The yolk protein is then accumulated by receptor-mediated endocytosis. A pathway of the yold protein and factors determining its routing in the oocyte have been studied.

Atypical rotavirus among diarrhoeic children living in Belém, Brazil

Atypical rotaviruses were detected in faeces from two diarrhoeic children living in Belém, Pará, Brazil. Rotavirus particles were detected by electron microscopy and the RNA electrophoresis showed patterns which were compatible with group C rotaviruses. Tests for the presence of group A antigen by enzyme-linked-immunosorbent assay (ELISA) were negative. The two children had three successive rotavirus infection and in both cases the atypical strains were excreted at the time of the third infection, causing a mild and short-lasting disease.

Stability of isoenzyme and kinetoplast DNA (k-DNA) patterns in successively cloned Trypanosoma cruzi populations

Four Trypanosoma cruzi strains from zymodermes A, B, C and D were successively clonedon BHI-LIT-agar-blood BLAB). Twenty clones from the first generation (F1), 10 from The second (F2) and 4 from the third (F3) from the strains A138, B147 and C23 were isolated. The D150 strain provied 29 F1 and F2 clones. The strains and clones had their isoenzyme and K-DNA patterns determined. The clones from A138, Bl47 and C231 strains presented isoemzyme and K-DNA patterns identical between thewmselves and their respective parental strains. Therefore showing the homogenety and stability of isoenzyme and K-DNA patterns after successive cloning. The Dl50 strain from zymodeme D (ZD) showed heterogeneity. Twenty-eight out of 29 clones of the first generation were of zymodeme A and only one was of zymodeme C, confirming previous reports that ZD strains consisted of ZA and ZC parasite populations. The only D150 strain clone of zymodeme C showed a K-DNA pattern identical to its parental strain. The remining clones although similar among themselves were different from the parental strain. Thus the T. cruzi strains had either homonogeneus or heterogeneous populations. The clones produced by successive cloning provided genetically homonogeous populations. Their experimental use will make future results more reliable and reproducible.

Kinetics of antigen specific and non-specific polyclonal B-cell responses during lethal Plasmodium yoelii malaria

In order to study the kinetics and composition of the polyclonal B-cell activation associated to malaria infection, antigen-specific and non-specific B-cell responses were evaluated in the spleens of mice infected with Plasmodium yoelii 17 XL or injected with lysed erythrocytes or plasma from P. yoelii infected mice or with P. falciparum culture supernatants. Spleen/body weigth ratio, numbers of nucleated spleen cells and Immunoglobulin-containing and Immunoglobulin-secreting cells increased progressively during the course of infection,in parallel to the parasitemia. A different pattern of kinetics was observed when anti-sheep red blood cell and anti-trinitrophenylated-sheep red blood cell plaque forming cells response were studied: maximum values were observed at early stages of infection, whereas the number of total Immunoglobulin-containing and Immunoglobulin-secreting cells were not yet altered. Conversely, at the end of infection, when these latter values reached their maximum, the anti-sheep red blood cell and anti-trinitrophenylated-sheep red blood cell specific responses were normal or even infranormal. In mice injected with Plasmodium-derived material, a higher increase in antigen-specific PFC was observed, as compared to the increase of Immunoglobulin-containing and Immunoglobulin-secreting cell numbers. This suggested a "preferential" (antigen-plus mitogen-induced) stimulation of antigen-specific cells rather than a generalized non-specific (mitogen-induced) triggering of B-lymphocytes. On the basis of these and previous results, it is suggested that polyclonal B-cell activation that takes place during the course of infection appears as a result of successive waves of antigen-specific B-cell activation.

Trypanosoma cruzi: metacyclogenesis in vitro - I. Changes in the properties of metacyclic trypomastigotes maintained in the laboratory by different methods

In this work we have studied the modifications in the biological properties of Trypanosoma cruzi when the parasite is maintained for a long time in axenic culture. The studies were done with a clone from an avirulent strain (Dm30L) and a non-cloned virulent strain (EP) of T. cruzi. Both parasiteswere maintained, for at least three years, by successive triatomine/mouse alternate passage (control condition), or by serial passage in axenic medium (culture condition), or only in the mouse (mouse condition). The comparison between parasites of culture and control condition showed that metacyclogenesis capacity was reduced in the former and that the resulting metacyclics displayed an attenuatedvirulence. In order to compare the virulence of metacyclics from the urine of the insect vector, Rhodnius prolixus were infected by artificial feeding with parasites of the control or culture condition. After three triatomine/triatomine passages, there was observed an almost identical biological behavior for these parasites, hence indicating that the maintenance of T. cruzi for a long time in axenic culture affects the differentiation capacity and the virulence of the parasite. Additionally, it was demonstrated that it is possible to maintain T. cruzi exclusively through passages in the invertebrate host.

Ultrastructural aspects of spermatogonesis in Metamicrocotyla macracantha (Alexander, 1954) Koratha, 1955 (Monogenea; Microcotylidae)

Ultrastructural aspects of spermatogenesis, spermiogenesis and of the mature spermatozoon of a microcotylid monogenean Metamicrocotyla macracantha parasite from Mugil liza, are described. The irregularly-shaped spermatogonia divides by successive mitoses, forming the primary spermatocytes, identified by the presence of synaptonemal complexes in their nuclei. The spermatids formed by meiotic cell divisions of the secondary spermatocytes, differentiate into a mature spermatozoon. Cross sections of the head and the middle region of mature spermatozoa show the nucleus with strong condensed chromatin, the mitochondria with short cristae, peripheral microtubules and two axonemes with a 9+1 pattern, confirming the characteristics of this genus.

Characterization of subpopulations (clones and subclones) of the 21 SF strain of Trypanosoma cruzi after long lasting maintenance in the laboratory

Several studies have shown a clonal structure of Trypanosoma cruzi and its possible correlation with the behavioral heterogeneity of the parasite strains. In the present study, the 21 SF strain, that have been maintained in laboratory by successive passages in mice, for more than 15 years, showing a stability of biological and isoenzymic characteristics has been cloned, with the objective of establishing the characters of its clones and subclones. With the technique of isolation of a single parasite from the blood of infected mice, 5 clones and 14 subclones have been obtained. After four passages into mice, inoculum of 10(5) was obtained for each clone and subclone and inoculated into mice weighing 10 to 12 g. These were used for the study of the biological behavior of the clones: evolution of parasitemia, morphology of blood forms and host mortality. For isoenzymic characterization, the clones and subclones were analyzed for ALAT, ASAT, GPI and PGM enzymes. Results have shown that the 5 clones and the 14 subclones disclosed a biological behavior similar to the parental strain, with minor variability of the parasitemic profiles and also the same isoenzymic patterns. These results confirm the stability of the 21 SF strain and indicate a clonal homogeneity of its populations. This is compatible with the hypothesis that the T. cruzi strains represent an equilibrium of either homogenous or heterogeneous populations.

Trypanosoma cruzi: Maintenance in Culture Modify Gene and Antigenic Expression of Metacyclic Trypomastigotes

In this study we examined whether the maintenance of Trypanosoma cruzi by long-time in axenic culture produces changes in gene expression and antigenic profiles. The studies were made with a Dm30L-clone from a low-virulent strain and a non-cloned virulent EP-strain of T. cruzi. Both parasites were maintained, for at least seven years, by successive alternate passage triatomine/mouse (triatomine condition), or by serial passage in axenic medium (culture condition). The comparison of the [35S]methionine metabolic labeling products of virulent and non-virulent parasites by 2D-SDS-PAGE, clearly indicates that the expression of metacyclic trypomastigotes (but not of epimastigotes) proteins have been altered by laboratory maintenance conditions. Western blot analysis of EP and Dm30L-epimastigotes using a serum anti-epimastigotes revealed that although most of antigens are conserved, four antigens are characteristics of triatomine condition parasites and three other are characteristics of culture condition parasites. Anti-metacyclics serum revealed significative differences in EP- and Dm30L-metacyclic trypomastigotes from triatomine condition. However, avirulent metacyclic forms were antigenically very similar. These results suggest that besides a possible selection of avirulent subpopulation from T. cruzi strains genetically heterogeneous when maintained by long time in axenic culture, changes in virulence might be due to post-translational modifications of the antigens induced by the absence of the natural alternability (vertebrate-invertebrate) in the life-cycle of T. cruzi

Study of Schistosoma mansoni isolates from patients with failure of treatment with oxamniquine

After three successive treatments with oxamniquine the continuing elimination of Schistosoma mansoni eggs was observed in patients, who came from various regions of Brazil, with different clinical forms of schistosomiasis. The objective of the present study was to determine the experimental behaviour of five different S. mansoni isolates in Swiss Webster mice that were submitted to treatment with the same drug. The experimental group with failure of treatment showed higher mean number of surviving male worms when it was compared to the group without failure of treatment. These date suggest the possibility of resistance to oxamniquine.

Studies on the virulence and attenuation of Trypanosoma cruzi using immunodeficient animals

Tissue invasion and pathology by Trypanosoma cruzi result from an interaction between parasite virulence and host immunity. Successive in vivo generations of the parasite select populations with increasing ability to invade the host. Conversely, prolonged in vitro selection of the parasite produces attenuated sublines with low infectivity for mammals. One such subline (TCC clone) has been extensively used in our laboratory as experimental vaccine and tested in comparative experiments with its virulent ancestor (TUL). The experiments here reviewed aimed at the use of immunodeficient mice for testing the infectivity of TCC parasites. It has not been possible to obtain virulent, revertant sublines by prolonged passaged in such mice.