Karla Consort Ribeiro

Pseudocysts in trichomonads--new insights.

Tritrichomonas foetus and Trichomonas vaginalis, parasitic protists of the urogenital tract, display a trophozoite and a pseudocyst stage. The ultrastructure of the trophozoite was compared with the pseudocyst form. The latter appears under unfavorable environmental conditions when the flagella are internalized, and a true cell wall is not formed. Although some authors consider this form as a degenerate stage, the cell behaves as a resistant form. Pseudocysts were found in natural culture conditions and also under induction by hydroxyurea or cycles of cooling and warming cultures. They were studied by light and scanning and transmission electron microscopy, using immunofluorescence and videomicroscopy. This report presents evidence that the trichomonad pseudocysts appear under stress conditions and that they are competent to divide. Pseudocysts differ from trophozoites in that: (1) the flagella are located in endocytic vacuoles and remain beating; (2) the axostyle and the costa are not depolymerized but present a curved shape; (3) the axostyle does not exhibit staining with antitubulin antibodies when the mitotic spindle is observed; (4) the mitotic process occurs within pseudocysts but differs from that described for trophozoites; (5) a nuclear canal is formed connecting the two spindle poles; and (6) the process is reversible if the cells are transferred to fresh medium.

Structure and division of the Golgi complex in Trichomonas vaginalis and Tritrichomonas foetus.

We present observations on the fine structure and the division process of the Golgi complex in the protists Trichomonas vaginalis and Tritrichomonas foetus, parasites of the urogenital tract of humans and cattle, respectively. The Golgi in trichomonads is a prominent structure, associated with striated parabasal filaments to which this organelle seems to be connected. We followed by immunofluorescence and electron microscopy the Golgi in interphasic and mitotic cells. Ultrastructural studies were performed using fast-freezing fixation, immunocytochemistry using antisera to the known adhesins AP65 and AP51, cytochemistry (acid phosphatase, Ca++-ATPase, zinc iodide-osmium tetroxide technique (ZIO), for analysis of distribution of the endoplasmic reticulum and Golgi complex, and Thiérys techniques), routine and serial thin-sections. Three-dimensional reconstruction, NBD-ceramide, fluorescent lectin (WGA) and nocodazole treatments were also used. We demonstrate that: (1) the Golgi in trichomonads is a single-copy organelle; (2) presents a fenestrated structure; (3) is formed by 8-12 saccules; (4) is connected to the parabasal filaments by thin filamentous bridges; (5) by cytochemistry, presents a positive reaction for the lectin WGA, Ca++-ATPase, acid phosphatase, ZIO and Thiérys techniques; (6) does not appear to break down at any point of the cell cycle; (7) elongates during the cell cycle by lateral growth; (8) is labeled by anti-glutamylated tubulin antibodies, but it is not fragmented by nocodazole treatment; (9) before mitosis, the already elongated Golgi ribbon undergoes progressive medial fission, cisternae by cisternae, starting at the cisternae adjacent to the cell surface and ending with the cis-most cisternae; (10) the Golgikinesis originates two small Golgi ribbons; (11) the Golgi is intensely labeled with the antisera to the AP65 and AP51 adhesins in T. vaginalis, thus seeming to be a key station in the production of adhesins.

Contributions of the Axostyle and Flagella to Closed Mitosis in the Protists Tritrichomonas foetus and Trichomonas vaginalis

Abstract Tritrichomonas foetus and Trichomonas vaginalis are protists that undergo closed mitosis: the nuclear envelope remains intact and the spindle remains extranuclear. Here we show, in disagreement with previous studies, that the axostyle does not disappear during mitosis but rather actively participates in it. We document the main structural modifications of the cell during its cell cycle using video enhanced microscopy and computer animation, bright field light microscopy, confocal laser scanning microscopy, and scanning and transmission electron microscopy. We propose six phases in the trichomonads cell cycle: an orthodox interphase, a pre-mitotic phase, and four stages during the cell division process. We report that in T. foetus and T. vaginalis: a) all skeletal structures such as the costa, pelta-axostyle system, basal bodies, flagella, and associated filaments of the mastigont system are duplicated in a pre-mitotic phase; b) the axostyle does not disappear during mitosis, otherwise playing a fundamental role in this process; c) axostyles participate in the changes in the cell shape, contortion of the anterior region of the cell, and karyokinesis; d) flagella are not under assembly during mitosis, as previously stated by others, but completely formed before it; and e) cytokinesis is powered in part by cell locomotion.

The mitotic spindle and associated membranes in the closed mitosis of trichomonads.

In the present work, we followed the several phases of Tritrichomonas foetus and Trichomonas vaginalis cell cycles using immunofluorescence, serial thin sections, three‐dimensional (3D) reconstruction, and transmission electron microscopy. In motile trichomonad cells or in pseudocyst forms, the nuclear envelope persists throughout mitosis, and the spindle is extranuclear. We found three types of spindle microtubules: pole‐to‐nucleus microtubules which are attached to the nuclear envelope, pole‐to‐pole microtubules forming a cylindrical, cytoplasmic groove on the nuclear compartment in pseudocysts of T. foetus cells, and pole‐to‐cytosol microtubules which extend freely into the cytoplasm. We demonstrated that: (1) in T. foetus, the spindle is assembled from an MTOC located at the base of the costa, underneath one of the basal bodies; (2) the spindle presents an unusual arc shape during the initial phases of mitosis in motile trophozoites; (3) the spindle microtubules are glutamylated, but not acetylated; (4) several membranes similar to those of the endoplasmic reticulum follow the spindle microtubules; (5) finger‐like projections extend from the nucleus towards the cell poles in pseudocysts and multinucleated cells; and (6) vesicles formed in between the two nuclear membranes are seen in the course of mitosis in both trophozoite and pseudocyst forms.

Nucleus behavior during the closed mitosis of Tritrichomonas foetus

We present observations on the fine structure and the division process of the nucleus in the protist Tritrichomonas foetus, parasite of the urogenital tract of cattle. The nucleus was followed by immunofluorescence and electron microscopy during interphase and mitosis. Conventional karyotyping coupled to image processing and bright field Panotic staining were used to follow nucleus modifications, chromosome number and condensation pattern along the whole cell cycle. Confocal laser scanning microscopy (CLSM) using DNA fluorescent probes, followed by image processing in the SURF‐Driver program, produced three‐dimensional reconstruction data of the mitotic nucleus under each phase of the division process. Immunocytochemistry in thin‐sections revealed the chromosome spatial arrangement after bromodeoxyuridine incorporation and immunogold labeling using anti‐DNA monoclonal antibodies. Our results indicate that: (1) the nucleus assumes different size and shapes along mitosis: it appears oval in interphase, becoming lobed or concave in prophase, then undergoing torsion and constriction, displaying an ‘S’ shape (metaphase). Next, it becomes elongated and it is finally separated in two nuclei at the transition of anaphase to telophase; (2) T. foetus nucleus harbors five chromosomes; (3) chromosomes become condensed in a pre‐mitotic phase; (4) the nucleolus persists during the mitosis.

Cellular cardiomyoplasty in large myocardial infarction: Can the beneficial effect be enhanced by ACE‐inhibitor therapy?

Cellular cardiomyoplasty with bone marrow derived stromal (MSC) and mononuclear (BMNC) cells has been shown to improve performance of infarcted hearts. We performed a comparative study with MSC and BMNC and tested the hypothesis that captopril treatment could enhance the beneficial effect of cell therapy in large myocardial infarctions.

P2×7 purinergic signaling in dilated cardiomyopathy induced by auto-immunity against muscarinic M2 receptors: autoantibody levels, heart functionality and cytokine expression

Autoantibodies against the M2 receptors (M2AChR) have been associated with Dilated Cardiomyopathy (DCM). In the heart, P2×7 receptors influence electrical conduction, coronary circulation and response to ischemia. They can also trigger pro-inflammatory responses and the development of neurological, cardiac and renal disorders. Here, P2×7−/− mice displayed an increased heart rate and ST segment depression, but similar exercise performance when compared to wild type (WT) animals. After immunization with plasmid containing M2AChR cDNA sequence, WT mice produced anti-M2AChR antibodies, while P2×7−/− mice showed an attenuated production. Despite this, WT and P2×7−/− showed left ventricle cavity enlargement and decreased exercise tolerance. Transfer of serum from M2AChR WT immunized mice to näive recipients led to an alteration in heart shape. P2×7−/− mice displayed a significant increase in the frequency of spleen regulatory T cells population, which is mainly composed by the FoxP3+CD25− subset. M2AChR WT immunized mice showed an increase in IL-1β, IFNγ and IL-17 levels in the heart, while P2×7−/− group produced lower amounts of IL-1β and IL-17 and higher amounts of IFNγ. These results pointed to previously unnoticed roles of P2×7 in cardiovascular and immune systems, and underscored the participation of IL-17 and IFNγ in the progress of autoimmune DCM.

Bone marrow mesenchymal cells improve muscle function in a skeletal muscle re-injury model.

Skeletal muscle injury is the most common problem in orthopedic and sports medicine, and severe injury leads to fibrosis and muscle dysfunction. Conventional treatment for successive muscle injury is currently controversial, although new therapies, like cell therapy, seem to be promise. We developed a model of successive injuries in rat to evaluate the therapeutic potential of bone marrow mesenchymal cells (BMMC) injected directly into the injured muscle. Functional and histological assays were performed 14 and 28 days after the injury protocol by isometric tension recording and picrosirius/Hematoxilin & Eosin staining, respectively. We also evaluated the presence and the fate of BMMC on treated muscles; and muscle fiber regeneration. BMMC treatment increased maximal skeletal muscle contraction 14 and 28 days after muscle injury compared to non-treated group (4.5 ± 1.7 vs 2.5 ± 0.98 N/cm2, p<0.05 and 8.4 ± 2.3 vs. 5.7 ± 1.3 N/cm2, p<0.05 respectively). Furthermore, BMMC treatment increased muscle fiber cross-sectional area and the presence of mature muscle fiber 28 days after muscle injury. However, there was no difference in collagen deposition between groups. Immunoassays for cytoskeleton markers of skeletal and smooth muscle cells revealed an apparent integration of the BMMC within the muscle. These data suggest that BMMC transplantation accelerates and improves muscle function recovery in our extensive muscle re-injury model.