Induction of cellular proliferation in a human astrocytoma cell line by a Trypanosoma cruzi-derived antigen: a mechanism of pathogenesis?

Trypanosoma cruzi can compromise the human central nervous system (CNS) during acute infection or reactivation in immune-suppressed hosts. Astrocytes have been identified as targets of T. cruzi's CNS infection in humans. Despite a high degree of parasitism and cellular lysis by T. cruzi in vitro the number of astrocytoma cells did not change when compared to uninfected cultures. This work evaluated cellular proliferation, changes in Major Histocompatibility Complex (MHC) expression as a reflection of antigen processing, and cytokine (IL-6 & IL-8) secretion in a human astrocytoma cell line exposed to a trypomastigote-derived antigen. Light microscopy was used to evaluate the number of cells; MHC molecule expression, cell cycle and cytokine secretion were assessed by flow cytometry. The number of astrocytoma cells increased proportional to the amount of antigen used and the percentage of cells in G2/M phase was higher when compared to control cultures. Antigen exposure increased expression of MHC class II, but not MHC class I in comparison to cultures incubated without antigen. Astrocytoma cell secretion of IL-6 and IL-8 was unaffected by antigen exposure. These results suggest the participation of a trypomastigote-derived mediator that induces astrocytoma cell proliferation without an inflammatory response; which may contribute to the pathogenesis of neurologic Chagas disease.

Afferent connections of the nucleus centralis amygdalae. A horseradish peroxidase study and literature survey.

The central nucleus of the amygdala has been shown to be involved in cardiovascular regulation and the integration of arousal. In this study, the afferent input was investigated in cat by microinjecting horseradish peroxidase (HRP) into the central nucleus and examining retrogradely-labelled cells in the brain. Retrograde labelling was found in the cortex next to the sulcus ectosylvius anterior, fissura lateralis Sylvii, sulcus rhinicus anterior and posterior, sulcus suprasylvius, and pyriform and entorhinal cortices as well as in the insula and claustrum. Each of the sub-nuclei of the amygdaloid complex exhibited retrogradely-labelled perikarya. Labelled cells were also found in the diagonal band of Broca, nucl. lateralis septi, and nucl. proprius striae terminalis (bed nucl. of stria terminalis). In the hypothalamus the area preoptica medialis and lateralis, nucl. dorsomedialis, paraventricularis, periventricularis, arcuatus and mammilaris medialis were labelled. The nucl. subthalamicus, zona incerta, peripeduncular system, substantia nigra, and nucl. interpeduncularis contained HRP-marked cells. In the thalamus labelled cells were observed in the nucl. reuniens, nucl. centroposterior lateralis, nucl. latero-posterior, nucl. posterior, nucl. centro-anterior, antero-dorsalis, antero-medialis, antero-lateralis, centrum mdianum, nucl. reticularis, nucl. rhomboideus, nucl. parafascicularis and subfascicularis. The area tegmentalis Tsai and the corpora geniculata also contained labelled cells. In the brain stem, HRP-marked cells could be detected in the brachium colliculi inferioris, aqueductal grey matter, locus coeruleus, nucl. parabrachialis, in various nuclei of the formatio reticularis, in the nucl. retrofascialis, nucl. solitarius, nucl. commissuralis, nucl. ambiguus and nucl. dorsalis n. vagi. The results were compared to other neuroanatomical studies and to functional studies of the amygdala.

The effect of halofuginone, Wellcome 993 C, oxytetracycline, and diminazene diaceturate on Babesia equi-infected lymphoblastoid cell cultures.

The efficacy of halofuginone (DL-trans-7-bromo-6-chloro-3,3-(3-hydroxy-2-piperidyl) acetonyl-4-(3H) quinazolinone), Wellcome 993 C (2-hydroxy-3-cyclohexyl-1,4-naphthoquinone), and oxytetracycline, all of which have been shown to have a schizonticidal effect in the treatment of bovine theileriosis, and the babesicidal drug diminazene diaceturate, were tested against the schizont stages of Babesia equi in cell culture. The in vitro test system measured DNA synthesis in treated and untreated cell lines. Halofuginone (0.02 microgram/ml) and Wellcome 993 C (5 micrograms/ml) suppressed the incorporation of tritiated thymidine by more than 80%. Oxytetracycline was less effective, while diminazene diaceturate showed no notable effect. The insufficient schizonticidal activity may explain the failure of diminazene diaceturate to cure B. equi infections. Schizonticidal drugs either alone, or in combination with known babesicidal drugs, should be tested in the chemotherapy of B. equi infections.

Use of schizont and piroplasm antigens of Babesia equi in the indirect fluorescent antibody and complement fixation tests.

Eight ponies infected with Babesia equi were investigated for their serological response to B. equi schizont and piroplasm antigen with the indirect fluorescent antibody test (IFAT) and complement fixation test (CFT). Piroplasm antigen was prepared from an infected splenectomized pony, while schizont antigen was produced from cultured lymphoid cells which contained B. equi macroschizonts. The IFAT detected a rise in antibody titres to schizont antigen as well as to piroplasm antigen, but differences were obtained in the duration of antibody detection. Significant antibody titres to piroplasm antigen were detectable for a longer period post infection than to schizont antigen. The complement fixation test was not effective in detecting specific antibodies to schizont antigen in contrast to piroplasm antigen. The schizont antibody titres were in general extremely low and not detectable in 3 horses. Neither test showed any serological cross-reaction with B. caballi and B. bigemina antiserum using schizont antigen.

Cell-mediated immune response to Babesia equi-transformed lymphoblastoid cells in vitro.

The capacity of equine peripheral blood lymphocytes (PBL) to proliferate in the presence of Babesia equi-transformed lymphoblastoid stimulator cells was tested in an autologous as well as in an allogenic one way mixed lymphocyte reaction (MLR). It was found that both autologous and allogeneic responder lymphocytes incorporated high amounts of 3H-thymidine. The incorporation of 3H-thymidine was lower in MLR using as stimulator cells lymphocytes from which the cell line had previously been established, than when using parasitized culture cells as stimulator. Proliferation of PBL was achieved only with intact infected cells, whereas sonified lymphoblastoid cells did not have this effect. During the course of an infection with B. equi, PBL from six ponies were tested for their capacity to inhibit the in vitro growth on culture cells, when applying cell co-cultivation. Starting from day 18 p.i., PBL from older animals inhibited culture schizont growth more effectively than PBL from young ponies. On day 35 p.i., only PBL from two of the older ponies showed an inhibitory effect. Influences of effector cells on target cells and possible mechanisms of their recognition are discussed.

Fine structure of Babesia equi Laveran, 1901 within lymphocytes and erythrocytes of horses: an in vivo and in vitro study.

The development of Babesia equi (Piroplasmia) in the vertebrate host was studied by electron microscopy. The tick-borne sporozoites initiated an exoerythrocytic schizogony in lymphocytes in vivo and in vitro. In lymphocyte cultures the schizonts of B. equi lay as fissured bodies directly within the host cell forming numerous cytomeres. These cytomeres were bordered by a single membrane and contained polymorphous nuclei. Merozoite differentiation was initiated at several places by protrusions appearing at the periphery of the cytomeres. The cytoplasm of the schizont merged progressively into the young merozoites. The mature merozoites were pear-shaped (2.0 X 1.5 microns), bounded by a pellicle and had an apical complex without conoid. Numerous merozoites were observed entering erythrocytes in vitro. In vivo, lymphocytes containing B. equi schizonts were seen in capillaries within lymph nodes from the 12th day onwards after attachment of infected ticks. At the same time some erythrocytes were found that contained B. equi merozoites. Development of B. equi in the vertebrate host shows many similarities to the corresponding development of Theileria parasites. This makes a new discussion of the taxonomic position of B. equi necessary.

Babesia equi (Laveran 1901) 1. Development in horses and in lymphocyte culture.

The vertebrate development of Babesia equi from infected Hyalomma marginatum in Morocco was investigated in vitro and in vivo. It was demonstrated that the sporozoites of B. equi initiate a phase of exo-erythrocytic schizogony in lymphocytes both in vitro and in vivo. On the other hand, sporozoites did not invade erythrocytes in vitro. The complete vertebrate life cycle of B. equi was simulated in vitro, from the invasion of lymphocytes by sporozoites, to the development of macro- and microschizonts, the invasion of erythrocytes by merozoites, and the subsequent intra-erythrocytic schizogony. In the equine host, macro- and microschizonts were detected in lymph node biopsies during the febrile phase on days 12 to 14 after application of ticks. As a result, the taxonomic position of B. equi appears to be uncertain.

Immunological aspects of Theileria annulata infection calves. 2. Production of macrophage migration inhibition factor (MIF) by sensitized lymphocytes from Theileria annulata-infected calves.

Peripheral blood lymphocytes (PBLs) from calves infected experimentally with a stabilate of Theileria annulate, produced a factor which inhibited the migration of macrophages. This was first demonstrated against schizont antigen in some animals on day 8 post infection while in the others on day 12. The highest activity of the macrophage migration inhibition factor (MIF) was recorded on day 12 in some of the infected animals and on day 16 in the others, whereas on day 30 or more MIF was demonstrable. The MIF was also demonstrated against piroplasm antigen. 64 days after the first infection, the recovered calves were challenged with the homologous strain. It was found that PBLs from all challenged calves were able to produce MIF against both, schizont and piroplasm antigen. These results indicate occurrence of sensitized lymphocytes as effector cells of cell-mediated immunity to T. annulata infection.

Immunological aspects of Theileria annulata infection in calves. 1. E., EA, and EAC rosette forming cells in calves infected with T. annulata.

Peripheral blood lymphocytes (PBLs) from normal and Theilaria annulata-infected calves were assayed for E, EA, and EAC rosette formation. The E rosette forming cells showed the first increase on day 8 of the infection. This continued in all animals till day 14 the maximum increase was recorded. After that they showed reduced percentages of E rosettes, reaching normal values (as obtained before the infection) on day 24 to 30. When the recovered animals were challenged 64 days after the first infection, an increase in the percentage of E rosette formation was again observed. Although a moderate increase in the percentage of EAC rosette formation was recorded the tendency was the same as for E rosettes in regard to the course of the infection. In contrast to E rosettes, EAC rosettes remained constant during the challenge infection. The EA rosette forming cells generally had the same tendency as E rosettes during both the first and the challenge infection.

Influence of Trypanosoma infection on the formation of E, EA, and EAC rosettes with peripheral blood lymphocytes from calves.

Peripheral blood lymphocyte (PBL) from calves infected with T. brucei and T. congolense or T. congolense alone were tested for their capacity to form E, EA, and EAC rosettes. The results obtained indicate that early in the infection a slight increase in the number of T and B cells has occurred as indicated by the increase in E and EAC rosette formation. At the same time EA rosette forming cells were also increased and persisted during the whole time of the infection. The EA rosette forming cells were more likely null cells, because later in the infection neither E nor EAC rosette forming cells showed any increase.