Cytochemical study of different stages in the life cycle of Toxoplasma gondii. I. Nucleic acids and proteins in endozoites]

Using the Feulgen technique in addition to the methyl green-pyronin and gallocyanin-chromalum staining, nucleic acids were detected in Toxoplasma gondii of strains SS-119 and RH DNA was revealed in the nuclei of both intracellular and free individuals examined on various days (2--6) after mouse inoculation. A high RNA content in the cytoplasm of endozoites is a most characteristic feature of this stage. However, no definite nucleolus has been demonstrated in the endozoite nucleus. Using the Fast green and Alcian blue techniques, resp., histones were detected in the endozoite nuclei whose locality corresponded to that of Feulgen-positive material. The Acrolein-Schiff method located aldehyde groups of protein in endozoites, the detected stuff being confined mainly to the nuclear and perinuclear areas of the parasite. Tannofilic protein seems to screen the endozoite body, no difference between nuclear and cytoplasmic staining being seen. Tryptophan and tyrosin were not detected in the endozoites of Toxoplasma. The results obtained on Toxoplasma endozoites are compared with the metabolic patterns seen in the host cells of the peritoneal exudate and with previous literature data.     

Cytochemical study of different stages in the life cycle of Toxoplasma gondii. VII. Oxidation--reduction enzymes in the cyst forms]

Dehydrogenases of glycolysis, Kreb's cycle, and pentose-phosphate shunt were detected in cystozoites of Toxoplasma gondii strain SS-119 with various degrees of activity. A mixed oxidative metabolism may be postulated on this stage of the toxoplasma life cycle. Besides, the activity of cytochrome oxidase was detected in cystozoites; the addition of cytochrome c to the incubation medium significantly intensified the reaction intensity. Of interest seems the observation of a layer of higher enzymatic activity in the host brain tissue in the immediate neighbourhood with the cyst body. This may be regarded as the host cells' (or tissue') response to the presence of the parasite's alien body.     

Scanning Electron Microscopy of Toxoplasma gondii: Parasite Torsion and Host-Cell Responses during Invasion1

Scanning electron microscopy confirmed our previous finding that toxoplasmas actively invade mouse peritoneal cells that are inhibited from phagocytosis. The parasites entered cells with the conoid end first and sometimes showed a counter-clockwise torsion of the body during invasion. Counter-clockwise torsion was also noted in free toxoplasmas. Host-cell responses to active invasion varied with experimental conditions and with the type of host cell. Under adverse culture conditions for phagocytosis, normal macrophages formed rudimentary filopodia or lamellipodia around the tips of in vading toxoplasmas; macrophages subjected to hyperthermia before similar incubation with toxoplasmas showed little or no response to invasion. Normal and heat-treated lymphocytes showed little surface reaction to invasion, but occa ionally a flocculent collar was seen around the tip of an invading toxoplasma. Scanning electron microscopy provides clues to possil'e mechanisms of toxoplasma locomotion and host-cell invasion.     

Toxoplasma gondii: a simple method for titration of infectivity with monolayer cells

A simple method for assessing the infectivity of Toxoplasma gondii using primary cultured monolayer cells has been devised. Statistic analysis important for interpreting the results of such experimentation was made. In this method, the number of intracellular toxoplasmas and the percentage of confluency of the monolayer were measured. The latter value was used for conversion of the number of intracellular toxoplasmas per unit area to the percentage of infective toxoplasmas. A linear dose-response relationship between the number of intracellular toxoplasmas per unit area and that of toxoplasmas inoculated was demonstrated with primary cell monolayers from the lungs of two-day old inbred golden hamsters and the kidneys of newborn mice and newborn Wistar Imamichi strain rats. On the average, 37% of the toxoplasma organisms harvested from the peritoneal exudate of mice on the third or fourth day of infection were found to be infective. This value compares very favorably with the value 40% reported previously.     

Early recognized antigen (p34) of Toxoplasma gondii after peroral ingestion of tissue cyst forming strain (Me49 strain) in mice.

Serum from mouse orally ingested with tissue cyst forming strain (Me49) of Toxoplasma gondii was assayed by Western blot and immunofluorescene assay (IFA) to establish early responses in antigenicity of the parasite in mouse model of foodborne toxoplasmosis. Sera were collected weekly to blot the RH antigen transferred onto nitrocellulose paper after being separated by 12% SDS-PAGE. With the second week serum, 34 kDa protein (p34) was detected uniquely, and all antigens of T. gondii were detected with the sera from 3 or 4 weeks. p34 was not a member of the major surface membrane proteins and confirmed to be localized in the rhoptry by IFA. It was secreted into parasitophorous vacuolar membrane (PVM) during the entry into host cells. When applied to the human sera of which the ELISA absorbance was in negative range, 10.3% of sera detected p34, while all the ELISA positive sera detected the band. It has diagnostic usefulness of presumed T. gondii infection. We suggest the name of the p34 protein as ROP9.     

Acute toxoplasma infection of mice induces spleen NK cells that are cytotoxic for T. gondii in vitro

Murine spleen natural killer (NK) cells from normal and Toxoplasma-infected BALB/c mice were examined for their reactivity against RH strain tachyzoites in vitro. First, the effect of suspending medium on survival of extracellular RH tachyzoites was determined. Optimal parasite viability (by ethidium bromide-acridine orange staining) was observed when tachyzoites were incubated in phosphate-buffered saline (PBS) containing 10% horse serum (HS) for as long as 5 hr. In addition, parasite viability in PBS-HS correlated with subsequent infectivity, because freshly harvested and PBS-HS-incubated tachyzoites were equivalent in their ability to cause lethal infections in normal mice and to survive within normal mouse macrophages. Furthermore, viability and tumoricidal capacity of murine spleen NK cells incubated in PBS-HS was comparable to that of NK cells incubated in a standard cytotoxicity medium. Incubation of effector NK cells and target tachyzoites in PBS-HS in vitro revealed that spleen NK cells from 3-day Toxoplasma-infected mice had significantly greater cytotoxicity for extracellular RH tachyzoites than did control cells from uninfected mice. Moreover, Toxoplasma gondii-induced spleen NK cell toxoplasmacidal activity was significant at all effector to target cell ratios tested, and appeared to be mediated by direct contact between the host cell and the parasite. These in vitro results suggest that NK cells may be important in host defense against T. gondii.     

The relationship between the fate of the agent of toxoplasmosis, Toxoplasma gondii, in macrophages cultivated in vitro and the virulence of the parasites]

Comparative light microscopic and electron microscopic studies of development of a highly-virulent RH strain and a less virulent Lagrave strain of Toxoplasma cultivated in macrophages in vitro were made. Contrary to active multiplication of the highly virulent strain most of toxoplasmas of a less virulent strain disintegrated the first hours, degenerating completely in 24-48 hours after the penetration into the macrophages. Submicroscopic study showed no marked cytological changes of macrophages infected with a less virulent strain in comparison with the marked changes of the nuclei in macrophages infected with the RH strain. Disintegration of parasites within the phagosome was accompanied by a gradual transformation of an originally double (or triple) vacuolar membrane to a single membrane and by the disappearance of an additional layer of mitochondria and endoplasmic reticulum elements surrounding the vacuolar membrane. It is likely that the capacity of toxoplasma to develop in macrophages in vitro could become an additional marker to its virulence for albino mice.     

Modes of entry of Toxoplasma gondii trophozoites into normal mouse peritoneal macrophage and HeLa cell monolayers. A phase-contrast microcinematographic study (author's transl)]

The mode of entry of living trophozoites of Toxoplasma gondii (RH strain) into normal mouse peritoneal macrophage and HeLa cell monolayers was studied by phase-contrast microcinematography. The results have shown that Toxoplasma can enter into macrophages either by phagocytosis (Figs. 1 and 2) and/or by active penetration (Fig. 3). Only the latter process was observed with normally non-phagocytic HeLa cells (Fig. 4). During this process the parasites actively moved towards the host-cells by flexion and penetrated them always through their sharpest end. Active penetration was a rapid phenomenon (about 20 s at 37 degrees C) and was accompanied by a series of morphological changes, i.e., elongation of the anterior end, contraction and swelling of the parasite body. Contrasting with phagocytosis, toxoplasmas which had penetrated into the cell were not immediately isolated from the host-cytoplasm by a microscopically discernable vacuole. The nature of the process of penetration (pressure and/or perforation of the plasma membrane) is discussed.     

Reversal of elastase-induced pulmonary emphysema and promotion of alveolar epithelial cell proliferation by simvastatin in mice

Besides lowering cholesterol, statins exert multiple effects, such as anti-inflammatory activity and improvement of endothelial cell function. We examined whether simvastatin (SS) protects against the development of elastase-induced pulmonary emphysema in mice by using mean linear intercepts of alveoli (Lm) as a morphometric parameter of emphysema. After injection of intratracheal elastase on day 0, C57BL/6 mice were treated daily with SS (SS+ group) or PBS (SS- group) for 2 wk. A 21% decrease in Lm on day 7 was observed in the SS+ group vs. the SS- group. Anti-inflammatory effects of SS were observed as a decrease in percentage of neutrophils up to day 3, and in hydroxyproline concentration on day 3, in bronchoalveolar lavage fluid (BALF). SS also increased the number of proliferating cell nuclear antigen (PCNA)-positive alveolar epithelial cells between days 3 and 14. To confirm the role of statins in promoting proliferation of alveolar cells, mice were treated with SS (SS+) vs. PBS (SS-) for 12 days, starting 3 wk after elastase administration. After SS treatment, Lm decreased by 52% and PCNA-positive alveolar epithelial cells increased compared with the SS- group. Concentrations of vascular endothelial growth factor in BALF and endothelial nitric oxide synthase protein expression in pulmonary vessels tended to be higher in the SS+ group vs. the SS- group in this protocol. In conclusion, SS inhibited the development of elastase-induced pulmonary emphysema in mice. This therapeutic effect was due not only to anti-inflammation but also to the promotion of alveolar epithelial cell regeneration, partly mediated by restoring endothelial cell functions.     

Dissemination of Toxoplasma gondii to immunoprivileged organs and role of Toll/interleukin-1 receptor signalling for host resistance assessed by in vivo bioluminescence imaging

Toxoplasma gondii infection can lead to life-threatening systemic disease in the immunocompromised individual and in the developing fetus. Despite intensive investigation in animal models of toxoplasmosis, the processes leading to systemic dissemination remain poorly characterized. In the present study, in vivo bioluminescence imaging (BLI) was applied to the Toxoplasma mouse model to study the dynamics of infection in real time. Photon emission analyses revealed rapid dissemination of parasites in the organism and dissemination to immunoprivileged organs (brain, eyes and testes). Spatio-temporal analysis by BLI in individual mice showed that the virulent RH strain (type I) and the non-virulent ME49/PTG strain (type II) disseminate widely, but the virulent RH strain (type I) exhibits a more dramatic expansion of parasite biomass. Assessment by BLI of the Toll/interleukin-1 receptor (TIR) signalling pathway in host resistance to T. gondii revealed that signal transduction to the adaptor protein MyD88 is probably mediated by Toll-like receptor(s) rather than by IL-1R or IL-18R signalling. However, TLR1(-/-), TLR2(-/-), TLR4(-/-), TLR6(-/-) and TLR9(-/-) animals did not exhibit increased susceptibility to infection. These results suggest that intricate mechanisms regulate TIR-mediated responses during Toxoplasma infection.     

Factors influencing the toxicity of salivary gland extracts of Ixodes holocyclus Neumann

A bioassay using mice was developed to compare the toxin content of extracts of salivary glands of I. holocyclus at various stages of feeding. The quantity of toxin increased rapidly from the third day of feeding. Toxin production continued and increased in ticks removed after 3–5 days on mice and held at 30°C at 92% RH for 24 h, whereas no toxin was detected in the salivary glands of ticks fed for 3 days and treated similarly. It is suggested that major physiological changes occur in the salivary glands of I. holocyclus on the third day, which once stimulated continue independently of feeding. Toxin production in ticks was not suppressed by passively immunizing mice with anti-tick toxin but was in ticks fed upon hosts with a previous experience of tick feeding.Thus, to obtain salivary glands containing high concentrations of toxin for chemical analysis, it is necessary for salivary glands to develop 5 days from the initial attachment of the tick to a host with no previous experience of tick feeding. This can be achieved by passively immunizing mice against toxin, thus enabling the tick to feed 5 days without killing the mouse or by keeping the tick for 24 h at 30°C at 92% RH following the death of the mouse on the fourth day.     

Toxoplasma gondii: Calcium lonophore A23187-mediated exit of trophozoites from infected murine macrophages

The Ca²⁺ ionophore A23187 consistently induced the exit of Toxoplasma gondii trophozoites from cultured macrophages which they had recently infected. Following exit of toxoplasmas, the host macrophages underwent degeneration. A23187 was active at concentrations higher than 0.25 μM and the activity reached a plateau at the concentration of 1.0 μM. Noninfected macrophages or those engulfing heat-killed toxoplasmas, or some other particles, were not affected by treatment with A23187. The toxoplasmas exiting host cells were capable of infecting and proliferating in normal macrophages. The A23187-mediated exit of toxoplasmas proceeded despite external Ca²⁺ and was enhanced by the addition of ethylene glycol bis(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid (EGTA) in the reaction mixture. On the other hand, the A23187-mediated exit of toxoplasmas was inhibited significantly by exogenous Mg²⁺.     

Use of coisogenic host blastocysts for efficient establishment of germline chimeras with C57BL/6J ES cell lines.

Gene targeting in embryonic stem (ES) cells allows the production of mice with specified genetic mutations. Currently, germline-competent ES cell lines are available from only a limited number of mouse strains, and inappropriate ES cell/host blastocyst combinations often restrict the efficient production of gene-targeted mice. Here, we describe the derivation of C57BL/6J (B6) ES lines and compare the effectiveness of two host blastocyst donors, FVB/NJ (FVB) and the coisogenic strain C57BL/6-Tyr(c)-2J (c2J), for the production of germline chimeras. We found that when B6 ES cells were injected into c2J host blastocysts, a high rate of coat-color chimerism was detected, and germline transmission could be obtained with few blastocyst injections. In all but one case, highly chimeric mice transmitted to 100% of their offspring. The injection of B6 ES cells into FVB blastocysts produced some chimeric mice. However; the proportion of coat-color chimerism was low, with many more blastocyst injections required to generate chimeras capable of germline transmission. Our data support the use of the coisogenic albino host strain, c2J, for the generation of germline-competent chimeric mice when using B6 ES cells.     

Staphylococcus lipolyticus sp. nov., a new cold-adapted lipase producing marine species

A cold-adapted lipase producing bacterium, designated SS-33^T, was isolated from sea sediment collected from the Bay of Bengal, India, and subjected to a polyphasic taxonomic study. Strain SS-33^T exhibited the highest 16S rRNA gene sequence similarity with Staphylococcus cohnii subsp. urealyticus (97.18 %), Staphylococcus saprophyticus subsp. bovis (97.16 %) and Staphylococcus cohnii subsp. cohnii (97.04 %). Phylogenetic analysis based on the 16S rRNA gene sequences showed that strain SS-33^T belongs to the genus Staphylococcus. Cells of strain SS-33^T were Gram-positive, coccus-shaped, non-spore-forming, non-motile, catalase-positive and oxidase-negative. The major fatty acid detected in strain SS-33^T was anteiso-C15:0 and the menaquinone was MK-7. The genomic DNA G + C content was 33 mol%. The DNA-DNA hybridization among strain SS-33^T and the closely related species indicated that strain SS-33^T represents a novel species of the genus Staphylococcus. On the basis of the morphological, physiological and chemotaxonomic characteristics, the results of phylogenetic analysis and the DNA-DNA hybridization, a novel species is proposed for strain SS-33^T, with the name Staphylococcus lipolyticus sp. nov. The strain type is SS-33^T (=MTCC 10101^T?=?JCM 16560^T). Staphylococcus lipolyticus SS-33^T hydrolyzed various substrates including tributyrin, olive oil, Tween 20, Tween 40, Tween 60, and Tween 80 at low temperatures, as well as mesophilic temperatures. Lipase from strain SS-33^T was partially purified by acetone precipitation. The molecular weight of lipase protein was determined 67 kDa by SDS-PAGE. Zymography was performed to monitor the lipase activity in Native-PAGE. Calcium ions increased lipase activity twofold. The optimum pH of lipase was pH 7.0 and optimum temperature was 30 °C. However, lipase exhibited 90 % activity of its optimum temperature at 10 °C and became more stable at 10 °C as compared to 30 °C. The lipase activity and stability at low temperature has wide ranging applications in various industrial processes. Therefore, cold-adapted mesophilic lipase from strain SS-33^T may be used for industrial applications. This is the first report of the production of cold-adapted mesophilic lipase by any Staphylococcus species.     

A comparison of rodent caging systems based on microenvironmental parameters.

Four different mouse caging systems were evaluated for microenvironmental temperature, carbon dioxide, relative humidity (RH) and ammonia levels during a 7-day testing period. All caging systems evaluated had polycarbonate bases and consisted of either a molded polyester (MP) filter lid, one of two different polycarbonate filter lids, or no filter lid which served as a control. At 50% macroenvironmental RH (study I), all systems maintained an intracage temperature of 75.5 degrees F +/- 0.5 degrees. Both polycarbonate systems averaged greater than 2200 ppm of carbon dioxide more than the MP system and the controls. When compared with RH in the control cages, RH levels averaged over 20% and 5 to 8% RH greater in the polycarbonate filter lid systems and the MP system, respectively. There were no appreciable ammonia levels in either the MP or control systems. In the polycarbonate filter lid systems, ammonia levels were detectable on day 4 and were greater than 200 ppm by day 6. At 20% macroenvironmental RH (study II), there was a proportional 15 to 30% RH decrease from study I levels. Ammonia levels were undetectable in any system until day 7 and averaged only 17 ppm in one of the polycarbonate systems. Minimal differences were observed in studies III, IV and V when pine shavings were used instead of hardwood chips, a CD-1 stock instead of a DBA/2J strain, and different grades of filter inserts in the polycarbonate systems, respectively.     

Response of the somatotropic axis to alterations in feed intake of channel catfish (Ictalurus punctatus)

To better understand the effects of reduced feeding frequency on the GH–IGF-I axis, channel catfish (Ictalurus punctatus), were either fed (Fed control, commercial diet fed daily), fed every other day (FEOD, commercial diet fed every other day), or not fed (Unfed, no feed). Pituitary GH mRNA increased whereas hepatic growth hormone receptor (GHR), IGF-I mRNA, and plasma IGF-I decreased in the FEOD and Unfed fish (P < 0.05). In another study, fish were either continually fed (Fed) or fasted and then re-fed (Restricted) to examine the physiological regulation of somatostatin-14 (SS-14) and SS-22 mRNA. Fasting increased (P < 0.05) levels of SS-14 mRNA in the hypothalamus and pancreatic islets (Brockmann bodies) at d 30 while re-feeding decreased SS-14 mRNA to control values in all tissues examined by d 45. Fasting had no effect on levels of SS-22 mRNA in the pancreatic islets whereas SS-22 mRNA was not detected in the stomach or hypothalamus. The results demonstrate that feeding every other day has similar negative impacts on components of the GH–IGF-I axis as fasting. The observed increase in SS-14 mRNA in the hypothalamus and pancreatic islets suggests a role for SS-14 in modulating the GH–IGF-I axis in channel catfish.     

Polyoma Pseudovirions II. Influence of Host Cell on Pseudovirus Production

The type of host cell influenced the relative amounts of pseudovirions and polyoma virions produced. The infection of primary mouse embryo cells resulted in the production of particles that were predominantly pseudovirions. Infection of baby mouse kidney or 3T3D cells yielded mainly infectious polyoma virus. The length of time that infection was allowed to continue also affected the amount of pseudovirions relative to polyoma virions. The longer the viral infection was allowed to proceed, the greater the quantity of pseudovirions produced. Pseudovirion production could be correlated with the fragmentation of host cell DNA to a size of approximately 3 x 10(6) daltons. The fragmentation of host cell DNA was much more extensive in primary mouse embryo cells than in the other cell types.     

Expressed Salmonella antigens within macrophages enhance the proliferation of CD4+ and CD8+ T lymphocytes by means of bystander dendritic cells

ATP-dependent Lon protease-deficient Salmonella enterica serovar Typhimurium (strain CS2022) appeared to invade successfully the mesenteric lymph nodes (MLN) and Peyer's patches (PP) of BALB/c mice and appeared to be easily eradicated by the host after oral immunization. As detected by flow cytometry, the population of major histocompatibility complex class I (MHC-I)-expressing macrophages and dendritic cells (DCs) was increased in the PP of mice immunized with CS2022 on day 6 after immunization. Thereafter, the population of splenic surface CD69(+) T lymphocytes prepared from mice immunized with CS2022 6 weeks prior to measurement increased as a result of the administration of the extracellular vesicles of RAW264.7 macrophage-like cells derived by Salmonella challenge. In addition, the proliferation of CD8(+) and even of CD4(+)T cells isolated from mouse spleens immunized with CS2022 was enhanced after cocultivation with naive DCs in the presence of the extracellular vesicles. These findings indicate that the extracellular vesicles prepared from the Salmonella-challenged macrophages carried salmonellae antigens to bystander DCs, thereby stimulating T-cell responses. Therefore, as antigen presentation after phagocytosis should be a central process in the T-cell activation that occurs in response to Salmonella infection, an oral immunization with CS2022 sufficiently induces T cell-mediated immunity in mice.