J. Howe

Experimental Blastocystis hominis infection in laboratory mice

Abstract Young (less than 8 weeks old) immunocompetent BALB/c mice became infected with Blastocystis hominis after inoculation of fecal cysts orally and of in vitro axenic-culture forms intracecally. This study confirmed that the fecal cyst was the form responsible for external transmission and that the mode of transmission was by the fecal-oral route. The infection was self-limiting and the infected BALB/c mice appeared normal except that some of them showed weight loss and lethargy. Both vacuolar and granular forms were found in the cecum, but only cyst forms were observed in the colon. Histological examination of the cecum and colon showed intense inflammatory-cell infiltration, edematous lamina propria, and mucosal sloughing. It is apparent that although B. hominis is not invasive, it is capable of causing pathogenesis in BALB/c mice.

Exposure to Titanium Dioxide Nanoparticles Induces Autophagy in Primary Human Keratinocytes

Understanding the mechanisms of cell-nanomaterial interactions is vital in harnessing the potential of using nanomaterials in biomedical applications. By immuno-labeling of LC3 and TEM analysis, it is found that titanium dioxide nanoparticles are internalized by human keratinocytes and induce autophagy. Autophagy appears to play a cytoprotective role in response to toxicity influence exerted by the nanoparticles.

Observations on the ultrastructure and viability of the cystic stage of Blastocystis hominis from human feces

This report describes the ultrastructure and viability of cysts of Blastocystis hominis from feces of infected patients. The cysts were round to ovoid, measured 2-5 μm in size, and contained a condensed cytoplasm that had vacuoles of varying sizes, four nuclei, and as many as six cristate mitochondria. The cell wall was rather electron-lucent. Surprisingly, chromatoid-like structures were found in the cytoplasm and nucleus of some of the cysts. These have not previously been reported in Blastocystis. The cysts can survive in water for up to 19 days at normal temperatures but are fragile at extreme temperatures and in common disinfectants.

Ultrastructure of Blastocystis hominis cysts

Blastocystis hominis cysts were concentrated from human faeces by repeated washing in distilled water and centrifugation on Ficoll-Paque. This procedure gave a concentrated suspension of cysts without yielding any non-cystic forms. The cysts were examined by both transmission (TEM) and scanning electron microscopy (SEM). Cysts were surrounded by a fibrillar layer that appeared porous in SEM. Many naked cysts without the fibrillar layer were also visible in SEM. They were variable in shape but mostly circular to oval. The diameter of the cyst without the fibrillar layer was 3.5 μm in both TEM and SEM. The nuclear structure was typical ofBlastocystis and exhibited multiple mitochondria with poorly developed cristae. Glycogen was present in small to large clumps in the cytoplasm. The cyst wall was 5–100 nm thick and was bounded internally by an electron-dense plasmalemma.

In vivo and in vitro studies on the antiviral activities of viperin against influenza H1N1 virus infection

Influenza A virus has caused a number of pandemics in past decades, including the recent H1N1-2009 pandemic. Viperin is an interferon (IFN)-inducible protein of innate immunity, and acts as a broad-spectrum antiviral protein. We explored the antiviral activities and mechanisms of viperin during influenza virus (IFV) infection in vitro and in vivo. Wild-type (WT) HeLa and viperin-expressing HeLa cells were infected with influenza A/WSN/33/H1N1 (WSN33) virus, and subjected to virological, light and electron microscopic analyses. Viperin expression reduced virus replication and titres, and restricted viral budding. Young and old viperin-knockout (KO) mice and WT control animals were challenged with influenza WSN33 at lethal doses of 10(3) and 10(4) p.f.u. via the intratracheal route. Lungs were subjected to histopathological, virological and molecular studies. Upon lethal IFV challenge, both WT and KO mice revealed similar trends of infection and recovery with similar mortality rates. Viral quantification assay and histopathological evaluation of lungs from different time points showed no significant difference in viral loads and lung damage scores between the two groups of mice. Although the in vitro studies demonstrated the ability of viperin to restrict influenza H1N1 virus replication, the viperin-deficient mouse model indicated that absence of viperin enhanced neither the viral load nor pulmonary damage in the lungs of infected mice. This may be due to the compensation of IFN-stimulated genes in the lungs and/or the influenza non-structural protein 1-mediated IFN antagonism dampening the IFN response, thereby rendering the loss of viperin insignificant. Nevertheless, further investigations that exploit the antiviral mechanisms of viperin as prophylaxis are still warranted.

A multiple fission-like mode of asexual reproduction in Blastocystis hominis

A non-axenic and an axenic isolate ofBlastocystis hominis have been induced to form cysts in vitro using an encystation medium. The morphology of the parasite at different time points was observed by scanning electron microscopy. In day-2 cultures the cysts were spherical and had a non-uniform, coarse outer surface around the body. A deep, pore-like opening was seen in some of the parasites. Most of the cysts from day-4 and day-6 cultures ruptured, revealing small, uniformly sized spherical bodies occurring in grape-like clusters. Acridine orange staining confirmed that these bodies were the progeny ofBlastocystis hominis. A multiple fission-like reproduction process giving rise to many daughterBlastocystis occurs within the cyst.

Ultrastructural changes during in vitro encystment of Blastocystis hominis

The morphological changes occurring inBlastocystis hominis at different time points following in vitro encystment were studied by electron microscopy. The following stages of the parasite were sequentially seen: (a) the amoebic form, which was irregular in shape, with a majority of the organelles being concentrated at the condensed cytoplasmic region; (b) the pre-cystic form, which was rounded and had an electron-dense material forming a homogeneous wall around the central body; and (c) the cystic form, which had a very prominent, thick osmiophilic electron-dense wall, within which there were many inclusions and possibly reproductive granules. The amoebic form appeared to be an intermediate stage between the vacuolar form and the pre-cystic form, as this stage allowed the parasite to ingest bacteria to enhance encystment. The pre-cystic stage had previously been shown in experimental infection to be infective. The role of the cystic stage in producing infection is currently being investigated.

Ultrastructural and Phylogenetic Studies on Blastocystis Isolates from Cockroaches

ABSTRACT. Four Blastocystis isolates from cockroaches were established and these isolates were morphologically confirmed as Blastocystis organisms by light and/or electron microscopy. As these isolates were morphologically indistinguishable from Blastocystis isolated from other animals, phylogenetic analyses were conducted using their small subunit ribosomal RNA genes. A analyses of these sequences with previously reported ones that had been classified into nine Blastocystis clades indicated the presence of a new clade that comprised only Blastocystis organisms from cockroaches (clade X). A clade comprised of amphibian and reptilian Blastocystis organisms (clade IX) was located at the basal position of the Blastocystis tree together with the common ancestor of Proteromonas and Protoopalina, clade X emerged after the divergences of these two basal clades and its branching position was clearly supported by bootstrap analysis.

Linoleic and linolelaidic acids differentially influence proliferation and apoptosis of MOLT-4 leukaemia cells.

The effects of varying concentrations of linoleic acid and its transisomer linolelaidic acid on the proliferation the ultrastructural morphology of MOLT‐4 T‐lymphoblastic leukaemia cells were investigated. At 2 and 4 days after exposure to the fatty acids, the cells were counted by flow cytometry and observed by electron microscopy. After 4 days of treatment, linoleic acid was growth stimulatory at concentrations of 200μm or less, but was markedly inhibitory at 400μm. In contrast, linolelaidic acid stimulated proliferation at concentrations of 100 and 200μm, but inhibited cell growth at 400μm. Cells treated with 400μm linoleic acid displayed dense accumulations of characteristic lipid globules and glycogen granules, and exhibited ultrastructural evidence of apoptosis including vacuolization, membrane blebbing and chromatin margination at the nuclear periphery. These results support the notion that geometrical isomerism and concentration of polyunsaturated fatty acids influence the proliferative destiny of cancer cells. Reverse transcription polymerase chain reaction (RT‐PCR) analysis revealed a previously documented larger alternatively spliced p53 gene transcript in MOLT‐4 cells cultured under reduced serum conditions. However, only wild‐type p53 transcripts were amplified by RT‐PCR of MOLT‐4 cells exposed to phytohaemagglutinin, linoleic acid or linolelaidic acid.

Do Blastocystis hominis colony forms undergo programmed cell death

Ultrastructural observations were made on colony forms of the protozoan parasite, Blastocystis hominis. Cross-sections of entire colonies were observed by TEM. Cells within a colony were heterogeneous in morphology, consisting of vacuolar, amoeboid, multivacuolar and unusual forms. Dying cells appeared to be in the process of fragmenting into numerous membrane-bound vesicles, giving rise to empty spaces within the colony. Interestingly, older colonies appeared to show cell fragmentation which resulted in larger, membrane-bound structures. Numerous cytoplasmic inclusions were present in the central vacuole of some cells, with many containing mitochondria. Amoeboid forms were observed to harbour small membrane-bound vesicles in endosome-like compartments. Other unusual features included margination of chromatin material and distinct blebbing of nuclei. These ultrastructural features suggest that B. hominis colony forms perhaps undergo a form of programmed cell death.