Deborah J. Stenzel

Expression of Vaccinia Recombinant Hpv 16 L1 and L2 Orf Proteins in Epithelial-Cells Is Sufficient for Assembly of Hpv Virion-Like Particles

A recombinant vaccinia virus termed pLC201VV was designed to coexpress the L1 and L2 late genes of human papillomavirus type 16 (HPV16). Synthesis of the L1 and L2 proteins occurred in cells infected with pLC201VV, and 40-nm virus-like particles with a density of 1.31 g/ml were produced in the nuclei of cells synthesizing both L1 and L2, but not in cells synthesizing either protein alone. Virus-like particles were partially purified from infected cells by sucrose gradient sedimentation and shown to consist of capsomeres similar to HPV and contain glycosylated L1 viral capsid protein. The production of HPV-like particles using recombinant vaccinia virus should be useful for biochemical studies and could provide a safe source of material for the development of a vaccine.

Synthesis and assembly of infectious bovine papillomavirus particles in vitro

Bovine papillomavirus type 1 (BPV-1) virions were produced in vitro using vaccinia virus (VV) recombinants expressing the BPV-1 L1 and L2 capsid proteins. Particles morphologically resembling papillomaviruses were observed in the nucleus of cells infected with a VV recombinant for the BPV-1 L1 protein, and greater numbers of similar particles were seen in the nuclei of cells infected with a VV double recombinant for L1 and L2. Virus-like particles (VLPs) assembled in cells infected with the VV double recombinant for BPV-1 L1 and L2, and not those assembled in cells infected with the VV recombinant for BPV-1 L1 alone, were able to package BPV-1 DNA. Transcription of the BPV-1 E1 viral open reading frame was observed after a mouse fibroblast cell line was exposed to VLPs produced using a BPV-1 L1/L2 VV recombinant in a cell line containing episomal BPV-1 DNA. E1 transcription was not observed when the VLPs were pre-incubated with antibodies to the capsid protein of BPV-1. This system should allow an in vitro approach to the definition of the BPV-1 cellular receptor.

Blastocystis in Humans and Animals: Morphology, Biology, and Epizootiology

Publisher Summary This chapter reviews the biology and epizootiology of Blastocystis. The various experimental attempts to induce different forms of Blastocystis hominis (B. hominis) in culture are discussed. The morphology of B. hominis is presented as determined by various techniques, such as light microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Binary fission is the only proven method of reproduction for B. hominis. B. hominis cells dividing by binary fission are seen commonly by light microscopy and less frequently by electron microscopy. The cells divide into two approximately equal portions, with the distribution of organelles into both halves. The description of various forms of Blastocystis is discussed. Some of these forms include forms seen in vivo, fresh faecal form, cyst form, vacuolar form, granular form, and amoeboid form. The various Blastocystis spp. that exist in invertebrates, amphibia, reptiles, birds, and a variety of mammals, including rats, guinea pigs, cats, monkeys, and apes, are also reviewed.

Ultrastructural Study of Chlamydia pneumoniae In a Continuous-Infection Model

ABSTRACT We have established an in vitro model of long-term continuousChlamydia pneumoniae infection in HEp-2 cells. Using transmission electron microscopy, we demonstrated the presence of spontaneous abnormal chlamydial inclusions similar in appearance to the persistent chlamydial forms induced in vitro by treatment with cytokines or antibiotics or by nutrient deprivation.

Ureaplasma parvum and Ureaplasma urealyticum are detected in semen after washing before assisted reproductive technology procedures

OBJECTIVE To investigate the prevalence of ureaplasmas in semen and washed semen and to explore their effect on semen andrology variables. DESIGN Prospective study. SETTING In vitro fertilization (IVF) unit of a private hospital. PATIENT(S) Three hundred forty-three men participating in an assisted reproductive technology (ART) treatment cycle. MAIN OUTCOME MEASURE(S) The prevalence of ureaplasmas in semen and washed semen tested by culture, polymerase chain reaction assays, and indirect immunofluorescent antibody assays. RESULT(S) Ureaplasmas were detected in 73 of 343 (22%) semen samples and 29 of 343 (8.5%) washed semen samples. Ureaplasmas adherent to the surface of spermatozoa were demonstrated by indirect immunofluorescent antibody testing. Ureplasma parvum serovar 6 (36.6%) and U. urealyticum (30%) were the most prevalent isolates in washed semen. A comparison of the semen andrology variables of washed semen ureaplasma positive and negative groups demonstrated a lower proportion of nonmotile sperm in men ureaplasma positive for washed semen. CONCLUSION(S) Ureaplasmas are not always removed from semen by a standard ART washing procedure and can remain adherent to the surface of spermatozoa.

A cyst-like stage of Blastocystis hominis

A cyst-like form of Blastocystis hominis is described in stools and in culture. This form is more common in stored stools than fresh material. A cyst wall is secreted under the surface coat of the cell, and the surface coat and cell debris subsequently separate from the cyst. Whether this stage can withstand adverse environmental conditions and is infective to a new host remain to be determined.

The ultrastructural architecture of the adult Schistosoma japonicum tegument

The tegument of the adult blood fluke Schistosoma japonicum is in direct contact with the host blood and immune systems. A comprehensive understanding of the ultrastructure of the tegument is crucial to the understanding of how the parasite maintains itself within the mammalian host. Important functions such as nutritional uptake and immune evasion are suspected functions of the tegument and this review discusses these aspects and presents some insights into some of these crucial functions. Transmission electron microscopy has allowed the identification of ultrastructural features of the adult S. japonicum, some of which differ from the reported features of other schistosome species. Morphological differences within the tegument of the adult S. japonicum are noted between sexes, among different regions of the worms and between aspects along the length of the parasite. Differences included variations in the ultrastructure, size and number of tegumental bodies and mitochondria within the matrix, and differences in the relative area of the apical surface of the tegument. Functions of the various components of the tegument matrix and specialised functions of different regions of the male and female parasites are discussed based on ultrastructural findings and previously reported biochemical and molecular data.

Schistosoma japonicum: Immunolocalization of paramyosin during development

This paper describes the localization of paramyosin immunoreactivity in Schistosoma japonicum and represents the first comparative immunolocalization study among schistosome adult, cercariae and lung schistosomula by electron microscopy. A polyclonal antibody was utilized to immunolabel paramyosin or paramyosin-like proteins. Paramyosin was localized within the muscle layer of all 3 developmental stages. Furthermore, paramyosin was localized within granules of the post-acetabular glands of cercariae, and within the tegument matrix and surface of lung schistosomules. Adults and cercariae did not display any detectable paramyosin on the surface or within the tegument. The possible functions of paramyosin within S. japonicum and the relevance of these findings in relation to the reported protective properties of paramyosin as an anti-schistosome vaccine target molecule are discussed.

Ultrastructure of Blastocystis hominis in human stool samples

A study of the ultrastructure of Blastocystis hominis in human stools found morphological differences between the organisms seen and those present in laboratory cultures. B. hominis found in stool samples showed little morphological variation with storage time before fixation, but were consistently smaller (approximately 5 microns in diameter), with a thicker surface coat than the cultured organisms. The large central vacuole, characteristic of the cultured organisms, and accepted as standard morphology of B. hominis, was rarely observed in organisms present in stool samples. Instead, a number of small vacuoles, or possibly a network of interconnected vacuoles, were noted. After short-term culture, organisms from these samples appeared with the typical vacuolated morphology. No large vacuoles were present in organisms obtained at colonoscopy. These results suggest that the vacuolated form as previously described may be an artefact of culture conditions, and that the form of B. hominis present in the gastrointestinal tract is avacuolar.

A family of cathepsin B cysteine proteases expressed in the gut of the human hookworm, Necator americanus

mRNAs encoding cathepsin B-like cysteine proteases (CatBs) are abundantly expressed in the genomes of blood-feeding nematodes. Recombinant CatBs have been partially efficacious in vaccine trials in animal models of hookworm infection, supporting further investigation of these enzymes as new control tools. We recently described a family of four distinct CatBs (Na-CP-2, -3, -4, -5) from the human hookworm, Necator americanus. Here we show that these N. americanus CatBs form a robust clade with other hookworm CatBs and are most similar to intestinal CatBs from Haemonchus contortus. All four mRNAs (Na-cp-2, -3, -4 and -5) are up-regulated during the transition from a free-living larva to a blood-feeding adult worm and are also expressed in gut tissue of adult N. americanus that was dissected using laser microdissection microscopy. Recombinant Na-CP-3 was expressed in soluble, secreted form in the yeast Pichia pastoris, while Na-CP-2, -4 and -5 were expressed in insoluble inclusion bodies in Escherichia coli. Recombinant Na-CP-3 was not catalytically active when secreted by yeast but underwent auto-activation to an active enzyme at low pH in the presence of dextran sulphate. Activated Na-CP-3 digested gelatin and cleaved the fluorogenic substrate Z-Phe-Arg-aminomethylcoumarin (AMC) but not Z-Arg-Arg-AMC. Recombinant Na-CP-3 did not digest intact hemoglobin but digested globin fragments generated by prior hydrolysis with N. americanus aspartic hemoglobinases. Antibodies raised in mice to all four recombinant proteins showed minimal cross-reactivity with each other, and each antiserum bound to the intestine of adult N. americanus, supporting the intestinal expression of their mRNAs. These data show that N. americanus expresses a family of intestinal CatBs, many of which are likely to be involved in nutrient acquisition and therefore are potential targets for chemotherapies and vaccines.