Worldwide genomic diversity of the human papillomaviruses-53, 56, and 66, a group of high-risk HPVs unrelated to HPV-16 and HPV-18

Among more than 200 human papillomavirus (HPV) types presumed to exist, 18 "high-risk" HPV types are frequently found in anogenital cancer. The best studied types are HPV-16 and 18, which are only distantly related to one another and form two separate phylogenetic branches, each including six closely related types. HPV-30, 53, 56, and 66 form a third phylogenetic branch unrelated to HPV-16 and 18. Worldwide comparison of HPV-16 and 18 isolates revealed a distribution of variant genomes that correlated with the geographic origin and the ethnicity of the infected cohort and led to the concept of unique African, European, Asian, and Native American HPV-16 and 18 variants. Here, we address the question whether similar phylogenies are found for HPV-53, 56, and 66 by determining the sequence of the long control regions (LCR) of these HPVs in samples from Europe, Asia, and Africa, and from immigrant societies in North and South America. Phylogenetic trees calculated from point mutations and a few insertions/deletions affecting 2-4.2% of the nucleotide sequences were distinct for each of the three HPVs and divergent from HPV-16 and 18. In contrast to the "star-phylogenies" formed by HPV-16 and 18 variants, 44 HPV-53 isolates represented nine variants, which formed two deep dichotomic branches reminiscent of the beginning split into two new taxa, as recently observed for subtypes of HPV-44 and 68. A total of 66 HPV-56 isolates represented 17 variants, which formed three branches preferentially containing European, Asian, and African variants. Variants of a fourth branch, deeply separated from the other three, were characterized by a 25 bp insertion and created a dichotomy rather than star-like phylogeny. As it contained isolates from cohorts in all continents, it may have evolved before the spread of humans into all continents. 18 of 31 HPV-66 isolates represented the prototype clone, which was found in all parts of the world, while the remaining 13 clones formed 11 branches without any geographic association. Our findings confirm the notion of a quantitatively limited genomic diversity of each HPV type with some correlation to the geographic origin of the sample. In addition, we observed in some variants of these three HPV types mutations that affect the amino acid sequence of the E6 oncoproteins and the L1 capsid protein, supporting the possibility of immunogenic and oncogenic diversity between variants of any HPV type.     

Development of hatching blastocysts from immature human oocytes following in-vitro maturation and fertilization using a co-culture system

Recently, in-vitro maturation (IVM) of immature human oocytes recovered from non-stimulated follicles has been applied in the treatment of infertility. However, in previous reports, very few embryos cultured in conventional medium have reached the expanded blastocyst stage following in-vitro maturation and fertilization (IVM/IVF). The objective of this study was to investigate whether the developmental competence of human embryos following IVM/IVF could be enhanced by the use of a human ampullary cell co-culture system. Immature human oocytes were aspirated from small follicles at Caesarean section and then cultured in medium containing human menopausal gonadotrophin for 36 to 48 h, followed by insemination. Zygotes were randomly cultured either in conventional culture medium alone or in the co-culture system. Of 48 embryos cultured in conventional medium alone, all arrested at the 2-16- cell stage on day 3 after insemination. Of 46 embryos cultured in the co-culture system, 26 embryos (56.5%) arrested at the 2-16-cell stage. Six embryos (13%) developed to the morula stage. Fourteen embryos (30.4%) developed to expanded blastocysts and two blastocysts were hatching on day 7 after insemination. We conclude that co-culture significantly enhances the development of blastocysts in embryos resulting from IVM/IVF.      

Indications of the Protective Role of Natural Killer Cells in Human Cutaneous Leishmaniasis in an Area of Endemicity

The role of natural versus acquired immunity to Leishmania aethiopica infection in humans is the focus of our studies. We found in previous studies that mononuclear cells from nonexposed healthy Swedish donors responded to Leishmania antigen stimulation by proliferation and gamma interferon production. The main cell type responding was CD3⁻ CD16/56⁺ natural killer (NK) cells. These findings led us to suggest that the potential to produce a rapid, nonacquired NK cell response may be a protective phenotype. In order to test this hypothesis, an area in Ethiopia where Leishmania is endemic was selected, and peripheral blood mononuclear cells were obtained from individuals who had lived in the area most of their lives but had no evidence of past or present leishmaniasis. Their responses were compared with those of confirmed leishmaniasis patients from the same region with active lesions or cured leishmaniasis lesions. Cells from these donors were stimulated in vitro with L. aethiopica antigen. Responses were measured by proliferation, cytokine production, and phenotype analysis by fluorescence-activated cell sorting. The association of NRAMP1 alleles with the studied phenotype and susceptibility to L. aethiopica-induced leishmaniasis was also evaluated. The results show that Leishmania antigens can induce NK cell and CD8⁺-T-cell responses in vitro. This is clearly seen in proliferating cells from the cured (immune) individuals and the apparently protected controls from the area of endemicity. It contrasted with the reactivity of the patients, where some NK proliferation was coupled with enhanced CD4⁺-T-cell proliferation. We conclude from these observations that NK cells and CD8⁺ cells proliferating in response to Leishmania stimulation are involved in protection from and healing of (Ethiopian) cutaneous leishmaniasis; however, such mechanisms appear to be unrelated to the NRAMP1 host resistance gene.     

Killing of Aspergillus fumigatus by alveolar macrophages is mediated by reactive oxidant intermediates

Phagocytosis and mechanisms of killing of Aspergillus fumigatus conidia by murine alveolar macrophages (AM), which are the main phagocytic cells of the innate immunity of the lung, were investigated. Engulfment of conidia by murine AM lasts 2 h. Killing of A. fumigatus conidia by AM begins after 6 h of phagocytosis. Swelling of the conidia inside the AM is a prerequisite for killing of conidia. The contributions of NADPH oxidase and inducible nitric oxide synthase to the conidicidal activity of AM were studied using AM from OF1, wild-type and congenic p47phox(-/-) 129Sv, and wild-type and congenic iNOS(-/-) C57BL/6 mice. AM from p47phox(-/-) mice were unable to kill A. fumigatus conidia. Inhibitors of NADPH oxidase that decreased the production of reactive oxidant intermediates inhibited the killing of A. fumigatus without altering the phagocytosis rate. In contrast to NADPH oxidase, nitric oxide synthase does not play a role in killing of conidia. Corticosteroids did not alter the internalization of conidia by AM but did inhibit the production of reactive oxidant intermediates and the killing of A. fumigatus conidia by AM. Impairment of production of reactive oxidant intermediates by corticosteroids is responsible for the development of invasive aspergillosis in immunosuppressed mice.     

Skeletal muscle contractility is preserved in COPD patients with normal fat‐free mass

Aim: Peripheral muscle dysfunction often occurs in patients with chronic obstructive pulmonary disease (COPD). The muscle dysfunction may be caused by a loss of force‐generating capacity, resulting from a loss of muscle mass, as well as by other alterations in contractile properties of skeletal muscle. Methods: The maximal isometric voluntary strength and fatigability were determined in hand‐grip and quadriceps muscles from nine male COPD patients (FEV₁ 30–50% predicted) and control subjects matched for fat‐free mass (FFM), physical activity level and age. Contractile properties and fatigability of the quadriceps muscle were also studied with electrically evoked isometric contractions. Results: The maximal voluntary force (MVC) and fatigability of the handgrip muscle did not differ between the COPD patients and control subjects. Also the MVC of the quadriceps muscle and the rate of force rise, contraction time, force–frequency relationship and fatigability, as determined with electrically evoked contractions, were similar in patients with COPD and control subjects. Conclusion: Skeletal muscle strength, contractile properties and fatigability are preserved in patients with moderate COPD and a normal FFM and activity level. This suggests that skeletal muscle dysfunction does not take place during moderate COPD until cachexia and/or a decline in physical activity occur.     

Response to a hepatitis B polypeptide vaccine in micelle form in a young adult population

Polypeptide micelles with relative molecular weights of 25,000 (p25) and 30,000 (gp30) daltons were prepared from native 22-nm hepatitis B surface antigen (HBsAg) particles. This p25/gp30 complex was alum-adsorbed, and three dosage levels (20 micrograms, 4 micrograms, and 0.8 micrograms) were administered at 0, 1, and 6 months to 51 human volunteers. Local and systemic reactions were clinically insignificant, and all vaccinees seroconverted, regardless of dose. As anticipated, antibody responses diminished as the dosage was reduced. Seroconversion rates and geometric mean antibody levels for the 20 micrograms dosage group were significantly better than those observed with a commercial vaccine and were comparable to those achieved after immunization with 40 micrograms of the intact 22-nm particles used to prepare the polypeptides. By 2 weeks, an anti-HBs response was elicited in 80% of the group receiving 20 micrograms of the polypeptide vaccine. This rapid response to immunization may be particularly beneficial for postexposure prophylaxis where the early development of immunity is advantageous.     

Inhibition of adhesion of enterotoxigenic Escherichia coli cells expressing F17 fimbriae to small intestinal mucus and brush-border membranes of young calves

Enterotoxigenic Escherichia coli strains expressing F17 fimbriae bind to the intestinal mucosa of young calves. F17 fimbriae recognize receptors present in the mucus layer and the brush-border membranes from duodenum, jejunum and ileum. The adhesion of E. coli F17 can be inhibited by several glycoproteins. Adhesion is also inhibited by pretreatment of mucus and brush-border membranes with sodium metaperiodate. The use of glycoconjugates as potential adhesion-blockers is further discussed.     

Optimal heparin surface concentration and antithrombin binding capacity as evaluated with human non-anticoagulated blood in vitro

Contact between blood and a biomaterial surface takes place in many applications and is known to activate the coagulation and complement systems. Heparin surface coatings have been shown to reduce blood activation upon contact with artificial surfaces. To establish the optimal heparin surface concentration, blood was incubated in a tubing loop model at 37 degrees C. The tubing was coated with different surface concentrations of heparin and rotated at three different velocities. We demonstrate that the blood compatibility of a surface with regard to coagulation, complement, and platelet activation can be improved by increasing the heparin surface concentration in the 6-12 pmol antithrombin/cm2 concentration interval. The binding of factor H is not influenced by the increased heparin surface concentration, suggesting that this factor is not the primary regulator of complement on heparin surfaces. In addition, the heparin coating has no effect on the complement activation that occurs on gas surfaces in extracorporeal circuits.     

Characterization of a gene encoding survival motor neuron (SMN)-related protein, a constituent of the spliceosome complex

Mutations in the gene encoding the Survival Motor Neuron (SMN) protein are responsible for autosomal recessive proximal spinal muscular atrophy (SMA). SMN orthologues have been identified in the nematode worm Caenorhabditis elegans and the yeast Schizosaccharomyces pombe but, to date, no human paralogues have been described. Here we describe identification and characterization of an SMN-related protein (SMNrp) gene that encodes a novel protein of 239 amino acids, which has recently been identified as a constituent of the spliceosome complex and designated SPF30. Significant similarity to the SMN protein is apparent only within a central region of SMNrp that represents a tudor domain. The SMNrp/SPF30 gene has been mapped to chromosome 10q23. It is differentially expressed, with abundant levels in skeletal muscle. An exclusively nuclear localization for SMNrp in cultured cells and muscle sections was revealed using GFP fusion constructs and thereafter confirmed with a polyclonal antibody raised against SMNrp. Overexpression of SMNrp as a fusion protein in HeLa cells in culture induced dose-dependent apoptosis with positive TUNEL staining. In addition to a possible role for this protein as a pro-apoptotic factor, SMN and its related protein share significant similarities in sequence and cellular function.