Trends in the epidemiology of human G1P[8] rotaviruses: a hungarian study.

Epidemiological trends of the globally most common rotavirus genotype, G1P[8], were investigated in Hungary during a 16-year period by sequencing and phylogenetic analysis of the surface antigens. Antigen shift among epidemiologically major G1P[8] strains was observed in 6 seasons, as indicated by changes in the sublineages of the G1 VP7 and the P[8] VP4 genes. The temporal clustering of some rotavirus VP4 and VP7 gene sublineages and the periodic emergence and/or resurgence of previously unrecognized rotavirus sublineages in the study population suggest a dynamic nature for these common strains. Recently established international strain surveillance networks may help to identify and track the spread of epidemiologically important rotavirus strains across countries and continents.

Progesterone induced blocking factor (PIBF) taken in early pregnancy predicts the pregnancy outcome in women undergoing in vitro fertilization procedure.

Earlier data suggest a relationship between PIBF concentrations and the outcome of pregnancy. The aim of the study was to compare serum and urine concentrations of PIBF in women with successful pregnancy after IVF with those of women without pregnancy after IVF procedure, and to evaluate the potential relation between PIBF and the outcome of pregnancy. Urine and serum were collected from 120 women, undergoing IVF. 87.5% of patients had primary infertility. 69.2% faced female causes of infertility: 10.8% tubal cause, 11.7% ovulation disorder, and 46.7% other causes of infertility. 30.8% of patients had male factor of infertility. Among non-pregnant women (42) mean concentrations of PIBF in urine and serum were significantly lower (15.8 ng/mL; 148.4 ng/mL) than in women with positive beta HCG value (78) (19.1 ng/mL; 225.9 ng/mL). In 49 patients pregnancy terminated with a term delivery, in 10 patients with pretem delivery, while in 19 patients the pregnancy terminated with a miscarriage. PIBF concentrations in urine (13.9 ± 2.8 ng/mL) and serum (124.6 ± 46.7 ng/mL) samples of women with miscarriage were significantly lower of those with preterm delivery (180.6 ± 54.4 ng/mL; 18.1 ± 4.4 ng/mL) and of those with term delivery (20.4 ± 8.5 ng/mL; 208.7 ± 114.3 ng/mL). Successful pregnancy after IVF procedure is predictable by measuring of urine and serum PIBF concentrations and could be important for predicting of early implantation and pregnancy outcome after IVF procedure and maybe to protect the risk pregnancy.

Genetic diversity and zoonotic potential of human rotavirus strains, 2003-2006, Hungary.

Rotavirus strain surveillance is being conducted in many countries before and after introduction of newly licensed vaccines to assess the impact of the vaccines on rotavirus strains. Here we describe a strain surveillance study in the Budapest area of Hungary (2003-2006) based on RNA profile analysis, genotyping by multiplex PCR and nucleotide sequencing. Among 1,983 G-typed rotaviruses we identified G1 (22%), G2 (4.8%), G3 (3.5%), G4 (18.5%), G6 (1.1%), G8 (<0.1%, n = 1), G9 (42%), and G12 (3.4%) specificities. Information on P genotype incidence was determined for a subset of samples (n = 814). In addition to the globally important strains, a variety of uncommon antigen combinations were also found, for example, P[9],G3; P[14],G6; or P[14],G8. Sequence and phylogenetic analysis of the VP7, VP4, VP6, and NSP4 genes of selected strains with uncommon antigen combinations demonstrated high similarity with certain bovine, porcine, feline, equine, and lapine rotaviruses, respectively. Continued surveillance is needed to assess the role of animal rotaviruses in human diseases.

Assignment of the group A rotavirus NSP4 gene into genotypes using a hemi-nested multiplex PCR assay: a rapid and reproducible assay for strain surveillance studies.

The rotavirus non-structural protein NSP4 has been implicated in a number of biological functions during the rotavirus cellular cycle and pathogenesis, and has been addressed as a target for vaccine development. The NSP4 gene has been classified into six genotypes (A-F). A semi-nested triplex PCR was developed for genotyping the major human NSP4 genotypes (A-C), which are common in human rotavirus strains but are also shared among most mammalian rotavirus strains. A total of 192 previously characterized human strains representing numerous G and P type specificities (such as G1P[8], G1P[4], G2P[4], G3P[3], G3P[8], G3P[9], G4P[6], G4P[8], G6P[4], G6P[9], G6P[14], G8P[10], G8P[14], G9P[8], G9P[11], G10P[11], G12P[6] and G12P[8]) were tested for NSP4 specificity by the collaborating laboratories. An additional 35 animal strains, including the reference laboratory strains SA11 (simian, G3P[2]), NCDV (bovine, G6P[1]), K9 and CU-1 (canine, G3P[3]), together with 31 field isolates (canine, G3P[3]; feline, G3P[9]; porcine, G2P[23], G3P[6], G4P[6], G5P[6], G5P[7], G5P[26], G5P[27], G9P[6] and G9P[7]) were also successfully NSP4-typed. Four human G3P[9] strains and one feline G3P[9] strain were found to possess an NSP4 A genotype, instead of NSP4 C, suggesting a reassortment event between heterologous strains. Routine NSP4 genotyping may help to determine the genomic constellation of rotaviruses of man and livestock, and identify interspecies transmission of heterologous strains.

PIBF+ extracellular vesicles from mouse embryos affect IL-10 production by CD8+ cells.

Earlier evidence suggests, that the embryo signals to the maternal immune system. Extracellular vesicles (EVs) are produced by all types of cells, and because they transport different kinds of molecules from one cell to the other, they can be considered as means of intercellular communication. The aim of this work was to test, whether the embryo is able to produce sufficient amounts of EVs to alter the function of peripheral lymphocytes. Embryo-derived EVs were identified by their Annexin V biding capacity, and sensitivity to Triton X dependent lysis, using flow cytometry. Transmission electron microscopy was used to detect EVs at the implantation site. Progesterone-induced blocking factor (PIBF) expression in embryo-derived EVs was demonstrated with immuno-electron microscopy. The % of IL-10 + murine lymphocytes was determined by flow cytometry. EVs were present in embryo culture media, but not in empty media. Mouse embryo-derived EVs adhere to the surface of both CD4+ and CD8+ murine peripheral T lymphocytes, partly, via phosphatidylserine binding. The number of IL-10+ murine peripheral CD8+ cells increases in the presence of embryo-derived EVS, and this effect is counteracted by pre-treatment of EVs with an anti-PIBF antibody, suggesting that the embryo communicates with the maternal immune system via EVs.

PIBF positive uterine NK cells in the mouse decidua.

Though uterine NK cells (u NK cells) contain cytotoxic granules, and selectively over- express the genes of perforin and granzymes, during normal pregnancy, they are not cytotoxic. Progesterone is indispensable for the establishment and maintenance of pregnancy both in humans and in mice. Mouse uterine NK cells do not express the classical progesterone receptor, yet progesterone affects the recruitment and function of uterine NK cells, the latter partly via the Progesterone-Induced Blocking Factor (PIBF). We demonstrated PIBF positive granulated cells in the mouse decidua. The aim of this study was to characterize these cells by lectin immunohistochemistry and anti-perforin reactivity. PIBF+ granulated cells were absent from the deciduae of alymphoid mice, but appeared in the decidua of those that had been reconstituted with bone marrow from male BALB/c mice. PIBF+ granulated cells bound the DBA lectin, suggesting their NK cell nature, and also contained perforin, which co-localized with PIBF in the cytoplasmic granules. In anti-progesterone treated mice all of the PIBF+ cells were perforin positive at g. d. 12.5, in contrast to the 54% perforin positivity of PIBF+ cells in untreated mice. CONCLUSION: The PIBF+ granulated cells in the decidua belong to the NK population, and PIPB co-localizes with perforin in the cytoplasmic granules.

Expansion of CD4 phenotype among CD160 receptor-expressing lymphocytes in murine pregnancy.

PROBLEM: CD160, a cell surface co-receptor, is capable of up- or downregulating cell proliferation, cytotoxicity or cytokine production on lymphocytes. Our aim was to investigate CD160+ lymphocytes in the periphery and at the maternal-foetal interface during murine pregnancy. METHOD OF STUDY: CD4+ , CD8+ and gamma/delta T-cell phenotype, TIM3 co-expression and cytotoxic activity of CD160+ lymphocytes of pregnant BALB/c mice were analysed by flow cytometry. RESULTS: The percentage of CD160+ lymphocytes in the decidua was unchanged compared to non-pregnant endometrium; however, the ratio of CD4+ cells within the CD160 population was significantly increased. The co-expression of TIM3 co-inhibitory molecule and cytotoxicity of CD160+ cells were increased in the decidua. CONCLUSION: The expansion of CD4-expressing CD160+ decidual lymphocytes is a new observation suggesting a potential regulatory role of T-cell function during mouse pregnancy. The altered immunological character of CD160+ lymphocytes could play a role in the maintenance of murine pregnancy.

The decidua-the maternal bed embracing the embryo-maintains the pregnancy.

The decidua has been known as maternal uterine tissue, which plays essential roles in protecting the embryo from being attacked by maternal immune cells and provides nutritional support for the developing embryo prior to placenta formation. However, there are questions that still remain to be answered: (1) How does the decidua supply nutrition and provide a physical scaffold for the growing embryo, before placental vascular connection is established? (2) How is the balance between preventing an anti-embryo immune response and protecting both embryo and mother from infections established? To understand basic personas in decidual tissues, we review the structure of the decidua composed of terminally differentiated uterine stromal cells, blood vessels, and a number of repertoire of uterine local immune cells, including the well-known uterine natural killer (uNK) cells and recently discovered innate lymphoid cells (ILCs). Decidual macrophages and uterine dendritic cells (DCs) are supposed to modulate adaptive immunity via balancing cytokines and promoting generation of regulatory T (Treg) cells. During decidualization, vascular and tissue remodeling in the uterus provide nutritional and physical support for the developing embryo. Secretion of various cytokines and chemokines from both the embryo and the decidual cells activates multiple signaling network between the mother and the embryo upon implantation. Defects in the decidual development during early pregnancy result in loss of pregnancy or complications in later gestational stage.

Identification of the novel lapine rotavirus genotype P[22] from an outbreak of enteritis in a Hungarian rabbitry.

Application of improved molecular techniques in the detection and characterization of rotavirus strains has led to the recent description of several new combinations, specificities, and genetic variants of the outer capsid genes, VP7 and VP4. In spite of the enormous diversity of mammalian rotavirus strains, the few lapine rotaviruses characterized to date, appear to carry a narrow range of such antigen combinations; only P[14], G3 and, based on a more recent study, P[22], G3 rotaviruses have proved to be epidemiologically important in rabbits. In the present study, we characterized a lapine group A rotavirus with a super-short electropherotype detected in an outbreak of fatal enteritis in a Hungarian commercial rabbitry. Based on sequence and phylogenetic analysis of the VP7, VP4, and NSP4 genes, our lapine strain is a P[22], G3 rotavirus that carries the NSP4 genotype shared by most lapine rotaviruses. Although the P[22] VP4 specificity has been newly identified, the relatively high sequence variation between our strain and those identified in Italy (89.1-90.4% nucleotide identity; region VP8*) implies that these strains diversified far before they were described for the first time, strongly suggesting that this genotype may have circulated in rabbitries or in nature without prior detection. We conclude that genotype P[22] lapine rotaviruses show a wider geographical dispersal than previously thought, although understanding their true epidemiological significance needs further investigation.

Progesterone induced blocking factor isoforms in normal and failed murine pregnancies.

PROBLEM: Progesterone induced blocking factor (PIBF) is required for successful pregnancy. Alternative splicing produces PIBF isoforms with different functions. The full-length (90 kDa) PIBF is involved in cell cycle regulation, whereas smaller secreted forms act as cytokines. We aim to examine the PIBF exon pattern and protein isoform profile in normal and failed murine pregnancies. METHOD OF STUDY: Pregnant Balb/c mice were killed on gestation days 12-14 or 17-19. Normal and resorbed fetuses, placentae, and uterine tissue were used for RNA and protein analysis with RT-PCR and Western blot, respectively. RESULTS: Late pregnancy and resorption were associated with lower expression of the N-terminal exons, together with significantly reduced production of the full-length protein. CONCLUSION: Reduced production of the full-length PIBF protein might result in disturbed cell cycle regulation and dysregulated trophoblast invasion, while the absence of PIBF isoforms containing exon 2-4 coded sequences might lead to the loss of local immunosuppression.

Emergence of serotype G12 rotaviruses, Hungary.

We describe the emergence of serotype G12 rotaviruses (67 [6.9%] of 971 specimens tested) among children hospitalized with rotavirus gastroenteritis in Hungary during 2005. These findings are consistent with recent reports of the possible global spread and increasing epidemiologic importance of these strains, which may have implications for current rotavirus vaccination strategies.