A mucosal vaccination approach for herpes simplex virus type 2.

An estimated 1 out of every 5 Americans is infected with herpes simplex virus type 2 (HSV-2). Efforts in developing a potent vaccine for HSV-2 have shown limited success. Here we describe a heterologous vaccination strategy for HSV-2 based on an intramuscular DNA prime followed by a liposome-encapsulated antigen boost delivered intranasally. Both portions of the vaccine express the immunogenic HSV-2 glycoprotein D. In female Balb/c mice, this heterologous immunisation regimen stimulated high titers of serum neutralising antibodies, a DNA priming dose dependent T helper type response, enhanced mucosal immune responses and potent protective immunity at the portal of entry for the virus: the vaginal cavity. A clear synergistic effect on immune responses and protection from infection was seen using this heterologous immunisation approach. Suboptimal DNA prime (0.5 µg) followed by the liposome boost resulted in an 80% survival rate when mice were infected 2 weeks after immunisation. A higher dose of DNA priming (5 µg) followed by the liposome boost resulted in sterilising immunity in 80% of mice. The vaccine induced durable protection in mice, demonstrated by a 60% survival rate when lethal infections were performed 20 weeks after the immunisation primed with 0.5 µg of DNA vaccine.

The 5' flanking region of the Rhesus monkey H3 (DMBT1) gene contains putative progesterone response elements.

DMBT1 (deleted in malignant brain tumors) encodes a large scavenger receptor cysteine rich (SRCR) protein with proposed tumor suppressor properties due to its frequent deletion or lack of expression in a variety of different tumors including endometrial cancers. The gene is alternatively spliced to produce a number of related proteins with suspected functions in mucosal inflammation and epithelial regeneration. Expression of DMBT1 has been demonstrated in a wide variety of cell types, mostly of epithelial origin, including tissues of the respiratory system, the alimentary system, brain, and reproductive system. We have previously identified a Rhesus monkey cDNA clone H3 (homologous to human DMBT1) as a progesterone-induced gene in Rhesus monkey endometrium during the secretory phase of the menstrual cycle. As an initial step in understanding the molecular mechanisms of H3 (DMBT1) regulation we have cloned and sequenced 1.5 kb of the 5'-flanking region expected to contain promoter sequences of the Rhesus monkey gene and identified six putative progesterone receptor binding sites in the 5'-upstream region.

Microarray profiling of progesterone-regulated endometrial genes during the rhesus monkey secretory phase.

BACKGROUND: In the endometrium the steroid hormone progesterone (P), acting through its nuclear receptors, regulates the expression of specific target genes and gene networks required for endometrial maturation. Proper endometrial maturation is considered a requirement for embryo implantation. Endometrial receptivity is a complex process that is spatially and temporally restricted and the identity of genes that regulate receptivity has been pursued by a number of investigators. METHODS: In this study we have used high density oligonucleotide microarrays to screen for changes in mRNA transcript levels between normal proliferative and adequate secretory phases in Rhesus monkey artificial menstrual cycles. Biotinylated cRNA was prepared from day 13 and days 21-23 of the reproductive cycle and transcript levels were compared by hybridization to Affymetrix HG-U95A arrays. RESULTS: Of approximately 12,000 genes profiled, we identified 108 genes that were significantly regulated during the shift from a proliferative to an adequate secretory endometrium. Of these genes, 39 were up-regulated at days 21-23 versus day 13, and 69 were down-regulated. Genes up-regulated in P-dominant tissue included: secretoglobin (uteroglobin), histone 2A, polo-like kinase (PLK), spermidine/spermine acetyltransferase 2 (SAT2), secretory leukocyte protease inhibitor (SLPI) and metallothionein 1G (MT1G), all of which have been previously documented as elevated in the Rhesus monkey or human endometrium during the secretory phase. Genes down-regulated included: transforming growth factor beta-induced (TGFBI or BIGH3), matrix metalloproteinase 11 (stromelysin 3), proenkephalin (PENK), cysteine/glycine-rich protein 2 (CSRP2), collagen type VII alpha 1 (COL7A1), secreted frizzled-related protein 4 (SFRP4), progesterone receptor membrane component 1 (PGRMC1), chemokine (C-X-C) ligand 12 (CXCL12) and biglycan (BGN). In addition, many novel/unknown genes were also identified. Validation of array data was performed by semi-quantitative RT-PCR of two selected up-regulated genes using temporal (cycle day specific) endometrial cDNA populations. This approach confirmed up-regulation of WAP four-disulfide core domain 2 (WFDC2) and SLPI during the expected window of receptivity. CONCLUSION: The identification of P-regulated genes and gene pathways in the primate endometrium is expected to be an important first step in elucidating the cellular processes necessary for the development of a receptive environment for implantation.

Role of progesterone in structural and biochemical remodeling of endometrium.

The endometrial response to the varying levels of ovarian steroids is exhibited as alterations in its form and function. These changes in endometrial morphology and physiology, especially those observed during the implantation window are prerequisites to support embryo attachment and invasion. However the state of endometrial receptivity to embryo results from an operative network of several molecular events triggered by estrogen, progesterone and probably some other factors, yet to be discovered. It is well established that estrogen and progesterone are the critical endocrine determinants of endometrial functions. However the precise delineation of hormone driven events and their interaction is yet to be ascertained. Several attempts have been made to understand these cascades, however most of these studies have been conducted in vitro using one or the other component of endometrial tissues. We have attempted to investigate in vivo morphological and biochemical/molecular changes in endometrium in response to neutralization of progesterone synthesis/ function in two primate animal models. In one of the models, ovariectomized rhesus monkeys, artificial menstrual cycles were simulated and subsequent effects on the _expression of various genes were investigated in presence and absence of sufficient progesterone levels. The results coincided with those observed in the endometrium of the other model, bonnet monkeys presenting normal hypothalamus-ovarian-pituitary function but displaying retarded endometrial growth due to blocked progesterone receptor. A significant decline was observed in the expression of transforming growth factor beta, transforming growth factor beta receptor, leukaemia inhibitory factor, whereas no remarkable changes were observed in the expression of estrogen receptor and progesterone receptors in response to neutralization of progesterone synthesis/function in these two animal models. Taking support from the inferences drawn from previously published in vitro studies and our data from in vivo studies conducted in these two models, we propose a hypothesis supporting a potential link between the expressions of transforming growth factor beta, leukaemia inhibitory factor, cyclooxygenases and integrins.

Temporal regulation of gene expression during the expected window of receptivity in the rhesus monkey endometrium.

Progesterone has been shown to regulate a number of genes and gene networks in the primate endometrium. This action of progesterone is essential to provide an appropriate milieu for embryo-endometrial communication that can lead to implantation and the successful initiation of pregnancy. A temporal regulation of endometrial genes is most likely required to achieve an appropriate state of receptivity in the primate endometrium. Using simulated menstrual cycles in the rhesus monkey, endometrial tissue was harvested at days that encompass the expected window of receptivity (4-10 days after the estradiol surge) and subsequently converted to cycle day-specific cDNA populations. Using differential display reverse transcriptase-polymerase chain reaction, 12 cDNA fragments were isolated and sequenced whose mRNA levels were elevated during this time frame. The temporal expression patterns of these mRNAs were confirmed by semiquantitative polymerase chain reaction. Two of these fragments exhibited high homology to previously characterized human genes: 1) secretory leukocyte protease inhibitor, also known as antileukoprotease, an endometrial neutrophil elastase inhibitor with antibacterial and anti-inflammatory properties; and 2) syncytin, also known as endogenous retrovirus W envelope protein, a highly fusogenic membrane glycoprotein that induces formation of giant syncytia and is believed to be important in decidual and placental development. The temporal regulation of these genes by progesterone supports their likely role in the orchestration of molecular and cellular events that are required to achieve a state of receptivity in the primate endometrium.

Gene expression in the rhesus monkey endometrium: differential display and laser capture microdissection.

The primate endometrium is a complex heterogeneous tissue that requires proper maturation to achieve a hospitable environment for implantation. Endometrial differentiation and maturation is primarily controlled through the action of progesterone during the secretory phase. Many of the genes and gene networks that are involved in this process are likely to be induced or inhibited in a temporal, spatial, and cell-type specific context within the endometrium. We have used several approaches to address these latter issues in the rhesus monkey endometrium. The use of differential display with hormonally distinct endometrial cDNA populations prepared from artificially controlled menstrual cycles has allowed us to identify different P-dependent mRNA regulatory patterns during simulated secretory phases. In addition, we have coupled differential display with laser capture microdissection to further study region and cell-type specific changes in the primate endometrium.

Assessment and application of laser microdissection for analysis of gene expression in the rhesus monkey endometrium.

We investigated the use of laser capture microdissection (LCM) to identify differences in gene expression between cell types or regions within the rhesus monkey endometrium. Different cell types were harvested from the two major regions of the endometrium during midsecretory phases (Days 21-23) of adequate artificial menstrual cycles: glandular epithelia (G) or stroma (S) from the functionalis (F) or the basalis (B). Amplification of the cDNA populations (primer-specific adaptors) was used to increase the amount of nucleic acid for further analysis. This single amplification step allowed us to detect the housekeeping genes (glyceraldehyde-3-phosphate dehydrogenase and 18S rRNA) and the cDNA smears in the samples. Using differential display reverse transcription polymerase chain reaction (DDRT-PCR), six fragments were selected, cloned, and sequenced based on their regional and cell type localization. Primer-specific PCR analysis subsequently confirmed the localization of three fragments: F1, highly expressed in the functionalis but not the basalis, was homologous (93% identical) to the human leukotriene B4 receptor BLT2; FS-1, highly expressed only in the stroma of the functionalis, had a 94% homology with an as yet uncharacterized gene (FLJ124360); and BG-1, primarily expressed only in the glandular epithelia of the basalis, showed a 98% homology with an uncharacterized bacterial artificial chromosome clone sequence. These LCM-generated cDNA populations coupled with DDRT-PCR can provide an important avenue for the identification of new or novel gene fragments that display cell type- or region-specific gene expression in the rhesus monkey endometrium.