scholarly journals 222.Characterisation of the infertility effect induced by a recombinant murine cytomegalovirus expressing murine zona pellucida 3

2004 ◽  
Vol 16 (9) ◽  
pp. 222
Author(s):  
M. L. Lloyd ◽  
J. M. Papadimitriou ◽  
S. O'Leary ◽  
M. A. Lawson ◽  
G. R. Shellam
Keyword(s):  
Zona Pellucida ◽  
Recombinant Virus ◽  
Fertility Control ◽  
High Titre ◽  
Autoimmune Response ◽  
Murine Cytomegalovirus ◽  
Post Inoculation ◽  
Primary Role ◽  
Primordial Follicles

A recombinant mouse virus, murine cytomegalovirus, that has been engineered to encode the fertility antigen murine zona pellucida 3 (mZP3), is being developed for fertility control in mice. A single inoculation of the recombinant virus induces complete infertility in female BALB/c mice which persists for the breeding life of the animal. The extent of this autoimmune response was unexpected especially as the incorporation of mZP3 appears to have immunologically attenuated the growth of the recombinant virus. The histological features of the infection are an initial depletion in tertiary follicles by 21 days post inoculation followed by a progressive depletion of primordial follicles, leading to an almost complete absence of follicles by 150 days post-infection. High titre, long lasting, zona pellucida-specific antibody is present in infertile BALB/c mice although infertility has not been linked with either a critical titre or a dominant immunoglobulin isotype. However, our evidence suggests that anti-ZP3 antibody plays a primary role in infertility since antibodies are detected in vivo bound to the zona pellucida of ovaries from recombinant virus-infected mice, and passively transferred antibody from infected animals induces infertility in the absence of recombinant virus. In addition, an experiment in which immunoglobulin-deficient mice remained fertile after inoculation with the recombinant virus indicates that antibody is crucial for the immunocontraceptive effect to occur. Other immune mechanisms are also being explored.

284. Leukocyte trafficking in the ovary of mice immunised with recombinant murine cyclomegalovirus expressing murine zona pellucida 3

2005 ◽  
Vol 17 (9) ◽  
pp. 119
Author(s):  
S. O'Leary ◽  
M. L. Lloyd ◽  
G. R. Shellam ◽  
S. Maddocks
Keyword(s):  
T Cells ◽  
Zona Pellucida ◽  
Post Infection ◽  
Recombinant Virus ◽  
Murine Cytomegalovirus ◽  
Research Centre ◽  
Post Inoculation ◽  
Cooperative Research ◽  
Primordial Follicles

Inoculation of female BALB/c mice with recombinant murine cytomegalovirus encoding murine zona pellucida antigen (MCMV-ZP3) confers infertility characterised by depletion in ovarian tertiary follicles by day 21 post inoculation followed by a progressive depletion in primordial follicles.1 Cell mediated immune responses begin as early as day 10 post immunisation with MCMV-ZP32 with the recruitment of leukocytes before serum antibody can be clearly detected in mice. The physiological mechanisms leading to infertility in inoculated mice are being progressively delineated with the role of leukocyte subsets implicated in early pathological changes in ovarian architecture. The aim of this study was to investigate the effect of MCMV-ZP3 infection on leukocytes including T cells recruited into the ovary following infection with recombinant virus. Fifteen BALB/c female mice were randomly allocated into three groups of five animals at 6 weeks of age. Group one received an injection of PBS, group two and three received intraperitoneal inoculations of 2 × 104 pfu of MCMV and MCMV-ZP3 respectively. Ovaries were retrieved at day 10, 21 and 35 post inoculation and one ovary from each mouse was sectioned for immunohistochemical analysis of resident leukocytes using mAb CD45 reactive with all leukocyte lineages and mAb for CD4 and CD8 positive T cells. MCMV-ZP3 inoculation increased the abundance of ovarian leukocytes including CD4 and CD8 positive T cells for all time points post immunisation except for CD8 positive T cells 21 days post infection (Table 1). These results suggest that leukocytes, including T cells, are involved in causing early changes in the ovary post infection with MCMV-ZP3 that lead to the depletion of existing ovarian follicles leading to life long infertility in mice. Further experiments are underway to investigate the role of antibody and changes in leukocyte populations in the ovary as the course of infection with recombinant virus progresses. This study is funded by the Cooperative Research Centre for Pest Animal Control. (1)Lloyd ML, et al. (2003). Biol. Reprod. 68, 2024–32.(2)O’Leary S, et al. (2004). Reprod. Fertil. Devel. 16(Supplement), 77.

scholarly journals 223.Ovarian pathology in mice following immunisation with recombinant murine cytomegalovirus expressing murine zona pellucida 3

2004 ◽  
Vol 16 (9) ◽  
pp. 223
Author(s):  
S. O'Leary ◽  
M. L. Lloyd ◽  
G. R. Shellam ◽  
S. Maddocks
Keyword(s):  
Zona Pellucida ◽  
Murine Cytomegalovirus ◽  
Research Centre ◽  
Post Inoculation ◽  
Leukocyte Infiltration ◽  
Cooperative Research ◽  
Cx43 Expression ◽  
Wild Mice ◽  
Primordial Follicles ◽  
Ovarian Pathology

Immunocontraception is a promising biological control for wild mice in Australia, having the potential to reduce the socioeconomic cost of plagues with minimal environmental impact. Inoculation of BALB/c mice with recombinant murine cytomegalovirus encoding murine zona pellucida antigen (mCMV-ZP3) confers total infertility characterised by depletion in ovarian tertiary follicles by Day 21 post inoculation followed by a progressive depletion in primordial follicles (1). The mechanisms underlying ovarian pathology are largely unknown but are likely to involve antibody mediated and cell mediated immune responses. The immune pathology may also be facilitated by acute responses involving antibody binding to ZP in growing follicles resulting in recruitment of inflammatory cells and oocyte destruction. The aim of this study was to investigate the effect of mCMV–ZP3 infection on leukocyte infiltration and expression of oocyte-derived signalling molecules in ovarian tissue. Fifteen BALB/c female mice were randomly allocated into three groups of 5 animals. Group one received an injection of PBS, group two and three received intraperitoneal inoculations of 2�×�104�p.f.u. of mCMV and mCMV-ZP3 respectively. Ovaries were retrieved at Day 7 post inoculation and one ovary from each mouse was sectioned for immunohistochemical analysis of resident leukocytes using mAb CD45 reactive with all leukocyte lineages. The other ovary was processed for real time quantitative RT-PCR analysis of growth and differentiation factor 9 (GDF-9) and connexin 43 (Cx43) expression. mCMV-ZP3 inoculation increased the abundance of ovarian leukocytes (P�=�0.08), significantly increased expression of Cx43 mRNA (p<0.05), but did not alter GDF-9 mRNA expression. These results suggest that changes in expression of ovarian regulators due to ZP3 immunisation begins early after recombinant MCMV infection in mice, and implicates leukocyte infiltration in the mechanism leading to permanent ovarian failure. Further experiments are underway to investigate the dynamics of leukocyte trafficking and expression of oocyte-derived signals as the course of infection progresses. This study is funded by the Cooperative Research Centre for Pest Animal Control. (1) Lloyd, M. L., et al. (2003). Biology of Reproduction 68(6): 2024-32.

scholarly journals Deletion of the R78 G Protein-Coupled Receptor Gene from Rat Cytomegalovirus Results in an Attenuated, Syncytium-Inducing Mutant Strain

1999 ◽  
Vol 73 (9) ◽  
pp. 7218-7230 ◽  
Author(s):  
Patrick S. Beisser ◽  
Gert Grauls ◽  
Cathrien A. Bruggeman ◽  
Cornelis Vink
Keyword(s):  
G Protein ◽  
Recombinant Virus ◽  
Murine Cytomegalovirus ◽  
Striking Difference ◽  
Predicted Amino Acid Sequence ◽  
G Protein Coupled Receptor ◽  
Recombinant Viruses ◽  
G Protein Coupled

ABSTRACT The rat cytomegalovirus (RCMV) R78 gene belongs to an uncharacterized class of viral G protein-coupled receptor (GCR) genes. The predicted amino acid sequence of the R78 open reading frame (ORF) shows 25 and 20% similarity with the gene products of murine cytomegalovirus M78 and human cytomegalovirus UL78, respectively. The R78 gene is transcribed throughout the early and late phases of infection in rat embryo fibroblasts (REF) in vitro. Transcription of R78 was found to result in three different mRNAs: (i) a 1.8-kb mRNA containing the R78 sequence, (ii) a 3.7-kb mRNA containing both R77 and R78 sequences, and (iii) a 5.7-kb mRNA containing at least ORF R77 and ORF R78 sequences. To investigate the function of the R78 gene, we generated two different recombinant virus strains: an RCMV R78 null mutant (RCMVΔR78a) and an RCMV mutant encoding a GCR from which the putative intracellular C terminus has been deleted (RCMVΔR78c). These recombinant viruses replicated with a 10- to 100-fold-lower efficiency than wild-type (wt) virus in vitro. Interestingly, unlike wt virus-infected REF, REF infected with the recombinants develop a syncytium-like appearance. A striking difference between wt and recombinant viruses was also seen in vivo: a considerably higher survival was seen among recombinant virus-infected rats than among RCMV-infected rats. We conclude that the RCMV R78 gene encodes a novel GCR-like polypeptide that plays an important role in both RCMV replication in vitro and the pathogenesis of viral infection in vivo.

scholarly journals Kisspeptin level in the aging ovary is regulated by the sympathetic nervous system

2017 ◽  
Vol 232 (1) ◽  
pp. 97-105 ◽  
Author(s):  
Daniela Fernandois ◽  
Gonzalo Cruz ◽  
Eun Kyung Na ◽  
Hernán E Lara ◽  
Alfonso H Paredes
Keyword(s):  
Adrenergic Receptor ◽  
Follicular Development ◽  
Sympathetic Nerves ◽  
Female Rats ◽  
Reproductive Aging ◽  
Beta Adrenergic Receptor ◽  
Beta Adrenergic ◽  
Primordial Follicles ◽  
Follicular Cyst

Previous work has demonstrated that the increase in the activity of sympathetic nerves, which occurs during the subfertility period in female rats, causes an increase in follicular cyst development and impairs follicular development. In addition, the increase in ovarian sympathetic activity of aged rats correlates with an increased expression of kisspeptin (KISS1) in the ovary. This increase in KISS1 could participate in the decrease in follicular development that occurs during the subfertility period. We aimed to determine whether the blockade of ovarian sympathetic tone prevents the increase in KISS1 expression during reproductive aging and improves follicular development. We performed 2 experiments in rats: (1) an in vivo blockade of beta-adrenergic receptor with propranolol (5.0 mg/kg) and (2) an ovarian surgical denervation to modulate the sympathetic system at these ages. We measured Kisspeptin and follicle-stimulating hormone receptor (FSHR) mRNA and protein levels by qRT-PCR and western blot and counted primordial, primary and secondary follicles at 8, 10 and 12 months of age. The results showed that ovarian KISS1 decreased but FSHR increased after both propranolol administration and the surgical denervation in rats of 8, 10 and 12 months of age. An increase in FSHR was related to an increase in the number of smaller secondary follicles and a decreased number of primordial follicles at 8, 10 and 12 months of age. These results suggest that intraovarian KISS1 is regulated by sympathetic nerves via a beta-adrenergic receptor and participates locally in ovarian follicular development in reproductive aging.

scholarly journals Topical Application of Double-Stranded RNA Targeting 2b and CP Genes of Cucumber mosaic virus Protects Plants against Local and Systemic Viral Infection

Plants ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 963
Author(s):  
Maria C. Holeva ◽  
Athanasios Sklavounos ◽  
Rajendran Rajeswaran ◽  
Mikhail M. Pooggin ◽  
Andreas E. Voloudakis
Keyword(s):  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Topical Application ◽  
Plant Virus ◽  
Plant Protection ◽  
Post Inoculation ◽  
Tobacco Plants ◽  
Double Stranded Rna

Cucumber mosaic virus (CMV) is a destructive plant virus with worldwide distribution and the broadest host range of any known plant virus, as well as a model plant virus for understanding plant–virus interactions. Since the discovery of RNA interference (RNAi) as a major antiviral defense, RNAi-based technologies have been developed for plant protection against viral diseases. In plants and animals, a key trigger of RNAi is double-stranded RNA (dsRNA) processed by Dicer and Dicer-like (DCL) family proteins in small interfering RNAs (siRNAs). In the present study, dsRNAs for coat protein (CP) and 2b genes of CMV were produced in vitro and in vivo and applied onto tobacco plants representing a systemic solanaceous host as well as on a local host plant Chenopodium quinoa. Both dsRNA treatments protected plants from local and systemic infection with CMV, but not against infection with unrelated viruses, confirming sequence specificity of antiviral RNAi. Antiviral RNAi was effective when dsRNAs were applied simultaneously with or four days prior to CMV inoculation, but not four days post inoculation. In vivo-produced dsRNAs were more effective than the in vitro-produced; in treatments with in vivo dsRNAs, dsRNA-CP was more effective than dsRNA-2b, while the effects were opposite with in vitro dsRNAs. Illumina sequencing of small RNAs from in vivo dsRNA-CP treated and non-treated tobacco plants revealed that interference with CMV infection in systemic leaves coincides with strongly reduced accumulation of virus-derived 21- and 22-nucleotide (nt) siRNAs, likely generated by tobacco DCL4 and DCL2, respectively. While the 21-nt class of viral siRNAs was predominant in non-treated plants, 21-nt and 22-nt classes accumulated at almost equal (but low) levels in dsRNA treated plants, suggesting that dsRNA treatment may boost DCL2 activity. Taken together, our findings confirm the efficacy of topical application of dsRNA for plant protection against viruses and shed more light on the mechanism of antiviral RNAi.

NE turnover in genetically hypertensive rats of Lyon strain. I. Brain nuclei

1988 ◽  
Vol 255 (4) ◽  
pp. H729-H735 ◽  
Author(s):  
M. Sautel ◽  
J. Sacquet ◽  
M. Vincent ◽  
J. Sassard
Keyword(s):  
Blood Pressure ◽  
Hypothalamic Nucleus ◽  
Primary Role ◽  
Hypertensive Rats ◽  
Brain Areas ◽  
Brain Nuclei ◽  
Genetically Hypertensive Rats ◽  
Low Blood Pressure ◽  
Genetically Hypertensive

Several indirect evidences of alterations in the central catecholaminergic structures were obtained in genetically hypertensive rats. Because they could be of pathogenetic value, we measured, in the present work, the in vivo turnover (TO) of norepinephrine (NE) in brain areas of 5- and 22-wk-old genetically hypertensive (LH) rats of the Lyon strain, and their simultaneously selected normotensive (LN) and low blood pressure (LL) controls. Among the changes observed, the increased TO of NE in the A2 and A6 regions of 5-wk-old LH rats and its decrease in the posteroventral hypothalamic nucleus of 22-wk-old LH animals appeared likely to compensate for hypertension. On the contrary, the decreased TO of NE in the anterior hypothalamic nucleus observed at 5 wk and in the A6 and A1 areas at 22 wk of age in LH rats could participate in the development or the maintenance of hypertension. Above all, it was postulated that the increased TO of NE found in the A7 region of 5-wk-old LH rats could play a primary role in the pathogenesis of hypertension in the Lyon model.

scholarly journals Enhancing the Nrf2 Antioxidant Signaling Provides Protection Against Trichloroethene-mediated Inflammation and Autoimmune Response

2020 ◽  
Vol 175 (1) ◽  
pp. 64-74 ◽  
Author(s):  
Nivedita Banerjee ◽  
Hui Wang ◽  
Gangduo Wang ◽  
M Firoze Khan
Keyword(s):  
Oxidative Stress ◽  
T Cells ◽  
Molecular Mechanisms ◽  
Inflammatory Responses ◽  
Autoimmune Response ◽  
Mediated Effects ◽  
Antioxidant Gene Expression ◽  
Responsive Element

Abstract Trichloroethene (trichloroethylene, TCE) and one of its reactive metabolites dichloroacetyl chloride (DCAC) are associated with the induction of autoimmunity in MRL+/+ mice. Although oxidative stress plays a major role in TCE-/DCAC-mediated autoimmunity, the underlying molecular mechanisms still need to be delineated. Nuclear factor (erythroid-derived 2)-like2 (Nrf2) is an oxidative stress-responsive transcription factor that binds to antioxidant responsive element (ARE) and provides protection by regulating cytoprotective and antioxidant gene expression. However, the potential of Nrf2 in the regulation of TCE-/DCAC-mediated autoimmunity is not known. This study thus focused on establishing the role of Nrf2 and consequent inflammatory responses in TCE-/DCAC-mediated autoimmunity. To achieve this, we pretreated Kupffer cells (KCs) or T cells with/without tert-butylhydroquinone (tBHQ) followed by treatment with DCAC. In both KCs and T cells, DCAC treatment significantly downregulated Nrf2 and HO-1 expression along with induction of Keap-1 and caspase-3, NF-κB (p65), TNF-α, and iNOS, whereas pretreatment of these cells with tBHQ attenuated these responses. The in vitro findings were further verified in vivo by treating female MRL+/+ mice with TCE along with/without sulforaphane. TCE exposure in mice also led to reduction in Nrf2 and HO-1 but increased phospho-NF-κB (p-p65) and iNOS along with increased anti-dsDNA antibodies. Interestingly, sulforaphane treatment led to amelioration of TCE-mediated effects, resulting in Nrf2 activation and reduction in inflammatory and autoimmune responses. Our results show that TCE/DCAC mediates an impairment in Nrf2 regulation. Attenuation of TCE-mediated autoimmunity via activation of Nrf2 supports that antioxidants sulforaphane/tBHQ could be potential therapeutic agents for autoimmune diseases.

scholarly journals STEM-20. RADIO-SENSITIZING GLIOBLASTOMA CANCER STEM CELLS BY INHIBITION OF FACT

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi237-vi238
Author(s):  
Miranda Montgomery ◽  
Abigail Zalenski ◽  
Amanda Deighen ◽  
Sherry Mortach ◽  
Treg Grubb ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dna Damage ◽  
Cancer Stem Cells ◽  
Combination Therapy ◽  
High Rate ◽  
Primary Role ◽  
Cancer Cell Growth ◽  
Treatment Paradigm

Abstract Glioblastoma (GBM) has a particularly high rate of recurrence with a 5-year overall survival rate of approximately 5%. This is in part due to a sub-population of cancer stem cells (CSC), which are both radioresistant and chemotherapeutically resistant to conventional treatments. Here we investigated CBL0137, a small molecule form of curaxin, in combination with radiotherapy as a means to radiosensitize CSCs. CBL0137 sequesters FACT (facilitates chromatin transcription) complex to chromatin, which leads to activation of p53 and inhibition of NF-κB. This sequestering of FACT results in cytotoxicity especially within tumor cells and prevents FACT from performing its primary role as a histone chaperone, as well as inhibits its part in the DNA damage response pathway. We show that when combined with radiotherapy, CBL0137 administration limited the ability of CSCs to identify and repair damaged DNA. CSCs treated in vitro with CBL0137 and irradiation showed an increased inhibition of cancer cell growth and decreased viability compared to irradiation or drug alone. Combination therapy also showed more DNA damage in the CSCs than with either agent alone. Based on our in vitro evidence for the efficacy of combination therapy to target CSCs, we moved forward to test the treatment in vivo. Using a subcutaneous model, we show that the amount of CD133+ cells (a marker for GMB CSCs) was reduced in irradiation plus CBL0137 compared to either treatment alone. Survival studies demonstrated that irradiation plus CBL0137 compared to irradiation alone or CBL0137 alone increase lifespan. Here we show the ability of CBL0137, in combination with irradiation, to target patient GBM CSCs both in vitro and in vivo. This work establishes a new treatment paradigm for GBM that inclusively targets CSCs and may ultimately reduce tumor recurrence.

In vivo survival of transferred sheep embryos following puncture of the zona pellucida and in vitro culture

1994 ◽  
Vol 35 (1-2) ◽  
pp. 81-89 ◽  
Author(s):  
P.A. Pugh ◽  
J.G. Thompson ◽  
K. Logan ◽  
H.R. Tervit
Keyword(s):  
In Vitro Culture ◽  
Zona Pellucida

scholarly journals Adhesion molecules during somitogenesis in the avian embryo.

1987 ◽  
Vol 104 (5) ◽  
pp. 1361-1374 ◽  
Author(s):  
J L Duband ◽  
S Dufour ◽  
K Hatta ◽  
M Takeichi ◽  
G M Edelman ◽  
...  
Keyword(s):  
Adhesion Molecules ◽  
Tissue Remodeling ◽  
Cell Aggregation ◽  
Avian Embryo ◽  
Primary Role ◽  
Calcium Dependent ◽  
Tissue Dissociation ◽  
Spatio Temporal

In avian embryos, somites constitute the morphological unit of the metameric pattern. Somites are epithelia formed from a mesenchyme, the segmental plate, and are subsequently reorganized into dermatome, myotome, and sclerotome. In this study, we used somitogenesis as a basis to examine tissue remodeling during early vertebrate morphogenesis. Particular emphasis was put on the distribution and possible complementary roles of adhesion-promoting molecules, neural cell adhesion molecule (N-CAM), N-cadherin, fibronectin, and laminin. Both segmental plate and somitic cells exhibited in vitro calcium-dependent and calcium-independent systems of cell aggregation that could be inhibited respectively by anti-N-cadherin and anti-N-CAM antibodies. In vivo, the spatio-temporal expression of N-cadherin was closely associated with both the formation and local disruption of the somites. In contrast, changes in the prevalence of N-CAM did not strictly accompany the remodeling of the somitic epithelium into dermamyotome and sclerotome. It was also observed that fibronectin and laminin were reorganized secondarily in the extracellular spaces after CAM-mediated contacts were modulated. In an in vitro culture system of somites, N-cadherin was lost on individual cells released from somite explants and was reexpressed when these cells reached confluence and established intercellular contacts. In an assay of tissue dissociation in vitro, antibodies to N-cadherin or medium devoid of calcium strongly and reversibly dissociated explants of segmental plates and somites. Antibodies to N-CAM exhibited a smaller disrupting effect only on segmental plate explants. In contrast, antibodies to fibronectin and laminin did not perturb the cohesion of cells within the explants. These results emphasize the possible role of cell surface modulation of CAMs during the formation and remodeling of some transient embryonic epithelia. It is suggested that N-cadherin plays a major role in the control of tissue remodeling, a process in which N-CAM is also involved but to a lesser extent. The substratum adhesion molecules, fibronectin and laminin, do not appear to play a primary role in the regulation of these processes but may participate in cell positioning and in the stabilization of the epithelial structures.

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