PreImplantation Factor and Endocrinology of Implantation and Establishment of Early Pregnancy: A Contemporary View.

The earliest stages of pregnancy are marked by countless changes in the maternal environment. A specific coordination of activity is required for a successful pregnancy, starting early in the menstrual cycle. Early establishment of maternal-fetal crosstalk is critical for the progression of pregnancy. Many factors, both maternal and fetal derived, play specific and important roles immediately following fertilization, through implantation and beyond. Here we present a review of some of the key factors involved with a focus on PreImplantation Factor (PIF), a small peptide secreted only by competent embryos, which carries an important role required for pregnancy progression.

Progressive aggregation despite chaperone associations of a mutant SOD1-YFP in transgenic mice that develop ALS.

Recent studies suggest that superoxide dismutase 1 (SOD1)-linked amyotrophic lateral sclerosis results from destabilization and misfolding of mutant forms of this abundant cytosolic enzyme. Here, we have tracked the expression and fate of a misfolding-prone human SOD1, G85R, fused to YFP, in a line of transgenic G85R SOD1-YFP mice. These mice, but not wild-type human SOD1-YFP transgenics, developed lethal paralyzing motor symptoms at 9 months. In situ RNA hybridization of spinal cords revealed predominant expression in motor neurons in spinal cord gray matter in all transgenic animals. Concordantly, G85R SOD-YFP was diffusely fluorescent in motor neurons of animals at 1 and 6 months of age, but at the time of symptoms, punctate aggregates were observed in cell bodies and processes. Biochemical analyses of spinal cord soluble extracts indicated that G85R SOD-YFP behaved as a misfolded monomer at all ages. It became progressively insoluble at 6 and 9 months of age, associated with presence of soluble oligomers observable by gel filtration. Immunoaffinity capture and mass spectrometry revealed association of G85R SOD-YFP, but not WT SOD-YFP, with the cytosolic chaperone Hsc70 at all ages. In addition, 3 Hsp110's, nucleotide exchange factors for Hsp70s, were captured at 6 and 9 months. Despite such chaperone interactions, G85R SOD-YFP formed insoluble inclusions at late times, containing predominantly intermediate filament proteins. We conclude that motor neurons, initially "compensated" to maintain the misfolded protein in a soluble state, become progressively unable to do so.

Intranasal immunization with recombinant vesicular stomatitis virus expressing murine cytomegalovirus glycoprotein B induces humoral and cellular immunity.

Cytomegalovirus is a leading cause of morbidity and mortality among neonatal and immunocompromised patients. The use of vaccine prophylaxis continues to be an effective approach to reducing viral infections and their associated diseases. Murine cytomegalovirus (mCMV) has proven to be a valuable animal model in determining the efficacy of newly developed vaccine strategies in vivo. Live recombinant vesicular stomatitis viruses (rVSV) have successfully been used as vaccine vectors for several viruses to induce strong humoral and cellular immunity. We tested the ability of intranasal immunization with an rVSV expressing the major envelope protein of mCMV, glycoprotein B (gB), to protect against challenge with mCMV in a mouse model. rVSV-gB-infected cells showed strong cytoplasmic and cell surface expression of gB, and neutralizing antibodies to gB were present in mice after a single intranasal vaccination of VSV-gB. After challenge with mCMV, recovery of live virus and viral DNA was significantly reduced in immunized mice. In addition, primed splenocytes produced a CD8+ IFNgamma response to gB. The ability to induce an immune response to a gene product through mucosal vaccination with rVSV-gB represents a potentially effective approach to limiting CMV-induced disease.

Motor deficits and altered striatal gene expression in aphakia (ak) mice.

Like humans with Parkinson's disease (PD), the ak mouse lacks the majority of the substantia nigra pars compacta (SNc) and experiences striatal denervation. The purpose of this study was to test whether motor abnormalities in the ak mouse progress over time, and whether motor function could be associated with temporal alterations in the striatal transcriptome. Ak and wt mice (28 to 180 days old) were tested using paradigms sensitive to nigrostriatal dysfunction. Results were analyzed using a linear mixed model. Ak mice significantly underperformed wt controls in rotarod, balance beam, string test, pole test and cotton shred tests at all ages examined. Motor performance in ak mice remained constant over the first 6 months of life, with the exception of the cotton shred test, in which ak mice exhibited marginal decline in performance. Dorsal striatal semi-quantitative RT-PCR for 19 dopaminergic, cholinergic, glutaminergic and catabolic genes was performed in 1- and 6-month-old groups of ak and wt mice. Preproenkephalin levels in ak mice were elevated in both age groups. Drd1, 3 and 4 levels declined over time, in contrast to increasing Drd2 expression. Additional findings included decreased Chrnalpha6 expression and elevated VGluT1 expression at both time points in ak mice and elevated AchE expression in young ak mice only. Results confirm that motor ability does not decline significantly for the first 6 months of life in ak mice. Their striatal gene expression patterns are consistent with dopaminergic denervation, and change over time, despite relatively unaltered motor performance.

Rat hepatocyte engraftment in severe combined immunodeficient x beige mice using mouse-specific anti-fas antibody.

BACKGROUND: Hepatocyte transplantation holds promise as a treatment for acute and chronic liver failure; however, robust model systems needed to study xenogeneic hepatocyte transfer are lacking. Severe combined immunodeficient x beige (SCID/bg) hybrid mice readily accept foreign tissue. Repopulation of C.B-17 SCID/bg mouse liver with rat hepatocytes was studied following induction of mouse hepatocyte apoptosis using an anti-mouse agonistic fas monoclonal antibody (Jo2 mAb) that does not engage xenogeneic fas. METHODS: SCID/bg mice were transplanted with 1 x 10(6) fresh adult rat hepatocytes intrasplenically and treated with various doses, routes and frequencies of Jo2 mAb. Rat cell repopulation was characterized by quantitative immunofluorescent antibody (q-IFA) staining specific for rat dipeptidyl peptidase type IV (DPP-IV) and leucine amino peptidase, amplification of rat genomic DNA using polymerase chain reaction and histopathological and serum biochemistry analyses. RESULTS: Analysis of liver sections from mice treated twice weekly for 12 weeks with 0.4 mg/kg Jo2 mAb intraperitoneally consistently demonstrated >50% rat hepatocytes in the parenchymal mass by q-IFA. Rat hepatocyte engraftment protected mice from Jo2 mAb-mediated liver hemorrhage and hepatocyte apoptosis. Serum liver enzyme levels did not increase in Jo2 mAb-treated mice that were highly engrafted with rat hepatocytes, in contrast to matched non-engrafted mice. At 12 weeks post-engraftment, minimal fibrosis and inflammation were apparent and liver architecture had returned to near normal. Jo2 mAb did not induce histopathological abnormalities in other tissues known to express fas antigen (i.e. heart, lung). CONCLUSIONS: This novel model represents a simple and robust system of xenogeneic hepatocyte transplantation that could be applied to studies of liver biology, regeneration and hepatocyte transplantation.

CD4+ T-cell reconstitution reduces cytomegalovirus in the immunocompromised brain.

Cytomegalovirus (CMV) infection is the most common opportunistic infection of the central nervous system in patients with human immunodeficiency virus or AIDS or on immunosuppressive drug therapy. Despite medical management, infection may be refractory to treatment and continues to cause significant morbidity and mortality. We investigated adoptive transfer as an approach to treat and prevent neurotropic CMV infection in an adult immunodeficient mouse model. SCID mice were challenged with intracranial murine CMV (MCMV) and reconstituted with MCMV- or vesicular stomatitis virus (VSV)-sensitized splenocytes, T cells, or T-cell subsets. T cells labeled with vital dye or that constitutively generated green fluorescent protein (GFP) were identified in the brain as early as 3 days following peripheral transfer. Regardless of specificity, activated T cells localized to regions of the brain containing CMV, however, only those specific for CMV were effective at clearing virus. Reconstitution with unsorted MCMV-immune splenocytes, enriched T-cell fractions, or CD4(+) cells significantly reduced virus levels in the brain within 7 days and also prevented clinical disease, in significant contrast with mice given VSV-immune unsorted splenocytes, MCMV-immune CD8(+) T cells, and SCID control mice. Results suggest CMV-immune T cells (particularly CD4(+)) rapidly cross the blood-brain barrier, congregate at sites of specific CMV infection, and functionally eliminate acute CMV within the brain. In addition, when CMV-immune splenocytes were administered prior to a peripheral CMV challenge, CMV entry into the immunocompromised brain was prevented. Systemic adoptive transfer may be a rapid and effective approach to preventing CMV entrance into the brain and for reducing neurotropic infection.

Complete protection from papillomavirus challenge after a single vaccination with a vesicular stomatitis virus vector expressing high levels of L1 protein.

We generated an attenuated, recombinant vesicular stomatitis virus (VSV) expressing high levels of the cottontail rabbit papillomavirus (CRPV) L1 protein from an upstream site in the VSV genome. Rabbits vaccinated once with this VSV-L1 recombinant produced high levels of anti-L1 antibody and were completely protected against papilloma formation after challenge with CRPV. In contrast, animals vaccinated only once with a VSV vector expressing lower levels of L1 from a downstream site in the VSV genome generated lower levels of L1 antibody and demonstrated only incomplete protection from papilloma formation after challenge. We conclude that the level of L1 protein expression is critical in generating complete immunity with a single-dose vaccine.

Systemic immune deficiency necessary for cytomegalovirus invasion of the mature brain.

Cytomegalovirus (CMV) is a significant opportunistic pathogen associated with AIDS and immunosuppressive therapy. Infection of the mature central nervous system (CNS) can cause significant pathology with associated neurological deficits, mental disorders, and cognitive impairment and may have potentially fatal consequences. Using genetically immunocompromised mice, we studied mechanisms of CMV invasion into, and behavior within, the CNS. Adult immunodeficient (nude and SCID) and control mice were peripherally infected with recombinant mouse CMV expressing a green fluorescent protein reporter gene. Control mice actively eliminated acute peripheral infection and were resistant to invasion of CMV into the brain. In contrast, virus infected brains of immunodeficient mice but only after a minimum of 21 days postinoculation. After inoculation, CMV was found in circulating leukocytes (MAC-3/CD45(+)) and in leukocytes within the brain, suggesting these cells as a possible source of CMV entry into the CNS. CNS infection was observed in many different cell types, including neurons, glial cells, meninges, ependymal cells, and cells of cerebral vessels. Infection foci progressively expanded locally to adjacent cells, resulting in meningitis, choroiditis, encephalitis, vasculitis, and necrosis; clear indication of axonal transport of CMV was not found. Regional distribution of CMV was unique in each brain, consisting of randomly distributed, unilateral foci. Testing whether CMV gained access to brain through nonspecific vascular disruption, vascular injections of a tracer molecule revealed no obvious disruption of the blood brain barrier in mice with CMV in the brain. Results indicate the importance of host adaptive immunity (particularly T cells) in controlling entry and dissemination of CMV into the brain and are consistent with the view that virus may be carried into the brain by circulating mononuclear cells that traffic through the blood brain barrier.

Intranasal vaccination with a recombinant vesicular stomatitis virus expressing cottontail rabbit papillomavirus L1 protein provides complete protection against papillomavirus-induced disease.

Immunizations with live recombinant vesicular stomatitis viruses (rVSV) expressing foreign viral proteins have successfully protected animals from challenges with several heterologous viruses. We developed an rVSV expressing the major capsid protein (L1) of cottontail rabbit papillomavirus (CRPV) and tested the efficacy of protection following CRPV challenge. An rVSV expressing L1 of CRPV (VSV-L1) was characterized for the protective ability afforded by intranasal, intradermal, or intramuscular vaccination in rabbits subsequently challenged with CRPV. Protein expression of L1 in VSV-L1 was confirmed by radioimmunoprecipitation assays. Nuclear localization of L1 was demonstrated by indirect immunofluorescence assays. Immunized rabbits elicited significant VSV neutralization and VLP-L1 enzyme-linked immunosorbent assay titers. VSV-L1 vaccination was not associated with weight loss or any other adverse clinical signs in the rabbit model. VSV shedding in nasal secretions occurred in some rabbits, peaking at 4 to 6 days after intranasal vaccination, with no further shedding after day 6. Specific humoral immunity to the L1 protein was consistently seen after a single VSV-L1 vaccination when administered through an intradermal or intramuscular route or after a boost via the intranasal route. Rabbits were completely protected from CRPV-induced papillomas after VSV-L1 vaccination and boost given intranasally or intramuscularly. Vaccination with VSV-L1 is a novel approach to prevent papillomavirus-induced disease and demonstrates a potential strategy for developing a human papillomavirus vaccine that can be given without injection.