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A variety of diseases, including some which affect the hemostatic and blood coagulation systems, result from dysfunction of specific cell types. Attempts to ameliorate such diseases by implantation of healthy donor cells typically fail due to immune rejection of the donor cells. One method to possibly overcome this limitation is to administer donor cells prior to immune competence, thus inducing immune tolerance to the donated cells. To accomplish this, donor cells must be administered in utero, relatively early in the developmental process. We describe herein studies in which engraftments of fetal hepatocytes and a hepatoblast line were successfully accomplished by in utero administration of cells in mice. This approach reversed the clinical outcome of Factor X (FX) deficiency, increasing the survival time of FX null mice. Further, engraftment of donor cells was observed in the liver, as well as the spleen, brain, interstitium of the gonad, and lung. These results demonstrate the potential for application of in utero administration of fetal or blast cells for correction of disease related to specific cell or tissue dysfunction. The potential for ectopic engraftment suggests that this approach requires further refinement. [ABSTRACT FROM AUTHOR]

Vaccination, as an approach to prostate cancer, has largely focused on immunotherapy utilizing specific molecules or allogeneic cells. Such methods are limited by the focused antigenic menu presented to the immune system and by immunotolerance to antigens recognized as “self”. To examine if a xenogeneic tissue vaccine could stimulate protective immunity in a human prostate cancer cell line, a vaccine was produced by glutaraldehyde fixation of harvested PAIII prostate cancer cells tumors (GFT cell vaccine) from Lobund-Wistar rats. Immunocompetent Ncr-Foxn1 mice were vaccinated with the GFT cell vaccine four times, 7 days apart. The control animals were either not vaccinated or vaccinated with media or glutaraldehyde-fixed PC346C human prostate cancer cells and adjuvant. About 8 days after the final boost, serum and spleens were harvested. The splenocytes were co-incubated with PC346C cells and then transplanted orthotopically into sygneneic immunodeficient nude mice. About 10 weeks later, the prostates were weighed and sampled for histolologic examination. The spleens were harvested from additional mice, and the splenocytes were cultured, either with or without pulsing by GFT cells, and the supernatants harvested 72 h later for cytokine analysis. Results showed that vaccination with GFT cells resulted in increased serum antibody to a PAIII cell lysate; reduced weight of the prostate/seminal vesicle complex and reduced incidence of prostate cancer in nude mice; increased splenocyte supernatant levels of TNF-α, IL-2, IFN-γ and IL-12, cytokines associated with Th1 immunity; and increased splenocyte supernatant levels of IL-4 and IL-10, cytokines associated with Th2 immunity. In summary, the results suggest that use of a xenogeneic tissue vaccine can stimulate protective immunity against human prostate cancer cells. [ABSTRACT FROM AUTHOR]

Abstract: FVIItTA/− mice are heterozygous for the FVII− null allele and a gene-targeted allele expressing very low levels of FVII. Approximately half the FVIItTA/− in a mixed C57Bl/6:129x1/SVJ background survive much longer than mice in a C57Bl/6 background, suggesting that genetic background affects the survival of mice expressing very low levels of FVII. A lineage of long-term surviving FVIItTA/− mice in a predominantly C57Bl/6 background was generated by selecting 16 weeks or older FVIItTA/− males and breeding them with C57Bl/6 FVII+/− females. It is postulated that these mice maintain 129x1/SVJ alleles at putative hemostatic modifier loci necessary for the survival of mice expressing very low levels of FVII. A SNP genomic scan was performed on DNA from long-term surviving FVIItTA/− mice. The scan identified 8 regions enriched for 129x1/SVJ sequences that might contain putative hemostatic modifier genes. [Copyright &y& Elsevier]

Presents research regarding the development of factor VII (FVII)-deficient mice embryos. Why there is surprise that the embryos developed normally; The mice which succumbed perinatally because of fatal hemorrhaging from normal blood vessels; Survival of embryos not depending on cellular receptor tissue factor (TF)-FVII-initiated fibrin formation; What this suggests about the role of TF during embryogenesis beyond fibrin formation.

Thromboembolic disease is a leading cause of morbidity and mortality worldwide. In the last several years there have been a number of studies attempting to identify mechanisms that stop thrombus growth. This paper identifies a novel mechanism related to formation of a fibrin cap. In particular, protein transport through a fibrin network, an important component of a thrombus, was studied by integrating experiments with model simulations. The network permeability and the protein diffusivity were shown to be important factors determining the transport of proteins through the fibrin network. Our previous in vivo studies in mice have shown that stabilized non-occluding thrombi are covered by a fibrin network (‘fibrin cap’). Model simulations, calibrated using experiments in microfluidic devices and accounting for the permeable structure of the fibrin cap, demonstrated that thrombin generated inside the thrombus was washed downstream through the fibrin network, thus limiting exposure of platelets on the thrombus surface to thrombin. Moreover, by restricting the approach of resting platelets in the flowing blood to the thrombus core, the fibrin cap impaired platelets from reaching regions of high thrombin concentration necessary for platelet activation and limited thrombus growth. The formation of a fibrin cap prevents small thrombi that frequently develop in the absence of major injury in the 60000 km of vessels in the body from developing into life threatening events. [ABSTRACT FROM AUTHOR]

Anticoagulant protein C (PC) is important not only for maintenance of normal hemostasis, but also for regulating the host immune response during inflammation. Because mice with a designed total genetic deficiency in PC (PC-/- mice) die soon after birth, attempts to dissect PC function in various coagulation/inflammation-based pathologies through use of mice with less than 50% of normal PC levels have not been successful to date. In the current investigation, we have used a novel transgenic strategy to generate different mouse models expressing 1-18% of normal PC levels. In contrast to PC-/- mice, mice with only partial PC deficiency survived beyond birth and also developed thrombosis and inflammation. The onset and severity of these phenotypes vary significantly and are strongly dependent on plasma PC levels. Our findings additionally provide the first evidence that maternal PC is vital for sustaining pregnancy beyond 7.5 days postcoitum, likely by regulating the balance of coagulation and inflammation during trophoblast invasion. These low PC-expressing transgenic mouse lines provide novel animal models that can be used to elucidate the importance of PC in maintenance of the organism and in disease. [ABSTRACT FROM AUTHOR]

Reducing expression of the tissue factor gene in prostate adenocarcinoma cells (PAIII) results in a cell line that, in vivo, mimics the growth of wildtype (wt) PAIII. However, instead of continuing to grow and metastasize as wt PAIII tumors do, tissue factor deficient PAIII (TFD PAIII) masses spontaneously regress after several weeks. Although whole cell vaccines are typically inactivated prior to administration to prevent proliferation within the host, numerous studies have suggested that exposure to live, attenuated, whole tumor cells, and the extracellular microenvironment they recruit, increases immunotherapeutic potential. Here, we provide support for this notion, and a strategy through which to implement it, by demonstrating that subcutaneous vaccinations with the TFD PAIII protect the Lobund–Wistar rat against subsequent wt PAIII cell challenge. TFD PAIII immunized rats suffered significantly less metastasis of wt PAIII challenge tumors compared to unvaccinated naïve controls rats. These results offer the intriguing possibility that the TFD PAIII vaccine is an effective system for the prevention and, possibly, the treatment of prostate cancer. [ABSTRACT FROM AUTHOR]

Activated protein C (APC) is a systemic anti-coagulant and anti-inflammatory factor. It reduces organ damage in animal models of sepsis, ischemic injury and stroke and substantially reduces mortality in patients with severe sepsis. It was not known whether APC acts as a direct cell survival factor or whether its neuroprotective effect is secondary to its anti-coagulant and anti-inflammatory effects. We report that APC directly prevents apoptosis in hypoxic human brain endothelium through transcriptionally dependent inhibition of tumor suppressor protein p53, normalization of the pro-apoptotic Bax/Bcl-2 ratio and reduction of caspase-3 signaling. These mechanisms are distinct from those involving upregulation of the genes encoding the anti-apoptotic Bcl-2 homolog A1 and inhibitor of apoptosis protein-1 (IAP-1) by APC in umbilical vein endothelial cells. Cytoprotection of brain endothelium by APC in vitro required endothelial protein C receptor (EPCR) and protease-activated receptor-1 (PAR-1), as did its in vivo neuroprotective activity in a stroke model of mice with a severe deficiency of EPCR. This is consistent with work showing the direct effects of APC on cultured cells via EPCR and PAR-1 (ref. 9). Moreover, the in vivo neuroprotective effects of low-dose mouse APC seemed to be independent of its anti-coagulant activity. Thus, APC protects the brain from ischemic injury by acting directly on brain cells. [ABSTRACT FROM AUTHOR]

The use of adipose stromal cells (ASC) for promoting repair of tissues is a promising therapy, but the mechanisms are not fully understood. We previously demonstrated tissue salvage by ASCs in mouse ischemia model, and showed ASCs secrete significant level of hepatocyte growth factor (HGF) which contributes to cell survival. To determine the specific contribution of HGF, we employed knockdown of HGF using small interfering RNA (siRNA). Human ASCs were transduced with a dual-cassette lentiviral construct expressing GFP gene and either a siRNA targeting HGF (iHGF), or a control sequence. Transduction of ASCs with iHGF (ASC-iHGF) reduced HGF levels by >80% (p<0.01). The proliferation of endothelial cells increased by 1.7-fold in growth-factor-deficient minimal medium (MM) when supplemented with a 1:1 mixture of control ASC conditioned medium (CM) (p<0.01). This growth stimulation was absent when ASC-iHGF CM was added. Angiogenic sprout formation by endothelial cells was 2.0-fold higher when cultured in ASC CM compared to MM (p<0.005). This stimulation was abolished when ASC-iHGF CM was used. The effect of iHGF on in vivo potency of ASC was assayed in a mouse hindlimb ischemia model. Relative perfusion (ischemic/non-ischemic limb) by day 15 was 84+4% in mice treated systemically with control ASCs, which was significantly greater (p<0.05) than both the saline (58+6%) and ASC-iHGF (69+5%) groups. These results indicate that secretion of HGF is necessary for the potency of ASCs both in vitro and in vivo. This supports the emerging concept that local factor secretion is a key element of cell-based angiogenesis and tissue support. [ABSTRACT FROM AUTHOR]