Hemorrhage is a common clinical manifestation in dengue patients. However, the pathogenic mechanism of dengue virus (DV)-induced hemorrhage awaits clarification. We established a mouse model of DV hemorrhage using immunocompetent C57BL/6 mice by injecting DV serotype 2 strain 16681 intradermally. While inoculation of 3 ؋ 10 9 PFU of DV induced systemic hemorrhage in all of the mice by day 3 of infection, one out of three of those injected with 4 ؋ 10 7 to 8 ؋ 10 7 PFU developed hemorrhage in the subcutaneous tissues. The mice that were inoculated with 4 ؋ 10 7 to 8 ؋ 10 7 PFU but that did not develop hemorrhage were used as a basis for comparison to explore the pathogenic mechanism of dengue hemorrhage. The results showed that mice with severe thrombocytopenia manifested signs of vascular leakage and hemorrhage. We observed that high viral titer, macrophage infiltration, and tumor necrosis factor alpha (TNF-␣) production in the local tissues are three important events that lead to hemorrhage. Immunofluorescence staining revealed that DV targeted both endothelial cells and macrophages. In addition, the production of high levels of TNF-␣ in tissues correlated with endothelial cell apoptosis and hemorrhage. By comparing TNF-␣ ؊/؊ to IgH ؊/؊ , C5 ؊/؊ , and wild-type mice, we found that TNF-␣ was important for the development of hemorrhage. In vitro studies showed that mouse primary microvascular endothelial cells were susceptible to DV but that TNF-␣ enhanced DV-induced apoptosis. Our mouse model illustrated that intradermal inoculation of high titers of DV predisposes endothelial cells to be susceptible to TNF-␣-induced cell death, which leads to endothelium damage and hemorrhage development. This finding highlights the contribution of the innate immune response to dengue hemorrhage.

Heterogeneity of cancer stem/progenitor cells that give rise to different forms of cancer has been well demonstrated for leukemia. However, this fundamental concept has yet to be established for solid tumors including breast cancer. In this communication, we analyzed solid tumor cancer stem cell markers in human breast cancer cell lines and primary specimens using flow cytometry. The stem/progenitor cell properties of different marker expressing-cell populations were further assessed by in vitro soft agar colony formation assay and the ability to form tumors in NOD/SCID mice. We found that the expression of stem cell markers varied greatly among breast cancer cell lines. In MDA-MB-231 cells, PROCR and ESA, instead of the widely used breast cancer stem cell markers CD44+/CD24-/low and ALDH, could be used to highly enrich cancer stem/progenitor cell populations which exhibited the ability to self renew and divide asymmetrically. Furthermore, the PROCR+/ESA+ cells expressed epithelial-mesenchymal transition markers. PROCR could also be used to enrich cells with colony forming ability from MB-361 cells. Moreover, consistent with the marker profiling using cell lines, the expression of stem cell markers differed greatly among primary tumors. There was an association between metastasis status and a high prevalence of certain markers including CD44+/CD24−/low, ESA+, CD133+, CXCR4+ and PROCR+ in primary tumor cells. Taken together, these results suggest that similar to leukemia, several stem/progenitor cell-like subpopulations can exist in breast cancer.

In this study, we explore a potential vaccine for human papillomavirus (HPV)-induced tumors, using heat shock protein as an adjuvant, a peptide vaccine for safety, and adeno-associated virus ( Cervical carcinoma remains one of the most common malignancies worldwide, with 500,000 new cases diagnosed each year (5) and 200,000 cervical cancer deaths annually (21). Human papillomavirus type 16 (HPV-16) is the predominant etiologic agent of cervical cancer and carries three transforming oncogenes, E5, E6, and E7 (10,20,45). Their products are thus unique tumor antigens and can be ideally used as tumor vaccines (7). Because E6 and E7 oncoproteins are consistently retained and expressed, the E6 and E7 oncogenes become more attractive targets for T-cell-based immunotherapy of cervical cancer. Evidence for the value of HPV antigen-directed immunotherapy against cervical cancer comes from the experimental and natural papillomavirus-associated tumors that can be controlled by immunization with E7 antigen. Previous studies have used different modes of immunization, such as (i) recombinant E7 vaccinia viruses (2,3,16,24,28,42), (ii) syngeneic cells transfected with E7 (8, 9), (iii) E7 proteinpulsed dendritic cells (12, 37), (iv) peptides corresponding to a cytotoxic T-lymphocyte (CTL) epitope in E7 with incomplete Freund's adjuvant (13), (v) E7 vaccine based on papillomavirus-like particles (17,25,31), and (vi) Salmonella enterica serovar Typhimurium expressing E7 epitope inserted into hepatitis B virus core (26). These studies demonstrate that CTLs are likely to be the most effective immunological effector mechanisms.Adeno-associated virus (AAV), a single-stranded virus, has been studied as a vector for gene therapy (29, 41). Classified as a defective human parvovirus, AAV has many natural features that are attractive for a human gene therapy vector, such as nonpathogenicity, targeted integrating capability, and a broad host range (human, simian, murine, canine, and avian). In sharp contrast to other viral vectors that have been used in vaccination, such as vaccinia virus or adenovirus, AAV vectors do not express any viral genes. The only viral DNA that must be included in an AAV vector is the 145-bp inverted terminal repeat. Since naked DNA is used for immunization, the only gene expressed by AAV vectors is that for the antigen itself.Since HPV-16 E7 is a transforming oncoprotein (20), dangerous side effects such as transformation are not anticipated with a protein vaccine. Peptide vaccination with a CTL epitope to prevent the outgrowth of a tumor is a safe and effective immunotherapeutic method (13). However, a peptide vaccine combined with a toxic adjuvant such as incomplete Freund's adjuvant sometimes can lead to rapid tumor growth through specific T-cell tolerance induction (36). Recently, Mycobacterium tuberculosis heat shock protein 70 (hsp70) has been used as an adjuvant-free carrier to stimulate the humoral and cellular response to a full-length human immunodeficiency virus p24 (33) that is covalently linked to hsp...

Like poliovirus infection, severe infection with enterovirus 71 (EV71) can cause neuropathology. Unlike poliovirus, EV71 is often associated with hand-foot-and-mouth disease (HFMD). Here we established three mouse models for experimental infection with the same clinical isolate of EV71. The NOD/SCID mouse model is unique for the development of skin rash, an HFMD-like symptom. While the NOD/SCID mice developed limb paralysis and death at near-100% efficiency, the gamma interferon receptor knockout (ifngr KO) and stat-1 knockout mice exhibited paralysis and death rates near 78% and 30%, respectively. Productive infection with EV71 depends on the viral dose, host age, and inoculation route. Levels of infectious EV71, and levels of VP1-specific RNA and protein in muscle, brain, and spinal cord, were compared side by side between the NOD/SCID and stat-1 knockout models before, during, and after disease onset. Spleen fibrosis and muscle degeneration are common in the NOD/SCID and stat-1 knockout models. The main differences between these two models include their disease manifestations and cytokine/ chemokine profiles. The pathology of the NOD/SCID model includes (i) inflammation and expression of viral VP1 antigen in muscle, (ii) increased neutrophil levels and decreased eosinophil and lymphocyte levels, and (iii) hair loss and skin rash. The characteristic pathology of the stat-1 knockout model includes (i) a strong tropism of EV71 for the central nervous system, (ii) detection of VP1 protein in the Purkinje layer of cerebellar cortex, pons, brain stem, and spinal cord, (iii) amplification of microglial cells, and (iv) dystrophy of intestinal villi. Our comparative studies on these new models with oral or intraperitoneal (i.p.) infection underscored the contribution of host immunity, including the gamma interferon receptor, to EV71 pathogenesis. IMPORTANCEIn the past decade, enterovirus 71 (EV71) has emerged as a major threat to public health in the Asia-Pacific region. Disease manifestations include subclinical infection, common-cold-like syndromes, hand-foot-and-mouth disease (HFMD), uncomplicated brain stem encephalitis, severe dysregulation of the autonomic nerve system, fatal pulmonary edema, and cardiopulmonary collapse. To date, no effective vaccine or treatment is available. A user-friendly and widely accessible animal model for researching EV71 infection and pathogenesis is urgently needed by the global community, both in academia and in industry.