Intra-Peritoneal Hyperthermia Combining α-Galactosylceramide in the Treatment of Ovarian Cancer

The purpose of this study was to investigate the anti-tumor effect and potential mechanisms of i.p. hyperthermia in combination with α-galactosylceramide (α-GalCer) for the treatment of ovarian cancer. In this study, immuno-competent tumor models were established using murine ovarian cancer cell lines and treated with i.p. hyperthermia combining α-GalCer. Th1/Th2 cytokine expression profiles in the serum, NK cell cytotoxicity and phagocytic activities of dendritic cells (DCs) were assayed. We also analyzed the number of CD8⁺/IFN-γ⁺ tumor specific cytotoxic T cells, as well as the tumor growth based on depletion of lymphocyte sub-population. Therapeutic effect on those ovarian tumors was monitored by a non-invasive luminescent imaging system. Intra-peritoneal hyperthermia induced significant pro-inflammatory cytokines expression, and sustained the response of NK and DCs induced by α-GalCer treatment. The combination treatment enhanced the cytotoxic T lymphocyte (CTL) immune response in two mouse ovarian cancer models. This novel treatment modality by combination of hyperthermia and glycolipid provides a pronounced anti-tumor immune response and better survival. In conclusion, intra-peritoneal hyperthermia enhanced the pro-inflammatory cytokine secretion and phagocytic activity of DCs stimulated by α-GalCer. The subsequent CTL immune response induced by α-GalCer was further strengthened by combining with i.p. hyperthermia. Both innate and adaptive immunities were involved and resulted in a superior therapeutic effect in treating the ovarian cancer.     

Antibody detection of SARS-CoV spike and nucleocapsid protein

Early detection and identification of SARS-CoV-infected patients and actions to prevent transmission are absolutely critical to prevent another SARS outbreak. Antibodies that specifically recognize the SARS-CoV spike and nucleocapsid proteins may provide a rapid screening method to allow accurate identification and isolation of patients with the virus early in their infection. For this reason, we raised peptide-induced polyclonal antibodies against SARS-CoV spike protein and polyclonal antibodies against SARS-CoV nucleocapsid protein using 6x His nucleocapsid recombinant protein. Western blot analysis and immunofluorescent staining showed that these antibodies specifically recognized SARS-CoV.     

Immune mechanism of the antitumor effects generated by bortezomib

Bortezomib, a proteasome inhibitor, is a chemotherapeutic drug that is commonly used to treat a variety of human cancers. The antitumor effects of bortezomib-induced tumor cell immunogenicity have not been fully delineated. In this study, we examined the generation of immune-mediated antitumor effects in response to treatment by bortezomib in a murine ovarian tumor model. We observed that tumor-bearing mice that were treated with bortezomib had CD8(+) T cell-mediated inhibition of tumor growth. Furthermore, the comparison of tumor cell-based vaccines that were produced from tumor cells treated or untreated with bortezomib showed vaccination with drug-treated tumor cell-based vaccines elicited potent tumor-specific CD8(+) T cell immune response with improved therapeutic antitumor effect in tumor-bearing mice. Conversely, the untreated tumor cell-based vaccines led to no appreciable antitumor response. Treatment of tumor cells with bortezomib led to the upregulation of Hsp60 and Hsp90 on the cell surface and promoted their phagocytosis by dendritic cells (DCs). However, cell surface expression of Hsp60, instead of Hsp90, is the more important determinant of whether bortezomib-treated tumor cells can generate tumor-specific CD8(+) T cells. CD11c(+) DCs that were treated with bortezomib in vitro had enhanced phagocytic activities. In addition, CD11c(+) DCs from bortezomib-treated tumor-bearing mice had increased maturation. At lower concentrations, bortezomib had no inhibitory effects on T cell proliferation. Taken together, our data indicate that bortezomib can render tumor cells immunogenic by upregulating the cell surface expression of heat shock protein 60 and heat shock protein 90, as well as improve DC function, which results in potent immune-mediated antitumor effects.     

Improvement of cell lysis activity of immobilized lysozyme with reversibly soluble-insoluble polymer as carrier

Summary Hen egg white lysozyme was immobilized by covalent binding to a polymer showing reversibly soluble-insoluble characteristics with pH change. The retention of the specific activity of the immobilized enzyme can be as high as 41% of that of the free enzyme. The immobilized enzyme could be used in repeated batch lysis of M. lysodeikticus cells and to enhance the release of intracellular proteins 1.4 folds when compared with batch operation.     

Cytotoxic T-lymphocyte responses to human papillomavirus type 16 E5 and E7 proteins and HLA-A*0201-restricted T-cell peptides in cervical cancer patients

Previously, we found that human papillomavirus type 16 (HPV-16) E5 protein is a tumor rejection antigen and can induce cytotoxic T-lymphocyte (CTL) activity. Therefore, in this study, human leukocyte antigen A*0201 (HLA-A*0201)-restricted human CTL epitopes of HPV-16 E5 protein were identified using a bioinformatics approach, and the abilities of these predicted peptides to induce an immune response in HLA-A*0201 transgenic mice were confirmed by assaying E5-specific CTLs and in vitro-generated CTLs from normal peripheral blood T lymphocytes of HLA-A2-positive human donors. Second, the CTL responses to HLA-A*0201 CTL epitopes (E5 63-71 and E7 11-20) were examined in HPV-16-infected patients with HLA-A2. Third, the effect of HLA-A-type alleles on CTL activities in response to the entire E5 and E7 proteins was examined in cervical cancer patients. E5 and E7 peptides (but not the whole proteins) stimulated E5- and E7-specific CTL recall responses in HPV-16- and HLA-A2-positive cervical cancer patients, and HPV-16 E5 and E7 proteins stimulated na?ve T cells in HPV-16-negative cervical cancer patients with HLA-A11 and -A24 haplotypes. In summary, this is the first demonstration that E5 63-71 is an HLA-A*0201-restricted T-cell epitope of HPV-16 E5.     

HLA-A*0201 T-cell epitopes in severe acute respiratory syndrome (SARS) coronavirus nucleocapsid and spike proteins

The immunogenicity of HLA-A*0201-restricted cytotoxic T lymphocyte (CTL) peptide in severe acute respiratory syndrome coronavirus (SARS-CoV) nuclear capsid (N) and spike (S) proteins was determined by testing the proteins' ability to elicit a specific cellular immune response after immunization of HLA-A2.1 transgenic mice and in vitro vaccination of HLA-A2.1 positive human peripheral blood mononuclearcytes (PBMCs). First, we screened SARS N and S amino acid sequences for allele-specific motif matching those in human HLA-A2.1 MHC-I molecules. From HLA peptide binding predictions (http://thr.cit.nih.gov/molbio/hla_bind/), ten each potential N- and S-specific HLA-A2.1-binding peptides were synthesized. The high affinity HLA-A2.1 peptides were validated by T2-cell stabilization assays, with immunogenicity assays revealing peptides N223-231, N227-235, and N317-325 to be the first identified HLA-A*0201-restricted CTL epitopes of SARS-CoV N protein. In addition, previous reports identified three HLA-A*0201-restricted CTL epitopes of S protein (S978-986, S1203-1211, and S1167-1175), here we found two novel peptides S787-795 and S1042-1050 as S-specific CTL epitopes. Moreover, our identified N317-325 and S1042-1050 CTL epitopes could induce recall responses when IFN-gamma stimulation of blood CD8+ T-cells revealed significant difference between normal healthy donors and SARS-recovered patients after those PBMCs were in vitro vaccinated with their cognate antigen. Our results would provide a new insight into the development of therapeutic vaccine in SARS.     

Pigment epithelium-derived factor protects retinal pigment epithelium from oxidant-mediated barrier dysfunction

Retinal pigment epithelium (RPE) cells form a monolayer at the blood-retina barrier between the retina and choriocapillaries. The barrier function may be damaged by multiple stresses to the cell, including the repeated exposure to oxidants that are generated by photoreceptor cell turnover. The purpose of our study was to document the protective effect of pigment epithelium-derived factor (PEDF), a tropic factor produced by the RPE, on H(2)O(2)-induced RPE barrier dysfunction. When assayed by a FITC-labeled dextran transepithelial flux, the increased permeability of the RPE barrier (induced by H(2)O(2)) was prevented by PEDF pretreatment. To further explore the mechanism leading to this permeability change, we investigated the distribution of cytoskeleton and junctional proteins. The redistribution of the two junctional proteins occludin, and N-cadherin and actin reorganization in RPE, induced by H(2)O(2), can be prevented by PEDF pretreatment. PEDF can also prevent H(2)O(2)-induced stress kinase p38/27-kDa heat shock protein signaling which is known to mediate actin rearrangement. These findings indicated that PEDF can stabilize actin, maintain normal membrane occludin and N-cadherin structure, and preserve the barrier function of RPE cells against oxidative stress.     

Renal ischemia/reperfusion injury inhibits differentiation of dendritic cells derived from bone marrow monocytes in rats

Dendritic cells (DCs) are impacted by surgical injury, exercise, and other physiological stressors. This study aims to determine whether renal I/R injury affects 1) the differentiation of myeloid DCs from bone marrow monocytes (BMMos) and the maturation and activation state of these DCs and 2) DC infiltration of kidney. Sprague-Dawley rats were subjected to I/R injury or sham-operated. Creatinine clearance was monitored daily during the 14 d of reperfusion that followed the ischemic insult. At 2 and 14 d of reperfusion, the following were assessed 1) properties of BMMo-derived DCs (i.e., the amount of generated DCs, differentiation state markers [CD11c, CD80, CD86, and Ia], and functional state [MLR and amount of IL-12 produced]), and 2) the presence of DCs in the kidney. Numbers of BMMo-derived DCs were significantly decreased in the I/R injured group (compared with the sham-operated group) at 2 d but not 14 d. A comparison of the their functionality found mixed lymphocyte response [MLR] and IL-12 production were similar in the two groups at both time points. Also, immunohistochemistry showed infiltrating DCs in the outer medulla of the I/R injured kidney at 2 d but not 14 d of reperfusion. Thus, I/R stress reduces the number of DCs differentiated from BMMos but not the functional activity of these DCs. This decrease may reflect a stress-induced downshift in the capacity of BMMos to differentiate into DCs and a parallel upshift in the capacity of DCs to infiltrate the kidney.