Treatment of hepatocellular carcinoma in a mouse xenograft model with an immunotoxin which is engineered to eliminate vascular leak syndrome.

Vascular leak syndrome (VLS) is the major dose-limiting toxicity of immunotoxin and interleukin-2 therapy. It has been evidenced that VLS-inducing molecules share a three-amino acid consensus motif, (x)D(y), which may be responsible for initiating VLS. Here we have constructed a recombinant immunotoxin (SMFv-PE38KDEL) by genetically fusing PE38KDEL to a single-chain antibody derived from SM5-1 monoclonal antibody, which has a high specificity for melanoma, hepatocellular carcinoma and breast cancer.

Structural basis for recognition of CD20 by therapeutic antibody Rituximab.

Rituximab is a widely used monoclonal antibody drug for treating certain lymphomas and autoimmune diseases. To understand the molecular mechanism of recognition of human CD20 by Rituximab, we determined the crystal structure of the Rituximab Fab in complex with a synthesized peptide comprising the CD20 epitope (residues 163-187) at 2.6-A resolution.

Eradication of hepatoma and colon cancer in mice with Flt3L gene therapy in combination with 5-FU.

We developed a recombinant defective adenovirus with an insert of gene encoding extracellular domain of mouse Flt3L (Ad-mFlt3L) under control of cytomegalovirus promoter to investigate the biological efficacy of Flt3L in combination with chemotherapeutical drug, 5-FU, in eliciting an effective anti-cancer immunity in mouse hepatoma and colon cancer model systems. The constructed Ad-mFlt3L efficiently infected hepatoma and colon cancer cells both in vitro and in vivo, leading to a high production of mFlt3L proteins in association with accumulation of DCs, NK cells and lymphocytes in local tumor tissues. Administration of Ad-mFlt3L can protect bone marrow injury caused by 5-Fu and stimulates proliferation and maturation of lymphocytes, APCs and NKs.

DRB genotyping in cynomolgus monkeys from China using polymerase chain reaction -sequence-specific primers.

Cynomolgus macaques are relevant models for human diseases and transplantation. In each case, a complete understanding of these models requires knowledge of the macaque major histocompatibility complex (MHC). Because of high polymorphism and multiple genes per haplotype, it has been difficult to develop a rapid typing method for cynomolgus monkey MHC class II.

TIP30/CC3 expression in breast carcinoma: relation to metastasis, clinicopathologic parameters, and P53 expression.

Metastasis is the most frequent cause of death in patients with breast cancer. Tip30/CC3 gene is a putative metastasis suppressor gene, which was first identified by a differential display analysis of messenger RNA from the highly metastatic human variant small cell lung carcinoma (SCLC) versus less metastatic classic SCLC cell lines. The aim of this study was to analyze the relationship between expression of TIP30/CC3 and clinical prognosis in 87 patients with surgically removed breast carcinoma.

Preparation and characterization of recombinant protein ScFv(CD11c)-TRP2 for tumor therapy from inclusion bodies in Escherichia coli.

Dendritic cells (DCs)-based immunotherapy represents an approach to the prevention and treatment of cancers. Targeting antigens to receptors on DCs can be expected to enhance immune response. We have constructed an expression vector pET32a(+)-ScFv(CD11c)-TRP2 based on a single-chain antibody fragment (ScFv) that targets the high affinity receptor CD11c which is expressed on murine DCs.

[Construction and application of retroviral vector system useful for high-efficiency transfection into human T cells].

Aim: To construct a retroviral vector system with high-efficiency transfection into human T cells and carrying a rapid screening label, and compare it with the traditional retroviral vector system.

Methods: Retroviral vector pCMMP-IRES-GFP was first constructed by inserting the internal ribosome entry site-green fluorescent protein (IRES-GFP) cDNA into retroviral vector pCMMP. The chimeric TCR gene was inserted into pCMMP-IRES-GFP and then co-transfected into packaging cell line 293T with other two assistant vectors pMD.

TIP30 inhibits growth of HCC cell lines and inhibits HCC xenografts in mice in combination with 5-FU.

Hepatocellular carcinoma (HCC) is an aggressive cancer with a poor prognosis. The specific cellular gene alterations responsible for hepatocarcinogenesis are not well known. Previous works showed that loss of TIP30, also called CC3, a putative tumor suppressor, increased the incidence of hepatocellular carcinoma in mice, and some clinical samples of human HCC tissues had aberrant expression of TIP30.

Concordant loss of melanoma differentiation antigens in synchronous and asynchronous melanoma metastases: implications for immunotherapy.

Because of its known heterogeneity, the analysis of antigen expression is crucial prior to the initiation of antigen-specific immunotherapy for melanoma. The melanoma differentiation antigens gp100, MART-1 and tyrosinase are involved in a common pathway of melanin synthesis. Peptides derived from these melanoma differentiation antigens are used in the immunotherapy of melanoma and antibodies recognizing these antigens are commonly applied to detect melanocytic lesions.

The monoclonal antibody SM5-1 recognizes a fibronectin variant which is widely expressed in melanoma.

Background: Previously we have generated the monoclonal antibody SM5-1 by using a subtractive immunization protocol of human melanoma. This antibody exhibits a high sensitivity for primary melanomas of 99% (248/250 tested) and for metastatic melanoma of 96% (146/151 tested) in paraffin embedded sections. This reactivity is superior to the one obtained by HMB-45, anti-MelanA or anti-Tyrosinase and is comparable to anti-S100.

Preparation and characterization of a monoclonal antibody against the protein LIGHT.

LIGHT (which is homologous to lymphotoxins, shows inducible expression, and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes [Genome Database designation, TNFSF14]), a newly identified member of the TNF superfamily, is up-regulated upon activation of T-cells. LIGHT plays an important role in the T-cell-mediated tumor and graft-versus-host disease via LIGHT/HVEM/LT beta R signaling. To prepare specific monoclonal antibody (MAb) against murine LIGHT, a fragment containing the extracellular domain of LIGHT was inserted into prokaryotic expression vector pET-32a(+).

Construction and characterization of a humanized anti-human CD3 monoclonal antibody 12F6 with effective immunoregulation functions.

12F6 is a murine anti-human CD3 monoclonal antibody, which competes with OKT3 for binding to human T cells and possesses more effective T-cell suppression and activation properties compared to OKT3. It thus exhibits the potential to be developed as an immunoregulation agent for manipulating T-cell functions and preventing acute allograft rejection. In an attempt to minimize the immunogenicity of murine 12F6 (m12F6) for potential clinical application, a humanized version of 12F6, denoted as hu12F6, was successfully constructed by complementary determining region (CDR) grafting and shown to maintain both T-cell activation and suppression activities similar to m12F6.

Differential expression of MART-1, tyrosinase, and SM5-1 in primary and metastatic melanoma.

The new monoclonal antibody SM5-1 has been shown to have significant advantages in immunohistochemistry of melanoma over currently used antibodies such as HMB-45 or anti-S100. In this study we compared the immunohistological staining pattern of SM5-1 with that of the more recently described antibodies A103 (anti-MART-1) and T311 (anti-Tyrosinase) in 344 paraffin-embedded melanoma specimens, consisting of 101 primary melanomas (77 SSM, 16 NM, 6 ALM, 2 LMM) and 243 melanoma metastases. The overall reactivity of SM5-1 for all the specimens was 92% (318/344) compared with 83% (285/344) for MART-1 and 71% (245/344) for Tyrosinase.

Effects of ribozyme targeting platelet-derived growth factor receptor beta subunit gene on the proliferation and apoptosis of hepatic stellate cells in vitro.

Background: Activation and proliferation of hepatic stellate cells (HSC) is essentially involved in the development and progression of hepatic fibrosis. The most potent growth factor for HSC is platelet-derived growth factor receptor (PDGF) and PDGF receptor beta subunit (PDGFR-beta) is the predominant signal transduction pathway of PDGF which is overexpressed in activated HSC. This study investigated the cleavage activity of hammerhead ribozyme targeting PDGFR-beta mRNA in HSC and the effect on biological characteristics of HSC.

Preparation and characterization of fusion protein truncated Pseudomonas Exotoxin A (PE38KDEL) in Escherichia coli.

Pseudomonas Exotoxin A (PE) and truncated PE have been used to prepare immunotoxin with monoclonal antibodies. Truncated Pseudomonas Exotoxin A (PE38KDEL) was expressed with the pET-32a(+) vector in Escherichia coli under control of a T7 promoter. The recombinant protein was purified by His-Ni(2+) metal affinity chromatography and gel filtration.

Treatment of hepatocellular carcinoma with adenoviral vector-mediated Flt3 ligand gene therapy.

Fms-like tyrosine kinase 3 ligand (Flt3L) plays an important role in development and activation of dendritic cells (DCs) and natural killer cells (NK). It has been shown that administration of either tumor cells transfected in vitro with Flt3L vectors or soluble Flt3L fusion protein in a high dose can enhance host antitumor immunity in animal model systems. In this study, we developed a recombinant defective adenovirus with an insert of gene encoding extracellular domain of mouse Flt3L (Ad-mFlt3L) under control of cytomegalovirus promoter and investigated its biological efficacy in eliciting tumor-specific immune response against hepatocellular carcinoma in mouse hepatoma model.

Murine mammary adenocarcinoma cells transfected with p53 and/or Flt3L induce antitumor immune responses.

Transfection of tumors with tumor-associated antigens (Ags) or cytokines can increase immunogenicity and slow down tumor growth. However, the effect of cotransfection with genes that encode a tumor-associated Ag, such as the tumor suppressor gene p53, and a cytokine has been rarely investigated. We report that transfection of 4T1 mammary tumor cells (p53-null) with the dendritic cell (DC) growth factor, fms-like tyrosine kinase 3 ligand (Flt3L), significantly delayed their growth in vivo, resulting in the rejection of 100% of the tumors formed by injection of tumor cells cotransfected with Flt3L and p53.

[Effects of tumor suppressing gene TIP30/CC3 on the growth of tumor cells].

Objective: To introduce the newly found gene TIP30/CC3 into a hepatoma cell line PLC/PRF/5 and select the stable expression clones. The growth and cell cycles were studied with the clones stably expressing TIP30/CC3 or anti-TIP30/CC3, and the effects of TIP30/CC3 gene on hepatoma cells were analyzed.

Methods: The internal expression of TIP30/CC3 protein was detected with Western blot, then TIP30/CC3 or anti-TIP30/CC3 cDNA was subcloned into a constitutive vector pcDNA3 followed by transfection into PLC/PRF/5.

[The expression of platelet-derived growth factor (PDGF) receptor-beta and its correlation with extracellular matrix in hepatic tissue in hepatic fibrosis rats].

Objective: To investigate the expression of PDGF receptor-beta and its correlation with extracellular matrix in hepatic tissue during hepatic fibrosis.

Methods: The model of hepatic fibrosis in rats was induced by carbon tetrachloride. PDGF receptor-beta subunit, collagen I, collagen III and a-SMA in hepatic tissues of these rats were examined using immunohistochemistry.

Posttraumatic stress disorder among professional and non-professional rescuers involved in an earthquake in Taiwan.

This study investigated the prevalence of posttraumatic stress disorder (PTSD) among professional and non-professional rescue workers involved in the 1999 Chi-Chi Earthquake in Taiwan. One month following the disaster, 252 rescue workers (167 professional rescue workers, 85 non-professional volunteers) were surveyed with the Chinese version of the Davidson Trauma Scale (DTS-C) and the Chinese version of the SPAN (SPAN-C). Non-professional rescuers had significantly higher scores than professional rescuers on both the DTS-C and the SPAN-C.

[Construction of adenovirus vector expressing TIP30 and its tumor suppressive effect in vitro and in vivo].

Objective: To construct an adenovirus vector expressing TIP30 gene (Ad-TIP30) and investigate its tumor suppressive effect in vitro and in vivo.

Methods: Ad-Easy system was used to construct Ad-TIP30 by recombination in E. coli.

Melanoma cells transfected to express CD83 induce antitumor immunity that can be increased by also engaging CD137.

Interactions between CD83 and its ligand(s) can up-regulate immune responses. M2-CD83 cells, derived by transfecting the M2 clone of mouse melanoma K1735 cells to express mouse CD83, were rejected by syngeneic mice, unless they were injected with a CD83Ig fusion protein. Rejection was mediated by CD4+ and CD8+ T cells plus natural killer cells, whereas rejection of M2-1D8 cells, which express anti-CD137 single-chain variable region fragments (scFv), occurs in the absence of CD8+ T cells.

TIP30 deficiency increases susceptibility to tumorigenesis.

TIP30, also called CC3 or Htatip2, is a putative metastasis suppressor that promotes apoptosis and inhibits angiogenesis. Although TIP30 has several characteristic features of a tumor suppressor in in vitro analyses, tumor development as a result of TIP30 inactivation has not been demonstrated in vivo, and abnormal expression of TIP30 in human cancer has not been reported. Using genetically engineered mice and cells deficient in TIP30, we show that TIP30-deficient mice have a high incidence of hepatocellular carcinoma and other tumors, and loss of TIP30 enhances susceptibility of fibroblasts to transformation by the SV40 large T antigen.

Regional, but not systemic recruitment/expansion of dendritic cells by a pluronic-formulated Flt3-ligand plasmid with vaccine adjuvant activity.

Regional recruitment of dendritic cells (DCs) by the local administration of granulocyte macrophage-colony stimulating factor (GM-CSF) or Flt3-ligand (Flt3L) has vaccine adjuvant activity. However, Flt3L, with its DC growth factor activity, has not been extensively studied as a vaccine adjuvant, particularly as a plasmid vector. We report that the intramuscular (IM) injection of a Flt3L plasmid (pNGVL-hFlex), when formulated in a pluronic carrier (SP1017, Supratek Pharma, Inc.

[Effect of ribozyme against platelet-derived growth factor receptor beta subunit mRNA on the biological characters of hepatic stellate cells].

Objective: To study the cleavage activity of hammerhead ribozyme targeting at platelet-derived growth factor receptor beta subunit (PDGFR- beta) mRNA in hepatic stellate cells (HSCs) and its effect on the biological characters of HSCs.

Methods: Expression vector of anti-PDGFR- beta ribozyme was constructed and transfected into rat-derived HSC-T6 cells with lipofectin. The positive cell clones were gained by G418 selection.