An In Vivo Mouse Model of Pelvic Recurrence of Human Colorectal Cancer.

Pelvic recurrence of colorectal cancer is a crucial problem because radical surgery can lead to excessive invasion. Novel therapeutic strategies are required instead of surgery. However, there are few suitable models because of the difficulty in transplanting and observing tumors in the pelvis. We have established an appropriate injection site suitable for the establishment of colorectal cancer pelvic recurrence that allows for the observation of tumor growth. DLD-1 cells stably expressing luciferase (DLD-1 clone#1-Luc) were inoculated into various points of female BALB/c nude mice and the engrafted cells were analyzed with an imaging system employing bioluminescent signals and computed tomography. Weekly analysis with the imaging system showed that a triangular area defined by the vagina, the anus, and the ischial spine was suitable for the engraftment of pelvic tumors. The imaging system was able to detect the engrafted tumor 7 days after the inoculation of cells. Weight loss was observed in our model, and overall survival was 21-42 days. Tumor involvement of adjacent organs was detected histopathologically, as is the case in the clinical situation. These findings suggest that this model is valid for evaluations of the therapeutic effects of novel treatments under development. It is hoped that this model will be used in preclinical research.

Comparative proteomic analysis of paclitaxel resistance-related proteins in human breast cancer cell lines.

Paclitaxel is widely used to treat various cancers; however, resistance to this drug is a major obstacle to breast cancer chemotherapy. To identify the proteins involved in paclitaxel resistance, the present study compared the proteomes of MCF-7 human breast cancer cells and its paclitaxel-resistant subclone MCF-7/PTX. Using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry, 11 upregulated and 12 downregulated proteins were identified in MCF-7/PTX cells compared with the parental cell line. These 23 proteins were functionally classified as stress-induced chaperones, metabolic enzymes and cytoskeletal proteins. The anti-apoptotic proteins, stress-70 protein, 78-kD glucose-regulated protein, peptidyl-prolyl cis-trans isomerase A (PPIA) and heterogeneous nuclear ribonucleoprotein H3, were also upregulated in MCF-7/PTX cells. Notably, knockdown of the stress-response chaperone PPIA using small interfering RNA in MCF-7/PTX cells restored their sensitivity to paclitaxel. These findings indicated that PPIA may have an important role in paclitaxel resistance in MCF-7/PTX cells.

Microglial Contact Prevents Excess Depolarization and Rescues Neurons from Excitotoxicity.

Microglia survey and directly contact neurons in both healthy and damaged brain, but the mechanisms and functional consequences of these contacts are not yet fully elucidated. Combining two-photon imaging and patch clamping, we have developed an acute experimental model for studying the role of microglia in CNS excitotoxicity induced by neuronal hyperactivity. Our model allows us to simultaneously examine the effects of repetitive supramaximal stimulation on axonal morphology, neuronal membrane potential, and microglial migration, using cortical brain slices from Iba-1 eGFP mice. We demonstrate that microglia exert an acute and highly localized neuroprotective action under conditions of neuronal hyperactivity. Evoking repetitive action potentials in individual layer 2/3 pyramidal neurons elicited swelling of axons, but not dendrites, which was accompanied by a large, sustained depolarization of soma membrane potential. Microglial processes migrated to these swollen axons in a mechanism involving both ATP and glutamate release via volume-activated anion channels. This migration was followed by intensive microglial wrapping of affected axons and, in some cases, the removal of axonal debris that induced a rapid soma membrane repolarization back to resting potentials. When the microglial migration was pharmacologically blocked, the activity-induced depolarization continued until cell death ensued, demonstrating that the microglia-axon contact served to prevent pathological depolarization of the soma and maintain neuronal viability. This is a novel aspect of microglia surveillance: detecting, wrapping, and rescuing neuronal soma from damage due to excessive activity.

Identification of phosphorylated serine-15 and -82 residues of HSPB1 in 5-fluorouracil-resistant colorectal cancer cells by proteomics.

To identify the proteins involved in 5-fluorouracil (5-FU) resistance, a comparison of the total and phosphorylated proteins between the human colorectal cancer (CRC) cell line DLD-1 and its 5-FU-resistant subclone DLD-1/5-FU was performed. Using 2-DE and MALDI-TOF/TOF-based proteomics, 17 up-regulated and 19 down-regulated protein spots were identified in the 5-FU-resistant DLD-1/5-FU cells compared with the parent cell lines. In DLD-1/5-FU cells, 7 anti-apoptotic proteins (HSPB1, proteasome subunit α-5, transitional endoplasmic reticulum ATPase, 14-3-3 ß, 14-3-3 γ, 14-3-3 σ, and phosphoglycerate kinase 1) were up-regulated and 4 proapoptotic proteins (cofilin-1, pyruvate kinase M2, glyceraldehyde-3-phosphate dehydrogenase, and nucleophosmin) were down-regulated. The results show that the acquired drug resistance of DLD-1/5-FU cells is caused by the prevention of drug-induced apoptosis, in particular through the enhanced constitutive expression of HSPB1 and its phosphorylated form. Short interfering RNA knockdown of endogenous HSPB1 in DLD-1/5-FU cells restored the sensitivity to 5-FU. Furthermore, MALDI-TOF/TOF and 2-DE Western blot analysis identified the phosphorylated residues of HSPB1 as Ser-15 and Ser-82 in the main (diphosphorylated) form and Ser-15, Ser-78, and Ser-82 in the minor (triphosphorylated) form. The current findings indicate that phosphorylated HSPB1 may play an important role in 5-FU resistance.

Inhibition of nitric oxide synthase in hyperdynamic circulation of rats with early or late cirrhosis secondary to common bile duct ligation.

BACKGROUND/AIMS: Preventing internal hemorrhage extends the lifespan of rats with chronic bile duct ligation (CBDL), a common animal model of portal hypertension. We investigated hemodynamics during the early and late stages of cirrhosis caused by CBDL. We also evaluated the hemodynamic influence of NO, which is the chief vasodilator in hyperdynamic syndrome, by administration of an NO synthase inhibitor (N(G)-nitro-L-arginine methyl ester: L-NAME; 10 mg/kg). ANIMALS/METHODS: In 24 Sprague-Dawley rats (9 sham rats and 15 CBDL rats), hemodynamics were assessed under conscious and unrestrained conditions 4 and 8 weeks after surgery. Before and 30 minutes after L-NAME administration, the cardiac index (CI) and regional blood flow were measured with the reference sample method using (141)Ce- and (113)Sn-microspheres (15 µm in diameter). RESULTS: A hyperdynamic systemic circulation and splanchnic hyperemia were observed after CBDL, and these changes increased with the progression of cirrhosis. L-NAME significantly diminished the hyperdynamic circulation and also reduced splanchnic hyperemia. In 4-week CBDL rats, a low hemoglobin concentration made an important contribution to the hyperdynamic circulation, and the portal collateral system collapsed when inflow to the portal territory was reduced by L-NAME treatment. In 8-week CBDL rats, systemic hemodynamics were closely linked to both the splanchnic circulation and the renal circulation before and after L-NAME administration, apart from hepatic artery blood flow. CONCLUSION: The distinctive hemodynamic changes of portal hypertension were found in 8-week CBDL rats. Thus, 8-week CBDL rats may be a better animal model of human portal hypertension than 4-week CBDL rats.

Comparative proteomic analysis of the ribosomes in 5-fluorouracil resistance of a human colon cancer cell line using the radical-free and highly reducing method of two-dimensional polyacrylamide gel electrophoresis.

Many auxiliary functions of ribosomal proteins (r-proteins) have received considerable attention in recent years. However, human r-proteins have hardly been examined by proteomic analysis. In this study, we isolated ribosomal particles and subsequently compared the proteome of r-proteins between the DLD-1 human colon cancer cell line and its 5-fluorouracil (5-FU)-resistant sub-line, DLD-1/5-FU, using the radical-free and highly reducing method of two-dimensional polyacrylamide gel electrophoresis, which has a superior ability to separate basic proteins, and we discuss the role of r-proteins in 5-FU resistance. Densitometric analysis was performed to quantify modulated proteins, and protein spots showing significant changes were identified by employing matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry. Three basic proteins (L15, L37 and prohibitin) which were significantly modulated between DLD-1 and DLD-1/5-FU were identified. Two proteins, L15 and L37, showed down-regulated expression in DLD-1/5-FU in comparison to DLD-1. Prohibitin, which is not an r-protein and is known to be localized in the mitochondria, showed up-regulated expression in DLD-1/5-FU. These 3 proteins may be related to 5-FU resistance.

Effect of chronic methylene blue administration on hypoxemia in rats with common bile duct ligation.

AIM: Acute administration of methylene blue (MB) can reverse hypoxemia in patients with hepatopulmonary syndrome (HPS). We evaluated the effect of chronic MB administration in common bile duct-ligated rats, which develop HPS by 5 weeks after surgery. METHODS: A total of 96 Sprague-Dawley rats were used, including 63 rats with common bile duct ligation (CBDL), 22 sham-operated rats and 11 normal control rats. MB (6 mg/kg) was injected s.c. once a day for 4 weeks. Evaluation of hemodynamics and intrapulmonary vascular dilatation (IPVD), as well as blood sampling for arterial blood gas analysis, were done under conscious and unrestrained conditions. Hemodynamics were assessed by the reference sample method using (141)Ce-microspheres (15 microm in diameter), and IPVD was also determined by i.v. injection of these microspheres. Histological examination of the lungs was done with hematoxylin-eosin staining and immunohistochemical staining for von Willebrand factor or vascular endothelial growth factor. RESULTS: Both the arterial oxygen tension and alveolar-arterial oxygen difference were significantly improved in MB-treated CBDL rats. The hyperdynamic circulation and splanchnic hyperemia seen in untreated CBDL rats were also alleviated by MB treatment. However, IPVD was not affected by MB. Histological examination of the lungs indicated that MB treatment reduced the proliferation of alveolar capillary vessels and angiogenesis, leading to improvement of arterial dysoxygenation. Hepatic synthetic and detoxification functions, as well as renal function, were not altered by MB treatment. CONCLUSION: Methylene blue may be a candidate treatment for HPS that does not cause deterioration of hepatic or renal function.

Proteomics-based identification of a tumor-associated antigen and its corresponding autoantibody in gastric cancer.

Identification of novel tumor-related antigens and autoantibodies will lead to early diagnosis of cancer and the development of more effective immunotherapies. The purpose of this study was to identify novel tumor antigens from the gastric cancer cell lines MkN-1, MkN-45 and KATOIII, and their related autoantibodies in sera of patients with gastric cancer using a proteomics-based approach. Proteins from the gastric cancer cell lines (MkN-1, MkN-45 and KATOIII) were separated by two-dimensional polyacrylamide gel electrophoresis, followed by Western blotting and antibody reaction with sera from patients with gastric cancer, healthy individuals and patients with other cancers. Positive spots were excised from Coomassie blue stained gels and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS). Sera from patients with gastric cancer yielded multiple spots, one of which was identified as the 78 kDa glucose-regulated protein (GRP78) by MALDI-TOF/TOF MS. Western blots against recombinant GRP78 showed reactivity in sera from 17/60 (28.3%) patients with gastric cancer and 0/20 (0.0%) of healthy individuals. Autoantibodies against GRP78 were found in 4/15 (26.7%) and 3/15 (20.0%) patients with esophageal and colon cancer, respectively. We identified for the first time an autoantibody against GRP78 in gastric cancer patients. The proteomic approach implemented in this study offers a powerful tool for identifying novel serum markers that may display clinical usefulness in cancer.

Histoculture drug response assay predicts the postoperative prognosis of patients with esophageal cancer.

Predicting response to chemotherapy would provide patients suffering from malignant tumor with not only more favorable outcomes, but also reduction of adverse events, and would enable chemotherapy tailored to individual patients. The purpose of this retrospective study was to evaluate the utility of histoculture drug response assay (HDRA) with the MTT endpoint. Subjects comprised 53 consecutive patients diagnosed with esophageal cancer, with 15 patients receiving preoperative chemoradiotherapy (CRT) followed by surgery (CRT group; n=15) and 38 patients undergoing surgery (surgery group; n=38), including 17 patients with histological lymph node metastasis who received postoperative chemotherapy. Tumor samples obtained from patients were used for HDRA with MTT endpoint and correlations of sensitivity from HDRA with MTT endpoint to clinical response to preoperative CRT, accuracy of in vitro sensitivity test, and clinical outcomes based on HDRA sensitivity were analyzed. HDRA was able to evaluate 379 of 424 assays (89.3%). In the CRT group, no significant correlation was confirmed between efficacy rate of 5-fluorouracil or cisplatin and histological findings in resected specimens after CRT. Efficacy rates of several anticancer agents using HDRA in the surgery group were observed in the range of 0.0-44.8%. On examination of clinical outcomes in the surgery group, in which patients with stage III received adjuvant chemotherapy, chemosensitivity-negative patients tended to display worse prognosis than chemosensitivity-positive patients. HDRA with MTT endpoint probably predicts the postoperative prognosis of patients with esophageal cancer.

Proteomic analysis of the basic proteins in 5-fluorouracil resistance of human colon cancer cell line using the radical-free and highly reducing method of two-dimensional polyacrylamide gel electrophoresis.

5-Fluorouracil (5-FU) is widely used for the treatment of patients with advanced colon cancers and it is the mainstay of chemotherapy. However, the acquisition of resistance to 5-FU is one of the most prominent obstacles to successful chemotherapy. The purpose of this study was to identify the novel biological basis of 5-FU resistance in colon cancer cells. This study is the first comparative proteomic analysis of basic proteins between the DLD-1 human colon cancer cell line and DLD-1/5-FU its 5-FU resistant sub-line using the radical-free and highly reducing method of two-dimensional polyacrylamide gel electrophoresis, which has a superior ability in the separation of basic proteins and the quantification of post-translational modification. A densitometric analysis was performed to quantify the modulated proteins, and protein spots showing significant changes were identified by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry. Six basic proteins significantly modulated between DLD-1 and DLD-1/5-FU were identified. All of them showed up-regulated expression in DLD-1/5-FU in comparison to DLD-1. The six identified spots, corresponding to five different proteins included heterogeneous nuclear ribonucleoprotein G, mitochondrial transcription factor A, histone H2B, histone H4 and ribosomal protein L3. Among the 5 basic proteins, several proteins are potentially related to 5-FU resistance by protecting the cells from DNA damage.

Proteomics-based identification of autoantibody against heat shock protein 70 as a diagnostic marker in esophageal squamous cell carcinoma.

Detection of novel tumor-related antigens and autoantibodies in cancer patients is expected to facilitate the diagnosis of early-stage malignant tumor and establish effective new immunotherapies. The purpose of this study was to identify novel tumor antigens in an esophageal squamous cell carcinoma (ESCC) cell line (TE-2) and related autoantibodies in sera from patients with ESCC using a proteomics-based approach. TE-2 proteins were separated by two-dimensional polyacrylamide gel electrophoresis, followed by Western blot analysis in which sera from patients with ESCC, healthy controls and patients with other cancers were tested for primary antibodies. Positive spots were excised from silver-stained gels and analyzed by matrix-assisted laser disorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). Sera from patients with ESCC yielded multiple spots, one of which was identified as heat shock protein 70 (Hsp70) by MALDI-TOF/TOF-MS. Concentrations of serum Hsp70 autoantibody were significantly higher for patients with ESCC (mean, 0.412+/-0.096 mg/ml) than for patients with gastric (0.236+/-0.112 mg/ml, P<0.001) or colon cancer (0.231+/-0.120 mg/ml, P<0.001) or healthy individuals (0.207+/-0.055 mg/ml, P<0.001) by enzyme-linked immunosorbent assay. We have identified an autoantibody against Hsp70 in ESCC patients. The proteomic approach implemented herein offers a powerful tool for identifying novel serum markers that may display clinical utility against cancer.

Correlation between thymidylate synthase and dihydropyrimidine dehydrogenase mRNA level and in vitro chemosensitivity to 5-fluorouracil, in relation to differentiation in gastric cancer.

PURPOSE: It has been suggested that the gene expression levels of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) help in the prediction of the response to 5-fluorouracil (5-FU) in vivo and in vitro in gastric cancers. METHODS: In this study, intratumoral TS and DPD gene expressions were evaluated with real time reverse transcriptional polymerase chain reaction technique to determine the correlation between the expression of these two genes and in vitro sensitivity to 5-FU, assessed by the histoculture drug response assay on 87 patients with gastric adenocarcinoma. RESULTS: The sensitivity to 5-FU did not show any difference in clinicopathological groups. DPD gene level was higher in undifferentiated (n = 39) than differentiated (n = 48) tumors (P = 0.043). In differentiated tumors, TS gene expression levels were higher in the tumors with relative resistance to 5-FU, while in undifferentiated cases, DPD mRNA levels were higher in tumors that showed resistance to 5-FU in vitro (P = 0.043 and 0.007, respectively). DPD also had significant predictive value for 5-FU sensitivity in undifferentiated cases [R(S) = -0.401, P = 0.011]. TS and DPD gene expression levels were more highly correlated in undifferentiated compared to differentiated cases [R(S) = 0.515 and 0.359, respectively]. CONCLUSIONS: Different gene expression might be responsible for 5-FU sensitivity in gastric cancers of different histologic origin.

Proteomics-based approach identifying autoantibody against peroxiredoxin VI as a novel serum marker in esophageal squamous cell carcinoma.

PURPOSE: Detection of novel tumor-related antigens and autoantibodies will aid in diagnosis of early-stage cancer and in development of more effective immunotherapies. The purpose of this study was to identify novel tumor antigens in an esophageal squamous cell carcinoma (ESCC) cell line (TE-2) and related autoantibodies in sera from patients with ESCC using a proteomics-based approach. EXPERIMENTAL DESIGN: TE-2 proteins were separated by two-dimensional PAGE, followed by Western blot analysis in which sera of patients with ESCC, healthy controls, and patients with other cancers were tested for primary antibodies. Positive spots were excised from silver-stained gels and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS). RESULTS: Sera from patients with ESCC yielded multiple spots, one of which was identified as peroxiredoxin (Prx) VI by MALDI-TOF/TOF MS. Western blot analysis against recombinant Prx VI showed reactivity in sera from 15 of 30 (50%) patients with ESCC and 2 of 30 (6.6%) healthy individuals. Autoantibody against Prx VI was found in sera from 1 of 30 (3.3%) patients with other types of cancer (colon cancer). CONCLUSION: We have identified for the first time an autoantibody against Prx VI in ESCC patients. The proteomic approach implemented here offers a powerful tool for identifying novel serum markers that may display clinical usefulness against cancer.

Oncolytic recombinant herpes simplex virus for treatment of orthotopic liver tumors in nude mice.

Cell-specific, replicating viruses are being developed as a new class of oncolytic agents. A novel approach to viral gene therapy with the use of replication-competent herpes simplex virus has been described; G92A is a replication-competent, multimutant oncolytic herpes simplex virus (HSV) that has been evaluated for anticancer effects and selectivity in the treatment of subcutaneous tumors. G92A replicates efficiently in albumin-producing tumor cell lines but not in non-albumin-producing tumor cell lines, whereas both types are equally susceptible to a non-tissue-specific recombinant HSV, hrR3. In this study, we analyzed the antitumoral efficacy of a single intrasplenic G92A or hrR3 injection in nude mice. In vivo, G92A replicated well in liver xenografts of human albumin-producing hepatoma cells (Hep3B) but not in liver xenografts of a non-albumin-producing malignant colon tumor cell line (HT29), whereas hrR3 replicated well in both tumor types. G92A effectively and selectively replicated throughout liver tumors without apparent hepatotoxicity and inhibited tumor growth, leading to a significantly increased survival time. By monitoring lacZ histochemical staining, we determined the oncolytic potential of recombinant HSV against liver tumors. Our results indicate that G92A warrants further investigation as a clinical therapy against malignant liver tumors.

Concomitant overexpression of cyclooxygenase-2 in HER-2-positive on Smad4-reduced human gastric carcinomas is associated with a poor patient outcome.

PURPOSE: The expression of cyclooxygenase-2 (COX-2) is known to be involved in gastric carcinogenesis and tumor progression, but little is known about the mechanisms responsible for the up-regulation of COX-2. We examined the involvement of two growth factor-signaling systems, HER-2 and transforming growth factor (TGF)-beta, in the induction of COX-2 in human gastric cancer tissue. EXPERIMENTAL DESIGN: COX-2 expression was detected by immunohistochemistry in surgical specimens obtained from 166 patients with advanced gastric cancer; possible correlations between the expression of COX-2 and the expression of HER-2, TGF-beta1, and Smad4, an intracellular mediator that transmits the TGF-beta signal, were then analyzed. RESULTS: COX-2 protein was overexpressed in 91 (54.8%) tumors; COX-2 overexpression was correlated with a differentiated histologic type, deep invasion, and positive lymph node metastasis. COX-2 was frequently overexpressed in HER-2-positive tumors (19 of 22, 86.4%) and in Smad4-reduced tumors (67 of 104, 64.4%) but irrelevant to the TGF-beta1 expression status. The expression levels of COX-2 and HER-2 and the reduction in Smad4 were all associated with a poor patient outcome. A multivariate analysis demonstrated a significantly poor outcome for the concomitant overexpression of COX-2 in patients with Smad4-reduced tumors. CONCLUSIONS: These results support the possibility that signal transduction via HER-2 and the TGF-beta/Smad system may be implicated in COX-2 expression and that the reduction of Smad4 may be, in part, of causal significance in the TGF-beta-initiated overexpression of COX-2, which is associated with a poor prognosis for patients with gastric cancer.

[Development of a novel gene therapy using survivin antisense expressing adenoviral vectors].

BACKGROUND: Survivin, a novel inhibitor of apoptosis, is undetectable in normal adult tissues but becomes notably expressed in the most common human cancers, and is recognized as a potential target in anticancer therapy. METHODS: In this study we evaluated a survivin antisense expressing replication-incompetent adenoviral vector under the control of the cytomegalovirus promoter (pAd.CMV-SAS) for cytoreductive effects in human HT-29 colon cancer cells in vitro and in vivo. RESULTS: Infection of tumor cells with pAd.CMV-SAS caused down-regulation of survivin expression and the potential for spontaneous apoptosis in tumor cells. In contrast, pAd.CMV-SAS did not affect cell viability of normal human cells including fibroblasts. In addition, infection of tumor cells with pAd.CMV-SAS resulted in an increase of the G0/G1 phase population in the cell cycle, and increased their sensitivity to chemotherapeutic drugs in vitro. The efficacies of pAd.CMV-SAS were inversely correlated with survivin expression level. In nude mice, pAd.CMV-SAS suppressed tumor formation, as well as decreased the tumor volumes to approximately 30% of the control tumors. Furthermore, it was confirmed that the anti-tumor efficacy of pAd.CMV-SAS was also enhanced in combination with chemotherapeutic drugs in vivo. CONCLUSIONS: These findings suggest that targeting of survivin using adenoviral antisense vectors may have a potential role in the selective therapy of colon cancer.

Prognostic value of tumor-infiltrating dendritic cells expressing CD83 in human breast carcinomas.

DCs are the most potent antigen-presenting cells that play a major role in initiating the antitumor immune response. Although the clinical significance of TIDCs has been investigated in a variety of human cancers, few studies have focused on the in situ maturation status of DCs. We have analyzed the maturation-specific significance of TIDCs in the prognosis of patients with breast carcinoma. We evaluated 130 breast carcinomas for the presence of TIDCs using immunohistochemistry with an anti-CD1a antibody for immature DCs and an anti-CD83 antibody for mature DCs. Intratumoral expression of immunosuppressive cytokines was also examined. All samples contained CD1a(+) TIDCs, and 82 (63.1%) samples contained CD83(+) TIDCs. The number of CD83(+) TIDCs was inversely correlated with lymph node metastasis and with tissue expression of VEGF and TGF-beta, whereas the number of CD1a(+) TIDCs was not. Kaplan-Meier analysis (log rank statistics) revealed a significant association of increasing number of CD83(+) TIDCs with longer relapse-free (p = 0.002) and overall (p < 0.001) survival. Furthermore, among patients with lymph node metastasis, the survival rate of those with larger numbers of CD83(+) TIDCs was significantly better than that of patients with fewer CD83(+) TIDCs. Multivariate analysis revealed that CD83(+) TIDCs had independent prognostic relevance in breast carcinomas. The infiltration of tumors by mature DCs expressing CD83 may be of great importance in initiating the primary antitumor immune response and is confirmed as an independent, immunologic prognostic parameter for survival in patients with breast cancer.

Alum augments the experimental allergenicity of Kunitz-type soybean trypsin inhibitor independent of the antigen-adsorption.

In order to inspect the significance of the adsorbing property in the adjuvant activity to enhance IgE production, we immunized BALB/c mice against Kunitz-type soybean trypsin inhibitor (KSTI), the most potent experimental allergen among soybean proteins, associated with Aluminum hydroxide (alum) or DEAE-Sephadex particles. The production of immunoglobulin isotypes was analyzed at the various amounts, 3-3,000 microg per mouse, of the antigen dosages. In our experiments, although alum did not adsorb KSTI significantly, it augmented the total and the antigen-specific IgE without affecting the optimal range of the antigen dosage. On the other hand, alum did not effectively enhance the production of the other immunoglobulin isotypes. The production of immunoglobulin isotypes other than IgE increased dose-dependently on the antigen. These results ensured our previous finding that another protein, ovalbumin, was used as the antigen. We also demonstrated that the adsorption of KSTI by DEAE-Sephadex in the immunizing vehicle resulted in the requirement of more KSTI for accomplishing the equal immunity in BALB/c mice compared to the control. Moreover, we demonstrated that, regardless of the inability to adsorb KSTI, alum exerted its adjuvant activity only when it was co-injected with the antigen. These results showed that some biochemical effect, other than adsorptive activity, to enhance the production of the antigen-specific IgE resides in alum.

Expression of HER2 in human gastric cancer cells directly correlates with antitumor activity of a recombinant disulfide-stabilized anti-HER2 immunotoxin.

BACKGROUND: Amplification of the human epidermal growth factor receptor 2 (HER2) gene and overexpression of the HER2 protein have been associated with an unfavorable prognosis. We determined the efficacy of an anti-HER2 immunotoxin, erb-38 [e23(dsFv)PE38], against human gastric cancer cells. METHODS: Immunotoxin was made by fusing the disulfide-stabilized Fv fragments (dsFv) of a monoclonal antibody e23 to a truncated mutant of M(r) 38 Pseudomonas exotoxin (PE38) that lacks its cell-binding domain. RESULTS: The immunotoxin-mediated cytotoxicity directly correlated with the expression levels of the HER2 gene and protein in human gastric cancer cells. Interestingly, MKN-45P cells, a variant line of MKN-45 producing peritoneal dissemination and ascites in vivo, expressed a higher level of HER2 and were more sensitive to erb-38 than MKN-45 cells. RFB-4, a control anti-CD22 immunotoxin, was cytotoxic against none of the tested human gastric cancer cells, also suggesting that the lysis mediated by erb-38 was specific for HER2 expression. Three consecutive iv injections of erb-38 at doses of 0.5 or 5 microg/body eradicated experimental liver metastases and peritoneal disseminations produced by MKN-45P in a dose-dependent manner. CONCLUSIONS: We conclude that an erb-38 anti-HER2 immunotoxin has specific antitumor activities against human gastric cancer cells overexpressing HER2.