Intratumoral interferon-alpha gene transfer enhances tumor immunity after allogeneic hematopoietic stem cell transplantation.

One of the major challenges in the treatment of solid cancers by allogenic hematopoietic stem cell transfer (alloHSCT) is the specific enhancement of antitumor immunity. Interferon (IFN) is a cytokine with pleiotropic biological functions including an immunomoduration, and our preclinical studies have shown that an intratumoral IFN-alpha gene transfer induced strong local tumor control and systemic tumor-specific immunity. In the present study, we examined whether the IFN-alpha gene transfer could enhance recognition of tumor-associated antigens by donor T cells and augment the antitumor activity of alloHSCT. First, when a mouse IFN-alpha adenovirus vector (Ad-mIFN) was injected into subcutaneous xenografts of syngeneic renal and colon cancer cells, tumor growth was significantly suppressed in a dose-dependent manner. A significant tumor cell death and infiltration of immune cells was recognized in the Ad-mIFN-injected tumors, and the dendritic cells isolated from the tumors showed a strong Th1-oriented response. The antitumor effect of Ad-mIFN was then examined in a murine model of minor histocompatibility antigen-mismatched alloHSCT. The intratumoral IFN-alpha gene transfer caused significant tumor suppression in the alloHSCT recipients, and this suppression was evident not only in the gene-transduced tumors but also in simultaneously inoculated distant tumors which did not receive the vector injection. A cytotoxicity assay showed specific tumor cell lysis by donor T cells responding to IFN-alpha. Graft-versus-host disease was not exacerbated serologically or clinically in the mice treated with IFN-alpha. This combination strategy deserves evaluation in future clinical trials for human solid cancers.

Extracellular matrix interacts with interferon alpha protein: retention and display of cytotoxicity.

We have been investigating the efficacy of an intratumoral interferon (IFN)-alpha gene transfer against solid cancers, and found that when the gene is transduced into the subcutaneous tumors, IFN-alpha concentration is markedly increased in the injected tumor but not in the serum. To explain this effective confinement of IFN-alpha to target tissues, we hypothesized that the extracellular matrix in the tumors interacts with IFN-alpha. In this study, a solid-phase-binding assay and immunoprecipitation demonstrated that the IFN-alpha binds directly to matrix proteins. Immunohistochemical staining showed a co-localization of IFN-alpha with pericellular fibronectin. In addition, matrix-bound IFN-alpha protein transduced intracellular signaling and potentiated its cytotoxic activity, suggesting that the retention of IFN-alpha protein on extracellular matrix is likely to play a role in its in vivo biological activity. The data suggest a therapeutic advantage of the intratumoral IFN-alpha gene transfer over the conventional parenteral therapy both in the safety and efficacy.

Allogeneic MHC gene transfer enhances an effective antitumor immunity in the early period of autologous hematopoietic stem cell transplantation.

PURPOSE: In autologous hematopoietic stem cell transplantation (HSCT), lymphopenia-induced homeostatic proliferation of T cells is driven by the recognition of self-antigens, and there is an opportunity to skew the T-cell repertoire during the T-cell recovery by engaging tumor-associated antigens, leading to a break of tolerance against tumors. However, the homeostatic proliferation-driven antitumor responses seem to decline rapidly in association with tumor growth. We hypothesized that a tumor-specific immune response induced by an immune gene therapy could enhance and sustain homeostatic proliferation-induced antitumor immunity. EXPERIMENTAL DESIGN: The antitumor effect of allogeneic MHC (alloMHC) gene transfer was examined at the early phase of the immune reconstitution after syngeneic HSCT. RESULTS: Syngeneic HSCT showed significant tumor growth inhibition of syngeneic colon cancer cells within a period of 30 days; however, the tumor then resumed rapid growth and the survival of the mice was not prolonged. In contrast, when the alloMHC plasmid was intratumorally injected at the early phase after syngeneic HSCT, the established tumors were markedly regressed and the survival of recipient mice was prolonged without significant toxicities, whereas no survival advantage was recognized in recipient mice injected with a control plasmid. This tumor suppression was evident even in the other tumors that were not injected with the alloMHC plasmid. The antitumor response was characterized by the development of tumor-specific T cell- and natural killer cell-mediated cytotoxicities. CONCLUSION: The results suggest the efficacy and safety of integrating intratumoral alloMHC gene transfer with an autologous HSCT for the treatment of solid cancers.

Allogeneic MHC gene transfer enhances antitumor activity of allogeneic hematopoietic stem cell transplantation without exacerbating graft-versus-host disease.

Enhancement of the specific antitumor activity of allogeneic hematopoietic stem cell transplantation (alloHSCT) against solid cancers is a major issue in the clinical oncology. In this study, we examined whether intratumoral allogeneic MHC (alloMHC) gene transfer can enhance the recognition of tumor-associated antigens by donor T cells and augment the antitumor activity of alloHSCT. In minor histocompatibility antigen-mismatched alloHSCT (DBA/2-->BALB/c: H-2(d)) recipients, alloMHC gene (H-2K(b)) was transduced directly into a s.c. tumor of CT26 colon cancer cells. Because CT26 cells have an aggressive tumorigenicity in syngeneic BALB/c mice, an H-2K(b) gene transfer provides only a limited antitumor effect after syngeneic (BALB/c-->BALB/c) HSCT. By contrast, the H-2K(b) gene transfer caused significant tumor suppression in the alloHSCT recipients, and this suppression was evident not only in the gene-transduced tumors but also in simultaneously inoculated distant tumors without gene transduction. In vitro cytotoxicity assay showed specific tumor cell lysis by donor T cells responding to the H-2K(b) gene transfer. Graft-versus-host disease was not exacerbated serologically or clinically in the treated mice, demonstrating that alloMHC gene transfer enhances the antitumor effects of alloHSCT without exacerbating graft-versus-host disease. This combination strategy has important implications for the development of therapies for human solid cancers.

Picosecond amorphization of SiO2 stishovite under tension.

It is extremely difficult to realize two conflicting properties-high hardness and toughness-in one material. Nano-polycrystalline stishovite, recently synthesized from Earth-abundant silica glass, proved to be a super-hard, ultra-tough material, which could provide sustainable supply of high-performance ceramics. Our quantum molecular dynamics simulations show that stishovite amorphizes rapidly on the order of picosecond under tension in front of a crack tip. We find a displacive amorphization mechanism that only involves short-distance collective motions of atoms, thereby facilitating the rapid transformation. The two-step amorphization pathway involves an intermediate state akin to experimentally suggested "high-density glass polymorphs" before eventually transforming to normal glass. The rapid amorphization can catch up with, screen, and self-heal a fast-moving crack. This new concept of fast amorphization toughening likely operates in other pressure-synthesized hard solids.

Magnetic resonance imaging study of the ventricle-brain ratio in parents of schizophrenia subjects.

Structural abnormalities found in probands with schizophrenia have been reported to occur to some degree in their unaffected relatives. However, there has yet to be a study that has focused on brain changes of parents of schizophrenics who are not the presumed obligate carriers. Using MRI, the authors studied the ventricle-brain ratio (VBR) of 9 pairs of parents of schizophrenics and 18 age- and sex-matched healthy controls. VBRs of the unaffected parents of schizophrenics were significantly larger than those of the controls. Our results suggest that large VBRs aggregate in the parents of schizophrenics and may serve as an indicator of vulnerability to the disorder.

Intraperitoneal injection of adenovirus expressing antisense K-ras RNA suppresses peritoneal dissemination of hamster syngeneic pancreatic cancer without systemic toxicity.

We examined the antitumor effect and safety of the adenovirus-mediated expression of antisense K-ras RNA in two peritoneal dissemination models of pancreatic cancer. First, we found that the infection of an adenovirus vector expressing antisense human K-ras RNA (AxCA-AS) induced significant apoptosis in vitro in human pancreatic cancer cells with K-ras mutation. Second, the intraperitoneal (ip) injection of AxCA-AS effectively suppressed the growth of human pancreatic cancer cells in the peritoneal cavity of nude mice. Third, in the hamster syngeneic peritoneal dissemination model, the ip injection of an adenovirus expressing antisense hamster K-ras RNA significantly suppressed the peritoneal growth of hamster pancreatic cancer cells, and no significant systemic toxicity was observed in the treated hamsters. This study suggests a feasibility of the development of a therapeutic strategy against pancreatic cancer based on the adenovirus-mediated transduction of an antisense K-ras construct.

Obstetric complications in siblings of Japanese schizophrenics: data from the Maternal and Child Health Handbook.

Although the precise etiology of schizophrenia remains unknown, the development of schizophrenia has been associated with a history of obstetric complication (OC). Furthermore, some studies show structural and functional brain abnormalities in the unaffected siblings of schizophrenics. In this study the perinatal histories of 18 unaffected siblings of schizophrenics and 15 unrelated healthy controls, as detailed in their mothers' Maternal and Child Health Handbook records, were retrospectively analyzed. Records were scored for obstetric complication by the method developed by [Parnas, J., Schulsinger, F., Teasdale, T.W., Shulsinger, H., Feldman, P.M., Mednick, S.A., 1982. Perinatal complications and clinical outcome within the schizophrenia spectrum. Br. J. Psychiatry 140, 416-420]. The authors found the sibling group had greater pregnancy and birth complication (PBC) frequency, severity and total scores than the control population.

Large increase in fracture resistance of stishovite with crack extension less than one micrometer.

The development of strong, tough, and damage-tolerant ceramics requires nano/microstructure design to utilize toughening mechanisms operating at different length scales. The toughening mechanisms so far known are effective in micro-scale, then, they require the crack extension of more than a few micrometers to increase the fracture resistance. Here, we developed a micro-mechanical test method using micro-cantilever beam specimens to determine the very early part of resistance-curve of nanocrystalline SiO2 stishovite, which exhibited fracture-induced amorphization. We revealed that this novel toughening mechanism was effective even at length scale of nanometer due to narrow transformation zone width of a few tens of nanometers and large dilatational strain (from 60 to 95%) associated with the transition of crystal to amorphous state. This testing method will be a powerful tool to search for toughening mechanisms that may operate at nanoscale for attaining both reliability and strength of structural materials.

Effects of age and the CYP2D6*10 allele on the plasma haloperidol concentration/dose ratio.

The authors studied the effect of aging and the CYP2D6*10 polymorphism on the plasma haloperidol (HAL) concentration after chronic administration of HAL. Subjects were 110 Japanese patients (66 male) treated orally with HAL. Venous blood was obtained from each patient for determination of the HAL concentration/dose (C/D) ratio (the plasma concentration of HAL divided by the daily dose of HAL per kilogram body weight) and for CYP2D6 genotyping. Overall, there was a significant linear correlation between the HAL C/D ratio and age. In subgroup analyses, the correlation was significant for patients with non-2D6*10 homozygous genotypes, but not for those with the 2D6*10 homozygous genotype. Overall, the HAL C/D ratio was significantly higher in older subjects (at least 50 years old) than younger ones (less than 50 years old). The ratio was significantly higher in older than in younger subjects for patients with non-2D6*10 homozygous genotypes, but not for those with the 2D6*10 homozygous genotype. Our results indicate that the effect of age on the HAL C/D ratio depends upon the CYP2D6*10 genotype. Because there are racial differences in the CYP2D6 genotype, further studies should investigate age effects on the HAL C/D ratio in different patient populations.

Effects of smoking and cytochrome P450 2D6*10 allele on the plasma haloperidol concentration/dose ratio.

OBJECTIVE: This study was carried out to evaluate the influence of CYP2D6 polymorphism and smoking on the plasma clearance of haloperidol (HAL) levels, accounting for the antipsychotic dose, body weight, and coadministration of other drugs. METHODS: Subjects were 110 Japanese patients (66 male, 44 female) diagnosed with schizophrenia, dementia, or mood disorder and treated orally with HAL. Venous blood was obtained from each patient to determine the HAL concentration/dose (C/D) ratio (plasma concentration of HAL divided by the daily dose of HAL per body weight) and for CYP2D6 genotyping. RESULTS: There was no significant difference in the HAL C/D ratio between nonsmokers and smokers. In patients with a non-2D6*10 homozygous genotype, smokers had a significantly lower HAL C/D ratio than nonsmokers, whereas smokers with a 2D6*10 homozygous genotype had a significantly higher HAL C/D ratio than those with a non-2D6*10 homozygous genotype. CONCLUSION: Our results suggest that the effect of smoking on the HAL C/D ratio depends on the CYP2D6*10 genotype.

Fracture-induced amorphization of polycrystalline SiO2 stishovite: a potential platform for toughening in ceramics.

Silicon dioxide has eight stable crystalline phases at conditions of the Earth's rocky parts. Many metastable phases including amorphous phases have been known, which indicates the presence of large kinetic barriers. As a consequence, some crystalline silica phases transform to amorphous phases by bypassing the liquid via two different pathways. Here we show a new pathway, a fracture-induced amorphization of stishovite that is a high-pressure polymorph. The amorphization accompanies a huge volume expansion of ~100% and occurs in a thin layer whose thickness from the fracture surface is several tens of nanometers. Amorphous silica materials that look like strings or worms were observed on the fracture surfaces. The amount of amorphous silica near the fracture surfaces is positively correlated with indentation fracture toughness. This result indicates that the fracture-induced amorphization causes toughening of stishovite polycrystals. The fracture-induced solid-state amorphization may provide a potential platform for toughening in ceramics.

Development of peritoneal tumor-targeting vector by in vivo screening with a random peptide-displaying adenovirus library.

The targeting of gene transfer at the cell-entry level is one of the most attractive challenges in vector development. However, attempts to redirect adenovirus vectors to alternative receptors by engineering the capsid-coding region have shown limited success, because the proper targeting ligands on the cells of interest are generally unknown. To overcome this limitation, we have constructed a random peptide library displayed on the adenoviral fiber knob, and have successfully selected targeted vectors by screening the library on cancer cell lines in vitro. The infection of targeted vectors was considered to be mediated by specific receptors on target cells. However, the expression levels and kinds of cell surface receptors may be substantially different between in vitro culture and in vivo tumor tissue. Here, we screened the peptide display-adenovirus library in the peritoneal dissemination model of AsPC-1 pancreatic cancer cells. The vector displaying a selected peptide (PFWSGAV) showed higher infectivity in the AsPC-1 peritoneal tumors but not in organs and other peritoneal tumors as compared with a non-targeted vector. Furthermore, the infectivity of the PFWSGAV-displaying vector for AsPC-1 peritoneal tumors was significantly higher than that of a vector displaying a peptide selected by in vitro screening, indicating the usefulness of in vivo screening in exploring the targeting vectors. This vector-screening system can facilitate the development of targeted adenovirus vectors for a variety of applications in medicine.

Embryonic stem cell transplantation correlates with endogenous neurogenin 3 expression and pancreas regeneration in streptozotocin-injured mice.

Pancreatic beta cell regeneration remains poorly understood, yet stimulation of adult beta cell neogenesis could lead to therapies for type 1 and type 2 diabetes. We studied the effect of embryonic stem (ES) cell transplantation on pancreas regeneration following beta cell injury. Female Balb/c nude mice were treated with streptozotocin to induce hyperglycemia and received an ES cell transplant 24 hr later beneath the renal capsule. Transplantation of ES cells prevented hyperglycemia in a subset of mice, maintaining euglycemia and mild glucose tolerance up to 5 weeks. Pancreata of euglycemic mice showed histological evidence of beta cell regeneration and expression of pancreas and duodenum transcription factor-1 (PDX-1) and neurogenin 3 (Ngn3) in ductal epithelium. Cell tracing analysis indicated that significant beta cell neogenesis from progenitor cells occurred between 2 to 3 weeks following injury in ES cell-transplanted mice but not in sham-transplanted animals. Significantly, whereas pancreas-localized ES cells or their derivatives were adjacent to sites of regeneration, neogenic pancreatic epithelia, including Ngn3+ cells, were endogenous. In conclusion, transplanted ES cells can migrate to the injured pancreas. Transplantation is associated with enhanced endogenous regeneration characterized by expression of Ngn3 and increased beta cell differentiation from endogenous progenitor cells.

Local interferon-alpha gene therapy elicits systemic immunity in a syngeneic pancreatic cancer model in hamster.

The interferon (IFN) protein is a cytokine with pleiotropic biological functions that include induction of apoptosis, inhibition of angiogenesis and immunomodulation. We previously examined the two antitumor mechanisms, taking advantage of the fact that IFN-alpha did not show cross-species activity in its in vivo effect. In a nude mouse subcutaneous xenograft model using human pancreatic cancer cells, the expression of human IFN-alpha effectively induced cell death of human pancreatic cancer cells, whereas mouse IFN-alpha augmented antitumor immunity by stimulation of natural killer cells. Here, we extended our investigation to a syngeneic pancreatic cancer model, so that the integrated antitumor activity of local IFN-alpha gene therapy, including the antiproliferative, proapoptotic, antiangiogeneic and immunomodulatory effects, can be evaluated rigorously. When a recombinant hamster IFN-alpha adenovirus was injected into syngeneic subcutaneous tumors of hamster pancreatic cancer (PGHAM-1) cells in Syrian hamster, tumor growth was significantly suppressed due to cell death and T cell- and natural killer cell-mediated antitumor immunity. Moreover, in this case, tumor regression was observed not only for the injected subcutaneous tumors but also for the untreated tumors both in the peritoneal cavity and at distant sites. No significant systemic toxicity was observed in the treated hamsters. Moreover, the subcutaneous rechallenge of PGHAM-1 cells was rejected in three of four cured hamsters from the initial tumor challenge. This study further demonstrated that local IFN-alpha gene therapy is a promising therapeutic strategy for pancreatic cancer, due to its multiple mechanisms of antitumor activity and its lack of significant toxicity.

CYP2D6*10 alleles do not determine plasma fluvoxamine concentration/dose ratio in Japanese subjects.

OBJECTIVE: The purpose of the present study was to investigate whether plasma fluvoxamine (FV) concentration is associated with CYP2D6*10 allele polymorphisms. METHODS: Subjects were 46 Japanese patients (21 males) carrying neither *3, *4 nor *5 alleles and treated orally using FV. Venous blood was obtained from each patient for determination of FV concentration/dose (C/D) ratio (plasma concentration of FV divided by daily dose of FV per body weight) and CYP2D6 genotyping. RESULTS: No significant differences in FV C/D ratio were found between subjects with no (n=13), one (n=18) or two (n=15) *10 alleles. CONCLUSION: Our results indicate that CYP2D6*10 genotypes do not exert significant effects on FV C/D ratio. As CYP2D6 genotypes differ with ethnic background, further studies should be conducted in different populations.

Interferon-alpha and antisense K-ras RNA combination gene therapy against pancreatic cancer.

Interferon alpha (IFN-alpha) is used worldwide for the treatment of a variety of cancers. For pancreatic cancer, recent clinical trials using IFN-alpha in combination with standard chemotherapeutic drugs showed some antitumor activity of the cytokine, but the effect was not significant enough to enlist pancreatic cancer as a clinically effective target of IFN-alpha. In general, an improved therapeutic effect and safety are expected for cytokine therapy when given in a gene therapy context, because the technology would allow increased local concentrations of this cytokine in the target sites. In this study, we first examined the antiproliferative effect of IFN-alpha gene transduction into pancreatic cancer cells. The expression of IFN-alpha effectively induced growth suppression and cell death in pancreatic cancer cells, an effect which appeared to be more prominent when compared with other types of cancers and normal cells. Another strategy we have been developing for pancreatic cancer targets its characteristic genetic aberration, K-ras point mutation, and we reported that the expression of antisense K-ras RNA significantly suppressed the growth of pancreatic cancer cells. When these two gene therapy strategies are combined, the expression of antisense K-ras RNA significantly enhanced IFN-alpha-induced cell death (1.3- to 3.5-fold), and suppressed subcutaneous growth of pancreatic cancer cells in mice. Because the 2',5'-oligoadenylate synthetase/RNase L pathway, which is regulated by IFN and induces apoptosis of cells, is activated by double-strand RNA, it is plausible that the double-strand RNA formed by antisense and endogenous K-ras RNA enhanced the antitumor activity of IFN-alpha. This study suggested that the combination of IFN-alpha and antisense K-ras RNA is a promising gene therapy strategy against pancreatic cancer.

Expression profiles of pancreatic cancer cell lines infected with antisense K-ras-expressing adenoviral vector.

The point mutations of the K-ras gene occur in as high as 70-90% of the cases with adenocarcinoma of the pancreas and apparently represent one of the key and early events in the carcinogenesis. However, the specific influence of the K-ras activation on global gene expression profiles in pancreatic cancer cells has not been elucidated. In this study, to promote elucidation of the K-ras-triggered molecular cascade(s) in pancreatic cancer, four pancreatic cancer cell lines with K-ras point mutations were infected with an adenovirus vector expressing an antisense K-ras RNA (AxCA-AS), and the change of gene expression was analyzed by oligonucleotide-based microarrays containing 12,626 genes. Among the genes showing more than 2-fold differences in the expression levels between the control- and antisense-K-ras-transduced cells, 7 genes were commonly up-regulated and 4 genes were commonly down-regulated in three or all of the four pancreatic cancer cell lines transduced with AxCA-AS. The altered gene expression levels observed by microarrays were confirmed by real-time RT-PCR methods. Then, the expression of the 4 down-regulated genes was examined in the untransduced surgical specimens of pancreatic cancer. The G-protein coupled receptor RE2 and phenylethanolamine N-methyltransferase had negligible expression levels in all pancreatic cancers, whereas the syntaxin 1A and p120 catenin isoform were significantly up-regulated in pancreatic cancers containing K-ras mutations compared with a pancreatic cancer with wild type K-ras gene. The transcriptional regulation of those genes may be a part of the molecular cascades triggered by K-ras activation leading to the development and/or progression of pancreatic cancer.

Adenovirus-mediated gene transfer of interferon alpha inhibits hepatitis C virus replication in hepatocytes.

Recently we reported that on-site interferon (IFN)-alpha production in the liver using an adenovirus vector can achieve a substantial confinement of IFN-alpha in the target organ and can improve liver fibrosis in a rat liver cirrhosis model. However, the major therapeutic effect of IFN for hepatitis C virus (HCV)-associated liver diseases is its antiviral effect on HCV. As a prelude to the in vivo HCV infection experiment using a primate animal model, here we examined the antiviral effect of IFN-alpha gene transfer into HCV-positive hepatocytes in vitro. The non-neoplastic human hepatocyte cell line PH5CH8 was inoculated with HCV-positive serum. Successful in vitro HCV replication and thus the validity of this model was confirmed by a strong selection for HCV variants determined by sequence analysis of the hypervariable region and an increase of HCV RNA estimated by real time TaqMan RT-PCR. One day after the inoculation of HCV, PH5CH8 cells were infected with adenoviral vectors encoding human IFN-alpha cDNA. HCV completely disappeared 9 days after the adenoviral infection, which is linked to the increase of 2('),5(')-oligoadenylate synthetase activity, suggesting that IFN-alpha produced by gene transfer effectively inhibits HCV replication in hepatocytes. This study supports the development of IFN-alpha gene therapy for HCV-associated liver diseases.

A simple and efficient method for constructing an adenoviral cDNA expression library.

cDNA expression cloning is a powerful method for the identification of genes that are able to confer a selectable phenotype on specific cell types. An adenovirus vector is characterized by several advantages over plasmid DNA and retroviral vector-mediated gene transfer, such as broad host range and high infectivity. However, an expression cloning protocol using the adenovirus vector has not been reported. We describe here a simple and efficient method for constructing adenovirus cDNA expression libraries based on Cre-lox-mediated in vitro recombination between adenoviral shuttle plasmid cDNA libraries and adenoviral genomic DNA tagged with terminal protein. In a model experiment, EGFP clones present at the frequency of 0.003% in the shuttle plasmid library could be efficiently converted to adenoviral vector in a 6-cm dish under optimized conditions, indicating that high-complexity libraries harboring low-abundance cDNAs can be produced. The efficiency of this system was demonstrated by the isolation of cDNA for CD2 (frequency less than 1 in 0.3 x 10(4) transcripts in T cells) from the human T cells. This effective and versatile method can facilitate the functional identification of genes for a variety of purposes.