EGFR inhibitors augment antitumour helper T-cell responses of HER family-specific immunotherapy.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is a major cause of cancer-related morbidity and mortality worldwide. Epidermal growth factor receptor (EGFR)-targeted therapy is an attractive strategy alternative to conventional cancer treatments for HNSCC, but its efficacy remains controversial. T-cell-based immunotherapy has been proposed as a novel therapeutic approach to improve the clinical outcome for HNSCC. In this study, we report human epidermal receptor (HER) family epitopes that induced CD4 T-cell responses to HNSCC. The results provide support for a novel strategy to treat HNSCC by combining EGFR-targeted therapy with T-cell-based immunotherapy. METHODS: We evaluated the capacity of predicted CD4 T-cell peptide epitopes from EGFR to induce antitumour immune responses in vitro. In addition, EGFR inhibitors were evaluated for their ability to augment tumour MHC class II expression in HNSCC cell lines and subsequently increase T-cell recognition. RESULTS: Among several predicted peptide epitopes, EGFR875-889 elicited CD4 T-cell responses that were restricted by HLA-DR4, DR15, or DR53 molecules, indicating that the peptide functions as a promiscuous T-cell epitope. The peptide-reactive T cells responded to autologous dendritic cells loaded with EGFR-expressing tumour cell lysates, indicating that these epitopes are naturally processed. In addition, the CD4 T cells were capable of directly recognising and killing HNSCC cells expressing EGFR and the appropriate HLA class II molecule. T cells reactive with the EGFR875-889 epitope could be detected in the blood of HNSCC patients. EGFR875-889-reactive CD4 T cells were also able to recognise several peptide analogues derived from homologous regions of EGFR family members, HER-2, HER-3 and c-MET. Finally, we examined the effects of EGFR tyrosine kinase inhibition or EGFR-blocking antibodies on CD4 T-cell tumour reactivity. Treatment of tumour cells with the EGFR inhibitors enhanced tumour recognition by EGFR875-889-reactive T cells presumably due to the upregulation of HLA-DR expression in the HNSCC cells. CONCLUSION: We identified novel CD4 T-cell EGFR epitopes and amongst these, EGFR875-889 functions as a promiscuous helper T-cell epitope that can elicit effective antitumour T-cell responses against tumours expressing HER family members and c-MET. These observations should facilitate the translation of T-cell-based immunotherapy into the clinic for the treatment of HNSCC and provide a rational basis for EGFR inhibition, immune-targeted combination therapy.

A novel oncogenic pathway by TLS-CHOP involving repression of MDA-7/IL-24 expression.

BACKGROUND: Translocated in liposarcoma-CCAAT/enhancer binding protein homologous protein (TLS-CHOP) (also known as FUS-DDIT3) chimeric oncoprotein is found in the majority of human myxoid liposarcoma (MLS), but its molecular function remains unclear. METHODS: We knockdowned TLS-CHOP expression in MLS-derived cell lines by a specific small interfering RNA, and analysed the gene expression profiles with microarray. RESULTS: TLS-CHOP knockdown inhibited growth of MLS cells, and induced an anticancer cytokine, melanoma differentiation-associated gene 7 (MDA-7)/interleukin-24 (IL-24) expression. However, double knockdown of TLS-CHOP and MDA-7/IL-24 did not inhibit MLS cell growth. CONCLUSION: Repression of MDA-7/IL-24 expression by TLS-CHOP is required for MLS tumour growth, and TLS-CHOP may become a promising therapeutic target for MLS treatment.

Magnetic-field-induced shape recovery by reverse phase transformation.

Large magnetic-field-induced strains have been observed in Heusler alloys with a body-centred cubic ordered structure and have been explained by the rearrangement of martensite structural variants due to an external magnetic field. These materials have attracted considerable attention as potential magnetic actuator materials. Here we report the magnetic-field-induced shape recovery of a compressively deformed NiCoMnIn alloy. Stresses of over 100 MPa are generated in the material on the application of a magnetic field of 70 kOe; such stress levels are approximately 50 times larger than that generated in a previous ferromagnetic shape-memory alloy. We observed 3 per cent deformation and almost full recovery of the original shape of the alloy. We attribute this deformation behaviour to a reverse transformation from the antiferromagnetic (or paramagnetic) martensitic to the ferromagnetic parent phase at 298 K in the Ni45Co5Mn36.7In13.3 single crystal.

Therapeutic silencing of an endogenous gene by siRNA cream in an arthritis model mouse.

Recent advances of biotechnology have laid the groundwork for potent and specific molecular-targeting therapies including RNA interference. The largest remaining hurdle for widespread use of this technology in skin is an effective delivery system. Here, we demonstrate an effective topical delivery system using a cream formulation containing a small-interfering RNA (siRNA) that specifically targets osteopontin (OPN). OPN is a validated target in numerous inflammatory diseases, including rheumatoid arthritis (RA). The siRNA targeting OPN was incorporated into a cream formulation GeneCream that penetrates the stratum corneum, depositing siRNA in the epidermis, dermis, and to a lesser extent, subcutaneous tissue. In addition, when the OPN siRNA cream was topically applied to the skin of a collagen antibody-induced RA mouse model, the siRNA cream prevented the occurrence of severe, irreversible damage to bone and cartilage. Thus, the siRNA cream provides effective delivery of active OPN siRNA, suggesting this formulation may represent a platform technology for delivery of siRNAs for treating various disorders including RA.

Nuclear encoding of a chloroplast RNA polymerase sigma subunit in a red alga.

A chloroplast RNA polymerase sigma factor is encoded by a nuclear gene, sigA, in the red alga Cyanidium caldarium RK-1. The encoded protein functions as an RNA polymerase sigma factor in vitro and it is localized to the chloroplast in vivo. SigA shows high sequence similarity to the sigma factors of cyanobacteria, which is indicative of the ancestral endosymbiotic event and subsequent transfer of the sigA gene to the nuclear genome.

Arabidopsis NPL1: a phototropin homolog controlling the chloroplast high-light avoidance response.

Chloroplasts relocate their positions in a cell in response to the intensity of incident light, moving to the side wall of the cell to avoid strong light, but gathering at the front face under weak light to maximize light interception. Here, Arabidopsis thaliana mutants defective in the avoidance response were isolated, and the mutated gene was identified as NPL1 (NPH-like 1), a homolog of NPH1 (nonphototropic hypocotyl 1), a blue light receptor used in phototropism. Hence, NPL1 is likely a blue light receptor regulating the avoidance response under strong light.

Expression of G protein-coupled receptor kinase 4 is associated with breast cancer tumourigenesis.

G-protein-coupled receptor kinases (GRKs) comprise a family of seven mammalian serine/threonine protein kinases that phosphorylate and regulate agonist-bound, activated, G-protein-coupled receptors (GPCRs). GRKs and beta-arrestins are key participants in the canonical pathways leading to phosphorylation-dependent GPCR desensitization, endocytosis, intracellular trafficking and resensitization. Here we show that GRK4 isoforms are expressed in human breast cancer but not in normal epithelia. In addition, GRK4-over-expressing cells activated the mitogen-activated protein kinase (MAPK) mediated by ERK 1/2 and JNK phosphorylation in breast cancer-derived cell lines. Furthermore, suppression of beta-arrestins decreased GRK4-stimulated ERK 1/2 or JNK phosphorylations. These data indicate that high-level expression of GRK4 may activate MAPK signalling pathways mediated by beta-arrestins in breast cancer cells, suggesting that GRK4 may be implicated in breast cancer carcinogenesis.

Magnetic anisotropy in Ni-Fe-Ga-Co ferromagnetic shape memory alloys in the single-variant state.

The effects of the addition of Co on the magnetic anisotropy in Ni(55-x)Fe(18)Ga(27)Co(x) (x = 1-6) single-variant ferromagnetic shape memory alloys have been investigated. By the addition of Co from 1 to 6 at.%, the Curie temperature T(C) is increased from 318 to 405 K, keeping the martensitic transformation temperatures above room temperature. As a result, the value of the uniaxial magnetic anisotropy constant |K(u)| at 300 K increases with increasing x of the Co concentration and the martensite phase of Ni(49)Fe(18)Ga(27)Co(6) exhibits a relatively high value of |K(u)| = 1.15 × 10(5) J m(-3) at 300 K. With increasing Co concentration, on the other hand, the c axis changes from the magnetic easy axis to the hard axis at 4.2 K, that is, the sign of K(u) is reversed from positive to negative between 2 and 3 at.% Co. Furthermore, K(u) in Ni(53)Fe(18)Ga(27)Co(2) is positive below 100 K and negative above 100 K up to T(C), reducing the magnetic anisotropy around 200 K. From the present results, it is evident that the magnetic anisotropy of Ni(55-x)Fe(18)Ga(27)Co(x) (x = 1-6) single-variant ferromagnetic shape memory alloys is very sensitive to Co concentration and also temperature.

Stress-assisted large magnetic-field-induced strain in single-variant Co-Ni-Ga ferromagnetic shape memory alloy.

The magnetic anisotropy and the magnetic-field-induced strain (MFIS) in a single-variant Co(47.5)Ni(22.5)Ga(30.0) ferromagnetic shape memory alloy (FSMA) have been investigated. From the magnetization curves for the single crystal, the hard c-axis was confirmed, and the uniaxial magnetic anisotropy constant K(u) at 300 K was evaluated to be -1.07 × 10(6) erg cm(-3) for the single-variant Co(47.5)Ni(22.5)Ga(30.0) martensite phase. The magnitude of compressive shear stress for the variant rearrangement was estimated to be 6.0-7.5 MPa from the stress-strain curves. An assisted stress τ(assist) of 6.0 MPa was applied before applying a magnetic field, and then a magnetic stress τ(mag) of 0.3 MPa was added. As a result, a large MFIS of about 7.6 % was obtained at room temperature in the martensite phase of the single-variant Co(47.5)Ni(22.5)Ga(30.0).

Magnetic anisotropy of epitaxially grown Co and its alloy thin films.

We have performed a systematic study on the correlation between magnetic anisotropy energy (MAE) and crystal structures, such as lattice parameters, stacking fault densities, lattice strain, and so on, for epitaxially grown Co, Co-Pt, and Co-Pd alloy thin films, and have found that the MAE strongly depends on the axial ratio c/a of the hcp crystal lattice. As the c/a of hcp Co decreases down to ∼1.61 which is smaller than 1.622 for bulk Co, the MAE becomes significantly enhanced up to ∼10(6) J m(-3). Similar trends have also been verified for hcp Co-Pt and -Pd. These results, which are qualitatively consistent with the classic single-ion anisotropy model and the recent first principles calculation, suggest a new effective way to control the MAE of magnetic thin films.

Effects of serine protease inhibitor and prostaglandin I2 on liver transplantation from non-heart-beating rat donors.

OBJECTIVES: We sought to preserve the microcirculation as a keystone in liver transplantation from a non-heart-beating donor (NHBD). The purpose of this study was to investigate the cytoprotective effects of a serine protease inhibitor, nafamostat mesilate, and prostaglandin I2 (PGI2) on livers transplanted from NHBDs. METHODS: Male Wistar rats were used in five groups of nine rats each. In group 1, livers were retrieved from heart-beating donors (HB group); in group 2, livers were retrieved from NHBDs that had experienced agonal apnea (NHB group); in group 3, livers were retrieved in the same manner as in the NHBD group but were pretreated with nafamostat mesilate (NM), 0.2 mg/kg/h, (NM group); in group 4, livers were retrieved in the same manner as in the NHBD group but were pretreated with prostaglandin (PG) I2, 33 ng/kg/h for 30 minutes (PG group); and in group 5, livers were retrieved in the same manner as in the NHBD group but were pretreated with NM plus PG, (NM+PG group). Livers were perfused for 60 minutes with Krebs-Henseleit bicarbonate buffer after 6 hours of cold preservation, after which the perfusate and liver tissue were analyzed in one set of experiments. In another set of experiments, livers retrieved and after 1 hour of cold preservation were transplanted according to the Kamada method. RESULTS: In the NM+PG group, the values of interleukin-1beta, tumor necrosis factor-alpha, and thromboxane B2 were significantly lower than those in the NHB group. At histologic analysis, sinusoidal endothelial cells were well preserved in the NM+PG group. The number of survivors at 7 days after liver transplantation in the 5 groups were 9, 0, 1, 1, and 3, respectively. CONCLUSION: The serine protease inhibitor, NM, and PGI2 supported sinusoidal endothelial cells and preserved microcirculation.

In vivo EPR detection and imaging of endogenous nitric oxide in lipopolysaccharide-treated mice.

Nitric oxide (NO), a simple diatomic free radical, is known to play a critical physiological role in diverse organisms. An iron complex, with N-(dithiocarboxy)sarcosine (Fe-DTCS), has a high affinity for endogenous NO and can trap, stabilize, and accumulate it. The stable NO adduct thus formed is detectable at room temperature with electron paramagnetic resonance (EPR) spectrometry. We report in vivo EPR imaging of endogenous NO, trapped by an Fe-DTCS complex, in the abdomen of a live mouse. To our knowledge, this is the first report on EPR imaging of endogenous free radicals produced in vivo. This EPR imaging method will be useful for the noninvasive investigation of the spatial distribution of NO in pathologic organs or tissues.

Elevated activity of beta-hexosaminidase and sulfhydryl modification in the B-variant of human lung cancer.

Activities of beta-hexosaminidase A and beta-hexosaminidase B (Hex B) were measured both in human lung carcinoma and the adjacent normal tissues of 47 patients. The specific activity of total beta-hexosaminidase in the tumors was considerably higher than in the adjacent normal tissues, irrespective of histological types. In isoelectric focusing experiments, Hex B purified from normal lung exhibited a single peak with an isoelectric point (pI) of 7.9, while Hex B purified from adenocarcinoma contained two forms with pI 7.6 and 7.9. With respect to heat stability, Hex B from the normal lung was very stable at 52 degrees, while the tumor Hex B (mixture of pI 7.6 and 7.9 forms) was unstable. After treatment of the tumor enzyme with dithiothreitol, heat stability was restored. When the tumor pI 7.6 form was treated with dithiothreitol and subjected to polyacrylamide gel electrophoresis, the enzyme converted to a pI 7.9 form similar to that of the normal lung. Determination of the sulfhydryl group of the tumor pI 7.6 form under nondenaturing conditions showed that the enzyme had some easily reducible disulfide bonds on its surface. These findings indicate that the formation of mixed disulfide bonds in the tumor Hex B increases the net negative charge and results in the appearance of a heat-labile form.

Dioxin suppresses the checkpoint protein, MAD2, by an aryl hydrocarbon receptor-independent pathway.

The compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shown recently to be carcinogenic, but little is currently known about the molecular mechanism of TCDD affecting cell proliferation and carcinogenesis. In this report, we demonstrate that TCDD suppresses the expression of the checkpoint protein, Mad2. Suppression of Mad2 was also observed in aryl hydrocarbon receptor-deficient mouse embryonic fibroblasts, suggesting that TCDD suppresses Mad2 by a novel TCDD receptor signaling mechanism. In addition, HeLa cells treated with TCDD failed to arrest in mitosis after nocodazole treatment. The Mad2 protein plays a significant role in accurate chromosome segregation in mitotic cells. Our data suggest that TCDD may increase chromosomal instability through the suppression of Mad2 expression.

Modulation of substrate selectivity in plasma lipid transfer protein reaction over structural variation of lipid particle.

The modulation of substrate selectivity of human plasma LTP reaction is the subject of the present investigation. The moderate selectivity by a factor of 5 to 6 was observed in the LTP-catalyzed transfer of cholesteryl ester over triacylglycerol between plasma lipoproteins. On the other hand, the transfer of cholesteryl ester by LTP was highly selective over the negligible transfer of triacylglycerol, by a factor of 60 to 500, between the microemulsions with LDL size, regardless of the activators such as human and pig apolipoprotein (apo) A-I, human apo C-III and apo E that bound to the surface of the emulsion in equilibrium. The presence of free cholesterol in these microemulsions reduced slightly the rate of cholesteryl ester transfer but had no effect on triacylglycerol transfer. Other surface-active reagents such as cholic acid, Triton X-100 and Tween-20, did not have an effect on the triacylglycerol transfer either. Triacylglycerol transfer by LTP became measurable between such lipid particles as prepared by co-sonication of lipid with pig apo A-I and isolated as the mixed-microemulsions in the density of LDL and HDL. In these conditions, the substrate selectivity for cholesteryl ester over triacylglycerol was a factor of 6 to 16 mimicking the ratio in plasma lipoproteins. The conformation of pig apo A-I estimated by circular dichroism showed that its apparent helical content was further more induced when apo A-I was integrated into the mixed-microemulsion by co-sonication than the lipid-bound apo A-I in equilibrium. Apo A-I, thus integrated into lipid particles, was highly resistant to the denaturation by guanidine hydrochloride while the lipid-bound apo A-I in equilibrium was denatured as readily as the lipid-free protein. Thus, triacylglycerol transfer by LTP was induced by structural modulation of substrate-carrying lipid particles such as higher integration of apolipoproteins.

Increase in the glucosylated form of erythrocyte Cu-Zn-superoxide dismutase in diabetes and close association of the nonenzymatic glucosylation with the enzyme activity.

Human erythrocytes contain glucosylated and nonglucosylated Cu-Zn-superoxide dismutases which can be separated by boronate affinity chromatography. The percentage of the glucosylated form is significantly increased in the erythrocytes of patients with diabetes as compared to normal erythrocytes. The nonglucosylated form of Cu-Zn-superoxide dismutase, which was washed through the boronate column, was glucosylated in vitro upon exposure to radioactive or non-radioactive D-glucose. Incorporation of D-glucose into the protein was observed, and with the increase in glucosylation, the enzymatic activity decreased, indicating that the glucosylation of the enzyme led to a low active form. This is the first demonstration that superoxide dismutase is glucosylated in erythrocytes and that the glucosylation leads to the inactivation of the enzyme.

Circular dichroism of intermediate subviral particles of reovirus. Elucidation of the mechanism underlying the specific monovalent cation effects on uncoating.

1. Circular dichroic (CD) spectra of purified intermediate subviral particles of reovirus were determined in the presence of different monovalent cations. 2. The CD spectra reveal that reo intermediate subviral particles can exist in two conformationally different forms. The two forms are readily distinguished by comparison of their ellipticities in the wavelength regions 210 nm and 220 nm, with a Na+-induced form exhibiting a reduced negative ellipticity relative to a Cs+-induced form. 3. The transition between the Na+- and Cs+-induced forms is reversible by manipulation of the species of monovalent cation present and appears to be temperature independent. 4. Temperature variation studies on dilute suspensions of particles indicate that the Na+-induced form is stable, whereas the Cs+-induced from undergoes a second transition, temperature dependent and irreversible, to become a viral core. 5. A model is presented relating these observations to the known properties of reovirus uncoating and transcriptase activation.

Heme peptide as a model substance for halogenomethane activation and heme modification.

Octa-heme peptide (CHP) obtained from Candida krusei cytochrome c was tested for suicidal activation of halogenomethanes. Under anaerobic conditions, CHP was kept in the reduced state in the presence of NADPH and NADPH-cytochrome P-450 reductase. Addition of CBrCl3 to the reduced CHP caused spectral changes such as rapid disappearance of alpha and beta bands and gradual decrease in the gamma-peak height, accompanied by oxidation of NADPH. Heme content of the reaction mixture, determined as pyridine hemochrome, also decreased NADPH dependently. CCl4 was less effective than CBrCl3, while CHCl3 had almost no effect. N-tert-butyl-alpha-phenylnitrone (PBN) suppressed the CBrCl3-induced heme damage, and resulted in the formation of radical adduct .PBN-CCl3 as evidenced by ESR spectroscopy. Radical formation was also observed with CCl4. The CHP damage induced by CBrCl3 was also accompanied by the release of Br- about 11-12-times molar excess of CHP, whereas the release of CHCl3 was about 20% that of Br-.FD-MS assay of the product of CHP reaction suggested that 10 trichloromethyl radicals bonded with CHP. Thus, CBrCl3 undergoes single-electron reduction in the presence of reduced CHP to trichloromethyl radicals, which covalently bind to CHP molecules. Heme peptide may be a useful tool in the study of mechanisms involved in the destruction of cytochrome P-450 by halogenomethanes.

Two types of differentially photo-regulated nuclear genes that encode sigma factors for chloroplast RNA polymerase in the red alga Cyanidium caldarium strain RK-1.

A nuclear gene, sigA, that encodes a sigma factor for chloroplast RNA polymerase has previously been identified and characterized in the primitive red alga Cyanidium caldarium strain RK-1. Southern hybridization analysis indicated the presence of two additional sigma factor genes, which have now been cloned and shown to encode virtually identical proteins that are homologous to eubacterial sigma factors. These genes, which are also present in the nuclear genome, have therefore been named sigB and sigC. The substantial sequence similarity of sigB and sigC to sigA of the same strain as well as to cyanobacterial principal sigma factors and other chloroplast sigma factors strongly suggests that the nuclear genome of C. caldarium contains three genes that encode two types of chloroplast sigma factors. Each of the three recombinant Sig proteins showed sigma factor activity in vitro when combined with the Escherichia coli RNA polymerase core enzyme. Northern blot analysis revealed that, whereas the overall abundance of sigA transcripts was not affected by light, the amount of sigB and sigC mRNAs was greater in the light than in the dark. Thus, multiple sigma factors appear to contribute to light-regulated gene expression in the chloroplast.

Characterization of two plastid sigma factors, SigA1 and SigA2, that mainly function in matured chloroplasts in Nicotiana tabacum.

We have isolated and characterized two genes from Nicotiana tabacum, whose products function as putative sigma factors for plastid RNA polymerase. Since the amino acid sequence deduced from the DNA sequences of both genes showed highly similar to that of the SigA protein of Arabidopsis thaliana, we termed the corresponding genes sigA1 and sigA2, respectively. Transient expression assay using a green fluorescent protein (GFP) fusion construct indicated that the N-terminal region of the sigA2 gene product could function as a transit peptide for import into chloroplasts. The gel-blot analysis of RNAs revealed that the sum of the sigA1 and sigA2 transcripts fluctuated apparently with an endogenous rhythm after 12-h-light, 12-h-dark entrainment in photomixotrophically cultured tobacco cells. RT-PCR based northern analysis revealed that the sigA1 and sigA2 transcripts increased along with the cell growth in cultured cells, and were most abundant in mature leaves and shoot meristems with very young leaves in tobacco plants. Immunoblot analysis of the cell extracts of tobacco plants also supports this notion. These results suggest that the sigma factors encoded by sigA1 and sigA2 play a role in chloroplast development and regulation of gene expression in matured chloroplasts.