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.

The field and temperature dependence of the magnetic and structural properties of the shape memory compound Ni(1.84)Mn(1.64)In(0.52).

Magnetization and high resolution neutron powder diffraction measurements have been made on the magnetic shape memory alloy Ni(1.84)Mn(1.64)In(0.52). The compound undergoes a broad structural phase transition, which on heating starts at ∼150 K and finishes at ∼215 K. On cooling there is a ∼20 K hysteresis. The high temperature parent phase is cubic (a = 5.988 Å) with the L2(1) structure in which the excess Mn atoms occupy the vacancies on the Ni and In sites. The magnetic moment is located mainly on the Mn atoms with the same magnitude on both the 4a (Mn) and 4b (In) sites. The low temperature martensite is monoclinic with parameters a = 4.405(2), b = 5.553(2), c = 12.950(2) Å, ß = 86.47(10)° and space group P2/m. The magnetic properties of the martensitic phase are complex and indicate metamagnetic behaviour.

Influence of experimental esophageal acidification on sleep bruxism: a randomized trial.

UNLABELLED: The aim of this cross-over, randomized, single-blinded trial was to examine whether intra-esophageal acidification induces sleep bruxism (SB). Polysomnography with electromyogram (EMG) of masseter muscle, audio-video recording, and esophageal pH monitoring were performed in a sleep laboratory. Twelve healthy adult males without SB participated. Intra-esophageal infusions of 5-mL acidic solution (0.1 N HCl) or saline were administered. The frequencies of EMG bursts, rhythmic masticatory muscle activity (RMMA) episodes, grinding noise, and the RMMA/microarousal ratio were significantly higher in the 20-minute period after acidic infusion than after saline infusion. RMMA episodes including SB were induced by esophageal acidification. This trial is registered with the UMIN Clinical Trials Registry, UMIN000002923. ABBREVIATIONS: ASDA, American Sleep Disorders Association; EMG, electromyogram; GER, gastroesophageal reflux; LES, lower esophageal sphincter; NREM, non-rapid eye movement; REM, rapid eye movement; RMMA, rhythmic masticatory muscle activity; SB, sleep bruxism; SD, standard deviation; UES, upper esophageal sphincter.

Evidence for the involvement of calbindin D28k in the presenilin 1 model of Alzheimer's disease.

Pathological hallmarks of Alzheimer's disease include memory deficits, accumulation of amyloid beta (Abeta) plaques, the appearance of neurofibrillary tangles, and dysregulation of calcium homeostasis, which has been linked to mutations in the presenilin gene that code for presenilin (PS) proteins. PSs are a family of multi-pass transmembrane proteins where normal presenilins (PS1 and PS2) are highly localized in the endoplasmic reticulum (ER). Several past studies have explored alterations in long-term potentiation (LTP), a proposed molecular correlate of memory, and in behavioral tests of spatial memory in a variety of PS1 models. These reports suggest that calcium plays a role in these alterations, but mechanistic explanations for changes in LTP and in behavioral tests of memory are still lacking. To test the hypothesis that calcium-related mechanisms, such as changes in calcium buffering, are associated with alterations in LTP and memory, we utilized in vitro experimental paradigms of LTP in hippocampal slices obtained from the PS1-M146V transgenic mouse model of Alzheimer's disease (AD). We also used the in vivo Morris water maze (MWM), a test for hippocampal dependent spatial memory. In addition, we used cellular assays to explore molecular mechanisms. We confirm that PS1 mutations (M146V) enhance LTP. We also find increases in some parameters of the MWM, and alterations in other parameters, such as path length indicating impairment in cognitive functioning in PS1-M146V mice. In addition, these findings are observed in association with increased calbindin D28K expression in the CA1 hippocampus of PS1-M146V mice.

Magnetic and structural properties of the magnetic shape memory compound Ni(2)Mn(1.48)Sb(0.52).

Magnetization and high resolution neutron powder diffraction measurements on the magnetic shape memory compound Ni(2)Mn(1.48)Sb(0.52) have confirmed that it is ferromagnetic below 350 K and undergoes a structural phase transition at T(M)≈310 K. The high temperature phase has the cubic L2(1) structure with a = 5.958 Å, with the excess manganese atoms occupying the 4(b) Sb sites. In the cubic phase above ≈310 K the manganese moments are ferromagnetically aligned. The magnetic moment at the 4(a) site is 1.57(12) µ(B) and it is almost zero (0.15(9) µ(B)) at the 4(b) site. The low temperature orthorhombic phase which is only fully established below 50 K has the space group Pmma with a cell related to the cubic one by a Bain transformation a(orth) = (a(cub) + b(cub))/2; b(orth) = c(cub) and c(orth) = (a(cub) - b(cub)). The change in cell volume is ≈2.5%. The spontaneous magnetization of samples cooled in fields less than 0.5 T decreases at temperatures below T(M) and at 2 K the magnetic moment per formula unit in fields up to 5.5 T is 2.01(5) µ(B). Neutron diffraction patterns obtained below ≈132 K gave evidence for a weak incommensurate magnetic modulation with propagation vector (2/3, 1/3, 0).

Cochlear protection from acoustic injury by inhibitors of p38 mitogen-activated protein kinase and sequestosome 1 stress protein.

This study evaluated the protective role of p38 mitogen-activated protein kinase (p38 MAPK) inhibitors and sequestosome 1 (Sqstm1/A170/p62), a stress-induced signal modulator, in acoustic injury of the cochlea in mice. Two weeks after the exposure of mice to acoustic stress, threshold shifts of the auditory brainstem response (ABR) from the pre-exposure level and hair cell loss were evaluated. The activation of p38 MAPK was observed in cochlea by immunostaining 4 h after acoustic stress. To examine the role of p38 MAPK in tissue injury, its inhibitors were i.p. injected into male wild-type C57BL mice before the acoustic overexposure. The inhibitors SB202190 and SB203580 but not the inactive analogue SB202474 dose-dependently decreased the auditory threshold shift and outer hair cell loss induced by acoustic overexposure, suggesting the involvement of p38 MAPK in ototoxicity. We found that acoustic overexposure induced the up-regulation of Sqstm1 mRNA expression in the cochlea of wild-type mice and that SQSTM1-deficient mice exhibited an enhanced ABR threshold shift and hair cell loss, suggesting a role of SQSTM1 in the protection of tissue from acoustic stress.

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.

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.

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 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.

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.

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.

Decrease of 4,5,6,7-tetrachlorophthalide in paddy field soil after aerial application.

Variation in the fungicide, 4,5,6,7-tetrachlorophthalide (phthalide), in paddy field soil was investigated in order to evaluate its decrease after aerial application by a radio-controlled helicopter. The maximum concentrations of phthalide were 309-320 microg/kg dry, which were 83%-96% of the applied phthalide. The organic carbon normalized soil sorption coefficient (K(oc)) was calculated to be 94-96 mL/g at 1 h after the application. The calculated K(oc) values increased to 620-1,300 mL/g from 1 through 9 days after the application and then 4,700-7,200 mL/g 14 days after the application. The half-life of the phthalide was calculated to be 20-31 days.

Variation of 4,5,6,7-tetrachlorophthalide in water after aerial application to rice cultivation area.

Variation in the fungicide, 4,5,6,7-phthalide, in water was investigated in order to evaluate the runoff of the fungicide after aerial application to paddy fields by a radio-controlled helicopter. The survey was conducted for 4 months after the application. The average and maximum concentrations of phthalide were 3.7-4.4 microg/L and 30.5-33.8 microg/L in the paddy fields, 0.37-0.64 microg/L and 2.7-7.5 microg/L in the drainage channels, and 0.18 and 0.83 microg/L in a river, respectively. The runoff ratios of the aerially applied phthalide from the paddy fields into the drainage channels were calculated to be 1.7-2.4%.

Cobalt-base high-temperature alloys.

We have identified cobalt-base superalloys showing a high-temperature strength greater than those of conventional nickel-base superalloys. The cobalt-base alloys are strengthened by a ternary compound with the L1(2) structure, gamma' Co3(Al,W), which precipitates in the disordered gamma face-centered cubic cobalt matrix with high coherency and with high melting points. We also identified a ternary compound, gamma' Ir3(Al,W), with the L1(2) structure, which suggests that the Co-Ir-Al-W-base systems with gamma+gamma' (Co,Ir)3(Al,W) structures offer great promise as candidates for next-generation high-temperature materials.

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.

The human papillomavirus E6 and E7 inducible oncogene, hWAPL, exhibits potential as a therapeutic target.

Here we show that human papillomavirus (HPV) E6 and E7 oncoproteins induce hWAPL expression. In addition, small interfering RNA (siRNA) of hWAPL suppressed the growth of tumours derived from SiHa cells in nude mice. Thus, hWAPL may be one of the effective targets of uterine cervical cancer therapy.