The induction of antigen-specific CTL by in situ Ad-REIC gene therapy.

An adenovirus vector carrying the human Reduced Expression in Immortalized Cell (REIC)/Dkk-3 gene (Ad-REIC) mediates simultaneous induction of cancer-selective apoptosis and augmentation of anticancer immunity. In our preclinical and clinical studies, in situ Ad-REIC gene therapy showed remarkable direct and indirect antitumor effects to realize therapeutic cancer vaccines. We herein aimed to confirm the induction of tumor-associated antigen-specific cytotoxic T lymphocytes (CTLs) by Ad-REIC. Using an ovalbumin (OVA), a tumor-associated antigen, expressing E.G7 tumor-bearing mouse model, we investigated the induction and expansion of OVA-specific CTLs responsible for indirect, systemic effects of Ad-REIC. The intratumoral administration of Ad-REIC mediated clear antitumor effects with the accumulation of OVA-specific CTLs in the tumor tissues and spleen. The CD86-positive dendritic cells (DCs) were upregulated in the tumor draining lymph nodes of Ad-REIC-treated mice. In a dual tumor-bearing mouse model in the left and right back, Ad-REIC injection in one side significantly suppressed the tumor growth on both sides and significant infiltration of OVA-specific CTLs into non-injected tumor was also detected. Consequently, in situ Ad-REIC gene therapy is expected to realize a new-generation cancer vaccine via anticancer immune activation with DC and tumor antigen-specific CTL expansion.

Role of LL-37 in Oral Bacterial DNA Accumulation in Dental Plaque.

Dental plaque, a highly structured polymicrobial biofilm, persistently forms in the oral cavity and is a common problem affecting oral health. The role of oral defense factors in either collaborating or disrupting host-microbiome interactions remains insufficiently elucidated. This study aims to explore the role of LL-37, a critical antimicrobial peptide in the oral cavity, in dental plaque formation. Through immunostaining dental plaque specimens, we observed that LL-37 and DNA colocalized in the samples, appearing as condensed clusters. In vitro experiments revealed that LL-37 binds rapidly to oral bacterial DNA, forming high molecular weight, DNase-resistant complexes. This interaction results in LL-37 losing its inherent antibacterial activity. Further, upon the addition of LL-37, we observed a visible increase in the precipitation of bacterial DNA. We also discovered a significant correlation between the levels of the DNA-LL-37 complex and LL-37 within dental plaque specimens, demonstrating the ubiquity of the complex within the biofilm. By using immunostaining on dental plaque specimens, we could determine that the DNA-LL-37 complex was present as condensed clusters and small bacterial cell-like structures. This suggests that LL-37 immediately associates with the released bacterial DNA to form complexes that subsequently diffuse. We also demonstrated that the complexes exhibited similar Toll-like receptor 9-stimulating activities across different bacterial species, including Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia, and Streptococcus salivarius. However, these complexes prompted dissimilar activities, such as the production of IL-1ß in monocytic cells via both NLRP3 pathway-dependent and pathway-independent mechanisms. This study, therefore, reveals the adverse role of LL-37 in dental plaque, where it binds bacterial DNA to form complexes that may precipitate to behave like an extracellular matrix. Furthermore, the unveiled stimulating properties and species-dependent activities of the oral bacterial DNA-LL-37 complexes enrich our understanding of dental plaque pathogenicity and periodontal innate immune responses.

Fine needle aspiration using forward-viewing endoscopic ultrasonography.

BACKGROUND AND STUDY AIM: A prototype forward-viewing instrument has been developed for therapeutic endoscopic ultrasound (EUS)-guided fine needle aspiration (FNA). We had the opportunity to use this forward-viewing echo endoscope and to study its clinical usefulness, mainly for diagnostic EUS-FNA. PATIENTS AND METHODS: The prototype forward-viewing echo endoscope was used for 15 months between November 2006 and March 2010, in a study group comprising 47 consecutive patients. Diagnostic EUS-FNA was done in 38 patients and the diagnostic accuracy of the forward-viewing device was compared with that from an oblique-viewing echo endoscope in reference patients who were matched by disease and puncture route. Therapeutic EUS was done in nine patients (pseudocyst drainage in six; celiac ganglia neurolysis, biliary drainage, and pancreatic duct drainage in one each). RESULTS: Diagnostic EUS-FNA provided a correct diagnosis in 97.4 % (37/38 patients), which was not significantly different from the 94.7 % (36/38) in the reference patients. Lesions considered difficult to access with an oblique-viewing scope, such as those located at the fornix, or the head of the pancreas, or associated with strictures, were easily punctured, as were those located at the body or tail of the pancreas or at the porta hepatis. Treatment was successful in all nine patients who underwent therapeutic EUS procedures. None of the 47 patients had any complications. CONCLUSIONS: A forward-viewing echo endoscope that allows target sites to be punctured more perpendicularly with minimal effort, can be used for diagnostic EUS-FNA and this may be advantageous, depending on the site of target lesions.

Redirection of tumor metastasis by expression of E-selectin in vivo.

The selectin class of adhesion molecules plays a critical role in facilitating leukocyte adhesion to and subsequent transmigration of endothelium. On this basis, selectins have been suggested to promote tumor cell attachment to endothelium, thereby facilitating metastasis of certain types of tumors, although direct evidence for such a role is lacking. To explore this hypothesis, two sets of transgenic mice were developed: TgnES, which constitutively expresses cell surface E-selectin in all tissues, under the control of the beta-actin promoter; and TgnEsol, which expresses truncated, soluble E-selectin in the liver, under the control of the alpha 1 antitrypsin promoter. B16F10 melanoma cells were stably transfected with alpha(1,3/1,4) fucosyltransferase-specific cDNA (B16F10ft), allowing them to express E-selectin ligands or with hygromycin resistance selection vector only B16F10hygro). Normal mice injected with B16F10ft and B16F10hygro and transgenic mice injected with B16F10hygro developed lung tumors exclusively. In contrast, TgnES mice injected with B16F10ft cells developed massive infiltrating liver tumors. B16F10ft cells injected into TgnEsol mice also formed liver tumors, but these grew more slowly, with a well-delineated, noninfiltrating distinct histologic pattern. These observations provide direct evidence that expression of E-selectin can redirect metastasis of tumor cells expressing appropriate ligands in vivo.

Two missense mutations in SLC26A4 gene: a molecular and functional study.

Mutations in the SLC26A4 gene encoding pendrin, an anion transporter, are responsible for non-syndromic hearing loss (HL) (DFNB4) and Pendred syndrome (PS). PS is a genetic disorder that causes early HL and affects the thyroid gland. Here, we report eight Tunisian families affected with profound HL. Clinical investigations revealed goiter in few patients. Genotyping using microsatellite makers showed linkage to SLC26A4, and missense mutations p.L445W and p.M147T were identified by sequencing and polymerase chain reaction-restriction fragment length polymorphism. The p.L445W mutation segregated in seven families and haplotype analysis suggested its founder effect. In order to understand the molecular pathogenic mechanisms of p.L445W and p.M147T mutations, SLC26A4 wild-type and mutant cDNA constructs were transiently expressed in COS7 cells and several human cell lines including Thyroid 8305C cells. Reverse transcription-PCR, western blot and immunofluorescence demonstrated that these two mutations abolished complex glycosylation of pendrin and prevented its targeting to the plasma membrane.

A novel orexin antagonist from a natural plant was discovered using zebrafish behavioural analysis.

OBJECTIVE: Phenotypic screening is one of the most practical approaches to the identification of mediators of behaviour, since it is difficult to model brain function in vitro, at a cellular level. We used a zebrafish (Danio rerio) behavioural assay to discover novel, natural, neuroactive compounds. MATERIALS AND METHODS: A zebrafish behavioural assay was performed for seven natural compounds, obtained from plants. The behavioural profiles were compared to those of known psychoactive drugs. We characterised a natural compound exhibiting a behaviour profile similar to that of suvorexant, using in silico, in vitro and microarray expression analysis. RESULTS: The behavioural analysis performed in this study classified central nervous system drugs according to their mechanism. Zebrafish treated with a natural compound, 8b-(4'-Hydroxytigloyloxy) costunolide (8b), showed behaviour profiles similar to those of zebrafish treated with suvorexant, a known orexin antagonist. This behavioural assay was validated using in silico and in vitro assays, which revealed that the new compound was a dual orexin receptor antagonist. In addition, transcriptome analysis suggested that 8b might regulate the nuclear factor-κB (NF-κB) related pathway. CONCLUSIONS: We conclude that zebrafish phenotypic screening, combined with in silico assays and gene expression profiling, is a useful strategy to discover and characterize novel therapeutic compounds, including natural products.

Mir142 loss unlocks IDH2R140-dependent leukemogenesis through antagonistic regulation of HOX genes.

AML is a genetically heterogeneous disease and understanding how different co-occurring mutations cooperate to drive leukemogenesis will be crucial for improving diagnostic and therapeutic options for patients. MIR142 mutations have been recurrently detected in IDH-mutated AML samples. Here, we have used a mouse model to investigate the interaction between these two mutations and demonstrate a striking synergy between Mir142 loss-of-function and IDH2R140Q, with only recipients of double mutant cells succumbing to leukemia. Transcriptomic analysis of the non-leukemic single and leukemic double mutant progenitors, isolated from these mice, suggested a novel mechanism of cooperation whereby Mir142 loss-of-function counteracts aberrant silencing of Hoxa cluster genes by IDH2R140Q. Our analysis suggests that IDH2R140Q is an incoherent oncogene, with both positive and negative impacts on leukemogenesis, which requires the action of cooperating mutations to alleviate repression of Hoxa genes in order to advance to leukemia. This model, therefore, provides a compelling rationale for understanding how different mutations cooperate to drive leukemogenesis and the context-dependent effects of oncogenic mutations.

Relationship between the MRI and EMG measurements.

The purpose of this study was to investigate the effect of intensive eccentric exercise on hamstring muscles by using magnetic resonance imaging (MRI) and to elucidate the relationships between the changes in the electromyographic (EMG) parameters and in the transverse relaxation time (T2) of the hamstring muscles. Seven male volunteers performed eccentric knee flexion exercise, and the EMG activity of the hamstring muscles was simultaneously measured. Before and immediately after the exercise, the maximum isometric knee flexion torque was measured and MR images of the hamstring muscles were obtained. For all hamstring muscles, the EMG activity of the fifth set was significantly lower than that of the first set. For each subject, a significant correlation was detected between the percentage change in the value of the post-exercise T2 value and those of EMG signals during the exercise only for the semitendinosus (ST) muscle and not for the biceps femoris (BF) and the semimembranosus (SM) muscles. These results suggested that the EMG-activity reductions in the BF, ST, and SM muscles were due to neuromuscular fatigue, and moreover the reduction in the ST muscle was due to a failure in the E-C coupling, which was caused by excessive muscle-fiber damage.

Expression of Tumour Endothelial Marker 8 in Canine Mammary Gland Tumour Cells.

The aim of this study was to investigate the expression of tumour endothelial marker 8 (TEM8) in canine mammary gland tumours (MGTs) by immunohistochemistry and to evaluate the association between tumour cell TEM8 expression and tumour histological features, histological grades and expression of luminal and basal/myoepithelial cell markers. TEM8 expression was detected in >60 % of neoplastic epithelial cells in all simple adenomas (n = 25), simple carcinomas (n = 43) and invasive micropapillary carcinomas (n = 5) studied. Six of the 18 solid carcinomas studied showed TEM8 expression in >60% of carcinoma cells present in solid structures and in 12 of the 18 solid carcinomas, <30% of the luminal structure-forming carcinoma cells showed TEM8 expression. TEM8 expression in the neoplastic cells was not associated with histological malignancy in canine MGTs. TEM8+ tumour cells frequently showed the luminal-like phenotype cytokeratin (CK)19+/p63-/α-smooth muscle actin (SMA)-, while most TEM8- tumour cells exhibited the basal-like phenotype CK19-/p63+/αSMA-. These findings indicate that TEM8 may be involved in maintaining the characteristics of luminal cells in canine MGTs and that TEM8 would be useful in identifying the type of neoplastic epithelial cell in MGTs.

Percutaneous thermal ablation for renal cell carcinoma in patients with Birt-Hogg-Dubé syndrome.

PURPOSE: The purpose of this study was to analyze the outcome of patients with Birt-Hogg-Dubé (BHD) syndrome who underwent percutaneous thermal ablation of renal cell carcinoma (RCC). MATERIALS AND METHODS: Six patients with genetically proven BHD syndrome who underwent one or more sessions of percutaneous thermal ablation for the treatment of RCC were included. There were 4 men and 2 women, with a mean age of 57.3±7.5 [SD] years (range: 44-67years). A total of 29 RCCs (1-16 tumors per patient) were treated during 20 thermal ablation sessions (7 with radiofrequency ablation and 13 with cryoablation). Outcomes of thermal ablation therapy were assessed, including technical success, adverse events, local tumor progression, development of metastases, survival after thermal ablation, and changes in renal function. RESULTS: Technical success was achieved in all ablation sessions (success rate, 100%). No grade 4 or 5 adverse events were observed. All patients were alive with no distant metastasis during a median follow-up period of 54months (range: 6-173months). No local tumor progression was found. The mean decrease in estimated glomerular filtration rate during follow-up was 10.7mL/min/1.73m2. No patients required dialysis or renal transplantation. CONCLUSION: Radiofrequency ablation and cryoablation show promising results for the treatment of RCCs associated with BHD syndrome. Percutaneous thermal ablation may be a useful treatment option for this rare hereditary condition.

Interleukin 3 as a trophic factor for central cholinergic neurons in vitro and in vivo.

We have found that interleukin 3 (IL-3), a growth factor for hematopoietic cells, is a novel trophic factor for mouse and rat central cholinergic neurons. It enhanced neurite outgrowth and elevated choline acetyltransferase activity. The effect seems to be specific for cholinergic neurons, since somatostatin release and glutamic acid decarboxylase and 2',3'-cyclic nucleotide 3'-phosphodiesterase activities were not significantly influenced by IL-3. In vivo, IL-3 was infused into the lateral ventricles of rats after unilateral axotomy of the septohippocampal pathways. Two weeks later, the IL-3-treated animals showed significant numbers of acetylcholinesterase-positive neurons remaining in the septal region.

Acute Inflammatory Syndrome Paradoxically Induced by De Novo Purine Inhibitors Synthesis Before Renal Transplantation: A Case Report and Review of the Literature.

BACKGROUND: Mycophenolate mofetil (MMF) and mizoribine (MZR) are increasingly used as immunosuppressive agents for organ transplantation and chronic inflammation. We report a patient with rheumatoid arthritis who had an acute inflammatory syndrome triggered by preoperative immunosuppression therapy with both MMF and MZR. CASE REPORT: A 41-year-old woman with IgA nephropathy was referred to our department for living donor renal transplantation. She had rheumatoid arthritis that was adequately treated with prednisolone 5 mg once a day and salazosulfapyridine 2000 mg once a day. MMF 1000 mg twice a day was started for desensitization therapy. Three days later, the patient developed arthritis in the joints of her left hand and elevated inflammatory markers. On day 7, MMF was switched to MZR 150 mg 3 times a day. However, the symptoms extended to both shoulders and the joints of the right foot; MZR was discontinued. The arthritis and inflammatory markers improved. Two months later, the patient was rechallenged with MMF followed by MZR, resulting in a similar clinical course as previously. Tacrolimus (TAC) 3 mg twice a day and everolimus (EVL) 0.5 mg twice a day were introduced as alternative immunosuppressant therapies. No arthritis occurred. ABO-compatible living donor renal transplantation was successfully performed. The patient received TAC, EVL, prednisolone, rituximab, and basiliximab, and her postoperative course was uneventful without arthritis or rejection. At 9 months postoperatively, the serum creatinine was 0.79 mg/dL. CONCLUSIONS: Acute inflammatory syndrome is an extremely rare complication triggered by preoperative immunosuppression therapy. If antimetabolites cannot be used in immunologically high-risk patients, transplantation becomes very difficult. Clinicians should keep in mind this paradoxical reaction.

Identification and characterization of XPC-binding domain of hHR23B.

hHR23B was originally isolated as a component of a protein complex that specifically complements nucleotide excision repair (NER) defects of xeroderma pigmentosum group C cell extracts in vitro and was identified as one of two human homologs of the Saccharomyces cerevisiae NER gene product Rad23. Recombinant hHR23B has previously been shown to significantly stimulate the NER activity of recombinant human XPC protein (rhXPC). In this study we identify and functionally characterize the XPC-binding domain of hHR23B protein. We prepared various internal as well as terminal deletion products of hHR23B protein in a His-tagged form and examined their binding with rhXPC by using nickel-chelating Sepharose. We demonstrate that a domain covering 56 amino acids of hHR23B is required for binding to rhXPC as well as for stimulation of in vitro NER reactions. Interestingly, a small polypeptide corresponding to the XPC-binding domain is sufficient to exert stimulation of XPC NER activity. Comparison with known crystal structures and analysis with secondary structure programs provided strong indications that the binding domain has a predominantly amphipathic alpha-helical character, consistent with evidence that the affinity with XPC is based on hydrophobic interactions. Our work shows that binding to XPC alone is required and sufficient for the role of hHR23B in in vitro NER but does not rule out the possibility that the protein has additional functions in vivo.

Adenovirus vector carrying REIC/DKK-3 gene: neoadjuvant intraprostatic injection for high-risk localized prostate cancer undergoing radical prostatectomy.

As the First-In-Human study of in situ gene therapy using an adenovirus vector carrying the human REIC (reduced expression in immortalized cell)/Dkk-3 gene (Ad-REIC), we conducted neoadjuvant intraprostatic injections in patients with high-risk localized prostate cancer undergoing radical prostatectomy (RP). Patients with recurrence probability of 35% or more within 5 years following RP, as calculated by Kattan's nomogram, were enrolled. Patients received two ultrasound-guided intratumoral injections at 2-week intervals, followed by RP 6 weeks after the second injection. After confirming the safety of the therapeutic interventions with initially planned three escalating doses of 1.0 × 1010, 1.0 × 1011 and 1.0 × 1012 viral particles (vp) in 1.0-1.2 ml (n=3, 3 and 6), an additional higher dose of 3.0 × 1012 vp in 3.6 ml (n=6) was further studied. All four DLs including the additional dose level-4 (DL-4) were feasible with no adverse events, except for grade 1 or 2 transient fever. Laboratory toxicities were grade 1 or 2 elevated aspartate transaminase/alanine transaminase (n=4). Regarding antitumor activities, cytopathic effects (tumor degeneration with cytolysis and pyknosis) and remarkable tumor-infiltrating lymphocytes in the targeted tumor areas were detected in a clear dose-dependent manner. Consequently, biochemical recurrence-free survival in DL-4 was significantly more favorable than in patient groups DL-1+2+3.

The hydrophobic region of signal peptides is involved in the interaction with membrane-bound SecA.

The positive charges of signal peptides are important for the interaction with SecA, a translocation ATPase. To examine whether or not the hydrophobic region of signal peptides also interacts with SecA, we constructed model preproteins, proOmpF-Lpps, possessing no positively charged amino acid residues at the amino-terminus and different numbers of alanine/leucine residues in the hydrophobic region of signal peptides. When the hydrophobic stretch was sufficiently long, amino-terminal positively charged residues were not required for the translocation of preproteins across the cytoplasmic membrane of Escherichia coli both in vitro and in vivo. Chemical cross-linking between SecA and preproteins possessing no positively charged residues at the amino-terminus was observed only in the presence of liposomes containing acidic phospholipids. The degree of cross-linking increased as the length of the hydrophobic stretch increased irrespective of whether positively charged residues were present or not. A preprotein possessing no positively charged residues at the amino-terminus, which is competent in the presence of liposomes, competitively inhibited the cross-linking of wild-type proOmpF-Lpp with SecA under the same conditions. It is concluded that both the amino-terminal positive charges and central hydrophobic domains are involved in the interaction with SecA in the initial stage of translocation in addition to their possible roles in transmembrane movement of preproteins.

alpha- and beta-adrenergic pathways differentially regulate cell type-specific apoptosis in rat cardiac myocytes.

BACKGROUND: The apoptosis of cardiac myocytes may play a role in the development of heart failure. Norepinephrine is one of the factors activated in heart failure and can induce myocardial cell apoptosis in culture. However, it is unknown if alpha- and beta-adrenergic pathways coordinately or differentially regulate apoptosis and if this apoptotic pathway uses common or cell type-specific apoptotic signals. METHODS AND RESULTS: We stimulated cultured neonatal rat cardiac myocytes with an alpha(1)-adrenergic agonist (PE, phenylephrine), a beta-adrenergic agonist (isoproterenol [Iso]) or a membrane-permeable cAMP analogue (8-Br-cAMP) in serum-free conditions for 48 hours. Iso and 8-Br-cAMP markedly increased the number of TUNEL-positive cells (%TUNEL-positive nuclei >40%) compared with saline stimulation (<10%). DNA fragmentation was also confirmed by ladder formation in agarose gels. Apoptotic myocytes were characterized by cell shrinkage and nuclear condensation, consistent with morphological features of apoptosis. The Iso-induced apoptosis was almost completely inhibited by the protein kinase A-specific inhibitor KT5720. In contrast, PE inhibited 8-Br-cAMP-induced myocardial cell apoptosis. The apoptosis-inhibitory effect by PE was negated by the alpha(1)-adrenergic receptor antagonist prazosin and the MEK-1-specific inhibitor PD098059. Interestingly, although 8-Br-cAMP markedly induced apoptosis in cardiac myocytes, it completely blocked serum depletion-induced apoptosis in PC12 cells, a rat pheochromocytoma cell line. CONCLUSIONS: These findings indicate that alpha- and beta-adrenergic pathways differentially regulate myocardial cell apoptosis. The results also suggest that a cAMP- protein kinase A pathway is necessary and sufficient for beta-adrenergic agonist-induced apoptosis and that this apoptotic pathway is not functional in other cell types, for example, PC12 cells.

The role of endothelin-converting enzyme-1 in the development of alpha1-adrenergic-stimulated hypertrophy in cultured neonatal rat cardiac myocytes.

BACKGROUND: Accumulating evidence suggests that the local synthesis of endothelin-1 (ET-1) plays a role in the development of heart failure in vivo. We investigated the role of endothelin-converting enzyme-1 (ECE-1), which mediates the conversion of big ET-1 to mature ET-1, in the development of alpha1-adrenergic-stimulated hypertrophy in cultured neonatal rat cardiac myocytes. METHODS AND RESULTS: Phenylephrine (PE) induced the expression of ET-1 in rat cardiac myocytes and accelerated the conversion of big ET-1 to ET-1. The ECE-1 mRNA levels were markedly increased 3 hours after PE stimulation (3.6-fold compared with saline stimulation, P<0.005). A specific ECE-1 antagonist, FR901533, inhibited the PE-stimulated increase in protein synthesis rate by 45% (P<0.05). As genetic markers for the hypertrophic response, FR901533 inhibited the PE-stimulated transcriptional activities of the 3.5-kb beta-myosin heavy chain promoter by 79% (P<0.01) but did not affect that of the 3.4-kb atrial natriuretic factor (ANF) promoter. In Bio14.6 Syrian cardiomyopathic hamsters, ventricular ET-1 and ANF mRNA levels did not correlate at 2 different stages. CONCLUSIONS: ET-1-independent pathways may mediate activation of the ANF gene program in ventricular myocytes both in vitro and in vivo. These results also indicate that the conversion of big ET-1 to ET-1 in rat cardiac myocytes is required for the development of alpha1-adrenergic-stimulated hypertrophy and beta-myosin heavy chain gene transcription.