Clinical practice pattern in patients with advanced urothelial cancer who had progressed on pembrolizumab in the pre-enfortumab vedotin era.

OBJECTIVES: Pembrolizumab, an anti-PD-1 monoclonal antibody, revolutionized the treatment for advanced urothelial carcinoma. However, the standard treatment for patients after disease progression with pembrolizumab had not been established until the recent approval of enfortumab vedotin. We analyzed the treatment of these patients in the real world, and the patient background and outcomes. METHODS: We extracted data from 543 patients who experienced progressive disease after pembrolizumab initiation from a Japanese nation-wide cohort of platinum-refractory, metastatic urothelial carcinoma. RESULTS: The median overall survival of the 543 patients was 3.5 months (95% confidence interval 3.0-4.1). Of these, only 20.6% (n = 112) received chemotherapy as a subsequent systemic treatment after progressive disease. The regimen of chemotherapy was very diverse. The median overall survival was 11.9 months (95% confidence interval 9.2-14.7) for patients who received chemotherapy, compared to 2.4 months for those who did not receive chemotherapy (95% confidence interval 2.1-2.9; P < 0.0001). Patients who received subsequent chemotherapy were more likely to have better performance status, neutrophil-to-lymphocyte ratio <3, hemoglobin >11 mg/dL, and history of a single chemotherapeutic regimen at pembrolizumab initiation. CONCLUSIONS: This report highlights the real-world practice of the management after pembrolizumab treatment failure in the pre-enfortumab vedotin era, characterized by infrequent use of subsequent anticancer therapy comprising various regimens, reflecting the lack of a standard treatment. Clinical introduction of enfortumab vedotin is expected to improve treatment outcomes in this setting. The present study will provide important baseline data for evaluating the influence of enfortumab vedotin on clinical practices and outcomes.

[Evaluation of Patients with Localized Prostate Cancer in whom Long-Term Successful Androgen Deprivation Therapy was Ceased].

We evaluated the clinical course of patients with localized prostate cancer in whom long-term successful androgen deprivation therapy (ADT) was ceased. Study subjects were 24 patients with stage B prostate cancer who were initially treated with ADT for a median duration of 93 months. The median age at the cessation of ADT was 84 years. The median nadir serum prostate specific antigen (PSA) level was 0.022 ng/ml. The median duration of follow-up from the cessation of ADT was 31 months. During follow-up, five patients showed PSA elevation of ≥2 ng/ml from the nadir. Serum testosterone level was tested in 20 patients, and five showed testosterone recovery ≥0.5 ng/ml. Seven patients died from diseases other than prostate cancer, but there were no deaths caused by prostate cancer. This study demonstrated that long-term successful ADT for localized prostate cancer could be ceased with adequate follow-up evaluation.

Impact of the objective response to and number of cycles of platinum-based first-line chemotherapy for metastatic urothelial carcinoma on overall survival of patients treated with pembrolizumab.

OBJECTIVES: To investigate the impact of the number of cycles and objective response to chemotherapy on overall survival in patients with metastatic urothelial carcinoma treated with pembrolizumab. METHODS: This multicenter, retrospective study included 755 patients from 59 institutions with advanced, chemoresistant urothelial carcinoma who received pembrolizumab. The associations of the overall survival with the number of cycles and best objective response were investigated using Cox multiple regression analysis. RESULTS: Overall, 391 patients received standard first-line chemotherapy and pembrolizumab as a second-line treatment, and were included in the final analysis. Of the 391 patients, 185 received less than four cycles, 134 received four to six cycles and 72 received more than six cycles of first-line chemotherapy. An objective response (complete or partial response) to chemotherapy was observed in 145 patients (37.1%). Univariate analysis showed that the overall survival of patients who received more than six cycles or responded to chemotherapy (complete or partial response) was significantly longer than that of patients who received less than four cycles or did not respond to chemotherapy (stable or progressive disease). At multivariate levels, no correlations were observed between overall survival and the number of cycles of or the response to chemotherapy. CONCLUSIONS: Therapeutic benefit of pembrolizumab can be expected irrespective of the objective response to and number of cycles of platinum-based first-line chemotherapy.

[Two Cases of Renal Pelvic Rupture Due to Trauma in a Patient with Ureteropelvic Junction Stenosis].

A pathologic kidney with hydronephrosis is prone to rupture after minor trauma to the renal pelvis ; however, it is controversial whether drainage, such as nephrostomy and ureteral stenting, should be performed in this setting. Herein we report traumatic rupture of the renal pelvis in 2 patients with ureteropelvic junction stenosis at two centers. Case 1 : A 15-year-old boy sustained a blunt injury on his left back while playing football. His family physician referred the patient to our hospital for suspected left renal injury. Contrast-enhanced abdominal computed tomography showed left hydronephrosis and fluid accumulation in the left retroperitoneal space, which led to a diagnosis of rupture of the left renal pelvis. The patient was treated conservatively ; however, fluid accumulation around the kidney worsened. A ureteral stent was placed, and the patient's renal colic and imaging findings improved. Case 2 : A 13-yearold boy fell and bruised his abdomen while playing soccer. He was unable to walk because of pain, and was brought to our hospital by ambulance. Contrast-enhanced abdominal computed tomography showed left hydronephrosis and fluid accumulation in the left retroperitoneal space, which led to a diagnosis of rupture of the left renal pelvis. A ureteral stent was placed on the same day, and the patient's renal colic improved.

[A Case of Castration-Resistant Prostate Cancer after Prostatectomy Treated with Resection of A Solitary Metastatic Site].

A 72-year-old man underwent a bilateral nerve-sparing radical retropubic prostatectomy (RRP) with pelvic lymph node dissection 11 years earlier. His prostate specific antigen (PSA) value at diagnosis was 61.024 ng/ml. Bone scans and computed tomographic (CT) scans showed no metastasis. Pathological findings and stage were adenocarcinoma, Gleason score 4+3, positive surgical margin, pT3b, and no lymph node metastasis. The postoperative PSA nadir was 0.218 ng/ml, and salvage radiotherapy (SRT, total 66 Gy) was performed six months after RRP. However, the PSA value continued to rise after SRT. Therefore, androgen deprivation therapy (ADT) was started 11 months after SRT. Nine years postoperatively, the PSA value showed a continuous rise despite ADT, and prostate cancer was considered to be castration-resistant. Although he was taking enzalutamide, his PSA value rose to 18. 271 ng/ml. Repeated bone scans and CT scans were negative. Eleven years after RRP, the fluoro-2-deoxy-D-glucosepositronemissiontomography (FDG-PET) revealed a nodule lesiondorsal to the left pubic bone. The patient underwent a resection of the lesion. Three months after the resection, his PSA level declined to 0.038 ng/ml, thus ADT was discontinued. Thirteen months after the resection, PSA re-elevation was absent, and follow-up without ADT is ongoing.

[A Case of Emphysematous Pyelonephritis Complicated by Bacterial Endophthalmitis in which Multidisciplinary Therapy Led to Preservation of the Kidney].

A 49-year-old male with untreated diabetes mellitus type 2 experienced eyesight deterioration and general malaise, and was treated for uveitis and orbital cellulitis. Later, he was taken to a local hospital via ambulance for a consciousness disorder and was diagnosed with bilateral infectious endophthalmitis, a right ureteral stone, and emphysematous pyelonephritis. He was then referred to our hospital for further examination and treatment. We immediately initiated intravenous antibiotic therapy with meropenem and glycemic control with continuous subcutaneous insulin infusion, and placed a ureteral and percutaneous drain tube into the right ureter and the emphysema, respectively. We performed a diagnostic and therapeutic vitrectomy on the patient's left eye. Urinary, blood, and vitreous cultures were positive for Klebsiella aerogenes. Abdominal contrast-enhanced computed tomography showed bilaterally comparable renal contrast enhancement. On the 60th hospital day, we performed endoscopic combined intrarenal surgery (ECIRS) and completely removed the urinary stone. Although he lost light sensitivity in his right eye, his left eyesight improved, and his blood glucose level was adequately managed by oral medication. Three months after the surgery, he was discharged from our hospital and he showed no sign of recurrence of the infection at ten months after surgery.

Pembrolizumab for treating advanced urothelial carcinoma in patients with impaired performance status: Analysis of a Japanese nationwide cohort.

BACKGROUND: The benefits of pembrolizumab in patients with advanced urothelial carcinoma (UC) and impaired performance status (PS) remain unknown. This study assessed the safety and efficacy of pembrolizumab in patients with platinum-refractory UC and Eastern Cooperative Oncology Group PS ≥2 to identify which subgroups may benefit from this drug. METHODS: This retrospective nationwide cohort study collected clinicopathological information for 755 patients from 59 institutions. The overall response rate (ORR) and overall survival (OS) were compared among the patients with PS 0-1, 2, and 3-4. Multivariate analysis was conducted to identify factors predicting OS in patients with PS ≥2. RESULTS: The numbers of patients with PS 0-1, 2, and 3-4 were 602, 98, and 55, respectively; the ORRs in these groups were 29.5, 15.3, and 9.1%, respectively, and the median OS times were 14.3, 3.1, and 2.4 months, respectively. In multivariate Cox regression analysis, a neutrophil-lymphocyte ratio (NLR) ≥3.5 (hazard ratio [HR] = 1.897) and liver metastasis (HR = 2.072) were associated with OS in the PS ≥2 subgroup. The median OS of patients with PS ≥2 without either risk factor was 6.8 months, compared with 3.1 months for patients with one risk factor and 2.3 months for patients with both risk factors. CONCLUSIONS: PS ≥2 portended worse ORR and OS than PS ≤1 despite a comparable safety profile. Among the patients with impaired PS, patients with NLR <3.5 and no liver metastasis may most greatly benefit from pembrolizumab therapy.

Predictive factors and radiological findings of adrenohepatic adhesion during laparoscopic adrenalectomy.

Purpose: This retrospective study aimed to identify predictive factors and imaging features of adrenohepatic adhesion found during laparoscopic right adrenalectomy. Materials and Methods: Altogether, 77 patients underwent laparoscopic right adrenalectomy between January 2005 and December 2018. Adrenohepatic adhesion was defined as strict adhesion that required either partial adrenalectomy with coagulation of residual tissue or partial hepatectomy to accomplish complete resection. We assessed their surgical video records to determine if adrenohepatic adhesion was present. Age, sex, body mass index, tumor size, tumor diagnosis and radiological findings (attachment between the liver and the adrenal gland, diameters of the right and left adrenal veins and its ratio) were evaluated as preoperative variables. Results: Adrenohepatic adhesion was present in 11 of the 77 patients (14.3%). Age, sex, and body mass index were not statistically significant factors. Tumor size was significantly small in adhesion group (14.2 mm vs. 25.9 mm, p=0.02). Attachment to the liver and adrenal gland was frequently seen regardless of the adhesion. The mean right/left adrenal veins diameters ratio was significantly lower in the adhesion group (0.8 vs. 1.1, p=0.01). Multivariate logistic regression analysis demonstrated the right/left adrenal veins diameters ratio was the only significant predictor of adhesion. The sensitivity, specificity, negative predictive value and positive predictive value were 0.82, 0.76, 0.43, and 0.95 respectively when the optimal cutoff value for the ratio was 0.9 (area under the curve, 0.75; 95% confidence interval, 0.60-0.90). Conclusions: The right/left adrenal veins diameters ratio was possible predictor of adrenohepatic adhesion.

Proteinase-activated receptor 4 stimulation-induced epithelial-mesenchymal transition in alveolar epithelial cells.

BACKGROUND: Proteinase-activated receptors (PARs; PAR1-4) that can be activated by serine proteinases such as thrombin and neutrophil catepsin G are known to contribute to the pathogenesis of various pulmonary diseases including fibrosis. Among these PARs, especially PAR4, a newly identified subtype, is highly expressed in the lung. Here, we examined whether PAR4 stimulation plays a role in the formation of fibrotic response in the lung, through alveolar epithelial-mesenchymal transition (EMT) which contributes to the increase in myofibroblast population. METHODS: EMT was assessed by measuring the changes in each specific cell markers, E-cadherin for epithelial cell, alpha-smooth muscle actin (alpha-SMA) for myofibroblast, using primary cultured mouse alveolar epithelial cells and human lung carcinoma-derived alveolar epithelial cell line (A549 cells). RESULTS: Stimulation of PAR with thrombin (1 U/ml) or a synthetic PAR4 agonist peptide (AYPGKF-NH2, 100 muM) for 72 h induced morphological changes from cobblestone-like structure to elongated shape in primary cultured alveolar epithelial cells and A549 cells. In immunocytochemical analyses of these cells, such PAR4 stimulation decreased E-cadherin-like immunoreactivity and increased alpha-SMA-like immunoreactivity, as observed with a typical EMT-inducer, tumor growth factor-beta (TGF-beta). Western blot analyses of PAR4-stimulated A549 cells also showed similar changes in expression of these EMT-related marker proteins. Such PAR4-mediated changes were attenuated by inhibitors of epidermal growth factor receptor (EGFR) kinase and Src. PAR4-mediated morphological changes in primary cultured alveolar epithelial cells were reduced in the presence of these inhibitors. PAR4 stimulation increased tyrosine phosphorylated EGFR or tyrosine phosphorylated Src level in A549 cells, and the former response being inhibited by Src inhibitor. CONCLUSION: PAR4 stimulation of alveolar epithelial cells induced epithelial-mesenchymal transition (EMT) as monitored by cell shapes, and epithelial or myofibroblast marker at least partly through EGFR transactivation via receptor-linked Src activation.

Protease-activated receptor 4-mediated Ca2+ signaling in mouse lung alveolar epithelial cells.

Protease-activated receptor (PAR)-4 is a recently identified low-affinity thrombin receptor that plays a pathophysiological role in many types of tissues including the lung. Here, we showed for the first time that PAR4 mRNA and protein are expressed on primary cultured mouse lung alveolar epithelial cells by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemical analyses. In a fura 2-AM-loaded single epithelial cell, stimulation with thrombin (1 U/ml) and a PAR4 agonist peptide (AYPGKF-NH(2), 1-100 microM) increased intracellular Ca(2+) concentration ([Ca(2+)](i)), which consisted of an initial peak phase followed by a slowly decaying delayed phase, while a PAR1 agonist peptide, TFLLR-NH(2) (1-100 microM), induced a transient increase in [Ca(2+)](i). AYPGKF-NH(2) (10 microM)-induced [Ca(2+)](i) response was attenuated by a PAR4 antagonist peptide (tcY-NH(2)), a phospholipase C inhibitor, U-73122 (1-10 microM) or a Ca(2+)-ATPase inhibitor, thapsigargin (1 microM). Removal of extracellular Ca(2+) or an inhibitor of store-operated Ca(2+) entry, trans-resveratrol (1 microM) shortened the time to shut off the Ca(2+) response without any significant effects on the magnitude of the peak [Ca(2+)](i). Thus, stimulation of PAR4 appeared to mobilize Ca(2+) from intracellular stores in the initial peak response and to enhance Ca(2+) entry through the store depletion-operated pathway in the delayed phase. The latter mechanism probably contributed to the longer responsiveness of PAR4 stimulation.

Involvement of Rho signaling in PAR2-mediated regulation of neutrophil adhesion to lung epithelial cells.

Protease-activated receptor 2 (PAR2) has been implicated in the pathogenesis of airway inflammation. We report that epithelial PAR2 stimulation with trypsin (0.05-1 U/ml) or an agonist peptide (SLIGKV-NH2, 1-100 microM) for 0.5-3 h dose- and time-dependently enhanced neutrophil adhesion to alveolar type II epithelial cells (A549 cells) and that this stimulation also induced the formation of epithelial actin filaments. Both responses in neutrophil adhesion and epithelial actin reorganization were reduced by a Rho inhibitor, mevastatin and by a Rho-associated kinase (ROCK) inhibitor, Y-27632 ((R)-(+)-trans-N-(4-Pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide). Neutrophil adherence was also inhibited by an inhibitor of actin polymerization, cytochalasin D and a tyrosine kinase inhibitor, genistein. Further, the PAR2-mediated tyrosine phosphorylation of focal adhesion kinase (FAK), a major cytoskeleton protein, was detected, and this response was inhibited by mevastatin or Y-27632. These results suggest that PAR2 stimulation of alveolar epithelial cells enhances neutrophil adhesion presumably at least in part through Rho/ROCK signal-mediated actin cytoskeleton reorganization associated with the tyrosine phosphorylation of FAK.

Suppression by protease-activated receptor-2 activation of gastric acid secretion in rats.

Activation of protease-activated receptor-2 (PAR-2), a receptor activated by trypsin/tryptase, induces neurally mediated gastric mucus secretion accompanied by mucosal cytoprotection. In the present study, we investigated whether PAR-2 could modulate gastric acid secretion in rats. Messenger RNAs for PAR-2 and PAR-1 were detected in the gastric mucosa and smooth muscle. The PAR-2-activating peptide SLIGRL-NH(2), but not the inactive control peptide, when administered i.v., strongly suppressed gastric acid secretion in response to carbachol, pentagastrin or 2-deoxy-D-glucose in the rats with a pylorus ligation. The PAR-2-mediated suppression of acid secretion was resistant to cyclooxygenase inhibition or ablation of sensory neurons by capsaicin. Our results provide novel evidence that in addition to stimulating neurally mediated mucus secretion, activation of PAR-2 suppresses gastric acid secretion independently of prostanoid production or sensory neurons. These dual actions of PAR-2 would result in gastric mucosal cytoprotection.

Human T-cell leukemia virus type 1 Tax modulates interferon-alpha signal transduction through competitive usage of the coactivator CBP/p300.

We describe here Tax protein of human T-cell leukemia virus type 1 (HTLV-1) as an interferon (IFN)-alpha antagonist counteracting the transactivation function of IFN-stimulated gene factor 3 (ISGF3). Co-expression of Tax, but not the Tax mutant unable to bind to CBP, significantly inhibited the reporter gene expression directed by IFN-stimulated regulatory elements, despite that the formation of DNA-binding ISGF3 complex was unaffected. Gene activation induced by STAT2 transcription domain was also inhibited by expression of Tax. Furthermore, Tax-mediated transcriptional inhibition was reversed by overexpression of p300. These observations indicate that Tax interferes with IFN-alpha-induced JAK-STAT pathway by competition with STAT2 for CBP/p300 binding. Consistently, GST pull-down assay showed that Tax dose-dependently inhibited binding of STAT2 to p300. This study suggests that Tax may prevent IFN-alpha from exerting its antiviral, antiproliferative and proapoptotic effects, thereby contributing to persistent viral infection and HTLV-1-associated oncogenesis.

Helper virus-independent trans-replication of hepatitis C virus-derived minigenome.

We have previously described a synthetic T7-driven cDNA minigenome containing the antisense sequence of luciferase gene and internal ribosome entry site of encephalomyocarditis virus flanked by 5'- and 3'-end sequences of hepatitis C virus (HCV) that contain cis-acting replication elements. Synthesis of minus-strand RNA from the artificial minigenome was determined by using Huh-7 cells harboring autonomously replicating HCV subgenome as a helper for provision of functional replication components. To further confirm and extend these studies, we investigated here whether the minigenome replication system could be reconstituted by transfection of naïve Huh-7 cells with plasmid expressing nonstructural (NS) proteins. Reporter assay and Northern blot analysis revealed that trans-expression of NS proteins from 3 to 5 resulted in high level of luciferase activity and synthesized minus-strand RNA. The analogous result was also obtained with the minigenome derived from HCV 2a, and both HCV 1b- and 2a-derived NS protein were able to support the chimeric minigenomes whose 5'- or 3'-end was replaced by the respective region of the heterologous virus. These results provide a basis for establishing the reverse genetic system that is helpful to study cis- and trans-acting factors involved in HCV RNA replication.

Up-regulation of hepatitis C virus replication by human T cell leukemia virus type I-encoded Tax protein.

Co-infection of hepatitis C virus (HCV) with other blood-borne pathogens such as human T cell leukemia virus (HTLV) is common in highly endemic areas. Clinical evidence showing a correlation between HTLV-I co-infection and rapid progression of HCV-associated liver disease promoted us to investigate the effect of HTLV-I-encoded Tax protein on HCV replication. Reporter assay showed that HCV replicon-encoded luciferase expression was significantly augmented by co-transfection of the Tax-expressing plasmid. Further, HCV RNA replication in replicon cells was increased either by co-culture with cells stably expressing Tax protein (Huhtax) or by culture in the presence of Huhtax-conditioned medium, indicating that Tax could also modulate HCV replication of adjacent cells in a paracrine manner. Additionally, HCV replication in Huhtax exhibited a reduced responsiveness to interferon-alpha-induced antiviral activity. This study demonstrates the facilitation of HCV replication by Tax protein, which may partially account for severer clinical consequences of HCV-related disease in HCV/HTLV co-infected individuals.

Inhibition of hepatitis C virus replication by pol III-directed overexpression of RNA decoys corresponding to stem-loop structures in the NS5B coding region.

Increasing evidence has shown that the stem-loop (SL) structures in the NS5B coding region of hepatitis C virus (HCV) function as cis-replicating elements that are indispensable for viral replication. We have investigated whether small RNA molecules analogous to the SL structures could inhibit HCV replication. Reporter assays showed that both in vitro transcribed and pol III-directed transcripts corresponding to 5BSL3.1 and 5BSL3.2 efficiently inhibited HCV replicon-encoded luciferase expression. Mutagenesis studies revealed that mutation in 5BSL3.2 which debilitated its binding to NS5B also abolished the ability of 5BSL3.2 RNA to inhibit HCV replication, suggesting that SL RNA inhibits HCV by sequestering the replication complex. Further, adenoviral-mediated expression of the SL RNAs potently blocked the replication of HCV replicon in Huh-7 cells. Importantly, SL RNAs derived from HCV 2a, an evolutionarily distant genotype, were also shown to suppress the replication of HCV 1b replicon in spite of the genetic heterogeneity between the SL elements of the two viruses, implying the potential of SL RNA-based approach to inhibit a wide range of HCV isolates. These results suggest that SL RNA decoys may prove to be useful in the treatment of hepatitis C, which may be advantageous over other sequence-specific gene therapy modalities (such as antisense RNA and siRNA) in preventing the escape of genetic variants.

Exploiting cis-acting replication elements to direct hepatitis C virus-dependent transgene expression.

We describe here a novel targeting gene therapy strategy to direct gene expression responsive to hepatitis C virus (HCV). The goal was approached by engineering a construct containing the antisense sequence of the transgene and internal ribosome entry site of encephalomyocarditis virus flanked by 5'- and 3'-end sequences of HCV cDNA that contain cis-acting replication elements. Thus, expression of the transgene is only promoted when the minus-strand RNA has been synthesized by the functional replication machinery present in infected cells. Reporter assay and strand-specific reverse transcription-PCR showed selective transgene expression in Huh-7 cells harboring an autonomously replicating HCV subgenome but remaining silent in uninfected cells. Furthermore, using the cytosine deaminase suicide gene as a transgene coupled with recombinant adenovirus delivery, we demonstrated that cytosine deaminase was specifically expressed in replicon cells, resulting in marked chemosensitization of replicon cells to the cytotoxic effects of flucytosine. This new targeting strategy could be extended to other single-stranded RNA viruses encoding the unique RNA-dependent RNA polymerase that has no parallel in mammalian cells.

Protease-activated receptor-2 (PAR-2)-related peptides induce tear secretion in rats: involvement of PAR-2 and non-PAR-2 mechanisms.

Protease-activated receptor-2 (PAR-2) plays an extensive role in the regulation of digestive exocrine secretion. The present study examined whether PAR-2-related peptides could modulate tear secretion in rats and analyzed the underlying mechanisms. SLIGRL-NH(2), a PAR-2-activating peptide (PAR-2-AP) derived from mouse/rat PAR-2, when administered i.v. in combination with amastatin, an aminopeptidase inhibitor, evoked tear secretion, whereas LRGILS-NH(2), a PAR-2-inactive reversed peptide, had no such effect. In contrast, LSIGRL-NH(2), a partially reversed peptide known to be inactive with PAR-2, caused tear secretion equivalent to the effect of SLIGRL-NH(2). SLIGKV-NH(2), a human-derived PAR-2-AP, also induced significant tear secretion though to a lesser extent, whereas neither VKGILS-NH(2), a reversed peptide, nor LSIGKV-NH(2), a partially reversed peptide, produced any secretion. In desensitization experiments, after the first dose of SLIGRL-NH(2), the second dose of SLIGRL-NH(2) produced no tear secretion, whereas the response to LSIGRL-NH(2) was only partially inhibited by preadministration of SLIGRL-NH(2). Preadministration of LSIGRL-NH(2) abolished the response to subsequently administered LSIGRL-NH(2) but not SLIGRL-NH(2). The tear secretion induced by LSIGRL-NH(2) but not by PAR-2-APs was blocked by atropine or hexamethonium. Mast cell depletion due to repeated doses of compound 48/80 did not alter the effect of SLIGRL-NH(2) or LSIGRL-NH(2). Finally, IGRL-NH(2), a possible core structure of LSIGRL-NH(2), triggered tear secretion in an atropine-reversible manner. Our findings suggest that the PAR-2-APs SLIGRL-NH(2) and SLIGKV-NH(2) cause tear secretion, most likely via PAR-2 and that LSIGRL-NH(2), a PAR-2-inactive peptide, and IGRL-NH(2), its key structure, trigger tear secretion by stimulating parasympathetic nerves via an unidentified target molecule.

Autogenous translational inhibition of core protein: implication for switch from translation to RNA replication in hepatitis C virus.

Positive-stranded viruses use the genomic RNA as a common template for translation and RNA replication which proceed in inverse direction; a certain regulatory mechanism for translation control is probably required to coordinate these two antagonistic processes. Hepatitis C virus (HCV) core protein is a good candidate that might play a role in such a regulation. In this study, we further investigated whether HCV core protein modulates internal ribosome entry site (IRES)-directed translation. The inclusion of the core-coding sequence significantly suppressed translation initiated by HCV IRES in monocistronic and bicistronic reporter systems. The region mainly responsible for this inhibition was mapped to nt 441-473 of the core-coding sequence. This suppression was eliminated by frameshift mutations introduced into this region, suggesting that it is the core protein expressed in cis, rather than the core-coding nucleotide sequence that negatively modulates the efficiency of HCV IRES-dependent translation. Furthermore, the core protein provided in trans also specifically decreased the IRES activity in directing cap-independent translation both in transfected cells and in cell-free translation study. Consistently, a gel mobility shift assay showed a specific interaction between the core protein and HCV IRES-containing RNA transcript. These findings suggest that HCV core protein may down-regulate the cap-independent translation as a regulatory mechanism required for initiation of transcription.

Down-regulation of viral replication by adenoviral-mediated expression of siRNA against cellular cofactors for hepatitis C virus.

Small interfering RNA (siRNA) is currently being evaluated not only as a powerful tool for functional genomics, but also as a potentially promising therapeutic agent for cancer and infectious diseases. Inhibitory effect of siRNA on viral replication has been demonstrated in multiple pathogenic viruses. However, because of the high sequence specificity of siRNA-mediated RNA degradation, antiviral efficacy of siRNA directed to viral genome will be largely limited by emergence of escape variants resistant to siRNA due to high mutation rates of virus, especially RNA viruses such as poliovirus and hepatitis C virus (HCV). To investigate the therapeutic feasibility of siRNAs specific for the putative cellular cofactors for HCV, we constructed adenovirus vectors expressing siRNAs against La, polypyrimidine tract-binding protein (PTB), subunit gamma of human eukaryotic initiation factors 2B (eIF2Bgamma), and human VAMP-associated protein of 33 kDa (hVAP-33). Adenoviral-mediated expression of siRNAs markedly diminished expression of the endogenous genes, and silencing of La, PTB, and hVAP-33 by siRNAs substantially blocked HCV replication in Huh-7 cells. Thus, our studies demonstrate the feasibility and potential of adenoviral-delivered siRNAs specific for cellular cofactors in combating HCV infection, which can be used either alone or in combination with siRNA against viral genome to prevent the escape of mutant variants and provide additive or synergistic anti-HCV effects.