Effect of pirfenidone injection on ferret vocal fold scars: A preliminary in vivo study.

OBJECTIVES: This study examined the antifibrotic effect of pirfenidone (PFD), which has received regulatory approval in the United States and Japan for treatment of idiopathic pulmonary fibrosis, on the scarred ferret vocal fold (VF) in vivo. METHODS: Eight male ferrets were divided into two groups: saline and PFD. All animals underwent unilateral scarring under anesthesia. The right VF was electrocauterized with ablation of the entire lamina propria. PFD (1.0 mg/mL) or saline injections into right-side scarred VFs were performed (under an operating microscope) 4 weeks later. After an additional 4 weeks, the larynges were harvested for histological analysis. Prior to harvesting, the ferrets were re-anesthetized, and the VFs were observed and recorded using a rigid video laryngoscope. We immunohistochemically evaluated the expression of collagen types I and III, alpha-smooth muscle actin (α-SMA), and fibronectin in the entire lamina propria. We compared the affected areas (calculated using ImageJ software) between the treated (right) and untreated (left) sides within the same animals and between groups. RESULTS: Collagen type I (P = 0.0021) and α-SMA (P = 0.0021) expression levels were lower in the PFD group, but the collagen type III and fibronectin levels did not differ significantly between the two groups. CONCLUSION: PFD injection into the scarred VF is a potentially promising novel antifibrotic treatment. LEVEL OF EVIDENCE: NA Laryngoscope, 130:726-731, 2020.

Successful Desensitization with Crizotinib after Crizotinib-induced Liver Injury in ROS1-rearranged Lung Adenocarcinoma.

Crizotinib has been approved for patients with advanced lung adenocarcinoma harboring rearrangements of the c-ROS-1 (ROS1) and anaplastic lymphoma kinase (ALK) genes. We report a patient with ROS1-rearranged lung adenocarcinoma who developed a crizotinib-induced mixed/cholestatic type of liver injury. The patient discontinued crizotinib after 34 days due to liver toxicity. Twenty-four days later, when transaminases and C reactive protein (CRP) were normalized, crizotinib was resumed using an oral desensitization method. The patient was successfully treated for manageable recurrence of liver injury and has been able to continue the treatment.

Transplanting mouse induced pluripotent stem cells into mouse otocysts in vivo.

The otocyst is an attractive target for studying treatment strategies for genetic hearing loss and for understanding inner ear development. We have previously reported that trans-uterine supplemental gene therapy in vivo into the otocysts of mice, which had a loss of function mutation in a causative gene of deafness, was able to prevent putative hearing loss. We herein set out to clarify the feasibility of allogenic cell transplantation into the mouse otocysts in vivo. We transplanted naive mouse-derived induced pluripotent stem cells (miPSCs) into the otocysts of wild type mice or connexin (Cx) 30 deficient mice, at embryonic day 11.5 (E11.5). The transplanted m-iPSCs survived in the lumens of the inner ears at E13.5 and E15.5 in wild type mice. In the Cx30 deficient mouse, the transplanted cells survived similarly, with some of the transplanted cells migrating into the lining cells of the lumens of the inner ears at E13.5 and showing tumorigenic cell proliferation at E15.5. In addition, engrafted cells appear to be able to differentiate after the cell transplantation. Our results suggest that otocyst transplanted cells survived and differentiated. A Cx30 deficiency may facilitate cell migration. These findings may offer some hope for cell transplantation therapy for profound genetic hearing loss caused by a Cxs deficiency.

Protein transduction therapy into cochleae via the round window niche in guinea pigs.

Cell-penetrating peptides (CPPs) are short sequences of amino acids that facilitate the penetration of conjugated cargoes across mammalian cell membranes, and as such, they may provide a safe and effective method for drug delivery to the inner ear. Simple polyarginine peptides have been shown to induce significantly higher cell penetration rates among CPPs. Herein, we show that a peptide consisting of nine arginines ("9R") effectively delivered enhanced green fluorescent protein (EGFP) into guinea pig cochleae via the round window niche without causing any deterioration in auditory function. A second application, 24 hours after the first, prolonged the presence of EGFP. To assess the feasibility of protein transduction using 9R-CPPs via the round window, we used "X-linked inhibitor of apoptosis protein" (XIAP) bonded to a 9R peptide (XIAP-9R). XIAP-9R treatment prior to acoustic trauma significantly reduced putative hearing loss and the number of apoptotic hair cells loss in the cochleae. Thus, the topical application of molecules fused to 9R-CPPs may be a simple and promising strategy for treating inner ear diseases.

Neurogenesis of the spiral ganglion cells in the cochlea requires the transcriptional cofactor TIS21.

The molecular mechanisms controlling the proliferation and differentiation of spiral ganglion cells (SGCs) in the inner ear are still largely unknown. TIS21 is a transcriptional cofactor that shows antiproliferative, antiapoptotic, and prodifferentiative effects on neural progenitor cells. To investigate the function of TIS21 during SGC development, we analyzed SGC neurogenesis from embryonic day 13.5 (E13.5) to postnatal day 4 (P4) in Tis21-GFP knock-in mice, in which the protein-encoding exon of the Tis21 gene was replaced by EGFP. Through E13.5 to P4, we found fewer SGCs in homozygous Tis21-GFP knock-in mice than in wild-type mice. Our results suggest that TIS21 is required for development of SGCs. Deleting Tis21 may affect progenitor cells or neuroblasts at the beginning of cochlear-vestibular ganglion formation and would consequently lead to a decrease in the number of SGCs.

Mouse otocyst transuterine gene transfer restores hearing in mice with connexin 30 deletion-associated hearing loss.

Although numerous causative genes for hereditary hearing loss have been identified, there are no fundamental treatments for this condition. Herein, we describe a novel potential treatment for genetic hearing loss. Because mutations or deletions in the connexin (Cx) genes are common causes of profound congenital hearing loss in both humans and mice, we investigated whether gene supplementation therapy using the wild-type Cx gene could cure hearing loss. We first generated inner ear-specific connexin 30 (Cx30)-deficient mice via the transuterine transfer of Cx30-targeted short hairpin RNA (shRNA-Cx30) into otocysts. The inner ear-specific Cx30-deficient mice mimicked homozygous Cx30-deficient mice both histologically and physiologically. Subsequently, we cotransfected the shRNA-Cx30 and the wild-type Cx30 gene. The cotransfected mice exhibited Cx30 expression in the cochleae and displayed normal auditory functions. Next, we performed the transuterine transfer of the wild-type Cx30 gene into the otocysts of homozygous Cx30-deficient mice, thereby rescuing the lack of Cx30 expression in the cochleae and restoring auditory functioning. These results demonstrate that supplementation therapy with wild-type genes can restore postnatal auditory functioning. Moreover, this is the first report to show that Cx-related genetic hearing loss is treatable by in vivo gene therapy.

Primary duodenal lymphoma: successful rituximab treatment and evaluation by FDG-PET.

A 68-year-old woman has been suffering from chronic inactive hepatitis with hepatitis C virus. Upper endoscopic and pathological findings revealed the typical features of duodenal follicular lymphoma and Helicobacter pylori (H. pylori) infection with her stomach was confirmed by the histological and cultural examinations. No other abnormal findings suspicious for tumor formation were observed by the various examinations such as computed tomography, abdominal ultrasonography and 67Ga-citrate scintigraphy. Primary duodenal follicular lymphoma: stage I according to the Modified Ann Arbor classification was diagnosed by these procedures. The eradication therapy was effective for the withdrawal of H. pylori but not for lymphoma. Four months later, abdominal ultrasonography and magnetic resonance imaging showed the progression of abdominal lymph nodes. Positron emission tomography (PET) with 18F-fluorodeoxyglucose showed radioactive uptake in mesenteric lymph nodes. Systemic chemotherapy using rituximab was started, since these findings suggested the clinical progression from stage I to II2. After 2 courses of the therapy, endoscopic and histological improvement of duodenal lymphoma and lower uptake in mesenteric lymph nodes suggested us complete remission. Rituximab may be useful for duodenal follicular lymphoma and PET with 18F-fluorodeoxyglucose has a potential value for the evaluation of mesenteric lymph nodes spread.

Electron tomography reveals diverse conformations of integrin alphaIIbbeta3 in the active state.

We used electron tomography to determine the three-dimensional (3D) structure of integrin alphaIIbbeta3 in the active state. We found that we obtained better density maps when we reconstructed a 3D volume for each individual particle in the tilt series rather than to extract the particle-containing subvolumes from a 3D reconstruction of the entire specimen area. The 3D tomographic reconstructions of 100 particles revealed that activated alphaIIbbeta3 adopts many different conformations. An average of all the individual 3D reconstructions nicely accommodated the crystal structure of the alphaVbeta3 headpiece, confirming the locations assigned to the alpha- and beta-subunit in the density map. The most striking finding of our study is the structural flexibility of the lower leg of the beta-subunit as opposed to the conformational stability of the leg of the alpha-subunit. The good fit of the atomic structure of the betaI domain and the hybrid domain in the active state showed that the hybrid domain swings out, and most particles used for tomography are in the active state. Multivariate statistical analysis and classification applied to the set of 3D reconstructions revealed that more than 90% reconstructions are grouped into the classes that show the active state. Our results demonstrate that electron tomography can be used to classify complexes with a flexible structure such as integrins.

[Anti-allergic action effect of Pseudolarix amabilis Rehd. extract and its efficacy on atopic dermatitis].

Pseudolarix amabilis Rehd. extract was examined in vitro for antibacterial effects, anti-inflammatory effects, and inhibitory effects on histamine release. Pseudolarix amabilis Rehd. extract was also examined for efficacy on dermatitis in atopic dermatitis model mice (NC mice) and effects on keratinous moisture level and transepidermal water loss in miniature pigs. Pseudolarix amabilis Rehd. extract had antibacterial effects on Staphylococcus aureus, Candida albicans, and Streptococcus pyogenes; however this antibacterial effect varied with the temperature at which and conditions under which Pseudolarix amabilis Rehd. was extracted. Pseudolarix amabilis Rehd. extract at the final concentration of 2 mg/mL significantly inhibited the hyaluronidase activity; and at 0.005, 0.05, and 0.5 mg/mL, it also significantly inhibited the histamine release. In the mice in which atopic dermatitis had been induced, 28-day administration of Pseudolarix amabilis Rehd. extract at 4 and 400 mg/mL significantly inhibited aggravation of dermatitis without having effects on body weight. In the dorsal skin of miniature pigs, Pseudolarix amabilis Rehd. extract at 4 and 400 mg/mL significantly increased keratinous moisture level with the increase in the number of dosing days, and caused no changes in transepidermal water loss. From the above results, it is clear that Pseudolarix amabilis Rehd. extract inhibits both proliferation of bacteria and inflammation caused by antigens. Furthermore, it is suggested that Pseudolarix amabilis Rehd. extract will serve as a medicinal drug which effectively moistens the skin and prevents and heals dermatitis.

Matrix extracellular phosphoglycoprotein (MEPE) is highly expressed in osteocytes in human bone.

The matrix extracellular phosphoglycoprotein (MEPE) gene is highly expressed in tumors that cause oncogenic hypophosphatemic osteomalacia (OHO). MEPE is also known as one of the bone-tooth matrix proteins and is associated with bone mineralization. We developed a rabbit polyclonal antibody directed against recombinant human MEPE (rhMEPE) after cloning its cDNA from the cDNA library of a nasal tumor tissue causing OHO. Using this antibody, we analyzed the distribution of MEPE in human bones by immunohistochemistry. In bone specimens from normal subjects, MEPE was predominantly expressed by osteocytes and not by osteoblasts. In bone specimens from patients with osteomalacia, however, MEPE was focally expressed by deeply located osteocytes. We also compared the MEPE positivity of osteocytes in mineralized bone and non-mineralized osteoid obtained from patients with osteomalacia and osteoporosis. Among osteomalacia patients, MEPE positivity was seen in 87.5 +/- 8.6% of the osteocytes from mineralized bone compared with 7.8 +/- 6.4% of those from osteoid. Among osteoporosis patients, MEPE positivity was found in 95.3 +/- 0.5% of the osteocytes from mineralized bone compared with 4.9 +/- 5.7% of those from osteoid. MEPE was mainly expressed by osteocytes embedded in the matrix of mineralized bone from patients with osteomalacia or osteoporosis. Our data provide the first histological evidence that MEPE is predominantly expressed by osteocytes in human bone, with significant expression by osteocytes within mineralized bone.

Immunohistochemical detection of Fas and apoptosis in type-1 autoimmune hepatitis.

BACKGROUND/AIMS: Although Fas expression has been reported in liver with chronic viral hepatitis and primary biliary cirrhosis, little is known about Fas expression and apoptosis in type-1 autoimmune hepatitis. The aim of this study was to investigate whether the expression of Fas and apoptosis are found in liver with autoimmune hepatitis. The relationship between Fas expression and clinicopathological findings including the occurrence of apoptosis was also investigated. METHODOLOGY: Fas expression and apoptosis were immunohistochemically examined in liver tissues from 20 patients with autoimmune hepatitis and five control subjects using specific antibodies against Fas and single-stranded DNA. The grade of expression of Fas and apoptosis was evaluated and compared with histological findings and the results of liver function tests in each patient. RESULTS: Fas expression in hepatocytes was detected in all patients with autoimmune hepatitis, while Fas expression was not detected in control livers. The Fas-positive hepatocytes were particularly abundant in those areas facing piecemeal and confluent necrosis. In 30% of autoimmune hepatitis cases, bile-duct cells were faintly stained for Fas. A few hepatocytes positive for single-stranded DNA were found in the areas facing piecemeal necrosis and confluent necrosis. In 95% cases, many bile-duct cells were positive for single-stranded DNA. No relationship between the expression of Fas and single-stranded DNA was found in hepatocytes or bile-duct cells. However, the degree of Fas expression in hepatocytes significantly correlated with serum transaminase concentrations and was increased in parallel with the grade of activity but not with the stage of fibrosis. CONCLUSIONS: We have demonstrated that Fas expression is detected in hepatocytes of the liver with autoimmune hepatitis and that the level of Fas expression reflects the severity of inflammation in autoimmune hepatitis.

A new chemotherapeutic regimen for advanced unresectable hepatocellular carcinoma.

BACKGROUND/AIMS: The aim of this study was to evaluate the efficacy and toxicity of a novel chemotherapeutic regimen for advanced unresectable hepatocellular carcinoma. METHODOLOGY: Seventeen patients with unresectable hepatocellular carcinoma were treated by arterial infusion once a week of low-dose cisplatin (12 mg/m2) and doxorubicin (6 mg/m2) via a subcutaneously implanted injection port and by daily oral administration of 300 mg/day of UFT comprising 5-fluorouracil prodrug tegafur (FT) and uracil (U) at a ratio of 1:4. RESULTS: The median number of chemotherapy courses was 13 (range, 5-33). All patients were evaluated for response, toxicity, and survival. As assessed by conventional imaging criteria, there were 3 (17.6%) complete responses with disappearance of the primary tumor, tumor thrombosis of the portal vein and metastatic para-aortic lymph node swelling. In addition, there were 4 (23.5%) partial responses. Among 11 patients who had initially high alpha-fetoprotein levels (> 200 ng/mL), 5 (45%) had a > 50% drop after therapy. The overall tumor response rate (complete response + partial response) was 41% and the median survival was 7.1 (range; 4.2-25.1) months. As for toxicity, there was 1 treatment-related death due to septicemia caused by catheter-related infection. Myelosuppression and renal toxicity was relatively mild and transient. CONCLUSIONS: These results suggest that our low-dose chemotherapeutic regimen may be useful for the treatment of advanced unresectable hepatocellular carcinoma without worsening the quality of life of the patient.

Life-threatening severe immune thrombocytopenia during alpha-interferon therapy for chronic hepatitis C.

Although mild thrombocytopenia is a common adverse effect of interferon therapy, severe life-threatening thrombocytopenia is extremely rare. Here, we report a case of chronic hepatitis C patient that developed severe thrombocytopenia during alpha-interferon therapy, possibly due to an autoimmune mechanism. A 24-year-old female presented chronic hepatitis C in May, 1998. Based on the clinicopathological findings including a liver biopsy, administration of alpha-interferon was begun. In the fourth week of therapy, she experienced mild dyspnea and general fatigue. Complete blood count demonstrated thrombocytopenia (48,000/microL). Despite the immediate withdrawal of interferon, her platelet count further decreased to 1,100/microL. Bone marrow aspirate and elevated platelet-associated IgG antibodies were suggestive of immune thrombocytopenia. She was treated with intravenous and oral administration of steroids. Her platelet count returned to normal level 5 days later. Response to steroid treatment was consistent with the diagnosis of alpha-interferon-induced immune thrombocytopenia in this patient.

Expression of SPARC by activated hepatic stellate cells and its correlation with the stages of fibrogenesis in human chronic hepatitis.

Secreted protein, acidic and rich in cysteine (SPARC), which functions in tissue remodeling, has been reported to be expressed by myofibroblasts in liver cirrhosis and hepatocellular carcinoma. This study aimed to reveal its expression in chronic hepatitis. Immuno-light and electron microscopy demonstrated that SPARC was expressed by nerve fibers and hepatic stellate cells (HSCs) in the liver parenchyma and myofibroblasts in the fibrous septa. Reaction products were localized in the rough endoplasmic reticulum and nuclear envelope. Serial section analysis demonstrated that SPARC, platelet-derived growth factor receptor-beta, and alpha-smooth muscle actin were co-expressed by HSCs. Quantitative analysis demonstrated that, while SPARC-positive HSCs were sparse in control livers, they significantly increased in number in the livers with chronic hepatitis. There were, however, no significant differences in number among the grades of activity, the stages of fibrosis, or etiology (virus-infected or autoimmune, hepatitis B virus or hepatitis C virus). In liver cirrhosis, however, they significantly decreased in number. The present results indicate that SPARC is expressed by activated HSCs in chronic hepatitis, suggesting the involvement of SPARC in hepatic fibrogenesis after chronic injuries.

LIGHT, a member of the tumor necrosis factor ligand superfamily, prevents tumor necrosis factor-alpha-mediated human primary hepatocyte apoptosis, but not Fas-mediated apoptosis.

LIGHT is a member of tumor necrosis factor (TNF) superfamily, and its receptors have been identified as lymphotoxin-beta receptor (LTbetaR) and the herpesvirus entry mediator (HVEM)/ATAR/TR2, both of which lack the cytoplasmic sequence termed the "death domain." The present study has demonstrated that LIGHT inhibits TNFalpha-mediated apoptosis of human primary hepatocytes sensitized by actinomycin D (ActD), but not Fas- or TRAIL-mediated apoptosis. Furthermore, LIGHT does not prevent some cell lines such as HepG2 or HeLa from undergoing ActD/TNFalpha-induced apoptosis. This protective effect requires LIGHT pretreatment at least 3 h prior to ActD sensitization. LIGHT stimulates nuclear factor-kappaB (NF-kappaB)-dependent transcriptional activity in human hepatocytes like TNFalpha. The time course of NF-kappaB activation after LIGHT administration is similar to that of the pretreatment required for the anti-apoptotic effect of LIGHT. LIGHT inhibits caspase-3 processing on the apoptotic protease cascade in TNFalpha-mediated apoptosis but not Fas-mediated apoptosis. In addition, increased caspase-3 and caspase-8 activities in ActD/TNFalpha-treated cells are effectively blocked by LIGHT pretreatment. However, LIGHT does not change the expression of TNFRp55, TNFRp75, and Fas. These results indicate that LIGHT may act as an anti-apoptotic agent against TNFalpha-mediated liver injury by blocking the activation of both caspase-3 and caspase-8.

In situ detection of lipid peroxidation and oxidative DNA damage in non-alcoholic fatty liver diseases.

BACKGROUND/AIMS: Although oxidative stress is an important candidate in the pathogenesis of non-alcoholic fatty liver disease (NAFLD), the localization and pathological significance of oxidative stress-induced cellular damage in NAFLD remains unclear. METHODS: Hepatic expression of 4-hydroxy-2'-nonenal (HNE) and 8-hydroxydeoxyguanosine (8-OHdG), as reliable markers of lipid peroxidation and oxidative DNA damage, respectively, was immunohistochemically investigated in NAFLD and the results were compared with histological findings. RESULTS: While no HNE adducts were observed in control livers, they were frequently detected in NAFLD. In NASH, the localization of the adducts was in the cytoplasm of sinusoidal cells and hepatocytes with a predominance in zone 3. The grade of necro-inflammation as well as the stage of fibrosis significantly correlated with the HNE index. Regarding 8-OHdG, although no 8-OHdG expression was observed in normal liver and only a few in fatty liver, 11 of 17 cases (64.7%) with NASH exhibited nuclear expression of 8-OHdG in hepatocytes and sinusoidal cells in areas of active inflammation. The 8-OHdG index significantly correlated with the grade of necro-inflammation. CONCLUSIONS: Oxidative cellular damage occurs frequently in livers with NAFLD and may be associated with some clinico-pathological features of NAFLD including liver fibrosis and possibly, hepatocarcinogenesis.

Isolation and identification of EG-VEGF/prokineticins as cognate ligands for two orphan G-protein-coupled receptors.

Endocrine gland-derived vascular endothelial growth factor (EG-VEGF, identical to prokineticin 1) is a novel peptide recently identified as a selective mitogen for endocrine gland endothelial cells. The present study demonstrates that EG-VEGF/prokineticin 1 and a peptide closely related to EG-VEGF, prokineticin 2, are cognate ligands of two orphan G-protein-coupled receptors designated ZAQ (=EG-VEGF/PK-R1) and I5E (=EG-VEGF/PK-R2). EG-VEGF/prokineticin 1 and prokineticin 2 induced a transient increase in intracellular calcium ion concentration ([Ca(2+)](i)) with nanomolar potency in Chinese hamster ovary (CHO) cells expressing EG-VEGF/PK-R1 and -R2 and bind to these cells with high affinity and with different receptor selectivity. EG-VEGF/prokineticins provoke rapid phosphorylation of p44/42 MAP kinase and DNA synthesis in the bovine adrenal capillary endothelial cells (BACE). The mRNAs of both EG-VEGF/PK-R1 and -R2 were expressed in BACE. The identification of the receptors for EG-VEGF/prokineticins may provide a novel molecular basis for the regulation of angiogenesis in endocrine glands.