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Sarcomatoid mesothelioma is an aggressive malignancy that can be challenging to distinguish from benign spindle cell mesothelial proliferations based on biopsy, and this distinction is crucial to patient treatment and prognosis. A novel deep learning based classifier may be able to aid pathologists in making this critical diagnostic distinction. SpindleMesoNET was trained on cases of malignant sarcomatoid mesothelioma and benign spindle cell mesothelial proliferations. Performance was assessed through cross-validation on the training set, on an independent set of challenging cases referred for expert opinion (‘referral’ test set), and on an externally stained set from outside institutions (‘externally stained’ test set). SpindleMesoNET predicted the benign or malignant status of cases with AUC’s of 0.932, 0.925, and 0.989 on the cross-validation, referral and external test sets, respectively. The accuracy of SpindleMesoNET on the referral set cases (92.5%) was comparable to the average accuracy of 3 experienced pathologists on the same slide set (91.7%). We conclude that SpindleMesoNET can accurately distinguish sarcomatoid mesothelioma from benign spindle cell mesothelial proliferations. A deep learning system of this type holds potential for future use as an ancillary test in diagnostic pathology.

The site of the gastrointestinal (GI) tract where biopsies are most likely to be diagnostic of graft-versus-host disease (GvHD) remains controversial. Recent reports have indicated that biopsies from the rectosigmoid have sufficient sensitivity and specificity for diagnosing GI GvHD and can be obtained via a less invasive flexible sigmoidoscopy procedure. While GvHD histologic grades 1–3 have little correlation with patients’ symptoms and overall clinical grade, histologic grade 4 GvHD does correlate with severe clinical presentation and a poor prognosis. We examined cases of lower GI biopsies obtained via a complete colonoscopy with ileal intubation for the evaluation of GvHD within a 2-year period from patients who underwent stem cell transplantation. In our study cohort, grade 4 GvHD was significantly more likely to be identified in a terminal ileum biopsy than in a biopsy from another site in the lower GI tract. Significantly, 5 of 6 patients with histologic grade 4 GvHD diagnosed on ileal biopsies died from complication of severe GI GvHD. Given the poor prognosis of histologic grade 4 GvHD in the terminal ileum, the detection of this finding may serve to inform clinicians that escalation or modification of treatment may need to be considered. Furthermore, our findings suggest that terminal ileal biopsies may help to increase sensitivity for identifying patients at high risk for poor outcome of GvHD.

Anti-Müllerian hormone (AMH) downregulates the level of stem cell factor (SCF) via the cAMP/PKA signaling pathway in human granulosa cells (GCs). Little information is available on the molecular mechanism underlying the interaction. This study is aimed at determining whether AMH regulates expression of SCF via the cAMP-PKA-CREB signaling pathway in human GCs. In the present study, we verified the binding of cAMP-response element-binding protein (CREB) to promoter of SCF in human GCs. Furthermore, the effect of CREB was tested on the SCF promoter, and the site of CREB binding to SCF promoter was identified using truncations as well as assays of SCF-promoted mutation and CREB mutation. To investigate the correlation among AMH, SCF promoter, and CREB, pGL-Basic-SCF+CREB was transfected into overexpressed AMH GCs (AMH-high GCs), low expressed AMH GCs (AMH-low GCs), and normal GCs (GCs), respectively. Finally, immunofluorescence, double immunostaining, and Western blot were carried out in AMH-high and AMH-low GCs to confirm the AMH-mediated regulation of SCF expression by inhibiting the phosphorylation of CREB (pCREB) in GCs. Results indicated CREB interacted with SCF promoter and significantly enhanced the transcription level of SCF. The CREB binding site was localized at 318-321 bp of SCF gene promote. AMH inhibits the expression of SCF by phosphorylation of CREB via the PKA signaling pathway in GCs. These findings provide an in-depth understanding of the molecular mechanism underlying AMH suppressing the follicle growth, which would aid in the development of a novel therapy.

Chronic infection with liver flukes (such as Clonorchis sinensis) can induce severe biliary injuries, which can cause cholangitis, biliary fibrosis, and even cholangiocarcinoma. The release of extracellular vesicles by C. sinensis (CsEVs) is of importance in the long-distance communication between the hosts and worms. However, the biological effects of EVs from liver fluke on biliary injuries and the underlying molecular mechanisms remain poorly characterized. In the present study, we found that CsEVs induced M1-like activation. In addition, the mice that were administrated with CsEVs showed severe biliary injuries associated with remarkable activation of M1-like macrophages. We further characterized the signatures of miRNAs packaged in CsEVs and identified a miRNA Csi-let-7a-5p, which was highly enriched. Further study showed that Csi-let-7a-5p facilitated the activation of M1-like macrophages by targeting Socs1 and Clec7a; however, CsEVs with silencing Csi-let-7a-5p showed a decrease in proinflammatory responses and biliary injuries, which involved in the Socs1- and Clec7a-regulated NF-κB signaling pathway. Our study demonstrates that Csi-let-7a-5p delivered by CsEVs plays a critical role in the activation of M1-like macrophages and contributes to the biliary injuries by targeting the Socs1- and Clec7a-mediated NF-κB signaling pathway, which indicates a mechanism contributing to biliary injuries caused by fluke infection. However, molecules other than Csi-let-7a-5p from CsEVs that may also promote M1-like polarization and exacerbate biliary injuries are not excluded.

Background Various stimuli, including Clonorchis sinensis infection, can cause liver fibrosis. Liver fibrosis is characterized by the activation of hepatic stellate cells (HSCs) with massive production of extracellular matrix (ECM). Our previous study showed that the TGF-β1-induced Smad signaling pathway played a critical role in the activation of HSCs during liver fibrosis induced by worm infection; however, the mechanisms that modulate the TGF-β/Smad signaling pathway are still poorly understood. Accumulating evidence demonstrates that miRNAs act as an important regulator of activation of HSCs during liver fibrosis. Methods The target of miR-497 was determined by bioinformatics analysis combined with a dual-luciferase activity assay. LX-2 cells were transfected with miR-497 inhibitor and then stimulated with TGF-β1 or excretory/secretory products of C. sinensis (CsESPs), and activation of LX-2 was assessed using qPCR or western blot. In vivo, the mice treated with CCl4 were intravenously injected with a single dose of adeno-associated virus serotype 8 (AAV8) that overexpressed anti-miR-497 sequences or their scramble control for 6 weeks. Liver fibrosis and damage were assessed by hematoxylin and eosin (H&E) staining, Masson staining, and qPCR; the activation of the TGF-β/Smad signaling pathway was detected by qPCR or western blot. Results In the present study, the expression of miR-497 was increased in HSCs activated by TGF-β1 or ESPs of C. sinensis. We identified that Smad7 was the target of miR-497 using combined bioinformatics analysis with luciferase activity assays. Transfection of anti-miR-497 into HSCs upregulated the expression of Smad7, leading to a decrease in the level of p-Smad2/3 and subsequent suppression of the activation of HSCs induced by TGF-β1 or CsESPs. Furthermore, miR-497 inhibitor delivered by highly-hepatotropic (rAAV8) inhibited TGF-β/smads signaling pathway by targeting at Smad7 to ameliorate CCL4-induced liver fibrosis. Conclusions The present study demonstrates that miR-497 promotes liver fibrogenesis by targeting Smad7 to promote TGF-β/Smad signaling pathway transduction both in vivo and in vitro, which provides a promising therapeutic strategy using anti-miR-497 against liver fibrosis. Graphical Abstract

Background Celiac disease (CD) is characterized histologically by inflammation and villous atrophy. Villous atrophy is thought to result from a disruption of epithelial cellular proliferation and death. Epithelial cells in intestinal mucosa normally proliferate in the crypts and migrate towards the lumen, eventually dying. Apoptotic bodies in crypts are usually abnormal and are associated with certain disease states. The presence of crypt apoptosis in celiac disease has not been thoroughly examined by routine histologic assessment of crypt apoptotic body count (ABC). Methods We quantified the ABC in duodenal biopsies from celiac patients before and after initiation of a gluten-free diet (GFD). We examined twenty-three duodenal biopsies from adult patients with celiac disease at diagnosis and following GFD and determined the maximum ABC in 10 consecutive crypts. Fourteen biopsies from heartburn patients served as controls. Results Mean duration between paired biopsies was 2.9 (0.5–8.5) years. Mean maximum ABC in active celiac disease was 5.44 per crypt and decreased to 2.60 with GFD (p =

AimsSerous cystadenomata (SCAs) are benign pancreatic cystic neoplasms that present a diagnostic challenge despite many investigational approaches. Notwithstanding the promise of molecular diagnostics, these tests have limited accessibility in day-to-day surgical pathology practices. We aim to corroborate and build on recent evidence which suggests that positive α-inhibin immunohistochemistry (IHC) is a helpful adjunct in the biopsy confirmation of pancreatic SCA.MethodsWe retrospectively reviewed 22 fine-needle aspirates/biopsies from 14 patients (mean age 65 years, 47–83 years) with pancreatic multicystic lesions radiologically suspicious for SCA (location: 6 body, 2 head, 4 tail, 1 neck, 1 uncinate; cyst size: mean 3.7 cm, 2.0–7.6 cm), as well as an additional 10 pancreatic resection specimens with confirmed SCA; α-inhibin IHC was performed on all cell blocks, biopsy slides and representative resection specimen sections. Where available, associated cyst fluid was analysed for correlative vascular endothelial growth factor A (VEGF-A) and carcinoembryonic antigen levels.ResultsAn α-inhibin IHC sensitivity of 80% was observed in the cases with resection confirmed SCA. Of the fine-needle aspirate/biopsy specimens, 59% (13/22) contained epithelial cells strongly positive for α-inhibin. When selecting for specimens that exhibited distinct strips of epithelium, the α-inhibin strong positivity rate increased to 73% (8/11). VEGF-A values were supportive of false-negative α-inhibin IHC in three cases and true-negative α-inhibin IHC in one case.ConclusionThis study postulates a diagnostic algorithm to confirm pancreatic SCA which may help to decrease unnecessary follow-up endoscopy/surgical resection and would decrease the associated morbidity, mortality and financial costs in patients with this otherwise benign condition.

Background Microglia activation induced by α-synuclein (α-syn) is one of the most important factors in Parkinson’s disease (PD) pathogenesis. However, the molecular mechanisms by which α-syn exerts neuroinflammation and neurotoxicity remain largely elusive. Targeting metabotropic glutamate receptor 5 (mGluR5) has been an attractive strategy to mediate microglia activation for neuroprotection, which might be an essential regulator to modulate α-syn-induced neuroinflammation for the treatment of PD. Here, we showed that mGluR5 inhibited α-syn-induced microglia inflammation to protect from neurotoxicity in vitro and in vivo. Methods Co-immunoprecipitation assays were utilized to detect the interaction between mGluR5 and α-syn in microglia. Griess, ELISA, real-time PCR, western blotting, and immunofluorescence assays were used to detect the regulation of α-syn-induced inflammatory signaling, cytokine secretion, and lysosome-dependent degradation. Results α-syn selectively interacted with mGluR5 but not mGluR3, and α-syn N terminal deletion region was essential for binding to mGluR5 in co-transfected HEK293T cells. The interaction between these two proteins was further detected in BV2 microglia, which was inhibited by the mGluR5 specific agonist CHPG without effect by its selective antagonist MTEP. Moreover, in both BV2 cells and primary microglia, activation of mGluR5 by CHPG partially inhibited α-syn-induced inflammatory signaling and cytokine secretion and also inhibited the microglia activation to protect from neurotoxicity. We further found that α-syn overexpression decreased mGluR5 expression via a lysosomal pathway, as evidenced by the lysosomal inhibitor, NH4Cl, by blocking mGluR5 degradation, which was not evident with the proteasome inhibitor, MG132. Additionally, co-localization of mGluR5 with α-syn was detected in lysosomes as merging with its marker, LAMP-1. Consistently, in vivo experiments with LPS- or AAV-α-syn-induced rat PD model also confirmed that α-syn accelerated lysosome-dependent degradation of mGluR5 involving a complex, to regulate neuroinflammation. Importantly, the binding is strengthened with LPS or α-syn overexpression but alleviated by urate, a potential clinical biomarker for PD. Conclusions These findings provided evidence for a novel mechanism by which the association of α-syn with mGluR5 was attributed to α-syn-induced microglia activation via modulation of mGluR5 degradation and its intracellular signaling. This may be a new molecular target for an effective therapeutic strategy for PD pathology.

Mice with different genetic backgrounds have various susceptibilities to infection with Clonorchis sinensis, although the mechanisms underlying are largely unknown. Toll-like receptor 4 (TLR4) as one of the most important pattern recognition receptors (PPRs) is essential for the invasion, survival, pathogenesis, and elimination of worms. The roles played by TLR4 in C. sinensis infection may vary due to the different genetic backgrounds of mice. In the present study, a relatively resistant mouse strain-C57BL/10 to C. sinensis was used for investigation on the possible roles of TLR4 in the biliary injuries and peribiliary fibrosis. TLR4 wild type (TLR4wild) and TLR4 defective (TLR4def) mice were orally infected with 45 metacercariae of C. sinensis, and all C. sinensis-infected mice and non-infected groups were anesthetized on day 28 post-infection. The liver and serum from each mouse were collected for assessment of the biliary injuries and biliary fibrosis. Meanwhile, hepatic leukocytes were isolated and detected for the activation of M1 or M2 macrophage using flow cytometry. The hepatic type 1 immune response and type 2 immune responses -relative molecules were also evaluated using ELISA and quantitative PCR. The data showed that TLR4def aggravated liver inflammatory cell infiltrations, bile duct proliferation, biliary and hepatocellular injuries, and ECM deposition in C. sinensis-infected mice, compared with TLR4wild mice when they were intragastrically administered with the same amounts of C. sinensis metacercaria. Furthermore, the M2-like macrophages and type 2 immune responses were significantly predominant induced in TLR4def mice, compared with that of TLR4wild mice following C. sinensis infection. But the type 1 immune response were significantly decreased in TLR4def mice, compared with TLR4wild mice after C. sinensis infection. These data demonstrate that TLR4 deficiency exacerbates biliary injuries and peribiliary fibrosis caused by C. sinensis in C57BL/10 strain mice, which is contributed by augments of type 2 immune responses and decrease pro-inflammatory responses.

Context.— Serosal involvement (pT4a category) and lymphovascular invasion have prognostic significance in colorectal carcinoma, but are subject to interobserver variation in assessment. Objectives.— To provide the first large-scale assessment of interobserver variability in pT4a category and lymphovascular invasion reporting in real-world practice and to explore the impact of information from guidelines on variability in reporting these features. Design.— Analysis of 1555 consecutive synoptic reports of colorectal carcinoma was performed using multivariate logistic regression. Interobserver variability before and after the presentation of guideline information was assessed using an image-based survey. Results.— Significant differences in the odds of reporting pT4a versus pT3 category, detecting lymphovascular invasion of any type, and detecting large vessel invasion were identified among hospital sites and for individual pathologists compared with the median pathologist at the same site. Consistent with these results, interobserver agreement was only moderate in the image-based survey regarding T4a staging and lymphovascular invasion (all κ ≤ 0.57). The provision of information from guidelines did not tend to increase interobserver agreement in the survey, though responses in favor of using an elastic stain increased following recommendations for their use. However, when observers were provided with elastic-stained images, interobserver agreement remained only moderate (κ = 0.55). Conclusions.— Real-world reporting of pT4a category and lymphovascular invasion shows substantial variability at both local and regional levels. Our study underscores the need to address these features in quality initiatives, and provides a novel method through which existing synoptic data can be harnessed to monitor reporting patterns and provide individualized feedback.

Repeated implantation failure (RIF) is a common endocrine disease that causes female infertility and the etiology is unknown. The abnormal expression of key proteins and hormones at the maternal-fetal interface affected the maternal-fetal communication and leads to adverse pregnancy outcomes. The expression of anti-Mullerian hormone (AMH) and AMH receptor II (AMHRII) was observed in the endometrium. This study aimed to investigate the expression of AMH and AMHRII at the human endometrium, decidual tissue, and blastocyst. Furthermore, the expression of AMH and AMHRII were examined in the RIF patients using immunohistochemistry and quantitative real-time PCR to test the AMHRII expression. The results demonstrated that AMH and AMHRII were present in healthy endometrium and AMHRII was highly expressed in mid-luteal phase. In addition, AMHRII expression was detected throughout the pregnancy and AMHRII's highest expression was in the second trimester. AMHRII was expressed in the blastocysts; however, AMH was not observed. The positive expression rate for AMHRII was significantly higher in the endometrium from RIF. Estrogen receptor (ER), insulin-like growth factor binding protein 1(IGFBP1), and prolactin (PRL) were significantly less expressed in RIF with high expression of AMHRII. The apoptosis was significantly higher in patients with high expression of AMHRII than in patients with normal expression of AMHRII. Our data suggests that AMHRII had an effect on RIF via the AMH and AMHRII signaling pathway. It participated in the development of RIF by interfering with endometrial decidualization and apoptosis.

Oxidative stress aggravates mitochondrial injuries and accelerates the proliferation of vascular smooth muscle cells (VSMCs), which are important mechanisms contributing to vascular remodeling in hypertension. We put forward the hypothesis that Astaxanthin (ATX), known to possess strong features of antioxidant, could attenuate vascular remodeling by inhibiting VSMC proliferation and improving mitochondrial function. The potential effects of ATX were tested on spontaneously hypertensive rats (SHRs) and cultured VSMCs that injured by angiotensin II (Ang II). The results showed that ATX lowered blood pressure, reduced aortic wall thickness and fibrosis, and decreased the level of reactive oxygen species (ROS) and H2O2 in tunica media. Moreover, ATX decreased the expression of proliferating cell nuclear antigen (PCNA) and ki67 in aortic VSMCs. In vitro, ATX mitigated VSMC proliferation and migration, decreased the level of cellular ROS, and balanced the activities of ROS-related enzymes including NADPH oxidase, xanthine oxidase, and superoxide dismutase (SOD). Besides, ATX mitigated Ca2+ overload, the overproduction of mitochondrial ROS (mtROS), mitochondrial dysfunction, mitochondrial fission, and Drp1 phosphorylation at Ser616. In addition, ATX enhanced mitophagy and mitochondrial biosynthesis by increasing the expression of PINK, parkin, mtDNA, mitochondrial transcription factor A (Tfam), and PGC-1α. The present study indicated that ATX could efficiently treat vascular remodeling through restraining VSMC proliferation and restoring mitochondrial function. Inhibiting mitochondrial fission by decreasing the phosphorylation of Drp1 and stimulating mitochondrial autophagy and biosynthesis via increasing the expression of PINK, parkin, Tfam, and PGC-1α may be part of its underlying mechanisms.

Due to limitations in early diagnosis and treatments of Parkinson's disease (PD), it is necessary to explore the neuropathological changes that occur early in PD progression and to design neuroprotective therapies to prevent or delay the ongoing degeneration process. Metabotropic glutamate receptor 5 (mGlu5) has shown both diagnostic and therapeutic potential in preclinical studies on PD. Clinical trials using mGlu5 negative allosteric modulators to treat PD have, however, raised limitations about the neuroprotective role of mGlu5. It is likely that mGlu5 has different regulatory roles in different stages of PD. Here, we investigated a protective role of cystic fibrosis transmembrane conductance regulator-associated ligand (CAL) in the progression of PD by differential regulation of mGlu5 expression and activity to protect against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity. Following treatment with 6-OHDA, mGlu5 and CAL expressions were elevated in the early stage and reduced in the late stage, both in vitro and in vivo. Activation of mGlu5 in the early stage by (RS)-2-chloro-5-hydroxyphenylglycine, or blocking mGlu5 in the late stage by 2-methyl-6-(phenylethynyl) pyridine, increased cell survival and inhibited apoptosis, but these effects were significantly weakened by knockdown of CAL. CAL alleviated 6-OHDA-induced neurotoxicity by regulating mGlu5-mediated signaling pathways, thereby maintaining the physiological function of mGlu5 in different disease stages. In PD rat model, CAL deficiency aggravated 6-OHDA toxicity on dopaminergic neurons and increased motor dysfunction because of lack of regulation of mGlu5 activity. These data reveal a potential mechanism by which CAL specifically regulates the opposite activity of mGlu5 in progression of PD to protect against neurotoxicity, suggesting that CAL is a favorable endogenous target for the treatment of PD.

Metabotropic glutamate receptor (mGlu)5 regulates microglia activation, which contributes to inflammation. However, the role of mGlu5 in neuroinflammation associated with Parkinson's disease (PD) remains unclear. Triptolide (T10) exerts potent immunosuppressive and anti-inflammatory effects and protects neurons by inhibiting microglia activation. In this study, we investigated the role of mGlu5 in the anti-inflammatory effect of T10 in a lipopolysaccharide (LPS)-induced PD model. In cultured BV2 cells and primary microglia, blocking mGlu5 activity or knocking down its expression abolished T10-inhibited release of proinflammatory cytokines induced by LPS. Moreover, T10 up-regulated mGlu5 expression decreased by LPS through enhancing mRNA expression and protein stability. T10 also reversed the reduction in mGlu5 membrane localization and modulated receptor-mediated mitogen-activated protein kinase activity induced by LPS. Pharmacological inhibition of signaling molecules increased nitric oxide level and inducible nitric oxide synthase (iNOS), tumor necrosis factor-α, and interleukin (IL)-1β and -6 transcript levels that were downregulated by treatment with T10. Consistent with these in vitro findings, blocking mGlu5 attenuated the anti-inflammatory effects of T10 in an LPS-induced PD model and blocked the decreases in the number and morphology of ionized calcium binding adaptor molecule 1-positive microglia and LPS-induced iNOS protein expression caused by T10 treatment. Besides, mGlu5 mediated the effect of T10 on microglia-induced astrocyte activation in vitro and in vivo. The findings provide evidence for a novel mechanism by which mGlu5 regulates T10-inhibited microglia activation via modulating protein expression of the receptor and its intracellular signaling. The study might contribute to the biological effects of Chinese herbs as an approach for protecting against neurotoxicity in PD.

Six undescribed pentacyclic triterpenoids including four triterpenoid aglycones, 1β,2a,3β,23-tetrahydroxyurs-12-en-28-ursolic acid, 2a,3a,6β,19α,23-pentahydroxyurs-12-en-28-ursolic acid, 2α,3α,20β,23-tetrahydroxyurs-12-en-28-ursolic acid and 1β,2a,3β,23-tetrahydroxyurs-12,20(30)-dien-28-ursolic acid, and two triterpenoid glucosides, 2a,3a,23-trihydroxy-12,20(30)-dien-28-ursolic acid 28-O-β-d-glucopyranoside and 1-oxo-3β,23-dihydroxyolean-12-en-28-oic acid 28-O-β-d-xylopyranoside, along with 5 known triterpenoids were isolated from a CH3Cl-soluble extract of the leaves of Cyclocarya paliurus. Their structures were established on the basis of chemical and spectroscopic approaches. These compounds were assessed for their antioxidant effects on FFA-induced hepatic steatosis in HepG2 cells. The results revealed that three saponins and two aglycones markedly increased SOD activity and reduced MDA level.

Numerous epidemiological studies suggested that there is a variable cancer risk in patients with Parkinson's disease (PD). However, the underlying mechanisms remain unclear. In the present study, the role of metabotropic glutamate receptor 5 (mGluR5) has been investigated in 6-hydroxydopamine (6-OHDA)-induced PD combined with liver cancer both in vitro and in vivo. We found that PD cellular model from 6-OHDA-lesioned MN9D cells suppressed the growth, migration, and invasion of Hepa1-6 cells via down-regulation of mGluR5-mediated ERK and Akt pathway. The application of 2-methyl-6-(phenylethyl)-pyridine and knockdown of mGluR5 further decreased the effect on Hepa-1-6 cells when co-cultured with conditioned media. The effect was increased by (S)-3,5-dihydroxyphenylglycine and overexpression of mGluR5. Moreover, more release of glutamate from 6-OHDA-lesioned MN9D cells suppressed mGluR5-mediated effect of Hepa1-6 cells. Application of riluzole eliminated the increased glutamate release induced by 6-OHDA in MN9D cells and aggravated the suppressive effect on Hepa-1-6 cells. In addition, the growth of implanted liver cancer was inhibited in 6-OHDA induced PD-like rats, and was associated with increased glutamate release in the serum and down-regulation of mGluR5 in tumor tissue. Collectively, these results indicate that selective antagonism of glutamate and mGluR5 has a potentially beneficial effect in both liver cancer and PD, and thus may provide more understanding for the clinical investigation and further an additional therapeutic target for these two diseases.

Anorectal melanoma is a rare disease that carries a poor prognosis. To date, limited genetic analyses confirmed KIT mutations as a recurrent genetic event similar to other mucosal melanomas, occurring in up to 30% of anorectal melanomas. Importantly, a subset of tumors harboring activating KIT mutations have been found to respond to c-Kit inhibitor-based therapy, with improved patient survival at advanced tumor stages. We performed comprehensive targeted exon sequencing analysis of 467 cancer-related genes in a larger series of 15 anorectal melanomas, focusing on potentially actionable variants based on gain- and loss-of-function mutations. We report the identification of oncogenic driver events in the majority (93%) of anorectal melanomas. These included variants in canonical MAPK pathway effectors rarely observed in cutaneous melanomas (including an HRAS mutation, as well as a BRAF mutation resulting in duplication of threonine 599), and recurrent mutations in the tumor suppressor NF1 in 20% of cases, which represented the second-most frequently mutated gene after KIT in our series. Furthermore, we identify SF3B1 mutations as a recurrent genetic event in mucosal melanomas. Our findings provide an insight into the genetic diversity of anorectal melanomas, and suggest significant potential for alternative targeted therapeutics in addition to c-Kit inhibitors for this melanoma subtype.

Objective To observe the effects of Huannao Yicong Formula (还脑益聪方, HYF) on learning and memory and it’s regulating effect on γ-secretase related anterior pharynx defective 1 (APH-1), presenilin enhancer-2 (PEN-2) signaling pathway, so as to discuss and further clarify the mechanism of HYF on Alzheimer’s disease.

Neuroendocrine carcinomas (NECs) of the colorectum are rare but highly aggressive neoplasms. These tumors show some shared genetic alterations with colorectal adenocarcinomas, and most of them have adjacent glandular adenoma or adenocarcinoma components. However, genetic data on colorectal NECs still are sparse and insufficient for definite conclusions regarding their molecular origin. Based on morphological characterization, panel and whole-exome sequencing, we here present results from an in-depth analysis of a collection of 15 colorectal NECs with glandular components, 10 of which by definition were mixed adenoneuroendocrine carcinomas (MANECs). Among shared genetic alterations of both tumor components, we most frequently found TP53, KRAS and APC mutations that also had highest allele frequencies. Mutations exclusive to glandular or neuroendocrine components outnumbered shared mutations but occurred at lower allele frequencies. Our findings not only provide additional evidence for a common clonal origin of colorectal NECs and adjacent glandular tumor components, but strongly suggest their development through the classical adenoma-carcinoma sequence. Moreover, our data imply early separation of glandular and neuroendocrine components during malignant transformation with subsequent independent mutational evolution.

Immune checkpoint inhibitors such as programmed cell death-1 inhibitor pembrolizumab have been shown to be effective in metastatic malignancies such as advanced melanoma. Immune-related adverse effects on multiple organs have been described, such as colitis, skin rash, and hypothyroidism. We present the case of a 44-year-old man with advanced melanoma and recurrent lung metastases who developed symptoms of dyspepsia and gastroesophageal reflux disease after 1 month of therapy with pembrolizumab. Gastric biopsy showed histologic features consistent with lymphocytic gastritis, which was absent on a biopsy 2 months before initiation of therapy. Lymphocytic infiltrates likely secondary to increased autoimmunity after use of immunotherapy have been observed in the colon; however, such histologic findings in the upper gastrointestinal tract have yet to be described. Here, we present a case of lymphocytic gastritis in a patient treated with pembrolizumab, suggesting a new manifestation of toxicity.

Targeting mGluR5 has been an attractive strategy to modulate glutamate excitotoxicity for neuroprotection. Although human clinical trials using mGluR5 negative allosteric modulators (NAMs) have included some disappointments, recent investigations have added several more attractive small molecules to this field, providing a promise that the identification of more additional strategies to modulate mGluR5 activity might be potentially beneficial for the advancement of PD treatment. Here, we determined the role of the interacting partner CAL (cystic fibrosis transmembrane conductance regulator–associated ligand) in mGluR5-mediated protection in vitro and in vivo. In astroglial C6 cells, CAL deficiency blocked (S)-3, 5-dihydroxyphenylglycine (DHPG)-elicited p-AKT and p-ERK1/2, subsequently prevented group I mGluRs-mediated anti-apoptotic protection, which was blocked by receptor antagonist 1-aminoindan-1, 5-dicarboxylic acid (AIDA), and PI3K or MEK inhibitor LY294002 or U0126. In rotenone-treated MN9D cells, both CAL and mGluR5 expressions were decreased in a time- and dose-dependent manner, and the correlation between these 2 proteins was confirmed by lentivirus-delivered CAL overexpression and knockdown. Moreover, CAL coupled with mGluR5 upregulated mGluR5 protein expression by inhibition of ubiquitin-proteasome-dependent degradation to suppress mGluR5-mediated p-JNK and to protect against cell apoptosis. Additionally, CAL also inhibited rotenone-induced glutamate release to modulate mGluR5 activity. Furthermore, in the rotenone-induced rat model of PD, AAV-delivered CAL overexpression attenuated behavioral deficits and dopaminergic neuronal death, while CAL deficiency aggravated rotenone toxicity. On the other hand, the protective effect of the mGluR5 antagonist MPEP was weakened by knocking down CAL. In vivo experiments also confirmed that CAL inhibited ubiquitination-proteasome-dependent degradation to modulate mGluR5 expression and JNK phosphorylation. Our findings show that CAL protects against cell apoptosis via modulating mGluR5 activity, and may be a new molecular target for an effective therapeutic strategy for PD. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13311-019-00730-7) contains supplementary material, which is available to authorized users.

Background Non-alcoholic fatty liver disease (NAFLD) is the most prevalent form of chronic liver diseases. Cyclocarya paliurus (C. paliurus), an edible and medicinal plant in Chinese folk, has been demonstrated to ameliorate diabetes, obesity and lipid metabolism disorders. However, its effects on NAFLD and its potential molecular mechanism have not been clearly expounded. Purpose The present study was designed to explore the therapeutic potential of triterpenic acids-enriched fraction from C. paliurus (CPT), as well as its underlying mechanism in vivo and in vitro models of NAFLD. Methods The metabolic effects and possible molecular mechanism of CPT were examined using HepG2 cells and primary hepatocytes (isolated from C57BL/6 J mice) models of fatty liver induced by palmitic acid (PA) and a high fat diet mouse model. Results In high fat diet-induced C57BL/6 J mice, CPT significantly reduced liver weight index, serum alanine transaminase (ALT), aspartate transaminase (AST), triacylglycerol (TG), total cholesterol (TC) and hepatic TG, TC levels. Moreover, CPT dramatically decreased the contents of blood glucose, insulin, and insulin resistance (HOMA-IR) index. Meanwhile, CPT significantly increased the tyrosine phosphorylation level of IRS and the uptake of 2-deoxyglucose (2DG) in PA-induced HepG2 cells and primary hepatocytes fatty liver models. Furthermore, in PA-induced HepG2 cells and primary hepatocytes, CPT significantly decreased the number of lipid droplets and intracellular TG content. In addition, mechanism investigation showed that CPT increased the phosphorylation of phosphoinositide 3-kinase (PI3K), protein kinase B (Akt) and glycogen synthase-3β (GSK3β) in vivo and in vitro models, which were abrogated by PI3K inhibitor LY294002 in vitro models. Conclusion These findings indicate that CPT may exert the therapeutic effects on NAFLD via regulating PI3K/Akt/GSK3β pathway.

In 2011, a new classification of lung adenocarcinoma was published. Since then there have been multiple studies regarding observer variability in predominant subtype determination, with levels of agreement generally in the weak to moderate range. In the 2011 and subsequent WHO 2015 classification, a recommendation was also made to visually assess and record the percentage of each subtype in 5% increments. The present study was initiated to determine the reproducibility of such gestalt assessments and to compare these data to a formal morphometric assessment. Five experienced pathologists reviewed multiple single images of 25 adenocarcinomas, taken at 2× and 10×, and estimated the percentage of lepidic, acinar, papillary, micropapillary, and solid components in 5% increments. After 2 months all the pathologists again reviewed the same images presented to them in a different order. We found that there was poor reproducibility within observers at 2× power using a 5% evaluation, but that this improved using 10% or 25% cutoffs. Use of 10× magnification allowed weak to moderate reproducibility at 5% increments, and this was again improved using 10% or 25% cutoffs. Correlation with morphometric assessment was poor except for the papillary and micropapillary subtypes. Differences among pathologists were generally low except for the acinar and, to a lesser degree, lepidic subtypes, which showed a wide spread of data. When estimating tumor subtype proportions, use of a 10× objective, and utilization of 10% or preferably 25% cutoffs provides a greater degree of consistency than a 5% cutoff.

Anabolic steroids are well-known to cause liver injury, which may manifest with jaundice and elevated liver enzymes. Selective androgen receptor modulators (SARMs) have been developed to enhance muscle bulk without the side effects associated with exogenous androgen steroids. We report a case of significant cholestatic liver injury associated with a SARM, ostarine (enobosarm), similar to that associated with anabolic steroids. Liver injury from SARMs has not been reported frequently, and we speculate that this may be seen more often as the consumption of SARMs increases in the athletic market.

A 20-year-old woman presented with abdominal pain and MRI findings of intussusception of the distal small bowel with no identifiable lead point and no visualisation of the appendix. A diagnostic laparoscopy succeeded in manually reducing the intussusception but was unable to find any candidate lead point. Intraoperatively, hyperperistalsis was observed throughout the small bowel which seemed prone to transient intussusception. Incidental appendectomy revealed an uninflamed appendix with Enterobius vermicularis (pinworm) infestation, the most common parasite present in appendectomy specimens worldwide. Although intussusception in young adults is an uncommon occurrence, the unique nature of this case is amplified by the concurrent finding of E. vermicularis infection of the appendix in an adolescent in western Canada, a phenomenon normally observed in paediatric populations with higher incidence in tropical areas. Although the mechanism of intussusception in this patient remains unclear, it is hypothesised that E. vermicularis colonisation acted as an irritant stimulating intestinal hypercontractility with resulting intussusception. Successful medical eradication of the pinworm in this individual may prevent future recurrence.

Background: Atopic dermatitis (AD) is an inflammatory skin disease characterized by chronic recurrent dermatitis with profound itching. Most patients have personal and/or family history of atopic diseases. Several criteria have been proposed for the diagnosis of AD. Although the clinical features of childhood AD have been widely studied, there has been less large-scale study on adult/adolescent AD. The aim of this study was to investigate the clinical features of adult/adolescent patients with chronic symmetrical eczema/AD and to propose Chinese diagnostic criteria for adult/adolescent AD. Methods: A hospital-based study was performed. Forty-two dermatological centers participated in this study. Adult and adolescent patients (12 years and over) with chronic symmetrical eczema or AD were included in this study. Questionnaires were completed by both patients and investigators. The valid questionnaires were analyzed using EpiData 3.1 and SPSS 17.0 software. Results: A total of 2662 valid questionnaires were collected (1369 male and 1293 female). Of all 2662 patients, 2062 (77.5%) patients had the disease after 12 years old, while only 600 (22.5%) patients had the disease before 12 years old, suggesting late-onset eczema/AD is common. Two thousand one hundred and thirty-nine (80.4%) patients had the disease for more than 6 months. One thousand one hundred and forty-four (43.0%) patients had a personal and/or family history of atopic diseases. One thousand five hundred and forty-eight (58.2%) patients had an elevated total serum IgE and/or eosinophilia and/or positive allergen-specific IgE. Based on these clinical and laboratory features, we proposed Chinese criteria for adult/adolescent AD. Of all 2662 patients, 60.3% were satisfied with our criteria, while only 48.2% satisfied with Hanifin Rajka criteria and 32.7% satisfied with Williams criteria, suggesting a good sensitivity of our criteria in adult/adolescent AD patients. Conclusion: Late-onset of eczema or AD is common. The clinical manifestations of AD are heterogeneous. We have proposed Chinese diagnostic criteria for adolescent and adult AD, which are simple and sensitive for diagnosis of adult/adolescent AD.

Fibrolamellar carcinoma is a rare primary hepatocellular malignancy arising in noncirrhotic livers of young individuals. Patients commonly present with a large solitary liver mass and nonspecific symptoms. Characteristic histologic features include large polygonal cells with oncocytic cytoplasm and prominent nucleoli separated into trabeculae and cords by dense parallel bands of collagen. Important differential diagnoses include classical hepatocellular carcinoma and intrahepatic cholangiocarcinoma, which may be distinguished by a judicious panel of immunohistochemical studies, including cytokeratin 7, CD68, and hepatocyte paraffin 1 (HepPar-1). In addition, fibrolamellar carcinomas are characterized by activation of protein kinase A. Prognosis of fibrolamellar carcinoma is similar to classical hepatocellular carcinoma occurring in the absence of liver cirrhosis and is strongly correlated with tumor resectability. Other treatment options include liver transplant, chemotherapy, and hepatic artery embolization. In this article, we review the clinical features, gross and microscopic pathology, molecular genetics, differential diagnosis, treatment, and prognosis of this rare and interesting tumor.

Background Multiple risk factors contribute to the progression of Parkinson’s disease, including oxidative stress and neuroinflammation. Epidemiological studies have revealed a link between higher urate level and a lower risk of developing PD. However, the mechanistic basis for this association remains unclear. Urate protects dopaminergic neurons from cell death induced by oxidative stress. Here, we investigated a novel role of urate in microglia activation in a lipopolysaccharide (LPS)-induced PD model. Methods We utilized Griess, ELISA, real-time PCR, Western blot, immunohistochemistry, and immunofluorescence to detect the neuroinflammation. For Griess, ELISA, Western blot, and immunofluorescence assay, cells were seeded in 6-well plates pre-coated with poly-l-lysine (PLL) and incubated for 24 h with the indicated drugs. For real-time PCR assay, cells were seeded in 6-well plates pre-coated with PLL and incubated for 6 h with the indicated drugs. For animal experiments, rats were injected with urate or its vehicle twice daily for five consecutive days before and after stereotaxic surgery. Rats were killed and brain tissues were harvested after 4 weeks of LPS injection. Results In cultured BV2 cells and rat primary microglia, urate suppressed proinflammatory cytokine production and inducible cyclooxygenase 2 and nitric oxide synthase expression to protect dopaminergic neurons from the toxic effects of activated microglia. The neuroprotective effects of urate may also be associated with the stimulation of anti-inflammatory factors interleukin 10 and transforming growth factor β1. Intracellular urate level was increased in a dose-dependent manner upon co-treatment with urate and LPS as compared with LPS alone, an effect that was abrogated by pretreatment with probenecid (PBN), an inhibitor of both glucose transporter 9 and urate transporter 1 (URAT1). PBN also abolished the anti-inflammatory effect of urate. Consistent with these in vitro observations, the number of tyrosine hydroxylase-positive neurons was decreased and the loss of motor coordination was reversed by urate administration in an LPS-induced rat model of PD. Additionally, increased plasma urate level abolished the reduction of URAT1 expression, the increase in the expression of interleukin-1β, and the number of ionized calcium-binding adaptor molecule 1-positive microglia along with changes in their morphology. Conclusions Urate protects neurons against cytotoxicity induced by microglia activation via modulating urate transporter-mediated intracellular urate level. Electronic supplementary material The online version of this article (10.1186/s12974-018-1175-8) contains supplementary material, which is available to authorized users.

Older asthmatic patients may develop fixed airway obstruction and clinical signs of chronic obstructive pulmonary disease (COPD). We investigated the added value of pathological evaluation of bronchial biopsies to help differentiate asthma from COPD, taking into account smoking, age, and inhaled corticosteroid (ICS) use. Asthma and COPD patients (24 of each category) were matched for ICS use, age, FEV1, and smoking habits. Five pulmonary and five general pathologists examined bronchial biopsies using an interactive website, without knowing patient information. They were asked to diagnose asthma or COPD on biopsy findings in both a pairwise and randomly mixed order of cases during four different phases, with intervals of 4–6 weeks, covering a maximal period of 36 weeks. Clinically concordant diagnoses of asthma or COPD varied between 63 %-73 %, without important differences between pairwise vs randomly mixed examination or between general vs pulmonary pathologists. The highest percentage of concordant diagnoses was in young asthmatic patients without ICS use and in COPD patients with ICS use. In non ICS users with fixed airway obstruction, a COPD diagnosis was favored if abnormal presence of glands, squamous metaplasia, and submucosal infiltrate was present and an asthma diagnosis in case of abnormal presence of goblet cells. In ICS users with fixed airway obstruction, abnormal presence of submucosal infiltrates, basement membrane thickening, eosinophils, and glands was associated with asthma. Histological characteristics in bronchial biopsies are reproducibly recognized by pathologists, yet the differentiation by histopathology between asthma and COPD is difficult without information about ICS use. Electronic supplementary material The online version of this article (doi:10.1007/s00428-015-1824-6) contains supplementary material, which is available to authorized users.

Thioredoxin 1 (Trx1) is known to play an important role in protecting against cell death. However, the mechanism for control of Trx1 in cell death resulting from DNA damage has not been fully investigated. In this study, we used the DNA-damaging agent methyl methanesulfonate (MMS) to investigate the protective effects of Trx1 against DNA damage and cell death in HEK293 cells. We found that MMS application caused dose-dependent changes in the Trx1 redox state determined by redox western blotting. At lower concentrations, both reduced and oxidized Trx1 were observed, whereas the reduced band was fully oxidized at the higher concentration. Trx1 overexpression and small interfering RNA knockdown in cells revealed that reduced Trx1 after exposure to lower doses of MMS attenuated DNA damage, assessed by comet assay, and level of the DNA-damage marker histone γ-H2AX, possibly through scavenging intracellular ROS and an increase in p21 protein level via enhancing its stability. However, oxidized Trx1 lost its protective ability to DNA damage in response to higher concentration of MMS. Corresponding to the redox state control of Trx1, cell death induced by different dose of MMS was also found, by inhibiting phosphorylations of p38 and 4E-BP1. These results indicate that reduced Trx1 plays important protective roles against MMS-induced DNA damage and cell death, suggesting that cell protection is regulated by the intracellular redox state. Control of the redox state of Trx1 and its regulating proteins may offer a novel therapeutic strategy for the control of cancer.

Context Colorectal cancer is a heterogeneous disease resulting from different molecular pathways of carcinogenesis. Recent data evaluating the histologic features and molecular basis of the serrated polyp–carcinoma pathway have significantly contributed to more comprehensive classifications of and treatment recommendations for these tumors. Objective To integrate the most recent molecular findings in the context of histologic classifications of serrated lesions and their implications in diagnostic pathology and colorectal cancer surveillance. Data Sources Published literature focused on serrated polyps and their association with colorectal cancer. Conclusions Three types of serrated polyps are currently recognized: hyperplastic polyps, sessile serrated adenomas/polyps, and traditional serrated adenomas. The BRAF V600E mutation is one of the most frequent molecular abnormalities identified in hyperplastic polyps and sessile serrated adenomas. In contrast, in traditional serrated adenomas, either BRAF V600E or KRAS mutations can be frequently identified. CpG methylation has emerged as a critical molecular mechanism in the sessile serrated pathway. CpG methylation of MLH1 often leads to reduced or lost expression in dysplastic foci and carcinomas arising in sessile serrated adenomas/polyps.

Clinical epidemiological studies have shown that there is a link between Parkinson's disease (PD) and cancer, but how PD regulates cancer development remains unknown. In our study, the effect of metabotropic glutamate receptor 5 (mGlu5) on hepatoma was explored in a rotenone-induced PD model both in vitro and in vivo. We found that conditioned media derived from MN9D dopaminergic neuronal cells by rotenone-induced toxicity inhibited the growth, migration, invasion and promoted apoptosis of Hepa1-6 cells, which corresponded with decreased expression of mGlu5. Furthermore, treatment with 2-methyl-6-(phenylethynyl)pyridine (MPEP), a mGlu5 antagonist and knockdown of mGlu5, further reduced ATP levels and migration distance, and increased cleavage of caspase-3 in Hepa1-6 cells. Additionally, we found that conditioned media derived from rotenone-treated MN9D dopaminergic neuronal cells enhanced reactive oxygen species (ROS) generation and JNK phosphorylation, which could be further increased by MPEP treatment, and attenuated by mGlu5 agonist, (RS)-2-Chloro-5-hydroxyphenylglycine (CHPG) and ROS scavenger, N-acetyl-l-cysteine (NAC). The results indicated that down-regulation of mGlu5 promoted cell apoptosis through the intracellular ROS/JNK signaling pathway in a rotenone-induced cellular PD model. These findings were confirmed in vivo in a rotenone-induced rat model of PD combined with diethylnitrosamine (DEN)-induced hepatoma. Expression of Ki67 was decreased, and the levels of caspase-3 and p-JNK were increased in this model, which was accompanied by a decrease in protein expression of mGlu5. The study suggest that negative regulation of mGlu5 may inhibit hepatoma development in a rotenone-induced PD model, and as such may help with our further understanding of the correlation between PD and cancer.

Context.— In advanced gastric, esophageal, and gastroesophageal junction adenocarcinomas (GE-GEJ-AC) that overexpress ERBB2 (erb-b2 receptor tyrosine kinase 2 or HER2), anti-HER2 monoclonal antibody therapy confers survival benefit. To select patients for treatment, HER2 expression and gene amplification are evaluated by immunohistochemistry (IHC) and in situ hybridization. Objective.— To determine whether GE-GEJ-AC tested for HER2 on biopsy specimens of a primary tumor show different IHC scores and/or HER2 amplification by in situ hybridization in matched resection specimens, potentially changing therapy eligibility. Design.— Immunohistochemistry and silver in situ hybridization were performed in biopsy and/or resection specimens from 100 patients. HER2 testing was performed in matched resection and biopsy specimens of 15 cases to determine whether GE-GEJ-AC with IHC scores of 0, 1+, and 2+ in biopsy and resection specimens had different IHC and silver in situ hybridization results. Results.— The IHC 3+ cases showed HER2 amplification in 4 of 5 cases (80%), and IHC scores of 0, 1+, and 2+ showed 3.5%, 14.3%, and 23.5% HER2 amplification by silver in situ hybridization. Among the 15 paired biopsy and resection specimens, 9 (60%) had at least pT2 stage GE-GEJ-AC with HER2 IHC scores of 0, 1+, or 2+ in the biopsy, and 2 of those 9 cases (22%) had IHC 3+ and HER2 amplification by silver in situ hybridization on the resection specimen. Conclusions.— Our data suggest that HER2 testing should be repeated on resection specimens of GE-GEJ-AC with HER2 IHC scores of negative (0 and 1+) or equivocal (2+) and in situ hybridization amplification negative biopsy specimen results to evaluate for HER2 heterogeneity when patients are being considered for anti-HER2 therapy.

Mucinous cystic neoplasm (MCN) is characterized by mucinous epithelial lining and subepithelial ovarian-like stroma and is a precursor to invasive carcinoma. Type 1 autoimmune pancreatitis (AIP), on the other hand, is a form of IgG4-related disease (IgG4-RD) and can present as a pancreatic mass. There is no known causal relationship between these two entities in the current literature. Here, we report a case of a middle-aged female patient who was found to have an enlarging pancreatic mass and subsequently underwent a distal pancreatectomy. Gross examination of the pancreatectomy specimen showed a 4.3 cm mass with a central 3.4 cm cystic component, confirmed to be an MCN on microscopy. The immediately adjacent peritumoral tissue showed storiform fibrosis, obliterative phlebitis, and focal IgG4-positive plasma cell–rich infiltrates (≥75/HPF) with an IgG4+/IgG+ ratio of 80%. The background pancreas was histologically normal and these characteristic AIP findings were limited to the pericystic tissue. Clinical workup with CT scan and MRI did not show any evidence of systemic IgG4-RD, nor was the serum IgG4 level ever elevated. To our knowledge, this is only the second reported case of a pancreatic MCN associated with peritumoral AIP-like changes in a patient without systemic IgG4-RD. Awareness of these potential AIP-like changes surrounding pancreatic neoplasms may be helpful in preventing misdiagnosis in needle biopsy and aspiration specimens. Keywords: Type 1 autoimmune pancreatitis, IgG4-related disease, IgG4-RD, IgG4-related pancreatitis, Mucinous cystic neoplasm, MCN, Sclerosing pancreatitis

Background: It has been reported that coronary slow flow (CSF) is associated with acute myocardial infarction, ventricular tachycardia, ventricular fibrillation, and even sudden cardiac death. Although studies concerning the etiopathogenesis of CSF are scarce, diffuse atherosclerosis and endothelial dysfunction are thought to play important roles. It has been suggested that a high plasma thrombomodulin (TM) level seems to play an important role in the pathogenesis of atherosclerosis and endothelial dysfunction. Objectives: We hypothesized that a high plasma TM level might be associated with CSF and aimed to research the relationship between plasma TM level and CSF. Methods: Fifty-two CSF patients with angiographically proven CSF and 44 cases with normal coronary flow were included in this study. Coronary flow velocity was determined by the thrombolysis in myocardial infarction (TIMI) frame count method. Plasma TM levels were measured in all the study subjects. Results: Plasma TM levels were significantly higher in the CSF group compared to the control group (3.9 ± 0.5 vs. 3.6 ± 0.3 ng/mL, p = 0.01). There was a positive relationship (r = 0.31, p = 0.002) between plasma TM level and mean TIMI frame count (TFC). Factors associated with mean TFC were plasma TM level (β = 0.206, p = 0.038) and red cell distribution width (β = 0.088, p = 0.009) in multiple linear regression analysis. Conclusions: Patients with CSF have a higher plasma TM level, and this may play an important role in the pathogenesis of CSF. An elevated plasma TM level may be a predictor of CSF. Future studies are needed to confirm these results.

Transcriptional changes accompany induced resistance of plants against insect feeding, and monitoring transcriptional reorganization triggered in response to herbivory is an essential step for deciphering the molecular basis of the resistance. To isolate up-regulated genes in tea leaves under mild infestation of green leafhopper (Empoasca vitis Gothe), a subtractive cDNA library was constructed using the suppression subtractive hybridization strategy. Subsequent differential screening, DNA sequencing analyses, and real-time PCR analyses identified 21 unique genes up-regulated by leafhopper feeding from the library. Among the genes likely to be directly involved in defense, those encoding pathogen-related protein and type 2 ribosome-inactivating protein were found in the subtractive library. In addition, genes involved in the CBL (calcineurin B-like)–CIPK (CBL-interacting protein kinases) pathway, cell wall fortification, secondary metabolite synthesis, water transport, and oxidative stress protection were identified in our experiment. Besides, a number of genes that have not been previously associated with defense responses against insects were isolated.

Sinonasal undifferentiated carcinoma (SNUC) is an aggressive malignancy of disputed histogenesis that arises in the sinonasal tract and has an extremely poor prognosis. Despite multimodality treatment with surgical resection, radiation, and chemotherapy, recurrence is common. The tumor spreads by direct local extension, but also metastasizes to lymph nodes, brain, lung and bone. To date, reports of metastasis to the skin have not been published. We report a case of a 58-year-old woman diagnosed with SNUC who underwent surgical resection of the tumor followed by chemoradiation. The tumor soon recurred, and she required wide re-excision. Two months after this procedure, she developed multiple dermal nodules in the head and neck region, clinically suspicious for metastases. Biopsy of a nodule from the right neck revealed a poorly differentiated carcinoma, with morphological and immunohistochemical findings consistent with a metastasis of the patient's known SNUC. We conclude that the skin may be a rare site of metastasis of SNUC, and in some cases may be the presenting sign of tumor recurrence despite aggressive multimodality treatment.

The diagnosis of sebaceous carcinoma presents an important challenge to both clinicians and pathologists, as many cases are initially misdiagnosed both clinically and histopathologically, potentially leading to adverse medical and legal outcomes. The distinction of sebaceous carcinoma from benign sebaceous proliferations and other tumors is therefore of utmost importance, and immunohistochemistry may be useful in this differential. We studied the expression of D2-40 (podoplanin) by immunohistochemistry to determine if it can aid in this differential diagnosis and to evaluate the possibility of lymphangiogenesis in sebaceous carcinoma. A total of 36 cases of sebaceous lesions, including 16 sebaceous carcinomas, 7 sebaceous adenomas, 6 sebaceomas, and 7 cases of normal glands and sebaceous hyperplasia, and 17 cases of basal cell carcinoma and 10 cases of squamous cell carcinoma, were also examined. We found no significant increase in tumor lymphangiogenesis by semiquantitative scoring of lymphovascular density per square millimeter of tumoral/peritumoral stroma in sebaceous carcinoma versus benign sebaceous proliferations. However, D2-40 staining showed a different pattern in the benign tumors, which were positive only in the basaloid cells (most pronounced in sebaceoma), versus sebaceous carcinoma, which was either negative or focally positive in a haphazard pattern in most cases, although some cases of basaloid sebaceous carcinomas showed strong positivity. We also found D2-40 to be only weakly and focally positive in basal cell carcinoma and weakly to moderately positive in squamous cell carcinoma, which showed increased staining with decreased differentiation. Therefore, overall, D2-40 is, of limited diagnostic utility in sebaceous lesions but may be useful in distinguishing sebaceoma and basaloid sebaceous carcinoma from basal cell carcinoma.

Glutamate excitotoxicity contributes to the development of Parkinson's disease (PD) and pharmacological blockade of metabotropic glutamate receptor 5 (mGluR5) has beneficial anti-akinetic effects in animal models of PD; however, the mechanism by which these antagonists alleviate PD symptoms is largely unknown. In our study, the effects of mGluR5 inhibition on DNA damage were investigated in a rotenone-induced model of PD. We first found that the selective mGluR5 antagonist, 2-methyl-6- (phenylethynyl) pyridine, prevented rotenone-induced DNA damage in MN9D dopaminergic neurons through a mechanism involving the downregulation of intracellular calcium release which was associated with a reduction in endoplasmic reticulum stress and reactive oxygen species (ROS)-related mitochondrial dysfunction. Interestingly, the ROS-related mitochondrial dysfunction was accompanied by an increase in expression of the antioxidant protein, Trx2. Treatment of cells with the calcium chelating agent 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or the ROS scavenger N-acetyl-L-cysteine, also reduced rotenone-induced DNA damage, while transfection of a dominant-negative form of Trx2 increased it. In addition, mGluR5 inhibition altered the expression profiles of proteins involved in DNA repair activity. Specifically, the expression of phosphorylated ERK (p-ERK) and CREB, as well as APE1 and Rad51 were elevated after rotenone stimulation and were subsequently downregulated following blockade of mGluR5. These findings were confirmed in vivo in a rotenone-induced rat model of PD. Inhibition of mGluR5 protected against neurotoxicity by mitigating oxidative stress-related DNA damage associated with 8-hydroxy-2'-deoxyguanosine production and also reduced p-ERK activity and Trx2 expression. These findings provide a novel link between mGluR5 and DNA damage in a model of PD, and reveal a potential mechanism by which mGluR5 mediates DNA damage in neurodegenerative diseases.

Inhalation of residual oil fly ash (ROFA), a component of ambient particulate matter, has been shown to increase pulmonary morbidity and impair lung defense mechanisms in exposed workers. Our objective was to evaluate the effect of ROFA preexposure on lung defense and injury after pulmonary challenge with a bacterial pathogen. Male Sprague-Dawley rats were dosed intratracheally at day 0 with saline (control) or ROFA (0.2 or 1 mg/100 g body weight). Three days later, a low (5 x 10(3)) or high (5 x 10(5)) dose of Listeria monocytogenes was instilled intratracheally into the ROFA- and saline-treated rats. Bronchoalveolar lavage was performed on the right lungs at days 6, 8, and 10. The recovered cells were differentiated, and chemiluminescence (CL) and nitric oxide (NO) production, two indices of alveolar macrophage (AM) function, were measured. At the same time points, the left lung and spleen were removed, homogenized, and cultured, and colony-forming units were counted after an overnight incubation. Exposure to ROFA and the high dose of L. monocytogenes led to marked lung injury and inflammation as well as to an increase in mortality, compared with rats treated with saline and the high dose of L. monocytogenes. Preexposure to ROFA significantly enhanced injury and delayed the pulmonary clearance of L. monocytogenes at both bacterial doses when compared to the saline-treated control rats. ROFA had no effect on AM CL but caused a significant suppression of AM NO production, as compared to the saline control rats. We have demonstrated that acute exposure to ROFA slowed the pulmonary clearance of L. monocytogenes. The suppression in AM NO production by ROFA pretreatment likely plays an important role. These results suggest that pulmonary exposure to ROFA may alter AM function and lead to increased susceptibility to lung infection in exposed populations.

This study is aimed at developing a high quality, validated finite element (FE) human head model for traumatic brain injuries (TBI) prediction and prevention during vehicle collisions. The geometry of the FE model was based on computed tomography (CT) and magnetic resonance imaging (MRI) scans of a volunteer close to the anthropometry of a 50th percentile male. The material and structural properties were selected based on a synthesis of current knowledge of the constitutive models for each tissue. The cerebrospinal fluid (CSF) was simulated explicitly as a hydrostatic fluid by using a surface-based fluid modeling method. The model was validated in the loading condition observed in frontal impact vehicle collision. These validations include the intracranial pressure (ICP), brain motion, impact force and intracranial acceleration response, maximum von Mises stress in the brain, and maximum principal stress in the skull. Overall results obtained in the validation indicated improved biofidelity relative to previous FE models, and the change in the maximum von Mises in the brain is mainly caused by the improvement of the CSF simulation. The model may be used for improving the current injury criteria of the brain and anthropometric test devices.

Study objectives To examine the lung defensemechanisms of both young and aged rats before and after pulmonarychallenge with a bacterial pathogen. Design Male Fischer 344 rats, either 2.5 months or 20 months of age, wereintratracheally inoculated with 5 × 10 3 ,5 × 10 4 , or 5 × 10 5 Listeriamonocytogenes , and the effects on mortality, lung inflammation, pulmonary bacterial clearance, alveolar macrophage (AM) function, and T-lymphocyte characterization were determined. Measurementsand results In noninfected control animals, the older rats hadlower numbers of AMs on lavage and a lower percentage of total T, CD4+, and CD8+ cells. No difference was observed between noninfected youngand old rats in AM function, assessing both chemiluminescence andnitric oxide (NO) production. After bacterial challenge, aged ratsexhibited an increase in mortality, pulmonary infection, and edema, andlung lesions, which were more extensive than those observed in theyounger rats. Interestingly, AM chemiluminescence was enhanced, while AM NO, a highly important antibacterial defense product, was abrogatedin the aged rats as compared to the young rats. Conclusions This study demonstrated that advanced age isassociated with alterations in lung defense mechanisms and increasedsusceptibility to pulmonary bacterial infection marked by elevatedmortality, slowed pulmonary bacterial clearance, and altered AMfunction, specifically a decrease in NO production. These observationsare indicative of reduced pulmonary defense function in an olderpopulation of rats.

In this study, we tested the hypothesis that exposure to diesel exhaust particles (DEP) may increase susceptibility of the host to pulmonary infection. Male Sprague-Dawley rats received a single dose of DEP (5 mg/kg), carbon black (CB, 5 mg/kg), or saline intratracheally. Three days later, the rats were inoculated intratracheally with approximately 5,000 Listeria monocytogenes and sacrificed at 3, 5, and 7 days postinfection, and we determined the number of viable Listeria in the left lobe of lungs. The remaining lungs underwent bronchoalveolar lavage (BAL) and the retrieved BAL cells were identified and counted. Luminol-dependent chemiluminescence, a measure of reactive oxygen species (ROS) formation, generated by BAL cells was monitored and the levels of nitric oxide and tumor necrosis factor (TNF)-[alpha] produced by macrophages in culture were determined. At 7 days postinfection, we excised the lung-draining lymph nodes and phenotyped the lymphocyte subpopulations. Exposure of rats to DEP, but not to CB, decreased the clearance of Listeria from the lungs. Listeria-induced generation of luminol-dependent chemiluminescence by pulmonary phagocytes decreased by exposure to DEP but not CB. Similarly, Listeria-induced production of NO by alveolar macrophages was negated at 3, 5, and 7 days after inoculation in DEP-exposed rats. In contrast, CB exposure had no effect on Listeria-induced NO production at 3 days after infection and had a substantially smaller effect than DEP at later days. Exposure to DEP or CB resulted in enlarged lung-draining lymph nodes and increased the number and percentage of CD4(+) and CD8(+) T cells. These results showed that exposure to DEP decreased the ability of macrophages to produce antimicrobial oxidants in response to Listeria, which may play a role in the increased susceptibility of rats to pulmonary infection. This DEP-induced suppression is caused partially by chemicals adsorbed onto the carbon core of DEP, because impaired macrophage function and decreased Listeria clearance were not observed following exposure to CB.

Dichloroacetic acid (DCA) and chloroform (CHCl 3 ) are both major by-products of drinking water chlorination and DCA increases the hepatotoxicity of CHCl 3 . In this study, we further characterized this effect and investigated DCA-induced alterations of CHCl 3 disposition and metabolism as a possible mechanism for this interaction. Both adult male and female Sprague-Dawley rats were gavaged with three doses (09:00, 16:00 and 09:00 the next morning) of DCA (each 2.45 mmol/kg), then challenged with an i.p. injection of CHCl 3 (3.12 or 9.35 mmol/kg). Hepatic damage was assessed 24 h after CHCl 3 administration as increased alanine aminotransferase (ALT), ornithine carbomyl transferase (OCT) and bilirubin in plasma. In a separate experiment, rats were pretreated with DCA or were given 14 CHCl 3 at the same dosages. The disposition of 14 C in various tissues and covalent binding of 14 CHCl 3 -derivatives to liver proteins and lipids were determined 1 h later. CHCl 3 -induced hepatotoxicity was significantly more severe in DCA-pretreated groups. ALT and OCT were more markedly elevated in DCA+CHCl 3 (3.12 mmol/kg) groups than NaCl+CHCl 3 animals. Plasma bilirubin content was elevated only in DCA+CHCl 3 groups and females were more susceptible to this effect. The responses of rats to DCA treatment were somewhat gender-different. DCA treatment increased total cytochrome P450 in females, but not in males. Hepatic glutathione concentration was elevated in males after DCA treatment, but not in females. In the present study we confirmed that DCA pretreatment potentiates the CHCl 3 -hepatotoxicity of both male and female rats. However, there was little evidence that DCA pretreatment significantly affected CHCl 3 disposition or increased CHCl 3 binding in vivo.

Previously, we showed that the pretreatment of dichloroacetic acid (DCA) increased CHCl3-induced hepatotoxicity in vivo and CHCl3 metabolism in vitro in both male and female rats. The effects of DCA on hepatic cytochrome P450-dependent monooxygenases were studied in this experiment. Male and female Sprague-Dawley rats were treated with DCA (each 2.45 mmol/kg) three times (9 AM, 4 PM, and 9 AM) and hepatic microsomes were prepared 3 hr after the last treatment (the same procedure as previous studies). After DCA treatment, the total content of cytochrome P450 (0.67 nmol/mg protein vs 0.79) and the activity of NADPH-dependent cytochrome P450 reductase (227 nmol/mg protein/min vs 332) were significantly increased in female rats, but they were unchanged in males (0.99 vs 0.98 for P450; 315 vs 311 for reductase). Induction of CYP2E1 was observed in both sexes, evidenced as increased activities of aniline and p-nitrophenol hydroxylases and increased CYP2E1 protein amount determined by immunoblot assay. In contrast, the CYP2B-related activity (dealkylation of pentoxyresorufin) and immunoreactive protein did not increase after DCA treatment in either males or females. The activity of ethoxyresorufin dealkylase was decreased in DCA-treated males compared to their controls (310 pmol/mg protein/min vs 229, p0.05), but it was not significantly affected in females. These data demonstrate that DCA treatment of both male and female rats altered the population of hepatic cytochrome P450. The results support the hypothesis that DCA increases CHCl3 metabolism, and therefore hepatotoxicity, by inducing CYP2E1.

Metabotropic glutamate receptors (mGlus) are G-protein-coupled receptors playing an important role in the central nervous system (CNS). Recently, mGlus have been identified in peripheral tissues, and aberrant expression or inhibition of the receptors functions in the development of certain cancers. However, the correlation of mGlu activity with hepatocellular carcinoma (HCC) remains unknown. In this study, we analyzed the effects of inhibiting mGlu5 activity in hepatocarcinoma cell lines and a xenograft model. Inactivation of mGlu5 with 2-Methyl-6-(phenylethyl)-pyridine (MPEP), a specific antagonist of the receptor, caused inhibition of cell growth, migration, and invasion of HepG2 and Bel-7402 cells, assessed by MTT assay, ATP production, wound healing, and Boyden chamber assay, respectively. Moreover, inhibition of tumor growth and the potential metastasis of hepatocellular carcinoma were also found in nude mice. Furthermore, mGlu5-mediated extracellular signal-regulated kinase (ERK) phosphorylation has been found to be partially involved in cell growth and migration, as detected by stimulation of (S)-3,5-Dihydroxyphenylglycine (DHPG), an agonist of the receptor, and blockage of MPEP and U0126, an inhibitor of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (MEK). These data indicate that inhibiting the activity of mGlu5 has the molecular potential to suppress oncogenic actions by blocking downstream effector molecules. The study suggests that mGlu5 activity may contribute to understanding the development of HCC.