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A key feature of lung fibrosis is the accumulation of myofibroblasts. Interleukin 13 (IL-13) is a pro-fibrotic mediator that directly and indirectly influences the activation of myofibroblasts. Transforming growth factor beta (TGF-β) promotes the differentiation of fibroblasts into myofibroblasts, and can be regulated by IL-13. However, IL-13’s downstream signaling pathways are not completely understood. We previously reported that the transcription factor Yin Yang 1 (YY1) is upregulated in fibroblasts treated with TGF-β and in the lungs of mice and patients with pulmonary fibrosis. Moreover, YY1 directly regulates collagen and alpha smooth muscle actin (α-SMA) expression in fibroblasts. However, it is not known if IL-13 regulates fibroblast activation through YY1 expression. We hypothesize that IL-13 up-regulates YY1 expression through regulation of AKT activation, leading to fibroblast activation. In this study we found that YY1 was upregulated by IL-13 in lung fibroblasts in a dose- and time-dependent manner, resulting in increased α-SMA. Conversely, knockdown of YY1 blocked IL-13-induced α-SMA expression in fibroblasts. Furthermore, AKT phosphorylation was increased in fibroblasts treated with IL-13, and AKT overexpression upregulated YY1, whereas blockade of AKT phosphorylation suppressed the induction of YY1 by IL-13 in vitro. In vivo YY1 was upregulated in fibrotic lungs from CC10-IL-13 transgenic mice compared to that from wild-type littermates, which was associated with increased AKT phosphorylation. Taken together, these findings demonstrate that IL-13 is a potent stimulator and activator of fibroblasts, at least in part, through AKT-mediated YY1 activation.

Immune-mediated lung injury is an important component of Pneumocystis pneumonia (PcP)-related immunorestitution disease (IRD). However, the individual contribution of CD4+and CD8+T cells to the pathophysiology of IRD remains undetermined. Therefore, IRD was modeled in severe combined immunodeficient mice, and specific T cell depletion was used to determine how T cell subsets interact to affect the nature and severity of disease. CD4+cells were more abundant than CD8+cells during the acute stage of IRD that coincided with impaired pulmonary physiology and organism clearance. Conversely, CD8+cells were more abundant during the resolution phase following P. carinii clearance. Depletion of CD4+T cells protected mice from the acute pathophysiology of IRD. However, these mice could not clear the infection and developed severe PcP at later time points when a pathological CD8+T cell response was observed. In contrast, mice depleted of CD8+T cells efficiently cleared the infection but developed more severe disease, an increased frequency of IFN-γ-producing CD4+cells, and a prolonged CD4+T cell response than mice with both CD4+and CD8+cells. These data suggest that CD4+T cells mediate the acute respiratory disease associated with IRD. In contrast, CD8+T cells contributed to neither lung injury nor organism clearance when CD4+cells were present, but instead served to modulate CD4 function. In the absence of CD4+cells, CD8+T cells produced a nonprotective, pathological immune response. These data suggest that the interplay of CD4+and CD8+T cells affects the ultimate outcome of PcP-related IRD.

To investigate clinicopathological characteristics of colorectal sessile serrated adenoma/polyp (SSA/P) and its differential diagnosis from other serrated lesions.Clinicopathological features of all cases of colorectal serrated lesions from 5 209 colorectal biopsy samples at Shanghai Tongji Hospital from 2008 to 2013 were reviewed. Three hundred and fifty-three cases of serrated lesions were erolled in the study. Morphological features of SSA/P were investigated with an emphasis on histologic criteria for diagnosis and a literature review was performed.Three hundred and fifty-three cases of serrated lesions were identified, including 25 SSA/P (7.1%), 278 hyperplastic polyp (HP, 78.8%), and 44 traditional serrated adenoma (TSA, 12.5%). Twenty-five patients with SSA/P consisted of 16 males and 9 females with a mean age of 62.2 years (aged 34-84 years) and the lesions involved sigmoid colon (14 cases), ascending colon (9 cases), rectum (1 case) and transverse colon (1 case). Grossly, the majority of SSA/P was sessile with an averaged size of 0.73 cm. Histologically, typical SSA/P had elongated crypts with prominent serration and distorted crypts architecture. The detection rates of crypts dilatation and branching in SSA/P and HP were 100% (25/25) and 24% (12/50, P0.01), 72% (18/25) and 4% (2/50, P0.01), respectively. Morphological features observed only in SSA/P included L-shaped crypts (48%, 12/25), pseudo infiltration of mucosa muscle (16%, 4/25), atypical nuclei (32%, 8/25), and increased mucus secretion (24%, 6/25).SSA/P microscopically shows prominent serration and abnormal architectures of crypts. Complete tissue sectioning and correct embedding are helpful for the diagnosis. SSA/P without cytological dysplasia should be distinguished from HP, especially those with only a few distorted crypts.

Patent foramen ovale and atrial septal aneurysm are associated with an increased risk of cryptogenic stroke and recurrent thromboembolic events. Percutaneous closure is a therapeutic option to medical therapy and surgical closure. We present the first case of endocarditis associated with a CardioSEAL device closing a patent foramen ovale.

Accurate lymph nodal staging of lung cancer is critical for determining the treatment options. With the help of (18)F-fluorodeoxyglucose positron emission tomography/computer tomography ((18)F-FDG-PET/CT), the clinician can rule out/in the regional lymph nodes positive for metastasis in the patients with lung cancer in a majority of cases. However, a small proportion of cases with false positivity of metastasis have been reported. Transbronchial needle aspirations and mediastinoscopic biopsies are still necessary to determine whether enlarged hypermetabolic mediastinal lymph nodes are positive for lung cancer metastasis. Here we report three intricate cases showing hypermetabolic activity in the mediastinal lymph nodes in the patients with pathologically diagnosed lung cancer on PET/CT. The first patient had squamous cell carcinoma in the left upper lobe of the lung with surrounding necrotizing granulomas and concurrent with silicosis and granulomatous inflammation in the lymph nodes; the second presented with symptoms of viral pneumonia, which was pathologically diagnosed as a lung adenocarcinoma, stage IA, concurrent with sarcoidosis involving the lymph nodes; the last case was diagnosed as squamous cell carcinoma in the right upper lobe of the lung, but lymph nodes showed reactive hyperplasia. These cases suggest that some cases are so complex that avid (18)F-FDG uptake in the mediastinal lymph nodes in the patients with pathologically diagnosed lung cancer should be carefully analyzed based on individual patients' clinical background.

Our in vitro studies revealed that a transcription factor, Forkhead box protein O1 (FoxO1), negatively regulates the expression of NaV1.5, a main α subunit of the cardiac Na(+) channel, by altering the promoter activity of SCN5a in HL-1 cardiomyocytes. The in vivo role of FoxO1 in the regulation of cardiac NaV1.5 expression remains unknown. The present study aimed to define the role of FoxO1 in the regulation of NaV1.5 expression and cardiac Na(+) channel activity in mouse ventricular cardiomyocytes and assess the cardiac electrophysiological phenotype of mice with cardiac FoxO1 deletion. Tamoxifen-induced and cardiac-specific FoxO1 deletion was confirmed by polymerase chain reaction (PCR). Cardiac FoxO1 deletion failed to result in either cardiac functional changes or hypertrophy as assessed by echocardiography and individual ventricular cell capacitances, respectively. Western blotting showed that FoxO1 was significantly decreased while NaV1.5 protein level was significantly increased in mouse hearts with FoxO1 deletion. Reverse transcription-PCR (RT-PCR) revealed that FoxO1 deletion led to an increase in NaV1.5 and Na(+) channel subunit β3 mRNA, but not β1, 2, and 4, or connexin 43. Whole patch-clamp recordings demonstrated that cardiac Na(+) currents were significantly augmented by FoxO1 deletion without affecting the steady-state activation and inactivation, leading to accelerated depolarization of action potentials in mouse ventricular cardiomyocytes. Electrocardiogram recordings showed that the QRS complex was significantly shortened and the P wave amplitude was significantly increased in conscious and unrestrained mice with cardiac FoxO1 deletion. NaV1.5 expression was decreased in the peri-infarct (border-zone) of mice with myocardial infarction and FoxO1 accumulated in the cardiomyocyte nuclei of chronic ischemic human hearts. Our findings indicate that FoxO1 plays an important role in the regulation of NaV1.5 and β3 subunit expressions as well as Na(+) channel activity in the heart and that FoxO1 is involved in the modulation of NaV1.5 expression in ischemic heart disease.

BACKGROUND Metastasis-associated in colon cancer-1 (MACC1), a newly identified regulator of HGF-MET signaling, may participate into the key steps of sporadic colorectal adenocarcinoma development. Given there are many pathogenetic distinctions between colitis-associated colorectal cancer (CAC) and sporadic colorectal adenocarcinomas, the potential roles of MACC1 in CAC carcinogenesis remain unknown. For the first time, we evaluated the expressions of MACC1 and MET in IBD-associated colitis, dysplasia, and adenocarcinoma. METHODS Expression was investigated by immunohistochemistry in tissue microarrays consisting of 13 normal colon, 11 active colitis, 9 dysplasia, 51 conventional CAC, 5 mucinous adenocarcinoma, and 1 signet ring cell adenocarcinoma specimens. The expression level of MACC1 or MET was evaluated with H-score system. RESULTS MACC1 expression was significantly higher in IBD-associated dysplasia than that in corresponding inflammatory or normal colonic tissue, and its level was further elevated from dysplasia to conventional CAC. Higher MACC1 expression was seen in a patient with CAC who had multifocal dysplasia or synchronous carcinoma. MACC1 overexpression (H-score >100) was seen in 67% of conventional CAC but in 0% of dysplasia and 0% of inflammation or normal colon. There was no difference of MACC1 expression found among well, moderately and poorly differentiated CAC. MET expressions in inflammation, dysplasia, and conventional CAC were statistically similar. No parallel expression of MACC1 and MET was detected in this study. MACC1 and MET expression was not increased in mucinous or signet ring cell carcinoma, 2 distinct variants of CAC. CONCLUSIONS Stepwise increase of MACC1 expression from IBD-associated colitis to dysplasia to adenocarcinoma suggests that MACC1 is strongly associated with conventional CAC tumorigenesis in a manner independent of MET. MACC1 may serve as a potential marker for early diagnosis of conventional CAC.

D2-40 is a new monoclonal antibody recognizing podoplanin and it is selective for lymphatic endothelium. Several studies have confirmed the reliability of D2-40 in the diagnosis of pleural malignant mesothelioma, but the evaluation of this antibody in other pleural neoplasms is limited. The aim of this study was to determine the diagnostic value of D2-40 in segregation of malignant mesothelioma from other pleural or lung neoplasms. Sixty-seven cases, including 36 pleural malignant mesotheliomas, 15 solitary fibrous tumors, 13 pleomorphic carcinomas, and 3 synovial sarcomas, were immunohistochemically studied using a D2-40 monoclonal antibody. Twenty-five (21 epithelioid and 4 biphasic) of 36 (69%) malignant mesotheliomas and 2 of 15 (13%) solitary fibrous tumors were positive for D2-40. The difference of D2-40 positivity between these 2 types of tumor was significant (P

Introduction: Almost all small-cell lung cancers (SCLC) and carcinoid tumors express neuroendocrine differentiation (NED), and 10% to 20% of non–small-cell lung cancers (NSCLC) are associated with NED. Although distinct clinical features and histology of SCLC and carcinoid tumors are well recognized, the clinical significance and the molecular basis of NED in NSCLC remain unclear. Methods: To explore the potential molecular pathway involved in NED of NSCLC and its clinical relevance, we conducted investigations using an NSCLC cell line (NCI-H157) as a NED induction model, and explored the potential intracellular signal transduction pathways involved in NED of NSCLC. We confirmed our findings using activators versus inhibitors to these signal transduction pathways in vitro. We also performed immunohistochemical stains of phospho-Erk1/2 of lung cancer specimens known to have NED and explored its clinical relevance. Results: We discovered that NED of NSCLC was associated with the activation of Erk1/2-mitogen-activated protein kinases (MAPK) signal transduction pathway, and the inhibition of the Akt signal transduction pathway. Using specific activator (Pb2+) and inhibitors (siRNA-Erk1/2 and U0126) to the Erk1/2-MAP-kinase pathway, as well as the inhibitor (LY294002) to the Akt pathway, we found that Erk1/2-MAP-kinase activation was essential for NED of NCI-H157 cells. Staining of Erk1/2-MAP-kinase pathway revealed a high rate of positivity in NSCLC tumors with NED when compared with other neuroendocrine lung tumors. Conclusions: To our knowledge, our findings are the first to describe the potential involvement of Erk/MAPK signal transduction pathway of NSCLC in the association with NED. Further investigation of the Erk/MAPK signal transduction pathway of NSCLC may yield discoveries in identifying specific molecular targets for the treatment of NSCLC with NED.

Parathyroid adenoma with prominent lymphocytic infiltrates is very rare, with only 8 previously reported cases in the English scientific literature. Cases with prominent lymphocytic infiltrates of ectopic (mediastinal) parathyroid adenomas have not been reported. The prominent lymphocytic infiltration may make the diagnosis difficult on frozen sections, even on permanent histologic sections. We herein describe a case of ectopic (mediastinal) parathyroid adenoma with prominent lymphocytic infiltration in a 29-year-old man with hyperparathyroidism for 4 years. Immunohistochemical studies were important in making such a diagnosis.

In the experiments here, the time- and voltage-dependent properties of the Ca2+-independent, depolarization-activated K+ currents in adult mouse ventricular myocytes were characterized in detail. In the majority (65 of 72, approximately 90%) of cells dispersed from the ventricles, analysis of the decay phases of the outward currents revealed three distinct K+ current components: a rapidly inactivating, transient outward K+ current, Ito,f (mean +/- SEM taudecay = 85 +/- 2 ms); a slowly (mean +/- SEM taudecay = 1,162 +/- 29 ms) inactivating K+ current, IK,slow; and a non inactivating, steady state current, Iss. In a small subset (7 of 72, approximately 10%) of cells, Ito,f was absent and a slowly inactivating (mean +/- SEM taudecay = 196 +/- 7 ms) transient outward current, referred to as Ito,s, was identified; the densities and properties of IK,slow and Iss in Ito,s-expressing cells are indistinguishable from the corresponding currents in cells with Ito,f. Microdissection techniques were used to remove tissue pieces from the left ventricular apex and from the ventricular septum to allow the hypothesis that there are regional differences in Ito,f and Ito,s expression to be tested directly. Electrophysiological recordings revealed that all cells isolated from the apex express Ito,f (n = 35); Ito,s is not detected in these cells (n = 35). In the septum, by contrast, all of the cells express Ito,s (n = 28) and in the majority (22 of 28, 80%) of cells, Ito,f is also present. The density of Ito,f (mean +/- SEM at +40 mV = 6.8 +/- 0.5 pA/pF, n = 22) in septum cells, however, is significantly (P0.001) lower than Ito,f density in cells from the apex (mean +/- SEM at +40 mV = 34.6 +/- 2.6 pA/pF, n = 35). In addition to differences in inactivation kinetics, Ito,f, Ito,s, and IK,slow display distinct rates of recovery (from inactivation), as well as differential sensitivities to 4-aminopyridine (4-AP), tetraethylammonium (TEA), and Heteropoda toxin-3. IK,slow, for example, is blocked selectively by low (10-50 microM) concentrations of 4-AP and by (/=25 mM) TEA. Although both Ito,f and Ito,s are blocked by high (100 microM) 4-AP concentrations and are relatively insensitive to TEA, Ito,f is selectively blocked by nanomolar concentrations of Heteropoda toxin-3, and Ito,s (as well as IK,slow and Iss) is unaffected. Iss is partially blocked by high concentrations of 4-AP or TEA. The functional implications of the distinct properties and expression patterns of Ito,f and Ito,s, as well as the likely molecular correlates of these (and the IK,slow and Iss) currents, are discussed.

Lu Xiao, Haiqing Bai, James Boyer, Bo Ye, Ning Hou, Haodong Xu, and Faqian Li Department of Pathology and Laboratory Medicine and Cardiovascular Research Institute, University of Rochester Medical Center, Rochester, NY, USA Backgrounds: Canonical Wnt signaling appears to have multiphasic and often antagonistic roles in cardiac development. The molecular mechanism for these opposing actions is not clear. We hypothesized that alternative splicing of TCF7L2, a nuclear interaction partner of beta-catenin is involved in the specificity of canonical Wnt signaling. Methods: RT-PCR were performed on embryonic (E16.5) and neonatal (day 8) hearts with primers spanning the end of first exon and the beginning of last exon and the products were cloned and sequenced. Result: There are totally 18 exons identified so far in TCF7L2. We sequenced 56 clones and 53 clones (29 from day 8) and (24 from E16.5) contained TCF7L2 sequences. No exon 6 or exon 17 was found in TCF7L2 transcripts of mouse hearts. Most clones (more than 80%) from E16.5 and day 8 hearts excluded exon 4. Both E16.5 and day 8 hearts had one clone with exon 9 deletion which does not change reading frame and another with alterations in exon 3 that lead to reading frame shift and premature stop codon. As reported in other organs, there were extensive alternative splicing in the C-terminal exons 14, 15 and 16. The inclusion of exon 14 was more frequently in day 8 (18 of 29, 62%) than in E16.5 (8 of 24, 33%) hearts. The peptide encoded by exon 14 has conserved functional motif. Additionally, this alternative exon usage can change the C-terminus of TCF7L2 to include or exclude the so-called E tail with two binding motifs for C-terminal binding protein. Conclusion: The isoform switch of TCF7L2 occurs in neonatal mouse hearts and may have a role in the terminal differentiation of cardiac myocytes during this period.

Backgrounds: Genetic variations of TCF7L2, a nuclear transcriptional factor in canonical Wnt signaling are associated with increased risks of Diabetes Mellitus. How TCF7L2 is regulated by diabetic conditions in the heart is poorly understood. Methods: TCF7L2 expression was investigated by Western blotting in HL-1 cells treated with 14 mM mannitol (M), 25 mM glucose (G), 10 nM insulin (I), and G plus I (G+I). In addition, TCF7L2 levels were examined by Western blotting in the heart of B6.Cg-m Leprdb/+ +/J (db) and C57Bl/6J (C57) mice 4 hours after intraperitoneal injections of 1.5 IU/kg insulin. Result: The expression of TCF7L2 was higher in db than C57 hearts. Insulin treatment decreased TCF7L2 in C57 hearts. Paradoxically, TCF7L2 was further increased by insulin in db hearts. In HL-l cells, TCF7L2 started to decrease at one hour and remained lower up to 24 hours after insulin treatment compared to untreated controls. In contrast, G increased TCF7L2 expression at 1, 6 and 24 hours after treatment relative to M. Insulin could decrease TCF7L2 at 1 and 6 hours, but failed to do so at 24 hours in G+I. At 48 hours, no difference in TCF7L2 expression was observed among different treatment groups. Conclusion: Insulin regulation of TCF7L2 is interfered by high glucose in vitro and misregulation of TCF7L2 in the heart of diabetic mice may contribute to the pathogenesis of diabetic cardiomyopathy.

Backgrounds: DNA damage occurs in cardiomyocytes during normal cellular metabolism and is significantly increased under cardiac stresses. How cardiomyocytes repair their DNA damage, especially DNA double strand breaks (DSBs), remains undetermined. We assessed DSBs caused by oxidative stress. More importantly, we investigated the spatiotemporal dynamics of DNA repair protein assembly/disassembly in DNA damage sites. Methods: Cultured neonatal rat cardiomyocytes were treated with different doses of hydrogen peroxide (H2O2) for 30 minutes to assess DNA damage response (DDR). To investigate the dynamics of DDR, cells were treated with 200 uM H2O2 and followed up to 72 hours. DSBs were evaluated by counting DNA damage foci after staining with antibody against histone H2AX phosphorylation at serine 139 (g-H2AX). The dynamics and posttranslational modification of DNA repair proteins were determined by Western blotting, immunolabeling, and confocal microscopy. Result: g-H2AX was proportionally increased to H2O2 dosage. Discrete nuclear g-H2AX foci were seen 30 minutes after hydrogen peroxide treatment with 50 uM, but became pannuclear when H2O2 was above 400 uM. At 200 uM of hydrogen peroxide, g-H2AX started to increase at 15 minutes and reached to highest levels at 60 minutes with up to 70 nuclear foci, started to decline at 2 hours, and returned to basal levels at 24 hours. DDR transducer kinase, ataxia telangiectasia mutated (ATM) was activated at 5 minutes with increased phosphorylation at serine 1981 (pATM) which started to decrease at 24 hours, but remained elevated up to 48 hours. Another DDR transducer kinase, ATM and Rad3-related (ATR) showed a biphasic activation at 30 minutes and 8 hours. ATM and ATR colocalized with g-H2AX. DNA damage mediator proteins such as MRN complex and p53BP1 were also recruited to sites of DNA damage at g-H2AX foci. Conclusions: DSBs and their repair have emerged as a new frontier of stress responses. Newly developed methods for studying g-H2AX and DNA repair protein dynamics can be explored to investigate DDR to oxidative stress in cardiomyocytes.

Primary ovarian carcinoids and metastatic tumors share similar morphologic features. Metastatic carcinoids must be excluded from primary ones for prognostic and therapeutic reasons. Gastrointestinal neuroendocrine (carcinoid) tumors are much more common with the majority arising from small intestine and appendix. The aim of this study is to evaluate the role of immunohistochemistry for CDX2 in differentiating primary ovarian from metastatic carcinoids of primary gastrointestinal origin. Thirty primary pure ovarian carcinoids, 16 primary ovarian carcinoids arising in association with benign teratomas, 10 ovarian carcinoids metastatic from primary gastrointestinal tract and 70 gastrointestinal neuroendocrine tumors were studied for the expression of CDX2 by immunohistochemistry. CDX2 expression revealed that 40 (57.1%) of 70 cases of gastrointestinal carcinoids and 9 (90%) of 10 ovarian metastatic carcinoids showed positive nuclear staining (diffuse or focal). On the other hand, 3 (18.8%) of 16 primary carcinoids with teratomatous elements showed weak positivity. Among the 70 gastrointestinal carcinoids, CDX2 was positive in 38 (90.5%) of 42 cases in the duodenum, small intestine, appendix, and only in 2 (11.8%) of 17 cases of colorectal carcinoids and none of the 11 cases in the stomach. It is concluded that CDX2 may be a useful marker to distinguish primary ovarian carcinoid from metastasis from small intestinal and appendiceal neuroendocrine tumors.

In the experiments here, the developmental expression of the functional Ca(2+)-independent, depolarization-activated K+ channel currents, Ito and IK, and of the voltage-gated K+ channel (Kv) alpha subunits, Kv1.2, Kv1.4, Kv1.5, Kv2.1, and Kv4.2 in rat ventricular myocytes were examined quantitatively. Using the whole-cell patch clamp recording method, the properties and the densities of Ito and IK in ventricular myocytes isolated from postnatal day 5 (P5), 10 (P10), 15 (P15), 20 (P20), 25 (P25), 30 (P30), and adult (8-12 wk) rats were characterized and compared. These experiments revealed that mean Ito densities increase fourfold between birth and P30, whereas IK densities vary only slightly. Neither the time- nor the voltage-dependent properties of the currents vary measurably, suggesting that the subunits underlying functional Ito and IK channels are the same throughout postnatal development. In parallel experiments, the developmental expression of each of the voltage-gated K+ channel alpha subunits, Kv1.2, Kv1.4, Kv1.5, Kv2.1, and Kv4.2, was examined quantitatively at the mRNA and protein levels using subunit-specific probes. RNase protection assays revealed that Kv1.4 message levels are high at birth, increase between P0 and P10, and subsequently decrease to very low levels in adult rat ventricles. The decrease in message is accompanied by a marked reduction in Kv1.4 protein, consistent with our previous suggestion that Kv1.4 does not contribute to the formation of functional K+ channels in adult rat ventricular myocytes. In contrast to Kv1.4, the mRNA levels of Kv1.2, Kv1.5, Kv2.1, and Kv4.2 increase (three- to five-fold) between birth and adult. Western analyses, however, revealed that the expression patterns of these subunits proteins vary in distinct ways: Kv1.2 and Kv4.2, for example, increase between P5 and adult, whereas Kv1.5 remains constant and Kv2.1 decreases. Throughout development, therefore, there is a mismatch between the numbers of Kv alpha subunits expressed and the functional voltage-gated K+ channel currents distinguished electrophysiologically in rat ventricular myocytes. Alternative experimental approaches will be required to define directly the Kv alpha subunits that underlie functional voltage-gated K+ channels in these (and other) cells. In addition, the finding that Kv alpha subunit protein expression levels do not necessarily mirror mRNA levels suggests that caution should be exercised in attempting functional interpretations of observed changes in mRNA levels alone.

Background— Diabetes mellitus is a major risk factor for cardiovascular mortality by increasing endothelial cell (EC) dysfunction and subsequently accelerating atherosclerosis. Extracellular-signal regulated kinase 5 (ERK5) is activated by steady laminar flow and regulates EC function by increasing endothelial nitric oxide synthase expression and inhibiting EC inflammation. However, the role and regulatory mechanisms of ERK5 in EC dysfunction and atherosclerosis are poorly understood. Here, we report the critical role of the p90 ribosomal S6 kinase (p90RSK)/ERK5 complex in EC dysfunction in diabetes mellitus and atherosclerosis. Methods and Results— Inducible EC-specific ERK5 knockout (ERK5-EKO) mice showed increased leukocyte rolling and impaired vessel reactivity. To examine the role of endothelial ERK5 in atherosclerosis, we used inducible ERK5-EKO-LDLR −/− mice and observed increased plaque formation. When activated, p90RSK associated with ERK5, and this association inhibited ERK5 transcriptional activity and upregulated vascular cell adhesion molecule 1 expression. In addition, p90RSK directly phosphorylated ERK5 S496 and reduced endothelial nitric oxide synthase expression. p90RSK activity was increased in diabetic mouse vessels, and fluoromethyl ketone-methoxyethylamine, a specific p90RSK inhibitor, ameliorated EC-leukocyte recruitment and diminished vascular reactivity in diabetic mice. Interestingly, in ERK5-EKO mice, increased leukocyte rolling and impaired vessel reactivity were resistant to the beneficial effects of fluoromethyl ketone-methoxyethylamine, suggesting a critical role for endothelial ERK5 in mediating the salutary effects of fluoromethyl ketone-methoxyethylamine on endothelial dysfunction. Fluoromethyl ketone-methoxyethylamine also inhibited atherosclerosis formation in ApoE −/− mice. Conclusions— Our study highlights the importance of the p90RSK/ERK5 module as a critical mediator of EC dysfunction in diabetes mellitus and atherosclerosis formation, thus revealing a potential new target for therapeutic intervention.

Colonic inertia is a frustrating motility disorder to patients, clinicians, and pathologists. The pathogenesis is largely unknown. The aims of this study were to: (1) characterize the expression of smoothelin, a novel smooth muscle-specific contractile protein expressed only by terminally differentiated smooth muscle cells, in the normal gastrointestinal (GI) tract; and (2) determine whether smoothelin is aberrantly expressed in patients with colonic inertia. A total of 57 resections of the normal GI tract (distal esophagus to left colon) were obtained from patients without GI motor dysfunction. Sixty-one colon resections were obtained from patients with a clinical diagnosis of colonic inertia. Smoothelin immunostaining was conducted on full-thickness tissue sections. In the nondysmotile controls, strong and diffuse cytoplasmic staining for smoothelin was observed in both the inner circular and outer longitudinal layers of the muscularis propria (MP) throughout the entire GI tract. The muscularis mucosae (MM) and muscular vessel walls were either completely negative or only patchily and weakly stained. The 1 exception to this pattern was observed in the distal esophagus, in which the MM was also diffusely and strongly stained. In cases with colonic inertia, a moderate to marked reduction of smoothelin immunoreactivity was observed in 15 of 61 (24.6%) colon resections, selectively seen in the outer layer of the MP. The data demonstrate that smoothelin is differentially expressed in the MP and MM of the normal GI tract and suggest that defective smoothelin expression may play a role in the pathogenesis of colonic inertia in a subset of patients. Copyright © 2012 by Lippincott Williams & Wilkins.

These multimedia modules have been developed to teach senior medical students on their surgical pathology elective the basic microscopic features of common surgical pathology diagnoses. The modules include normal histology and pathology organized into the following organ systems: breast, gastrointestinal, genitourinary, gynecologic, liver, and lung. The first component to this teaching set is a review of normal histology which may serve as a comparison for the histopathology component. A low- and high-power micrograph of each organ is provided. The student is tested with a quiz of micrographs of normal histology where they must identify the organ. Next, students review commonly encountered biopsies in each organ system. Under each diagnosis is listed the definition, etiology, symptoms/indications for biopsy, gross features, microscopic features, and a differential diagnosis. Radiologic features and pertinent additional stains are noted when appropriate. The goal of the surgical pathology rotation at this institution is to allow students to function as junior house officers, actively participating in previewing and sign-out. These teaching modules will provide basic clinical and microscopic descriptions to help the student preview and diagnose biopsy specimens on their own. The clinical implications of certain diagnoses as they pertain to aggressive management in neoplastic disease will also be mentioned. The modules are designed so students can easily navigate between slides, diagnoses, and differentials. Each image is provided with arrows and labels (which may be hidden) to guide the eye of the learner to recognize the patterns of each diagnosis. After completing a module, students will solidify their knowledge with a quiz. We plan to monitor the effectiveness of this assessment by having students take the quiz at the start and end of the rotation to compare the change in their score. These modules will provide an organized foundation on which the student may build his or her knowledge during slide review with senior residents or staff pathologists.

Histopathologic distinction among small-cell carcinoma (SCC), pancreatic endocrine tumor (PET), and gastrointestinal carcinoids metastasized to the liver in needle core biopsies can be extremely challenging because of limited material, crush artifact, and lack of detailed clinical history. In this study, a total of 61 surgically resected or biopsied specimens, including 27 SCCs (lung, 17; colon, 1; gallbladder, 2; stomach, 1; and unknown primary, 6), 18 gastrointestinal carcinoid tumors (GICTs) (stomach, 2; small intestine, 14; colon, 2), and 16 PETs were immunohistochemically examined for the expression of IMP3, TTF-1, CDX2, and NESP55 to evaluate their diagnostic value. The results showed that 24 (89%) of 27 SCCs exhibited strong cytoplasmic staining for IMP3 in 60% to 100% of the tumor cells. Eighteen (67%) SCCs were strongly and diffusely positive for TTF-1. In the remaining 9 TTF-1-negative SCCs (including 4 extrapulmonary cases), 7 showed strong and diffuse IMP3 expression. All SCCs were negative for CDX2 except for 1 case of colonic origin that showed strong CDX2 immunoreactivity. All 16 metastatic PETs were positively stained for IMP3 with 12 cases (75%) showing a diffuse and moderate-to-strong staining pattern while they were negative for TTF-1. Six PETs exhibited moderate-to-strong positivity for CDX2 with nuclear staining in 5% to 40% of tumor cells, and 5 showed a varying degree of positivity for NESP55. Three (17%) of 18 metastatic GICTs showed moderate IMP3 staining in 50% to 90% of the tumor cells, whereas CDX2 was expressed in 17 (94%) cases with moderate-to-strong staining in 50% to 100% of tumor cells. No NESP55 immunoreactivity was detected in metastatic SCCs and GICTs. In conclusion, a panel of these 4 markers is useful in segregating among SCC, PET, and GICT to help determine the primary site of hepatic metastasis.

Histologic evaluation of an endoscopic bile duct biopsy for malignancy is a known challenge. Our prior study has shown that the insulin-like growth factor-II mRNA binding protein-3 (IMP3), S100P, and the von Hippel-Lindau gene product (pVHL) are a useful immunopanel for the distinction between adenocarcinoma and benign biliary epithelium. To further evaluate the usefulness of the IMP3, S100P, and pVHL immunopanel to aid in the interpretation of bile duct biopsies, 16 histologically challenging bile duct biopsies that exhibited atypical histology or features suspicious for malignancy were immunohistochemically stained for IMP3, S100P, and pVHL. Clinical follow-up data were obtained for each case. The results showed that in the 11 cases that showed adenocarcinoma during follow-up, the following staining patterns in atypical/suspicious cells in the initial biopsies were observed: IMP3-positive/S100P-positive/pVHL-negative or reduced (n=6), IMP3-negative/S100P-positive/pVHL-negative or reduced (n=4), and IMP3-positive/S100P-negative/pVHL-negative (n=1). In the 5 follow-up-proven benign cases, 2 biopsies showed an IMP3-negative/S100P-positive/pVHL-positive pattern in atypical cells and 1 was negative for all 3 proteins. The remaining 2 biopsies exhibited an IMP3-positive/S100P-positive/pVHL-negative or reduced pattern in atypical cells that were histologically considered dysplastic on retrospective review. These observations reaffirm that bile duct adenocarcinoma frequently shows positive IMP3 and/or S100P staining with reciprocal negative or reduced pVHL expression. This staining pattern can also be seen in dysplastic epithelium in the absence of invasive carcinoma. On the contrary, benign biliary epithelium typically lacks IMP3 immunoreactivity and may retain normal pVHL expression. However, caution should be exercised when using this immunopanel in the interpretation of challenging bile duct biopsies, because S100P and pVHL stains may give rise to variable patterns that can be difficult to interpret.

Lung injury, whether induced by infection or caustic chemicals, initiates a series of complex wound-healing responses. If uncontrolled, these responses may lead to fibrotic lung diseases and loss of function. Thus, resolution of lung injury must be tightly regulated. The key regulatory proteins required for tightly controlling the resolution of lung injury have yet to be identified. Here we show that loss of deubiquitinase CYLD led to the development of lung fibrosis in mice after infection with Streptococcus pneumoniae. CYLD inhibited transforming growth factor-β-signalling and prevented lung fibrosis by decreasing the stability of Smad3 in an E3 ligase carboxy terminus of Hsc70-interacting protein-dependent manner. Moreover, CYLD decreases Smad3 stability by deubiquitinating K63-polyubiquitinated Akt. Together, our results unveil a role for CYLD in tightly regulating the resolution of lung injury and preventing fibrosis by deubiquitinating Akt. These studies may help develop new therapeutic strategies for preventing lung fibrosis., Lung injury initiates a series of wound-healing responses, which if unregulated, can lead to fibrosis. Li et al. show that the deubquitinase CYLD has a key role in the prevention of fibrosis by inhibiting transforming growth factor β-signalling through the direct deubiquitination of the protein kinase Akt.

Background Failed pediatric heart allografts with diastolic dysfunction exhibit severe epicardial fibrosis. The molecular mechanism underlying this process is poorly understood. Canonical Wnt/β-catenin signaling plays an important role in epithelial–mesenchymal transition and is implicated in fibrosing diseases. In this study, we tested the hypothesis that canonical Wnt/β-catenin signaling is activated in epicardial fibrosis of end-stage dysfunctional pediatric allografts. Methods Fourteen explanted heart grafts of 12 patients who had undergone 14 heart transplantations were used for immunohistochemical staining of β-catenin and its nuclear binding partners, T-cell factor/lymphoid enhancer factor family transcriptional factors. Fourteen age-matched native hearts from patients who had undergone first heart transplantation without evidence of epicardial fibrosis were used as controls. Results and conclusions Epicardial fibroblasts from explanted allografts demonstrated nuclear accumulation of β-catenin. These cells also showed nuclear positivity for T-cell factor 4. No T-cell factor 3 expression was present in the epicardium. T-cell factor 1 and lymphoid enhancer factor 1 were observed in lymphocytes, but not in other cell types of the epicardium. These findings suggest an association between canonical Wnt/beta-catenin signaling and epicardial fibrosis of failed pediatric heart allografts. Should activation of this pathway be shown to be causal to epicardial fibrosis in this setting, then inhibition of this pathway may help to prevent this devastating process.

Na(V)1.5 is a cardiac voltage-gated Na(+) channel αsubunit and is encoded by the SCN5a gene. The activity of this channel determines cardiac depolarization and electrical conduction. Channel defects, including mutations and decrease of channel protein levels, have been linked to the development of cardiac arrhythmias. The molecular mechanisms underlying the regulation of Na(V)1.5 expression are largely unknown. Forkhead box O (Foxo) proteins are transcriptional factors that bind the consensus DNA sequences in their target gene promoters and regulate the expression of these genes. Comparative analysis revealed conserved DNA sequences, 5'-CAAAACA-3' (insulin responsive element, IRE), in rat, mouse and human SCN5a promoters with the latter two containing two overlapping Foxo protein binding IREs, 5'-CAAAACAAAACA-3'. This finding led us to hypothesize that Foxo1 regulates Na(V)1.5 expression by directly binding the SCN5a promoter and affecting its transcriptional activity. In the present study, we determined whether Foxo1 regulates Na(V)1.5 expression at the transcriptional level and also defined the role of Foxo1 in hydrogen peroxide (H(2)O(2))-mediated Na(V)1.5 suppression in HL-1 cardiomyocytes using chromatin immunoprecipitation (ChIP), constitutively nuclear Foxo1 expression, and RNAi Foxo1 knockdown as well as whole cell voltage-clamp recordings. ChIP with anti-Foxo1 antibody and follow-up semi-quantitative PCR with primers flanking Foxo1 binding sites in the proximal SCN5a promoter region clearly demonstrated enrichment of DNA, confirming Foxo1 recruitment to this consensus sequence. Foxo1 mutant (T24A/S319A-GFP, Foxo1-AA-GFP) was retained in nuclei, leading to a decrease of Na(V)1.5 expression and Na(+) current, while silencing of Foxo1 expression by RNAi resulted in the augmentation of Na(V)1.5 expression. H(2)O(2) significantly reduced Na(V)1.5 expression by promoting Foxo1 nuclear localization and this reduction was prevented by RNAi silencing Foxo1 expression. These studies indicate that Foxo1 negatively regulates Na(V)1.5 expression in cardiomyocytes and reactive oxygen species suppress Na(V)1.5 expression through Foxo1.

Histological subtyping of pulmonary adenocarcinoma has recently been updated based on predominant pattern, but data on reproducibility are required for validation. This study first assesses reproducibility in subtyping adenocarcinomas and then assesses further the distinction between invasive and non-invasive (wholly lepidic) pattern of adenocarcinoma, among an international group of pulmonary pathologists. Two ring studies were performed using a micro-photographic image-based method, evaluating selected images of lung adenocarcinoma histologic patterns. In the first study, 26 pathologists reviewed representative images of typical and ‘difficult’ histologic patterns. A total number of scores for the typical patterns combined (n = 94) and the difficult cases (n = 21) were 2444 and 546, respectively. The mean kappa score (± s.d.) for the five typical patterns combined and for difficult cases were 0.77 ± 0.07 and 0.38 ± 0.14, respectively. Although 70% of the observers identified 12–65% of typical images as single pattern, highest for solid and least for micropapillary, recognizing the predominant pattern was achieved in 92–100%, of the images except for micropapillary pattern (62%). For the second study on invasion, identified as a key problem area from the first study, 28 pathologists submitted and reviewed 64 images representing typical as well as ‘difficult’ examples. The kappa for typical and difficult cases was 0.55 ± 0.06 and 0.08 ± 0.02, respectively, with consistent subdivision by the same pathologists into invasive and non-invasive categories, due to differing interpretation of terminology defining invasion. In pulmonary adenocarcinomas with classic morphology, which comprise the majority of cases, there is good reproducibility in identifying a predominant pattern and fair reproducibility distinguishing invasive from in-situ (wholly lepidic) patterns. However, more precise definitions and better education on interpretation of existing terminology are required to improve recognition of purely in-situ disease, this being an area of increasing importance.

Malignant melanoma is the deadliest form of skin cancer arising from the abnormal proliferation of epidermal melanocytes. With the incidence and mortality from this disease rising, accurate histopathologic diagnosis is crucial. Most cases of melanoma can be appropriately diagnosed based on morphologic criteria, but a subset of lesions including Spitz and dysplastic nevi, can be difficult to distinguish from malignant proliferations. In addition, even though current immunohistochemical stains tend to be rather sensitive and specific for most types of malignant melanoma (S-100, MelanA/MART-1, HMB-45 and tyrosinase), they are unable to distinguish malignant from benign melanocytes--a potential pitfall in architecturally and cytologically borderline cases, which can lead to inadequate treatment or surveillance. Also, the distinction between intranodal nevi and metastatic melanoma in sentinel lymph nodes is not morphologically straightforward in certain cases; however, the confirmative status of these lymph nodes is very important for clinical outcome and guiding treatment. Thus, finding a method to precisely distinguish melanoma from its benign mimickers is needed. Despite advances in melanoma investigation and research, no reliable diagnostic biomarkers have yet been identified. Another challenge in melanoma treatment is to determine patients’ prognosis. Currently, depth of invasion, tumor ulceration, and status of sentinel lymph node are three common objective measures of prognosis; with invasion greater than 1 mm, ulceration, and lymph node metastases portending a worse outcome. Other features, such as assignment of radial versus vertical growth phase, tumor infiltrating lymphocytes, and Clark's levels, are also useful for prognosis, but tend to be more subjective. Thus, an immunohistochemical marker predictive of disease progression and poorer prognosis would be useful, to identify melanomas with a more aggressive phenotype.

TTF-1 and napsin A are useful biomarkers for differentiating primary lung adenocarcinoma from metastatic tumors. Studies have shown, however, that TTF-1 and napsin A also can be expressed in extrapulmonary carcinomas, and that a small fraction of primary lung adenocarcinomas do not co-express these two markers. We attempted to determine whether a tissue-specific transcriptional factor, PAX8, can help determine primary sites of lung carcinomas. Immunohistochemical stains for PAX8, TTF-1 and napsin A were performed on 103 cases of metastatic lung carcinomas from a variety of origins and 120 cases of primary lung adenocarcinomas. Our data demonstrated that all 103 metastatic carcinomas were negative for napsin A, while 14 (13.6%; four thyroid, two endometrium, three colon, one prostate, one salivary adenoid cystic, two renal cell carcinomas, and one ovary) showed weak to strong TTF-1 nuclear staining in 5-60% of the tumor cells. All primary lung adenocarcinomas were negative for PAX8, whereas 46 (44.7%) metastatic carcinomas from the kidney (29/33), ovary (6/8), endometrium (5/5), endocervix (1/1), thyroid (4/5) and urinary tract (1/3) were positive for PAX8. Our data demonstrate that of combined use of PAX8, TTF-1 and napsin A is reliable to separate reliably lung primary from metastatic tumors.

Insulin-like growth factor II-messenger RNA-binding protein 3 (IMP3) is an oncofetal RNA-binding protein that promotes tumor cell proliferation by enhancing IGF-II protein synthesis and inducing cell adhesion and invasion by stabilizing CD44 mRNA. IMP3 expression has been studied in many human neoplasms with growing evidence that IMP3 is a biomarker of enhanced tumor aggressiveness. IMP3 expression has been correlated with a poorer phenotypic profile including increased risk of metastases and decreased survival. Only a few studies have examined IMP3 expression in lung cancers. We review here the literature concerning IMP3 expression in lung neoplasms, specifically adenocarcinoma, squamous cell carcinoma, and neuroendocrine tumors of the lung. IMP3 immunohistochemical expression was reported in 27-55% of cases of primary pulmonary adenocarcinoma and in 75-90% of cases of squamous cell carcinoma of the lung. In adenocarcinoma, IMP3 expression was reported to be correlated with more poorly differentiated histological grade, advanced stage of disease and lymph node metastases. IMP3 expression also may be a marker of high grade pre-invasive squamous lesions including high grade dysplasia and carcinoma in situ. In neuroendocrine tumors of the lung, IMP3 expression was expressed in all reported cases of large cell neuroendocrine carcinoma and small cell lung carcinoma, but expression was limited in carcinoid tumors. Overall, IMP3 appears to be a useful diagnostic marker for lung cancer pathology including for discriminating high grade neuroendocrine tumors and low grade carcinoids and for identifying high grade pre-invasive squamous lesions.

Differentiation of primary from metastatic adenocarcinoma in the lung can be challenging, and it demands sensitive and specific biomarkers, especially when the tissue for diagnosis is limited. Thyroid transcription factor-1 (TTF-1) has been considered a reliable marker for adenocarcinoma of lung origin. However, several recent studies have shown that TTF-1 immunostaining is also positive in adenocarcinomas arising in different organs including colon, endometrium, endocervix, and ovary. In addition, approximately 20% of lung primary adenocarcinomas are negative for TTF-1 immunostaining, and napsin A immunostaining has slightly higher sensitivity in detecting lung primary adenocarcinoma. We performed TTF-1 and napsin A immunostaining on 120 cases of primary lung adenocarcinomas and 37 cases of metastatic carcinomas in the lung. The results showed that 95 (79.2%) of 120 lung primary adenocarcinomas showed napsin A+/TTF-1+ double-positive immunostaining pattern. TTF-1⁻/napsin A+, TTF-1+/napsin A⁻, and TTF-1⁻/napsin A⁻ were seen in 8.3%, 3.3%, and 9.2% lung primary adenocarcinomas, respectively. Eight (21.6%) of the 37 metastatic carcinomas were positive for TTF-1 and they include clear-cell renal cell carcinomas completely negative for napsin A although napsin A was detected in 12 (80.0%) of 15 primary papillary and 3 (33.3%) of 9 primary clear-cell renal cell carcinomas. All renal epithelial neoplasms were TTF-1 negative. These findings indicate that double napsin A and TTF-1-positive immunostaining is highly specific for lung primary adenocarcinoma and the combination of these 2 biomarkers is warranted to help segregating primary lung adenocarcinoma from metastatic carcinoma in the lung.

Rationale: The differentiation of fibroblasts into myofibroblasts is a cardinal feature of idiopathic pulmonary fibrosis (IPF). The transcription factor Yin Yang 1 (YY1) plays a role in the proliferation and differentiation of diverse cell types, but its role in fibrotic lung diseases is not known. Objectives: To elucidate the mechanism by which YY1 regulates fibroblast differentiation and lung fibrosis. Methods: Lung fibroblasts were cultured with transforming growth factor (TGF)-β or tumor necrosis factor-α. Nuclear factor (NF)-κB, YY1, and α-smooth muscle actin (SMA) were determined in protein, mRNA, and promoter reporter level. Lung fibroblasts and lung fibrosis were assessed in a partial YY1-deficient mouse and a YY1f/f conditional knockout mouse after being exposed to silica or bleomycin. Measurements and Main Results: TGF-β and tumor necrosis factor-α up-regulated YY1 expression in lung fibroblasts. TGF-β–induced YY1 expression was dramatically decreased by an inhibitor of NF-κB, which blocked I-κB degradation. YY1 is significantly overexpressed in both human IPF and murine models of lung fibrosis, including in the aggregated pulmonary fibroblasts of fibrotic foci. Furthermore, the mechanism of fibrogenesis is that YY1 can up-regulate α-SMA expression in pulmonary fibroblasts. YY1-deficient (YY1+/−) mice were significantly protected from lung fibrosis, which was associated with attenuated α-SMA and collagen expression. Finally, decreasing YY1 expression through instilled adenovirus-cre in floxed-YY1f/f mice reduced lung fibrosis. Conclusions: YY1 is overexpressed in fibroblasts in both human IPF and murine models in a NF-κB–dependent manner, and YY1 regulates fibrogenesis at least in part by increasing α-SMA and collagen expression. Decreasing YY1 expression may provide a new therapeutic strategy for pulmonary fibrosis.

The presence of individual neuroendocrine cells in rare peripancreatic lymph nodes (LNs) suggests that neuroendocrine tumor or nested neuroendocrine cell proliferation can arise in situ from neuroendocrine cells native to any LN. However, it is very difficult to ascertain whether any neuroendocrine lesion in LNs is a primary tumor or a metastasis from adjacent organs. We encountered 4 cases of neuroendocrine proliferation in intrathoracic LNs (ILNs) of patients with primary lung adenocarcinoma. All patients had a single lung mass without mediastinal lymphadenopathy based on computed tomography and positron emission tomography imaging. Mediastinal staging was done by either mediastinoscopy or thoracotomy and none of them had metastasis from adenocarcinoma in any LN. One patient had three ILNs positive for neuroendocrine proliferation measuring 1.7, 1.8, and 4.0 mm, respectively and a minute tumorlet less than 1.0 mm in the lung. Three other patients had small areas of neuroendocrine proliferation no more than 1 mm in single ILN without any lung neuroendocrine lesion. Neuroendocrine cells in ILNs often formed nests of varying size with similar morphology to carcinoid tumorlet in the lung. Small clusters of neuroendocrine cells without any particular pattern were often seen together with these nests. These cells were positive for neuroendocrine markers: synaptophysin, chromogranin, and CD56. They were also positive for CK7 and TTF-1. It is interesting to note, single cells positive for neuroendocrine markers and TTF-1 were identified near or away from these neuroendocrine nests or clusters. These findings suggest that neuroendocrine lesion can be incidentally identified in ILNs. Close clinical follow-up is warranted as metastasis from or synchronous lesions in adjacent organs cannot be excluded.

Enhancer of zeste homolog 2, the catalytic subunit of polycomb repressive complex 2, is a histone methyltransferase and plays an important role in cell proliferation and cell cycle regulation. It has been shown to be overexpressed in a number of malignant neoplasms. This study aimed to determine the expression pattern of enhancer of zeste homolog 2 in neuroendocrine tumors of the lung and the potential of enhancer of zeste homolog 2 to serve as a biomarker to segregate carcinoids from high-grade neuroendocrine carcinomas. Fifty-four cases, including 25 typical carcinoids, 7 atypical carcinoids, 9 large-cell neuroendocrine carcinomas, and 13 small-cell lung carcinomas, were immunohistochemically studied using a monoclonal antibody against enhancer of zeste homolog 2. All 13 small-cell lung carcinomas demonstrated moderate to strong nuclear staining with 12 exhibiting more than 90% of tumor cells staining. All 9 large-cell neuroendocrine carcinomas were moderately to strongly positive for enhancer of zeste homolog 2, with 6 cases having staining in more than 80% of tumor cells. In contrast, all 25 typical carcinoids and 6 atypical carcinoids showed only rare scattered enhancer of zeste homolog 2-positive tumor cells, with 1 case of atypical carcinoid exhibiting moderate staining in 40% of tumor cells. A subsequent validation study of the 14 specimens of lung or mediastinal lymph node biopsy and fine-needle aspiration, including 6 small-cell lung carcinomas, 2 large-cell neuroendocrine carcinomas, 5 typical carcinoids, and 1 atypical carcinoid, was performed. Enhancer of zeste homolog 2 was diffusely and strongly positive in all small-cell lung carcinomas and large-cell neuroendocrine carcinomas, even with severe crush artifact, whereas it was only positive in rare tumor cells in carcinoids. These findings support the formulation that enhancer of zeste homolog 2 may play an important role in the regulation of biologic behavior of high-grade neuroendocrine carcinomas and as a diagnostically useful marker in distinguishing high-grade neuroendocrine carcinomas from carcinoids.

Histopathologic distinction between benign and malignant bile duct epithelial lesions on endoscopic biopsies can be extremely challenging because of limited material, crush artifact, and compounding inflammatory and/or reactive changes particularly after stent placement. In this study, a total of 72 endoscopic bile duct biopsies, including 40 adenocarcinomas and 32 benign cases, were immunohistochemically examined for the expression of S100P, von Hippel-Lindau gene product (pVHL), and IMP3 to evaluate their diagnostic value. The results showed that 36 adenocarcinomas (90%) exhibited strong nuclear and cytoplasmic staining for S100P, of which 30 (83.3%) showed diffuse immunoreactivity. Intermediate to strong cytoplasmic staining for IMP3 was demonstrated in 31 tumors (77.5%) (15 diffuse, 16 focal). Completely negative staining for pVHL was observed in 37 adenocarcinomas. In the remaining 3 tumors, focal (1) or diffuse (2) membranous and cytoplasmic pVHL immunoreactivity was detected. Twenty-eight tumors (70%) showed a S100P+/IMP3+/pVHL- staining pattern, 6 (15%) with a S100P+/IMP3-/pVHL- pattern, and 2 (5%) with a S100P-/IMP3+/pVHL- pattern. All 32 benign biopsies were completely negative for IMP3 with the exception of 2 cases with focal dysplasia where focal immunoreactivity was observed. Thirty benign biopsies (93.8%) were positive for pVHL with a diffuse staining pattern observed in 28 cases (93.3%). Eight benign biopsies (25%) showed focal S100P positivity. Twenty-two benign biopsies (68.8%) displayed a S100P-/IMP3-/pVHL+ staining pattern. In conclusion, an immunohistochemical panel consisting of S100P, pVHL, and IMP3 can be helpful in distinguishing adenocarcinoma from reactive epithelial changes on challenging bile duct biopsies. The findings of focal S100P and/or IMP3 expression with reciprocal loss of pVHL immunoreactivity in a few benign biopsies suggest a use of these markers in the detection of early epithelial dysplasia that may be beyond histologic recognition.

Rationale: Cyclic nucleotide phosphodiesterases (PDEs) through the degradation of cGMP play critical roles in maintaining cardiomyocyte homeostasis. Ca 2+ /calmodulin (CaM)-activated cGMP-hydrolyzing PDE1 family may play a pivotal role in balancing intracellular Ca 2+ /CaM and cGMP signaling; however, its function in cardiomyocytes is unknown. Objective: Herein, we investigate the role of Ca 2+ /CaM-stimulated PDE1 in regulating pathological cardiomyocyte hypertrophy in neonatal and adult rat ventricular myocytes and in the heart in vivo. Methods and Results: Inhibition of PDE1 activity using a PDE1-selective inhibitor, IC86340, or downregulation of PDE1A using siRNA prevented phenylephrine induced pathological myocyte hypertrophy and hypertrophic marker expression in neonatal and adult rat ventricular myocytes. Importantly, administration of the PDE1 inhibitor IC86340 attenuated cardiac hypertrophy induced by chronic isoproterenol infusion in vivo. Both PDE1A and PDE1C mRNA and protein were detected in human hearts; however, PDE1A expression was conserved in rodent hearts. Moreover, PDE1A expression was significantly upregulated in vivo in the heart and myocytes from various pathological hypertrophy animal models and in vitro in isolated neonatal and adult rat ventricular myocytes treated with neurohumoral stimuli such as angiotensin II (Ang II) and isoproterenol. Furthermore, PDE1A plays a critical role in phenylephrine-induced reduction of intracellular cGMP- and cGMP-dependent protein kinase (PKG) activity and thereby cardiomyocyte hypertrophy in vitro. Conclusions: These results elucidate a novel role for Ca 2+ /CaM-stimulated PDE1, particularly PDE1A, in regulating pathological cardiomyocyte hypertrophy via a cGMP/PKG-dependent mechanism, thereby demonstrating Ca 2+ and cGMP signaling cross-talk during cardiac hypertrophy.

Depending on the Breslow depth of the primary melanoma, sentinel lymph node biopsy is considered as standard of care for the staging of cutaneous melanoma, and is one of the most important prognostic factors. The histologic analysis of these specimens becomes difficult to interpret when benign intranodal nevic cells mimic metastases. Insulin-like growth factor-II messenger RNA (mRNA)-binding protein-3 (IMP3), also known as K homology domain-containing protein overexpressed in cancer or L523S, is a member of the insulin-like growth factor-II mRNA-binding protein family and has been shown to have diagnostic utility in distinguishing cutaneous melanoma from benign nevi. In this study, 43 sentinel lymph node biopsy specimens, including 13 with benign intranodal nevi and 30 with metastatic melanoma (two cases containing both benign nevi and metastatic melanoma), from 41 patients were immunohistochemically analyzed with a monoclonal antibody against IMP3. None of the benign intranodal nevi expressed IMP3, whereas 21 out of 30 (70%) of the lymph nodes containing metastatic melanoma did. It seems that IMP3 is helpful in distinguishing benign intranodal nevi from metastatic melanoma in sentinel lymph node biopsy specimens, and could be a valuable diagnostic adjunct in sentinel lymph node biopsy assessment in which questions arise as to the malignancy of the melanocytes present.

Insulin-like growth factor II mRNA binding protein 3 is an oncofetal protein that is expressed in multiple malignancies. This study aimed to determine the correlation of insulin-like growth factor II mRNA binding protein 3 expression with histologic grade of lung adenocarcinoma. Eighty-nine cases, including 11 atypical adenomatous hyperplasias, 10 pure bronchioloalveolar carcinomas, 36 well-differentiated adenocarcinomas and 41 moderately or poorly differentiated adenocarcinomas, were immunohistochemically studied using a monoclonal antibody against insulin-like growth factor II mRNA binding protein 3. Twenty-nine (70.7%) of 41 moderately to poorly differentiated adenocarcinomas were positive for insulin-like growth factor II mRNA binding protein 3, with 26 (89.7%) tumors demonstrating either a strong staining or staining in greater than 30% of tumor cells. Four (40.0%) of 10 bronchioloalveolar carcinomas and 13 (36.1%) of 36 well-differentiated adenocarcinomas exhibited insulin-like growth factor II mRNA binding protein 3 positivity with a variable degree and percentage of tumor cells staining. When bronchioloalveolar carcinomas were present in a pure form or as a component of adenocarcinomas, positive insulin-like growth factor II mRNA binding protein 3 staining was always patchy, with less than 20% of tumor cells stained. Overall, the frequency of positive insulin-like growth factor II mRNA binding protein 3 staining was lower in bronchioloalveolar carcinomas and well-differentiated adenocarcinomas compared to moderately/poorly differentiated adenocarcinomas (P < .01). No insulin-like growth factor II mRNA binding protein 3 signal was detected in any case of atypical adenomatous hyperplasia. These findings show that insulin-like growth factor II mRNA binding protein 3 is strongly and diffusely expressed in a large proportion of moderately/poorly differentiated lung adenocarcinomas, in particular in the solid component of mixed subtype adenocarcinomas, less frequently expressed in well-differentiated adenocarcinomas and bronchioloalveolar carcinomas, and negative in atypical adenomatous hyperplasias. The higher frequency of expression in moderately/poorly differentiated adenocarcinomas suggests that insulin-like growth factor II mRNA binding protein 3 expression may be associated with an aggressive biological behavior.

p90 ribosomal S6 kinase (p90RSK) is activated in cardiomyopathies caused by conditions such as ischemia/reperfusion injury and diabetes mellitus in which prolongation of cardiac repolarization and frequent arrhythmias are common. Molecular mechanisms underlying the electric remodeling in cardiac diseases are largely unknown. In the present study, we determined the role of p90RSK activation in the modulation of voltage-gated K + channel activity determining cardiac repolarization. Mice with increased cardiac p90RSK activity due to transgenic expression of p90RSK (p90RSK-Tg) had prolongation of QT intervals and of ventricular myocyte action potential durations. Fast transient outward K + current ( I to,f ), slow delayed outward K + current ( I K,slow ), and steady-state K + current ( I SS ) were significantly decreased in p90RSK-Tg mouse ventricular myocytes. mRNA levels of Kv4.3, Kv4.2, Kv1.5, Kv2.1, and KChIP2 from ventricles between p90RSK-Tg and nontransgenic littermate control mice were similar, as assessed by quantitative reverse transcriptase–polymerase chain reaction, indicating that p90RSK regulates voltage-gated K + channels through posttranslational modification. Kv4.3- and Kv1.5- rather than Kv4.2- and Kv2.1-encoded channels in HEK 293 cells were inhibited by p90RSK. In vitro phosphorylation analysis showed that Kv4.3 was phosphorylated by p90RSK at 2 conserved sites, Ser516 and Ser550 . p90RSK expression significantly inhibited Kv4.3- and Kv4.3 and KChIP2-encoded channel activities in HEK 293 cells, whereas p90RSK’s effects were blocked by amino acid mutation(s) at phosphorylation site(s) in Kv4.3. Hydrogen peroxide, a mediator of induced cardiac p90RSK activation in ischemia/reperfusion injury and diabetes mellitus, had effects similar to those of p90RSK on Kv4.3- or Kv4.3- and KChIP2-encoded channels. Fluoromethylketone, a specific p90RSK inhibitor, abolished hydrogen peroxide effects. These findings indicate that p90RSK activation is critical for reactive oxygen species-mediated inhibition of voltage-gated K + channel activity and leads to prolongation of cardiac repolarization.

Malignant mesotheliomas occur in the pleura, peritoneum, pericardium, and tunica vaginalis. The majority of tumors are pleural in origin. The typical pattern of spread is usually contiguous or via implantation. Hematogenous or lymphatic metastasis is not uncommon; however, metastasis to bone has rarely been well documented. This is a case report of malignant pleural mesothelioma metastatic to the femur with a pathologic fracture of femoral neck.

Small cell neuroendocrine carcinoma of the prostate is a rare variant of prostatic cancer that shares morphologic similarity with prostatic adenocarcinoma of Gleason 5 pattern. It has also been considered morphologically and immunohistochemically indistinguishable from small cell neuroendocrine carcinomas of other origins. CD44 is a cell-surface molecule proposed to identify cancer stem/progenitor cells in prostate cancer. We performed immunohistochemical study for CD44 expression in 11 cases of prostatic small cell neuroendocrine carcinoma and compared its patterns of expression with 73 cases of prostatic adenocarcinoma and 47 cases of small cell neuroendocrine carcinomas of other organs. Strong and diffuse membrane staining for CD44 was observed in 100% of the prostatic small cell neuroendocrine carcinomas. In conventional adenocarcinomas of the prostate, positive staining was only seen in rare, scattered tumor cells; and CD44 staining was negative in most of the small cell neuroendocrine carcinomas of nonprostate origin. The difference in CD44 expression between small cell neuroendocrine carcinomas of the prostate and those of other organs are statistically significant (P < .001). Our study demonstrates the utility of immunohistochemical staining for CD44 in distinguishing prostatic small cell neuroendocrine carcinoma from its mimickers including prostatic adenocarcinoma of Gleason 5 pattern and small cell neuroendocrine carcinomas of other organs. CD44 is the first marker that shows a high degree of tissue/organ specificity for small cell neuroendocrine carcinomas. Because CD44 is a putative marker of prostate cancer stem cells, the strong and diffuse expression of CD44 and the lack of expression of prostate luminal differentiation markers androgen receptor and prostatic specific antigen in prostatic small cell neuroendocrine carcinomas suggest that the tumor cells may retain cancer stem cell features.

Solitary fibrous tumor (SFT) is an unusual mesenchymal neoplasm that most often arises in the pleura; however, it has recently been described in a number of extrapleural sites. This report describes an extremely rare case of a benign SFT arising in the pancreas. A 41-year-old woman presented in the clinic with right upper abdominal pain. Subsequent ultrasonographic studies revealed a 1.5 × 1.5 × 1.4 cm hypoechoic mass within the pancreatic body, which was later confirmed on both helical computerized tomography and magnetic resonance imaging studies. An endocrine tumor was clinically suspected. Laparoscopic enucleation of the mass was performed. Microscopically, the tumor was composed of bland uniform spindle cells arranged between collagen bundles. On immunohistochemical studies, these spindle cells were positive for CD34 and bcl-2 but negative for cytokeratin (AE1+AE3 and Cam5.2), smooth muscle actin, desmin, S-100, and c-kit. Based on the light microscopic morphology and immunohistochemical staining profile, the diagnosis of SFT was rendered.

Summary Streptococcus pneumoniae ( S. pneumoniae ) causes high early mortality in pneumococcal pneumonia, which is characterized by acute lung injury (ALI). The molecular mechanisms underlying ALI and the high early mortality remain unknown. Despite recent studies that identify deubiquitinating enzyme cylindromatosis (CYLD) as a key regulator for T cell development, tumor cell proliferation, and NF-κB transcription factor signaling, its role in regulating bacteria-induced lethality, however, is unknown. Here, we showed that CYLD deficiency protected mice from S. pneumoniae pneumolysin (PLY)-induced ALI and lethality. CYLD was highly induced by PLY, and it inhibited MKK3-p38 kinase-dependent expression of plasminogen activator inhibitor-1 (PAI-1) in lung, thereby potentiating ALI and mortality. Thus, CYLD is detrimental for host survival, thereby indicating a mechanism underlying the high early mortality of pneumococcal pneumonia.

Transforming growth factor-beta (TGF-beta) family members are multifunctional growth factors involved in regulating diverse biological processes. Despite the critical role for TGF-beta in regulating cell proliferation, differentiation, migration and development, its role in regulating NF-kappaB-dependent inflammatory response still remains unclear. Here, we show that TGF-beta1 induces acetylation of NF-kappaB p65 subunit to synergistically enhance bacterium nontypeable Haemophilus influenzae-induced NF-kappaB activation and inflammatory response in vitro and in vivo. The TGF-beta1-induced acetylation of p65 is mediated via a Smad3/4-PKA-p300-dependent signaling pathway. Acetylation of p65 at lysine 221 by TGF-beta1 is critical for synergistic enhancement of bacteria-induced DNA-binding activity, NF-kappaB activation, NF-kappaB-dependent transcription of TNF-alpha and IL-1beta and interstitial polymorphonuclear neutrophil infiltration in vitro and in vivo. These studies provide new insights into the novel regulation of NF-kappaB by TGF-beta signaling.

K homology domain containing protein overexpressed in cancer (KOC) is a member of the insulin-like growth factor (IGF) messenger RNA-binding protein family and is expressed during embryogenesis and in certain malignancies. KOC, known as L523S and IGF messenger RNA-binding protein 3, was shown to be frequently expressed in high-grade neuroendocrine carcinomas of the lung in our immunohistochemical studies using a monoclonal antibody against human KOC. Specifically, all 10 small cell lung carcinomas (SCLCs) exhibited strong cytoplasmic staining, 9 with diffuse positivity and 1 with focal positivity. Among 14 large cell neuroendocrine carcinomas (LCNECs), 9 exhibited strong and diffuse cytoplasmic staining, and 5 cases showed focal immunoreactivity. In contrast, no KOC was detected in 21 typical and atypical carcinoids, except for one atypical carcinoid with oncocytic cells showing weak cytoplasmic staining. Although SCLCs exhibited a strong and diffuse staining pattern more frequently (90%) than LCNECs (64%), the difference did not reach statistical significance (P = .3408). Interestingly, our immunohistochemical studies demonstrated that IGF-II, reportedly regulated by KOC, was comparably expressed in SCLC, LCNEC, and typical and atypical carcinoids, irrespective of KOC expression status of the tumors. These results support the formulation that KOC may play an important role in the regulation of biologic behavior of high-grade neuroendocrine carcinomas. In addition, detection of KOC expression may be diagnostically useful in distinguishing high-grade neuroendocrine carcinomas from carcinoid tumors. Our findings of equivalent IGF-II expression in KOC-positive SCLC and LCNEC and KOC-negative carcinoid tumors suggest different regulatory mechanisms involved in the control of IGF-II expression in these tumors.

Squamous cell carcinoma evolving from squamous papilloma in both the upper and lower respiratory tract in the same patient is uncommon. The molecular mechanisms underlying the progression have not been well investigated. We herein describe a case of squamous cell carcinoma arising from respiratory papilloma in two independent occasions. The patient initially had oropharyngeal squamous cell carcinoma arising in a squamous papilloma at the age of 25 years. He subsequently developed squamous cell carcinoma in the left lower lobe of the lung, which was also associated with squamous papilloma, 8 years after the complete excision of the oropharyngeal lesion. Polymerase chain reaction-based broad-spectrum human papillomavirus DNA amplification and typing showed the presence of human papillomavirus type 11 DNA in both oropharyngeal and pulmonary tumors. Immunohistochemical studies showed that the expression status of p53, Rb, and p16 proteins was unaltered during tumor progression. These observations indicate that human papillomavirus 11-associated neoplastic transformation and tumor progression in the respiratory tract may not involve aberrant regulation of the p53 and Rb signaling pathways.

Previous studies have demonstrated a role for voltage-gated K + (Kv) channel α subunits of the Kv4 subfamily in the generation of rapidly inactivating/recovering cardiac transient outward K + current, I to,f , channels. Biochemical studies suggest that mouse ventricular I to,f channels reflect the heteromeric assembly of Kv4.2 and Kv4.3 with the accessory subunits, KChIP2 and Kvβ1, and that Kv4.2 is the primary determinant of regional differences in (mouse ventricular) I to,f densities. Interestingly, the phenotypic consequences of manipulating I to,f expression in different mouse models are distinct. In the experiments here, the effects of the targeted deletion of Kv4.2 (Kv4.2 −/− ) were examined. Unexpectedly, voltage-clamp recordings from Kv4.2 −/− ventricular myocytes revealed that I to,f is eliminated. In addition, the slow transient outward K + current, I to,s , and the Kv1.4 protein (which encodes I to,s ) are upregulated in Kv4.2 −/− ventricles. Although Kv4.3 mRNA/protein expression is not measurably affected, KChIP2 expression is markedly reduced in Kv4.2 −/− ventricles. Similar to Kv4.3, expression of Kvβ1, as well as Kv1.5 and Kv2.1, is similar in wild-type and Kv4.2 −/− ventricles. In addition, and in marked contrast to previous findings in mice expressing a truncated Kv4.2 transgene, the elimination I to,f in Kv4.2 −/− mice does not result in ventricular hypertrophy. Taken together, these findings demonstrate not only an essential role for Kv4.2 in the generation of mouse ventricular I to,f channels but also that the loss of I to,f per se does not have overt pathophysiological consequences.

Evidence is emerging that systemic metabolic disturbances contribute to cardiac myocyte dysfunction and clinically apparent heart failure, independent of associated coronary artery disease. To test the hypothesis that perturbation of lipid homeostasis in cardiomyocytes contributes to cardiac dysfunction, we engineered transgenic mice with cardiac-specific overexpression of fatty acid transport protein 1 (FATP1) using the α-myosin heavy chain gene promoter. Two independent transgenic lines demonstrate 4-fold increased myocardial free fatty acid (FFA) uptake that is consistent with the known function of FATP1. Increased FFA uptake in this model likely contributes to early cardiomyocyte FFA accumulation (2-fold increased) and subsequent increased cardiac FFA metabolism (2-fold). By 3 months of age, transgenic mice have echocardiographic evidence of impaired left ventricular filling and biatrial enlargement, but preserved systolic function. Doppler tissue imaging and hemodynamic studies confirm that these mice have predominantly diastolic dysfunction. Furthermore, ambulatory ECG monitoring reveals prolonged QT c intervals, reflecting reductions in the densities of repolarizing, voltage-gated K + currents in ventricular myocytes. Our results show that in the absence of systemic metabolic disturbances, such as diabetes or hyperlipidemia, perturbation of cardiomyocyte lipid homeostasis leads to cardiac dysfunction with pathophysiological findings similar to those in diabetic cardiomyopathy. Moreover, the MHC-FATP model supports a role for FATPs in FFA import into the heart in vivo.