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Objective: This meta-analysis was performed to evaluate the diagnostic efficacy of lung ultrasound (LUS) in cardiogenic pulmonary edema., Materials and Methods: An electronic search of databases, including MEDLINE, Embase, PubMed, and Web of Science, was performed to collect clinical studies on ultrasound diagnosis of cardiogenic pulmonary edema from inception to 23 March 2023. The number of patients with true-positive, true-negative, false-positive, and false-negative cardiogenic pulmonary edema diagnosed by LUS was collected, and the R package was used to analyze the diagnostic efficacy of LUS., Results: Nine pieces of literature were finally included with 2,097 participants, including 1,047 patients with cardiogenic heart failure. Across the nine included papers, the pooled sensitivity of LUS in the included studies was 0.92 (95% CI: 0.84, 0.97) with a maximum sensitivity of 0.99 (95% CI: 0.96 to 1.00) and a minimum of 0.59 (95% CI: 0.50, 0.68). The pooled specificity of the included studies was 0.87 (95% CI: 0. 82, 0.91) with a maximum specificity of 0.93 (95% CI: 0.90-0.95) and a minimum of 0.80 (95% CI: 0.67, 0.89). The pooled AUC was 0.93 (95% CI: 0.84 to 0.97), suggesting a high diagnostic value of LUS in cardiogenic pulmonary edema., Conclusions: Lung ultrasound offers a good diagnostic efficacy for cardiogenic pulmonary edema. Further standardization of the examination method is required to provide a reference for the clinical use of LUS.

Purpose: Prolactinoma is the most common type of pituitary adenoma. Most prolactinoma need medical treatment, but some of them are aggressive and require surgery. In previous decades, some miRNAs have been manifested as oncogenes or tumor suppressors. Consequently, miRNAs' abnormal expression involves tumorigenesis, invasion, and metastasis of different types of tumors, including pituitary tumors. The current study aim to explore the aggressiveness-associated miRNAs in prolactinoma and underlying molecular mechanisms based on the bioinformatic analysis and fundamental experiment studies., Methods: GSE46294 miRNA expression profile from the Gene Expression Omnibus (GEO) database was downloaded. Differentially expressed miRNAs (DEMs) were filtered from this data. Subsequently, the target genes of downregulated miRNAs were analyzed by Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. RT-qPCR, western blot, and CCK-8 assays were used to validate the effect of miR-137 on the proliferation of MMQ cells through AKT2. Finally, the binding site of rat miR-137 to AKT2 were predicted by Targetscan and Bibiserv database, and verified by double luciferase reporter assay., Results: Twenty-four changed DEMs (fourteen upregulated and ten downregulated) were identified. Target genes of downregulated DEMs were classified into three groups by GO terms. KEGG pathway enrichment analysis revealed these target genes enriched in the PI3K-Akt pathway. We also confirmed that miR-137 can target AKT2 and inhibit the proliferation of MMQ cells induced by AKT2., Conclusion: MiR-137 suppressed prolactinomas' aggressive behavior by targeting AKT2., (© 2022. The Author(s), under exclusive licence to Italian Society of Endocrinology (SIE).)

Objective: To summarize the clinical characteristics of inflammasomopathies, enhance the recognition of those diseases, and help to establish the early diagnosis. Methods: The clinical manifestations including fever, rash, systems involvement as well as laboratory results and genotypic characteristics of 35 children with inflammasomopathies diagnosed by the Department of Pediatrics, Peking Union Medical College Hospital, from January 1, 2008 to December 31, 2020 were analyzed retrospectively. Results: A total of 35 cases of inflammasomopathies were diagnosed, and 20 of them were boys while 15 were girls. Inflammasomopathies patients have early onset, the age of onset as well as diagnostic age were 1 (0,7) and 7 (3,12), respectively. Among those patients, 10 had familial mediterranean fever, 3 had mevalonate kinase deficiency, 15 cases had NLRP3 gene associated autoinflammatory disease, 4 cases had NLRP12-associated autoinflammatory disease, 2 cases had familial cold autoinflammatory syndrome 3, and 1 case had familial cold autoinflammatory syndrome 4. A total of 34 cases (97%) showed recurrent fever, 27 cases (77%) had skin rashes, while 11 cases (31%), 10 cases (29%), and 8 cases (23%) were presented with lymphadenopathy, hepatosplenomegaly and growth retardation, respectively. In terms of systemic involvement, there were 18 cases (51%), 12 cases (34%), 8 cases (23%), and 5 cases (14%) with skeletal, neurological, auditory, and renal involvement, respectively. Central nervous system involvement was seen only in NLRP3 gene associtated autoinflammatory diseases (12 cases), sensorineural deafness was seen in NLRP3 gene associtated autoinflammatory diseases (6 cases) and NLRP12 gene associated autoinflammatory diseases (2 cases), and abdominal pain was observed in familial Mediterranean fever (5 cases), mevalonate kinase deficiency (1 case) and NLRP12 gene related autoinflammatory diseases (1 case). In the acute inflammatory phase, the acute phase reactants (erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP)) of 35 cases (100%) were significantly increased. There were 21 cases received ferritin examination, and only 4 cases (19%) showed an increase of it. In terms of autoantibodies, among all 35 patients, 4 cases (11%) were positive for antinuclear antibodies (ANA). Conclusions: Fever, skin rash, and skeletal manifestations are the most common clinical features, accompanied with increased CRP and ESR, and negative results of autoantibodies such as ANA. The clinical manifestations of those diseases are complex and diverse, and it is prone to delayed diagnosis and treatment.

Objective: To summarize the clinical characteristics of type I interferonopathies and provide clues for early identification and diagnosis. Methods: Clinical data of 20 patients admitted to Department of Pediatrics, Peking Union Medical College Hospital and 5 patients admitted to Department of Rheumatology and Immunology, Shenzhen Children's Hospital from January 2016 to September 2021 were retrospectively analyzed. The data included gene results, clinical manifestations and auxiliary examination results. Results: Of the 25 cases, 12 were males and 13 were females. Age of onset ranged from 1 day to 11 years. And 84% of them had the onset before the age of 3 years. The cases consisted of 14 cases of Aicardi-Goutières syndrome (AGS), 6 cases of adenosine deaminase 2 deficiency (DADA2), 3 cases of stimulator of interferon genes (STING)-associated vasculopathy with onset in infancy (SAVI), and 2 cases of Spondyloenchondrodysplasia with immune dysregulation (SPENCDI). Eighteen patients (72%) experienced neurologic disorder, among whom 16 (64%) showed intracranial calcification, 11 (44%) had dystonia, 10 (40%) had leukodystrophy, 6 (24%) had epilepsy, 5 (20%) had brain atrophy and 5 (20%) had early-onset cerebrovascular events. Skin involvement occurred in 15 cases (60%), among whom 8 cases (32%) had chilblain-like rash, 4 cases (16%) had livedo reticularis, 3 cases (12%) had erythema, 2 cases (8%) had erythema nodosum and 2 cases (8%) had Raynaud's phenomenon. In addition, 12 cases (48%) had positive autoimmune antibodies, 10 cases (40%) manifested as developmental retardation, 8 cases (32%) experienced lung interstitial lesions, and 7 cases (28%) demonstrated thyroid dysfunction. And 1 died (4%) at 11 years of age. Conclusions: Type Ⅰinterferonopathies can involve multiple organs, and share the characteristics of systemic inflammatory and autoimmune diseases. The early-onset neurological symptoms (early-onset cerebrovascular events, intracranial calcification, leukodystrophy and cerebral atrophy), rashes (chilblain-like rash, livedo reticularis and erythema), positive autoimmune antibodies, developmental delay, interstitial lung disease and thyroid dysfunction may indicate type Ⅰ interferonopathies.

Aims: Plant tissues are the reservoirs of beneficial and harmful microbes that regulates plant growth. In the present study, we investigated the diversity, function and colonization of sugarcane roots associated with Bacillus spp., Methods and Results: A total of 20 Bacillus strains were isolated and identified by 16S rRNA gene sequencing, and their genetic diversity was examined by BOX, ERIC, REP, (GTG) 5 PCR techniques. Among all Bacillus isolates, 65% showed indole acetic acid-like compounds production, 50% solubilized phosphorus and 25% of the isolates were able to secrete siderophore. Moreover, all 20 Bacillus isolates showed antifungal activity against eight fungal pathogens and 11 of them (55%) antagonized tomato grey mold. Based on the plant growth-promoting traits and antifungal potential, isolate Y8 was selected for root and plant tissue colonization assays and a greenhouse-level sugarcane growth promotion study. Fluorescence microscopy results confirmed that isolate Y8 has a strong ability to colonize in the sugarcane root and leaves, and the root surface association of Y8 was confirmed by scanning electron microscopy. Furthermore, greenhouse experimental results demonstrated that Y8 has a significant effect on enhancing sugarcane biomass and root length., Conclusions: Endophytic Bacillus strains have growth-promoting properties and anti-fungal ability that can enhance plant fitness in an eco-friendly manner., Significance and Impact of the Study: Endophytic Bacillus strains would be a potential alternative to chemical fertilizer as well as a biocontrol agent in the future., (© 2019 The Society for Applied Microbiology.)

Objective: To explore the anti-apoptotic effect of perindopril on myocardial cells in mice with acute myocardial infarction (AMI)., Materials and Methods: A total of 48 mice were randomly divided into 4 groups before intervention, namely sham operation group (Sham group, n=12), AMI group (n=12), 1.5 mg/kg perindopril treatment group (Perindopril group, n=12), and 1.5 mg/kg perindopril treatment and Toll-like receptor-4 (TLR4) knockout group (TLR4-/-Perindopril group, n=12). Mice in the control group and AMI group were gavaged with normal saline, and those in the Perindopril group and TLR4-/-Perindopril group were gavaged with perindopril for 7 d. On the 4th day after drug administration, mice in the AMI group, Perindopril group and TLR4-/-Perindopril group were subjected to the ligation of the anterior descending coronary artery to induce AMI, and those in the Sham group underwent the same operation, but had a loose knot at the anterior descending coronary artery. At 24 h after the above operation, color echocardiography was performed on mice to observe changes in cardiac function. Then, the mice were sacrificed. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP)-biotin nick end labeling (TUNEL) assay was carried out to determine myocardial apoptosis. Immunohistochemistry and Western blotting technique were employed to detect the protein expression levels of B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), TLR4 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p50 in infarction zones. The messenger ribonucleic acid (mRNA) expression levels of TLR4 and NF-κB p50 in infarction zones were measured via Reverse Transcription-Polymerase Chain Reaction (RT-PCR)., Results: Perindopril could significantly reduce the number of apoptotic myocardial cells after AMI. Mouse echocardiography showed that ejection fraction (EF), left ventricular fractional shortening (FS), left ventricular end-systolic diameter (LVESD) and left ventricular end-diastolic diameter (LVEDD) of AMI mice in the Perindopril groups were markedly superior to those in the AMI group. AMI mice in the Perindopril group had decreased expression levels of Bax protein and TLR4 and NF-κB p50 mRNA and protein, as well as the Bax/Bcl-2 ratio. Knockout of TLR4 attenuated the effect of perindopril in alleviating myocardial apoptosis after AMI., Conclusions: Perindopril inhibits myocardial apoptosis in mice with AMI through the TLR4/NF-κB pathway.

Objective: To compare the 10 years risk for ischemic cardiovascular disease among Han, Uygur, Kazak nationality residents of Xinjiang Uygur Autonomous Region. Methods: From October 2007 to October 2010,14 618 adult (aged ≥35 years) Han ( n= 5 757),Uygur ( n= 4 767) and Kazak ( n= 4 094) residents were selected to join this study through the four-stage stratified cluster sampling method from 7 cities and regions of Xinjiang Uygur Autonomous Region. The 10 years risk for ischemic cardiovascular disease was calculated according to the 10 years ischemic cardiovascular disease risk assessment form modified with Chinese characteristics and compared among the residents of 3 nationalities. Results: (1) There were significant differences in age, body mass index, systolic blood pressure, diastolic blood pressure,fasting blood glucose,triglycerides,total cholesterol,low-density lipoprotein,high-density lipoprotein cholesterol, smoking history, and drinking history among Han, Uygur, Kazak nationality population (all P< 0.001). (2) There were significant differences in 10 years risk for ischemic cardiovascular disease between different gender and age group including 35-39, 40-44, 45-49, 50-54, 55-59, and ≥60 years old between Han, Uygur, Kazak nationality population (all P< 0.001). (3) There were significant differences in rates of 10%-20% and>20% of 10 years risk for ischemic cardiovascular disease between different gender in Han, Uygur, Kazak nationality population ( P values were 0.013 and <0.001, respectively). There were no significant differences in rates of <5% and 5%-9% of 10 years risk for ischemic cardiovascular disease between different gender in Han,Uygur,Kazak nationality population (all P> 0.05).(4) There were significant differences in detection rates of diabetes,hypertension,smoking,hypertriglyceridemia,and obesity in male and female Han,Uygur,Kazak nationality population with 10 years risk for ischemic cardiovascular disease ≥10% ( P< 0.01 or 0.05). Meanwhile,there was significant difference in detection rates of hypercholesteremia in male Han, Uygur, Kazak nationality adults( P< 0.001). There were no significant differences in detection rates of elevated low density lipoprotein cholesterol and reduced high density lipoprotein cholesterol in male and female Han,Uygur,Kazak nationality adults (all P> 0.05). Conclusion: There are gender and age differences in the 10 years risk for ischemic cardiovascular disease in ≥35 years old Han,Uygur,Kazak nationality adults from Xinjiang Uygur Autonomous Region.

Objective: To study the effects of chronic virus-mediated micro ribonucleic acid miR-27a on proliferation and migration capacities of liver cancer cells., Patients and Methods: A total of 60 patients with primary liver cancer from January 2015 to December 2016 were selected as observation group, 60 patients with chronic liver disease were selected as control group, and another 60 healthy subjects who received physical examination during the same period were selected as healthy group. All patients received serum miRNA detection. The different expressions of serum miRNAs in healthy people, patients with chronic liver disease, and patients with liver cancer were analyzed. The correlations of miR-27a with growth and proliferation of liver cancer MCC-7721 cells were studied., Results: The levels of miR-27a and miR-664b in subjects in control group and healthy group were significantly lower than those in observation group, but the expression levels of miR-30a were statistically elevated (p<0.05). The expression of serum miR-27a in liver cancer patients with higher tumor-node-metastasis (TNM) staging (stage III-IV, number of tumors >1, tumor size >5 cm, and vascular invasion) was significantly higher than those in patients with lower TNM staging (stage I-II, number of tumors =1, tumor size ≤5 cm, and no vascular invasion) (p<0.05). At 48 h and 72 h after transfection, the proliferation of liver cancer MCC-7721 cells was significantly enhanced compared to that in non-transfection group (p<0.05). The level of miR-27a was significantly upregulated in cisplatin-resistant liver cancer A549/CDDP cells compared with that in parental A549 cells. MiR-27a regulated epithelial-mesenchymal transition (EMT) and cisplatin resistance in vitro, while modulated the in vivo response of liver cancer cells to cisplatin. Further studies identified the Raf kinase inhibitor protein (RKIP) as a direct and functional target of miR-27a. The knockdown of RKIP by RNAi showed a similar effect to ectopic miR-27a expression, whereas the over-expression of RKIP weakened the function of miR-27a in liver cancer cells., Conclusions: MiR-27a participated in the proliferation and migration capacities of liver cancer cells and influenced the effect of cisplatin via targeting RKIP. The high expression of miR-27a is closely related to the malignant degree of liver cancer, which provides guidance for the diagnosis, targeted therapy, and prognostic evaluation of liver cancer.

Objective: To summarize the clinical characteristics and treatment efficacy of the first reported case of a Chinese boy with stimulator of interferon genes (STING) associated vasculopathy with onset in infancy (SAVI). Methods: Sanger sequencing of the gene TMEM173 was performed based on systemic evaluation and clinical analysis of a highly suspected SAVI child admitted to Peking Union Medical College Hospital. A literature search (search terms included 'STING''SAVI''autoinflammatory diseases' and 'interferonopathy') was conducted using Chinese literature database, EMBASE and PubMed to include recently published SAVI studies (searched from January 2010 to December 2017). Results: A 14-year-old boy who had a history of chronic dry cough along with decreased activity tolerance after birth presented with growth retardation, chilblain lesions on the ear, telangiectasia of multiple skin areas and long clubbed fingers. His C-reactive protein was 21 mg/L, erythrocyte sedimentation rate was 78 mm/1h, and IgG was 22.16 g/L. The high-resolution computed tomography (HRCT) revealed interstitial lung diseases and echocardiography showed pulmonary artery hypertension, with a level of 61 mmHg (1 mmHg=0.133 kPa). Genetic mutation of TMEM173 (c.463G>A, p.V155M) was confirmed by Sanger sequencing. His activity tolerance increased to some extent after treatment with tofacitinib at a dose of 5 mg twice a day. Our review yielded 8 publications (8 English and 0 Chinese) . To date 20 cases have been reported worldwide, who mostly presented with skin and lung involvement as well as growth retardation. Conclusions: SAVI has been included within the spectrum of interferonopathy, which is a kind of autoinflammatory diseases as well. Typical clinical features include chilblain skin lesions, interstitial lung disease, growth retardation, elevated IgG levels, and increased inflammation markers. Janus kinase (JAK) inhibitors may offer benefit for SAVI patients.

Objective: To evaluate the clinical efficacy between the minimally-Invasive surgical (MIS)-transforaminal lumbar interbody fusion (TLIF) technique associated with percutaneous pedicle screws in micro endoscopy discectomyand MIS-TLIF technique associated with both sides of the lower lumbar spine Wiltse approach in Quadrant channel with treatment of single segment herniation associated with lumbar instability syndrome. Methods: From January 2012 to January 2015, 75 cases that meet the inclusion and exclusion criteria were treated by retrospective study method, which were divided into two groups in Department of Orthopedics, the Affiliated Hospital of Putian University.Experimental group(30 patients) were treated with MIS-TLIF technique associated with percutaneous pedicle screws in microendoscopy discectomy, control group were treated with MIS-TLIF technique associated with both sides of the lower lumbar spine Wiltse approach in Quadrant Chanel.Compare operation time, blood loss, postoperativehospital stay, clinical efficacy, nailing accuracy, fusion rate, postoperative pain scoring of two groups. Results: The blood loss[(102.1±5.5) min vs(103.7±7.7) min, t =-0.586, P >0.05], postoperative blood loss, hospital stay[(44.6±5.2) ml and(57.2±5.3) ml, (7.3±1.6) d and(9.3±1.9) d; t =-5.813, -2.774, P <0.05], JOA score before and after surgery in same group were statistically significant( P <0.05), respectively.Patients of two groups compared with operation time, clinical efficacy, nailing accuracy[group A: 97.5%, group B: 95.7%; χ(2)=3.00, P >0.05.Postoperative 3 month , group A: 96.7%(29/30), group B: 94.3%(33/35; χ(2)=0.79, P >0.05], fusion rate[group A: 96.7%(29/30), group B: 94.3%(33/35), χ(2)=0.79, P >0.05], preoperative JOA score[(20.4±2.4)score and(7.9±1.0), (19.1±2.7)score and(7.8±1.2)score], postoperative JOA score were no statistically significant respectively, P >0.05. JOA score of both groups were statistically significant respectively Before and after operate.Excellent rate: group A; 84.4%(25/30), group B: 80.0%(28/35), χ(2)=0.43, P >0.05. Conclusion: MIS-TLIF technique associated with percutaneous pedicle screws in micro endoscopy discectomy relative to conventional minimally invasive spine surgery had many advantages: minimal damage, operation conveniently, precisely clinical effect, that is a kind of feasible and reliable minimally invasive surgery which is worth promoting.

Background: Although several new drugs have been approved in recent years, pulmonary arterial hypertension (PAH) remains a rapidly progressive disease with a poor prognosis. Ambrisentan, a selective endothelin type A antagonist, has been approved for treatment of PAH. This open label study assessed the efficacy and safety of ambrisentan in Chinese subjects with PAH., Methods: Eligible patients with PAH (World Health Organisation [WHO] functional class [FC] II orIII) were enrolled and received Ambrisentan (5 mg) once daily for a 12-week preliminary evaluation period, and a 12-week dose-adjustment period (dose titration to 10 mgallowed). Endpoints included: change from baseline in 6-Minute Walk Distance (6-MWD), N-Terminal Pro B-Type Natriuretic Peptide (NT-pro-BNP), WHO FC, Borg Dyspnoea Index (BDI), clinical worsening of PAH and incidences of adverse events (AE)., Results: One hundred thirty-three subjects (85 % women, mean age: 36 years) with PAH (WHOFC II or III) were enrolled and received ambrisentan (5 mg) once daily for a 12-week preliminary evaluation period, and a 12-week dose-adjustment period. Mean (SD) duration of drug exposure was 161.7 (27.13) days. Ambrisentan (average daily dose of 6.27 mg) significantly improved exercise capacity (6MWD) from baseline (mean: 377.1 m [m]) at week 12 (+53.6 m, p < 0.001) (primary endpoint). Improvement in exercise capacity was noted as early as week 4, and was sustained up to week 24 (+ 64.4 m, p < 0.001). NT-pro-BNP plasma levels decreased significantly (p < 0.001) at week 12 (-861.4 ng/L) and week 24 (-806 ng/L) from baseline (mean: 1600.7 ng/L). The WHO FC showed improvements for 44 subjects at week 12 and 51 subjects at week 24. BDI scores decreased significantly at week 12 (-0.3, p < 0.001) and week 24 (-0.2, p = 0.003) from baseline (mean: 2.5). Four patients died during the study (sudden cardiac death [n = 2], cerebral haemorrhage [n = 1], cardiac failure [n = 1]). Drug related adverse events occurred in 34.3 % of subjects; peripheral oedema (11.2 %) and flushing (8.2 %) occurred most frequently., Conclusion: Ambrisentan (5 and 10 mg, orally) significantly improved the exercise capacity in Chinese PAH subjects with a safety profile similar to that observed in global studies., Trial Registration: NCT No. (ClinicalTrials.gov): NCT01808313 ; Registration date (first time): February 28, 2013.

Allograft vasculopathy (AV) is characterized by diffuse stenoses in the vasculature of solid organ transplants. Previously, we developed two humanized models showing that alloantibody and ischemia reperfusion injury (IRI) exacerbated T cell-mediated AV in human arterial xenografts in vivo. Herein we examined a causal role for terminal complement activation in both settings. IRI, in contrast to alloantibody, elicited widespread membrane attack complex (MAC) assembly throughout the vessel wall. Both alloantibody and IRI caused early (24 h) and robust endothelial cell (EC) activation localized to regions of intimal MAC deposition, indicated by increases in nuclear factor kappa B (NF-κB)-inducing kinase, an MAC-dependent activator of noncanonical NF-kB, VCAM-1 expression and Gr-1+ neutrophil infiltration. Endothelial cell activation by alloantibody was inhibited by antimouse C5 mAb, but not by anti-C5a mAb or by control mAb, implicating MAC as the primary target of anti-C5 mAb. Antimouse C5 mAb significantly reduced alloantibody- and IRI-enhanced T cell infiltration and AV-like changes, including neointimal hyperplasia as well as intraluminal thrombosis in a subset of IRI-treated arterial grafts. These results indicate that increased AV lesion formation in response to either alloantibody or IRI is dependent on complement C5 activation and, accordingly, inhibition of this pathway may attenuate AV., (© Copyright 2016 The American Society of Transplantation and the American Society of Transplant Surgeons.)

The complement system plays a critical role in ischemia-reperfusion injury (IRI)-mediated delayed graft function (DGF). To better understand the roles of complement activation pathways in IRI in kidney transplantation, donor kidneys were treated ex vivo with terminal complement pathway (TP) inhibitor, anti-rat C5 mAb 18A10, or complement alternative pathway (AP) inhibitor TT30 for 28 h at 4°C pretransplantation in a syngeneic kidney transplantation rat model. All 18A10- and 67% of TT30-pretreated grafts, but only 16.7% of isotype control-pretreated grafts, survived beyond day 21 (p < 0.01). Inhibitor treatment in the final 45 min of 28-h cold ischemia (CI) similarly improved graft survival. Systemic posttransplant treatment with 18A10 resulted in 60% increased graft survival beyond day 21 (p < 0.01), while no TT30-treated rat survived > 6 days. Our results demonstrate that AP plays a prominent role during CI and that blocking either the AP or, more effectively the TP prevents ischemic injury and subsequent DGF. Multiple complement pathways may be activated and contribute to reperfusion injury; blocking the TP, but not the AP, posttransplant is effective in preventing reperfusion injury and increasing graft survival. These results demonstrate the feasibility of using complement inhibitors for prevention of DGF in humans., (© Copyright 2016 The American Society of Transplantation and the American Society of Transplant Surgeons.)

CD163 is a macrophage scavenger receptor with anti-inflammatory and pro-inflammatory functions. Here, we report that alveolar macrophages (AMΦs) from asthmatic subjects had reduced cell-surface expression of CD163, which suggested that CD163 might modulate the pathogenesis of asthma. Consistent with this, house dust mite (HDM)-challenged Cd163(-/-) mice displayed increases in airway eosinophils and mucous cell metaplasia (MCM). The increased airway eosinophils and MCM in HDM-challenged Cd163(-/-) mice were mediated by augmented CCL24 production and could be reversed by administration of a neutralizing anti-CCL24 antibody. A proteomic analysis identified the calcium-dependent binding of CD163 to Dermatophagoides pteronyssinus peptidase 1 (Der p1). Der p1-challenged Cd163(-/-) mice had the same phenotype as HDM-challenged Cd163(-/-) mice with increases in airway eosinophils, MCM and CCL24 production, while Der p1 induced CCL24 secretion by bone marrow-derived macrophages (BMMΦs) from Cd163(-/-) mice, but not BMMΦs from wild-type (WT) mice. Finally, airway eosinophils and bronchoalveolar lavage fluid CCL24 levels were increased in Der p1-challenged WT mice that received adoptively transferred AMΦ's from Cd163(-/-) mice. Thus, we have identified CD163 as a macrophage receptor that binds Der p1. Furthermore, we have shown that HDM-challenged Cd163(-/-) mice have increased eosinophilic airway inflammation and MCM that are mediated by a CCL24-dependent mechanism.

Background/objectives: Optimal obesity indices in predicting cardio-metabolic risk are less studied in Asian. We evaluated optimal waist-to-height ratio (WHtR) for predicting hypertension, dyslipidemia and diabetes in Han and Uygur populations in Xinjiang, a northwest part of China., Subjects/methods: This study involved 5603 Han and 4657 Uyghur participants. Anthropometric data, blood pressure, serum total cholesterol (TC), triglyceride, low-density lipoprotein, high-density lipoprotein and fasting glucose were determined. The cutoff values of WHtR were calculated; the relation between WHtR and prevalence of cardio-metabolic risks was evaluated., Results: There was a positive correlation between WHtR and blood pressure, TC, triglycerides and fasting glucose in both Han and Uygur participants (all P<0.001). The prevalence of hypertension, dyslipidemia and diabetes was higher with increased WHtR for both ethnic groups after adjusted by age. Calculated cutoff values of WHtR for predicting hypertension, dyslipidemia, diabetes or ⩾ 2 of these risk factors were 0.54 for both men and women in Han and 0.55 in male and 0.57 in female Uygur participants. A significant difference in blood pressure, triglycerides and fasting glucose between subgroups with WHtR either above or below the cutoff values was observed in both men and women of the two ethnicities., Conclusions: The optimal cutoff value of WHtR is a useful screen tool for predicting cardio-metabolic risks in Han and Uygur population in Xinjiang, northwest part of China.

Serum amyloid A (SAA) protein is not only an inflammatory factor but also an apolipoprotein that can replace apolipoprotein A1 (apoA1) as the major apolipoprotein of high-density lipoprotein cholesterol (HDL-C). However, the relationship between genetic polymorphisms of SAA and coronary artery disease (CAD) remains unclear. A total of four single nucleotide polymorphisms (rs12218, rs4638289, rs7131332, and rs11603089) of the SAA gene were genotyped using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method in two independent case-control studies, one of the Han population (1416 CAD patients and 1373 control subjects) and the other of the Uygur population (588 CAD patients and 529 control subjects). We found that the rs12218 CC genotype was more frequent among the CAD patients than among the controls in both the Han (8.3% vs. 4.8%, P < 0.001) and Uygur populations (15.5% vs. 11.3%, P < 0.05). After adjustments for confounding factors, such as sex, age, smoking, drinking, hypertension, diabetes, and serum levels of triglycerides, total cholesterol, HDL, and plasma SAA, the differences remained significant in the Han (CC vs. CT+TT, P < 0.001, OR = 3.863, 95% CI: 1.755-12.477) and Uygur groups (CC vs. CT+TT, P = 0.031, OR = 3.022, 95% CI: 1.033-8.840). Genetic polymorphisms in SAA1 are associated with CAD in the Han and Uygur populations in western China., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

This study was designed to estimate the prevalence of diabetes and impaired fasting glucose (IFG) in Xinjiang children in western China. Data were obtained from the Chun-Miao Project, a community-based, cross-sectional study designed to investigate the prevalence and risk factors of diabetes in children of the Chinese Uygur population in Xinjiang from February 2010 to May 2012. A total of 3644 children completed the survey and measurements of fasting glucose. Diabetes and IFG were defined using American Diabetes Association 2009 criteria. Overall, 0.7% of the 3644 Uygur children had IFG and 0.1% had diabetes. In the newborn to 8-year-old group, the prevalence of diabetes and IFG was 0.6 and 1.1%, respectively. In the 9-13-year-old group, the prevalence of diabetes and IFG was 0.1 and 0.7%, respectively. There was no evidence of IFG or diabetes in the 14-17-year-old group. Logistic regression analysis suggested that overweight and obesity were independent risk factors of diabetes in Uygur children of Xinjiang. The prevalence of diabetes and IFG in Uygur children was lower than that reported previously in children of other ethnicities in China.

Parafibromin, a tumor suppressor protein encoded by HRPT2/CDC73 and implicated in parathyroid cancer and the hyperparathyroidism-jaw tumor (HPT-JT) familial cancer syndrome, is part of the PAF1 transcriptional regulatory complex. Parafibromin has been implicated in apoptosis and growth arrest, but the mechanism by which its loss of function promotes neoplasia is poorly understood. In this study we report that a hypomorphic allele of hyrax (hyx), the Drosophila homolog of HRPT2/CDC73, rescues the loss-of-ventral-eye phenotype of lobe (Akt1s1). Such rescue is consistent with previous reports that hyx/parafibromin is required for the nuclear transduction of Wingless (Wg)/Wnt signals and that Wg signaling antagonizes lobe function. A screen using double hyx/lobe heterozygotes identified an additional interaction with orb and orb2, the homologs of mammalian cytoplasmic polyadenylation element binding protein (CPEB), a translational regulatory protein. Hyx and orb2 heterozygotes lived longer and were more resistant to starvation than controls. In mammalian cells, knockdown of parafibromin expression reduced levels of CPEB1. Chromatin immunoprecipitation (ChIP) showed occupancy of CPEB1 by endogenous parafibromin. Bioinformatic analysis revealed a significant overlap between human transcripts potentially regulated by parafibromin and CPEB. These results show that parafibromin may exert both transcriptional and, through CPEB, translational control over a subset of target genes and that loss of parafibromin (and CPEB) function may promote tumorigenesis in part by conferring resistance to nutritional stress.

Transcript depletion using small interfering RNA (siRNA) technology represents a potentially valuable technique for the treatment of cancer. However, delivering therapeutic quantities of siRNA into solid tumors by chemical transfection is not feasible, whereas viral vectors efficiently transduce many human tumor cell lines. Yet producing sufficient quantities of viral vectors that elicit acute and selective cytotoxicity remains a major obstacle for preclinical and clinical trials. Using the invertebrate Spodoptera frugiperda (Sf9) cell line, we were able to produce high titer stocks of cytotoxic recombinant adeno-associated virus (rAAV) that express short hairpin RNA (shRNA) and that efficiently deplete Hec1 (highly expressed in cancer 1), or Kntc2 (kinetochore-associated protein 2), a kinetochore protein directly involved in kinetochore microtubule interactions, chromosome congression and spindle checkpoint signaling. Depletion of Hec1 protein results in persistent spindle checkpoint activation followed by cell death. Because Hec1 expression and activity are only present in mitotic cells, non-dividing cells were not affected by rAAV treatment. On the basis of the results of screening 56 human tumor cell lines with three different serotype vectors, we used a tumor xenograft model to test the effects in vivo. The effects of the shHec1 vector were evident in sectioned and stained tumors. The experiments with rAAV-shRNA vectors demonstrate the utility of producing vectors in invertebrate cells to obtain sufficient concentrations and quantities for solid tumor therapy. This addresses an important requirement for cancer gene therapy, to produce cytotoxic vectors in sufficient quantities and concentrations to enable quantitative transduction and selective killing of solid tumor cells.

A critical advance in the technique of low-energy electron diffraction is presented and shown to enable determining detailed structures of nanomaterials, based on experimental methods that already exist or have been proposed. Our new cluster approach speeds up the computation to scale as n logn, rather than the current n3 or n2, with n the number of atoms, for example. Applications are illustrated for C60 molecules adsorbed on a Cu(111) surface, with and without coadsorbed metal atoms, exhibiting sensitivity to important structural features such as buckyball size and deformation.

To investigate the association between the expression of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) and the clinicopathological features in lepidic and invasive components of adenocarcinoma of the lung, we performed immunostaining for type IV collagen, various MMPs, and TIMPs in 27 cases of invasive adenocarcinomas and 5 cases of atypical adenomatous hyperplasia of alveolar epithelial cells (AAH). Mean extent of lepidic growth was 61% and the survival was significantly better in cases with 50% or more lepidic component. The preservation of type IV collagen in lepidic areas correlated inversely with lymphatic or vascular invasion (P = 0.02 and 0.002, respectively). Five-year survival was reduced in cases showing destruction of type IV collagen (P = 0.004) or expression of MMP-2 (P = 0.008) in lepidic areas. MMP-2 co-localized with MT-1-MMP (its activating enzyme) and TIMP-2 in neoplastic cells. Reactivity for other MMPs and TIMPs did not correlate with destruction of type IV collagen or prognosis. Type IV collagen was preserved in all cases of AAH. MMP-2, but not MT-1-MMP, was expressed in two of the five cases of AAH. Immunostaining for type IV collagen MMP-2 is useful in evaluating the prognosis of the lung.

Huntington's disease (HD) is characterized by the selective loss of striatal projection neurons. In early stages of HD, neurodegeneration preferentially occurs in the lateral globus pallidus (LGP) and substantia nigra (SN), two regions in which the axons of striatal neurons terminate. Here we report that in mice expressing full-length mutant huntingtin and modeling early stages of HD, neuropil aggregates form preferentially in the LGP and SN. The progressive formation of these neuropil aggregates follows intranuclear accumulation of mutant huntingtin and becomes prominent from 11 to 27 months after birth. Neuropil aggregates, but no intranuclear inclusions, were observed in the LGP and SN, suggesting that huntingtin aggregates are formed in the axons of striatal projection neurons. In the LGP and SN, we observed degenerated axons in which huntingtin aggregates were associated with dark, swollen organelles that resemble degenerated mitochondria. Neuritic aggregates also form in cultured striatal neurons expressing mutant huntingtin, block protein transport in neurites, and cause neuritic degeneration before nuclear DNA fragmentation occurs. These findings suggest that the early neuropathology of HD originates from axonal dysfunction and degeneration associated with huntingtin aggregates.

The 42 amino acid form of beta amyloid (Abeta42) plays a pivotal role in neurotoxicity and the activation of mononuclear phagocytes in Alzheimer's disease (AD). Our recent study revealed that FPRL1, a G-protein-coupled receptor, mediates the chemotactic and activating effect of Abeta42 on mononuclear phagocytes (monocytes and microglia), suggesting that FPRL1 may be involved in the proinflammatory responses in AD. We investigated the role of FPRL1 in cellular uptake and the subsequent fibrillar formation of Abeta42 by using fluorescence confocal microscopy. We found that upon incubation with macrophages or HEK293 cells genetically engineered to express FPRL1, Abeta42 associated with FPRL1 and the Abeta42/FPRL1 complexes were rapidly internalized into the cytoplasmic compartment. The maximal internalization of Abeta42/FPRL1 complexes occurred by 30 min after incubation. Removal of free Abeta42 from culture supernatants at 30 min resulted in a progressive recycling of FPRL1 to the cell surface and degradation of the internalized Abeta42. However, persistent exposure of the cells to Abeta42 over 24 h resulted in retention of Abeta42/FPRL1 complexes in the cytoplasmic compartment and the formation of Congo red positive fibrils in macrophages but not in HEK 293 cell transfected with FPRL1. These results suggest that besides mediating the proinflammatory activity of Abeta42, FPRL1 is also involved in the internalization of Abeta42, which culminates in the formation of fibrils only in macrophages.

This study is devoted to a detailed theoretical study of an inverse-electron demand Diels-Alder reaction (IDA) with 1,3,5-triazine as the diene and 2-aminopyrrole 1A(alpha) as the dienophile, which is a key step in a cascade reaction for the one-pot synthesis of purine analogues. Geometries were optimized with the B3LYP/6-31G* method and energies were evaluated with the MP2/6-311++G** method. This IDA reaction occurs through a stepwise mechanism, where the first step corresponds to the nucleophilic attack of 2-aminopyrrole to triazine to form a zwitterionic intermediate, which is in equilibrium with a neutral intermediate through a hydrogen transfer process, followed by a rate-determining ring-closure step. It is shown that the B3LYP method significantly overestimates the activation energy, whereas the MP2 method offers a reasonable activation barrier of 27.9 kcal/mol in the gas phase. The solvation effect has been studied by the PCM model. In DMSO, the calculated activation energy of the IDA reaction is decreased to 24.0 kcal/mol with a strong endothermicity of 17.4 kcal/mol due to the energy penalty of transforming two aromatic reactants into a nonaromatic IDA adduct. The possible stepwise [2+2] pathway is ruled out based on its higher activation and reaction energies than those of the [4+2] pathway. By comparing the IDA reactions of triazine to 2-aminopyrrole and pyrrole, we address two crucial roles of the alpha-amino substituent in lowering activation and reaction energies and controlling the reaction regiochemistry.

Background: Insulin-like growth factors (IGF-1 and IGF-2), the IGF-1 receptor (IGF-1R), and IGF-binding proteins (IGFBPs) are involved in normal pulmonary development and in the pathogenesis of smooth muscle cell tumors., Methods: To evaluate the role of the IGF system in lymphangioleiomyomatosis (LAM), we used immunohistochemical and in situ hybridization techniques to characterize the expression of IGF-1, IGF-2, IGF-1R, and IGFBP-2, -4, -5, and -6 in lung tissue from 18 LAM patients., Results: IGF-1, ICGF-2, IGF-1R, IGFBP-1, IGFBP-2, IGFBP-4, IGFBP-5, and IGFBP-6 were expressed by LAM cells. Reactivity and mRNA expression for IGF-2 were observed in LAM cells and resembled that found in normal smooth muscle cells during pulmonary development as well as in smooth muscle cell tumors. IGFBP-2, IGFBP-4, and IGFBP-6 were associated with spindle-shaped LAM cells, whereas IGFBP-5 was associated mainly with epithelioid LAM cells., Conclusions: These findings suggest that the IGFBPs modulate the effects of the IGFs on LAM cells. Thus, the patterns of localization and expression of components of the IGF system in LAM strongly suggest that these agents are involved in the proliferation of LAM cells.

A detailed mechanism for the Kulinkovich hydroxycyclopropanation reaction has been explored with density functional theory calculations on the reactions between R(1)COOMe and Ti(OMe)(2)(CH(2)CHR(2)) (R(1) and R(2) are hydrogen and alkyl groups). Addition of ester to titanacyclopropane is found to be fast, exothermic, and irreversible. It has a preference for the alpha-addition manifold over the beta-addition manifold in which its cycloinsertion transition states suffer from the steric repulsion between the R(2) and ester. The following intramolecular methoxy migration step is also exothermic with reasonable activation energy. The cyclopropane-forming step is the rate-determining step, which affords the experimentally observed cis-R(1)/R(2) diastereoselectivity in the alpha-addition manifold by generating cis-R(1)/R(2) 1,2-disubstituted cyclopropanol when R(1) is primary alkyl groups. On the contrary, the unfavored beta-addition manifold offers the diastereoselectivity contradicting the experimental observations. The effects of R(1) and R(2) on the regio- and stereoselectivity are also discussed.

Background: Pathogens infecting the arterial wall with resultant inflammation may contribute to atherogenesis. Human coronary artery smooth muscle cells (SMCs) infected with human cytomegalovirus (CMV) demonstrate a rapid increase in reactive oxygen species (ROSs), with activation of genes involved in viral replication and inflammation. Because estrogen appears to have antioxidant properties, we wished to determine whether this hormone attenuates SMC responses to CMV infection., Methods and Results: Using confocal microscopy and an intracellular fluorescent dye activated by ROSs, we found that 17beta-estradiol (0.1 to 10 nmol/L) and its stereoisomer 17alpha-estradiol (which has low affinity for the estrogen receptor) dose-dependently inhibited ROS generation in CMV-infected SMCs. These effects were not blocked by the estrogen receptor inhibitor ICI 182,780. 3-Methoxyestrone, which lacks the phenolic hydroxyl group, did not interfere with ROS generation. We found that 17beta-estradiol and 17alpha-estradiol, but not 3-methoxyestrone, prevented binding of nuclear factor (NF)-kappaB to DNA. Furthermore, in SMCs transfected with the reporter constructs 3XkappaB-CAT, MIEP-CAT, or ICAM-CAT, cotransfection with a CMV-IE72 expression plasmid caused promoter and CAT activation. Treatment with 17beta-estradiol and 17alpha-estradiol, but not 3-methoxyestrone, inhibited CAT activity and, in CMV-infected SMCs, prevented IE72 and ICAM-1 protein expression and cytopathic effects., Conclusions: These findings indicate that estrogen molecules with an A-ring hydroxyl group have estrogen receptor-independent anti-CMV effects at physiological concentrations by inhibiting ROS generation, NF-kappaB activation, NF-kappaB-dependent transcription, and viral replication. To the extent that chronic infection of the vascular wall with CMV contributes to atherogenesis, these antioxidant actions of estrogen may be of therapeutic importance.

Subcellular localization of cyclic nucleotide phosphodiesterases (PDEs) may be important in compartmentalization of cAMP/cGMP signaling responses. In 3T3-L1 adipocytes, mouse (M) PDE3B was associated with the endoplasmic reticulum (ER) as indicated by its immunofluorescent colocalization with the ER protein BiP and subcellular fractionation studies. In transfected NIH 3006 or COS-7 cells, recombinant wild-type PDE3A and PDE3B isoforms were both found almost exclusively in the ER. The N-terminal portion of PDE3 can be arbitrarily divided into region 1 (aa 1-300), which contains a large hydrophobic domain with six predicted transmembrane helices, followed by region 2 (aa 301-500) containing a smaller hydrophobic domain (of approximately 50 aa). To investigate the role of regions 1 and 2 in membrane association, we examined the subcellular localization of a series of catalytically active, Flag-tagged N-terminal-truncated human (H) PDE3A and MPDE3B recombinants, as well as a series of fragments from regions 1 and 2 of MPDE3B synthesized as enhanced green fluorescent (EGFP) fusion proteins in COS-7 cells. In COS-7 cells, the localization of a mutant HPDE3A, lacking the first 189 amino acids (aa) and therefore four of the six predicted transmembrane helices (H3A-Delta189), was virtually identical to that of the wild type. M3B-Delta302 (lacking region 1) and H3A-Delta397 (lacking region 1 as well as part of region 2) retained, to different degrees, the ability to associate with membranes, albeit less efficiently than H3A-Delta189. Proteins that lacked both regions 1 and 2, H3A-Delta510 and M3B-Delta604, did not associate with membranes. Consistent with these findings, region 1 EGFP-MPDE3B fusion proteins colocalized with the ER, whereas region 2 EGFP fusion proteins were diffusely distributed. Thus, some portion of the N-terminal hydrophobic domain in region 1 plus a second domain in region 2 are important for efficient membrane association/targeting of PDE3.

Previous studies suggest that estrogen may prevent expression of cell adhesion molecules implicated in vascular inflammation associated with atherosclerosis. We demonstrate the interaction and reciprocal interference of estrogen receptors (ERs) with p65, the nuclear factor-kappaB component, in smooth muscle cells that express ERalpha and ERss after exposure to 17ss-estradiol for 48 to 72 hours. ER and p65 do not associate directly, as shown by lack of coprecipitation, but instead compete for limiting amounts of p300, a close relative of the CREB-binding protein. Overexpressed p300 significantly reduced the inhibitory effect of ER on p65-dependent transcription as well as the inhibitory effect of p65 on ER-dependent transcription. These actions were ligand-dependent. The expression of both ER and nuclear factor-kappaB-dependent reporter genes was partially rescued from ER/p65 mutual inhibition by transient transfection of smooth muscle cells with a p300 expression vector. These actions of 17ss-estradiol may play an important role in the cytokine-induced expression of immune and inflammatory genes implicated in atherogenesis.

CIN85 is an 85-kDa adaptor protein whose functions in signaling pathways are presently unknown. Using the yeast two-hybrid screen, the B cell linker protein (BLNK) was identified as a binding partner of CIN85. Coimmunoprecipitation experiments using mammalian cells revealed that CIN85 directly bound to BLNK through its SH3 domains. Immunostaining analysis showed that CIN85 and BLNK were colocalized in the cytoplasm. These results indicate a potential role of CIN85 in the B cell receptor-mediated signaling pathway. It was also found that Crk-I, Crk-II, p130(Cas), p85-PI3K, Grb2, and Sos1 were components of CIN85 complexes. CIN85 interacted with itself through its coiled-coil region, resulting in formation of a tetramer. Both the coiled-coil region and SH3 domains of CIN85 were responsible for its subcellular localization. Our data suggest that CIN85 may serve for regulation of various signaling events through formation of its diverse complexes.

Background: Little is known of the morphology of the pneumocytes lining the parenchymal cysts in lymphangioleiomyomatosis (LAM)., Objective: To evaluate the structural characteristics of the alveolar epithelial cells in LAM., Methods: Immunohistochemical and electron microscopic studies were performed on lung tissue from 22 women with pulmonary LAM., Results: Epithelial cells that reacted with PE-10 (a mouse monoclonal antibody that recognizes the surfactant apoprotein A in type II pneumocytes) and TTF-1 (an antibody that identifies nuclear transcription factor found in type II pneumocytes) were the predominant cell type lining the surfaces of lesions of LAM and normal areas of lung. Scanning and transmission electron microscopic studies confirmed that these cells were type II pneumocytes as demonstrated by (1) apical microvilli, (2) electron-dense lamellar bodies, and (3) cytoplasmic projections that extended from the basal surfaces into the underlying connective tissue, where they made extensive contact with interstitial connective tissue cells. A few cells had morphologic characteristics of type I pneumocytes, including large flat surfaces lacking microvilli. Cells that appeared intermediate between type I and type II pneumocytes were observed occasionally., Conclusions: These observations and the reactivity of these cells with antibody to proliferating cell nuclear antigen demonstrate that extensive hyperplasia of type II pneumocytes is a major characteristic of LAM.

The bisdioxopiperazines, including dexrazoxane (ICRF-187), are catalytic or noncleavable complex-forming inhibitors of DNA topoisomerase II that do not produce DNA strand breaks. In this study we show that dexrazoxane inhibits the division of Chinese hamster ovary (CHO) cells resulting in marked increases in cell size (up to 80 microm in diameter), volume (up to 150-fold greater), and ploidy (as high as 32N). This last result indicates that the dexrazoxane-induced DNA reduplication was restricted to once per cell cycle. Kinetic analysis of the flow cytometry data indicated that the conversion between successively higher ploidy levels was progressively slowed at longer times of exposure to dexrazoxane. Both the protein and DNA content of dexrazoxane-treated CHO cells increased linearly over time in the same proportion. Light and electron microscopic studies of dexrazoxane-treated cells showed ring-like multilobulated nuclei. Immunohistochemical staining of dexrazoxane-treated cells showed that F-actin and acetylated alpha-tubulin were present in large, highly organized networks. Immunohistochemical staining of the dexrazoxane-treated CHO cells also showed that the topoisomerase II alpha colocalized with the DNA of the multilobulated nuclei. Staining of gamma-tubulin revealed that the dexrazoxane-treated cells contained multiple centrosomes, indicating that dexrazoxane prevents cytokinesis but not centrosome reduplication. It is concluded that dexrazoxane inhibits CHO cytokinesis in cells by virtue of its ability to inhibit topoisomerase II.

We describe the lesions of extrapulmonary lymphangioleiomyomatosis (LAM) affecting the lymph nodes of the mediastinum and retroperitoneum in 22 women (mean age +/- SD, 42.4+/-10.5 years). In most of these patients, the diagnosis of extrapulmonary LAM preceded that of pulmonary LAM, usually by 1 to 2 years. Eleven patients had distinct symptoms, including chylous pleural effusion and/or ascites, abdominal pain, and palpable abdominal masses. In the other 11 patients, the masses caused no symptoms. Well-circumscribed, encapsulated masses, measuring up to 20 cm in size, occurred in the mediastinum in 2 patients, the upper retroperitoneum in 15, extensive areas of the retroperitoneum in 2, and the pelvis in 3. The masses exceeding 3 cm in diameter contained large, multiple cysts filled with yellow-tan chylous fluid. Histologically, the masses were characterized by a proliferation of smooth muscle cells (LAM cells) arranged in fascicular, trabecular, and papillary patterns, which were associated with slit-like vascular channels. The LAM cells varied from small, spindle-shaped cells to large epithelioid cells. Immunohistochemical studies showed a strong reactivity of most LAM cells for alpha-smooth muscle actin and smooth muscle myosin heavy chain and a weak to moderate reactivity of a lesser number of cells for desmin and nonmuscle myosin heavy chain II-B. A reaction for HMB-45 and estrogen and progesterone receptors was observed mainly in epithelioid LAM cells. These patterns of reactivity are similar to those observed in pulmonary LAM. However, the chylous cysts are not a feature of pulmonary LAM and are thought to result from obstruction of lymphatics.

A review is presented of the clinical and morphological manifestations of lymphangioleiomyomatosis (LAM), a systemic disorder of unknown etiology that affects women. The clinical features include dyspnea, hemoptysis, recurrent pneumothorax, chylothorax, and chylous ascites. It is characterized by: 1) proliferation of abnormal smooth muscle cells (LAM cells) in pulmonary interstitium and along the axial lymphatics of the thorax and abdomen; 2) thin-walled pulmonary cysts, and 3) a high incidence of angiomyolipomas. The pulmonary cystic lesions have a characteristic appearance on high resolution computed tomography. The most specific method for diagnosing LAM is lung biopsy to demonstrate the presence of LAM cells, either by their characteristic histological appearance or by specific immunostaining with HMB-45 antibody. LAM cells differ in several important respects from the types of smooth muscle cells normally present in lung. Their reactivity with HMB-45 antibody is localized in stage I and stage II melanosomes. LAM cells show additional evidence of incomplete melanogenesis, and the significance of these observations remains to be determined. Two types of LAM cells are recognized: 1) small, spindle-shaped cells that are centrally located in the LAM nodules and are highly immunoreactive for matrix metalloproteinase-2 (MMP-2), its activating enzyme (MT-1-MMP), and proliferating cell nuclear antigen (PCNA), and 2) large, epithelioid cells that are distributed along the periphery of the nodules and show a high degree of immunoreactivity with HMB-45 antibody and with antibodies against estrogen and progesterone receptors. Types of treatment used for LAM include oophorectomy, administration of Lupron or progesterone and in very severe cases, pulmonary transplantation (following the onset of respiratory insufficiency, not relieved by O(2)).

Leakage of mitochondrial oxidants contributes to a variety of harmful conditions ranging from neurodegenerative diseases to cellular senescence. We describe here, however, a physiological and heretofore unrecognized role for mitochondrial oxidant release. Mitochondrial metabolism of pyruvate is demonstrated to activate the c-Jun N-terminal kinase (JNK). This metabolite-induced rise in cytosolic JNK1 activity is shown to be triggered by increased release of mitochondrial H(2)O(2). We further demonstrate that in turn, the redox-dependent activation of JNK1 feeds back and inhibits the activity of the metabolic enzymes glycogen synthase kinase 3beta and glycogen synthase. As such, these results demonstrate a novel metabolic regulatory pathway activated by mitochondrial oxidants. In addition, they suggest that although chronic oxidant production may have deleterious effects, mitochondrial oxidants can also function acutely as signaling molecules to provide communication between the mitochondria and the cytosol.

Purpose: To evaluate the possible roles of apoptosis in the murine retinopathy induced by coronavirus., Methods: Mice were inoculated with virus intravitreally. Mouse eyes harvested at varying times after inoculation were evaluated for apoptotic and immunologic events by hematoxylin and eosin staining, immunohistochemical staining, in situ terminal deoxynucleotidyltransferase dUTP nick-end labeling (TUNEL) assay, and electron microscopy. Isolated retinas were analyzed for infectious virus and for expression of apoptosis-associated genes., Results: The number of apoptotic events was significantly elevated in infected eyes from BALB/c and CD-1 mouse strains, reaching a maximum at days 6 through 10, and returning to normal levels at day 20. The majority of apoptotic cells were observed in the outer nuclear layer of the infected retina. In contrast, few apoptotic cells were observed in normal or mock-injected mouse eyes. Apoptotic events within the retina were associated with the presence of viral antigen, infiltration of CD8(+) T cells, and clearance of infectious virus. Reverse transcription-polymerase chain reaction (RT-PCR) analysis identified the upregulation of Fas ligand (FasL) and granzyme B mRNAs within the infected retinas. The development of apoptosis, regulative gene expression, and viral clearance were similar in both retinal degeneration-susceptible (BALB/c) and -resistant (CD-1) mice., Conclusions: Retinal apoptosis was associated with retinal inflammation, a decrease in infectious virus, and upregulation of genes associated with CTL killing. These studies indicate that retinal apoptosis may be one of the host mechanisms that contribute to limiting this retinal infection.

The retinoid-X receptor (RXR) regulates multiple hormonal pathways through heterodimerization with nuclear receptors such as the all-trans retinoic acid receptor (RAR). The orphan nuclear receptor NGFI-B (also called Nur77) can heterodimerize with RXR. Here we show that nerve growth factor (NGF) induces the phosphorylation of Ser 105 of NGFI-B in PC12 phaeochromocytoma cells, resulting in translocation of the NGFI-B-RXR heterodimer complex out of the nucleus using nuclear export signals within NGFI-B. As a consequence of the redistribution of RXR, the transcriptional activity of RXR-RAR is reduced. NGFI-B-mediated nuclear export of receptors may serve as a mechanism for crosstalk between NGF and retinoid pathways.

To understand the role of nonmuscle myosin II in cardiac and skeletal muscle, we used a number of polyclonal antibodies, three detecting nonmuscle myosin heavy chain II-B (NMHC II-B) and two detecting NMHC II-A, to examine the localization of these two proteins in fresh-frozen, acetone-fixed sections of normal human and mouse hearts and human skeletal muscles. Results were similar in both species and were confirmed by examination of fresh-frozen sections of human hearts subjected to no fixation or to treatment with either 4% p-formaldehyde or 50% glycerol. NMHC II-B was diffusely distributed in the cytoplasm of cardiac myocytes during development, but after birth it was localized to the Z-lines and intercalated discs. Dual labeling showed almost complete colocalization of NMHC II-B with alpha-actinin. Whereas endothelial cells, smooth muscle cells and fibroblasts showed strong immunoreactivity for NMHC II-A and NMHC II-B, cardiac myocytes only showed reactivity for the latter. The Z-lines of human skeletal muscle cells, in contrast to those of cardiac myocytes, gave positive reactions for both NMHC II-A and NMHC II-B. The presence of a motor protein in the Z-lines and intercalated discs raises the possibility that these structures may play a more dynamic role in the contraction/relaxation mechanism of cardiac and skeletal muscle than has been previously suspected., (Copyright 2000 Wiley-Liss, Inc.)

Laser scanning confocal fluorescence microscopy techniques were used to study the localization of von Willebrand factor (vWf; Factor VIII-related antigen) and thrombomodulin (transmembrane receptor for thrombin) in the microvascular endothelial cells in the normal human lung. Tissues were obtained from lobectomy specimens resected for solitary nodules (7 adenocarcinomas and 4 hamartomas) from 11 patients. The plasma membranes of the capillary endothelial cells in the alveolar zones (A-zones) showed red linear fluorescence for thrombomodulin. However, their cytoplasm was mostly unreactive for vWf. The microvessels which were located in the connective tissue (C-zones), including peribronchial, and subpleural areas and large vascular walls, consistently demonstrated band-like green fluorescence for vWf in their cytoplasm, and their plasma membranes usually lacked reactivity for thrombomodulin. Only a limited number of peribronchial capillaries measuring <10 microm in diameter showed a mosaic-like appearance, in which red fluorescence along the plasma membranes was found together with green fluorescence in the subjacent cytoplasm. In the juxtaalveolar (J-zones) microvessels located along the borders between A- and C-zones, and measuring up to 40 microm in diameter, the endothelial cells showed a mosaic-like pattern of distribution of the two antigens. However, the localization of thrombomodulin in the J-zone microvessels was separate and independent from that of vWf. The thrombomodulin-reactive cells were directly connected to the alveolar capillary endothelial cells. Heterogeneous patterns of distribution of thrombomodulin and vWf suggest that topographic differences of endothelial function occur to maintain a balance of coagulation and anticoagulation in the normal human lung.

Complete ablation of nonmuscle myosin heavy chain II-B (NMHC-B) in mice resulted in cardiac and brain defects that were lethal during embryonic development or on the day of birth. In this paper, we report on the generation of mice with decreased amounts of NMHC-B. First, we generated B(DeltaI)/B(DeltaI) mice by replacing a neural-specific alternative exon with the PGK-Neo cassette. This resulted in decreased amounts of NMHC-B in all tissues, including a decrease of 88% in the heart and 65% in the brain compared with B(+)/B(+) tissues. B(DeltaI)/B(DeltaI) mice developed cardiac myocyte hypertrophy between 7 months and 11 months of age, at which time they reexpressed the cardiac beta-MHC. Serial sections of B(DeltaI)/B(DeltaI) brains showed abnormalities in neural cell migration and adhesion in the ventricular wall. Crossing B(DeltaI)/B(DeltaI) with B(+)/B(-) mice generated B(DeltaI)/B(-) mice, which showed a further decrease of approximately 55% in NMHC-B in the heart and brain compared with B(DeltaI)/B(DeltaI) mice. Five of 8 B(DeltaI)/B(-) mice were born with a membranous ventricular septal defect. Moreover, 5 of 5 B(DeltaI)/B(-) mice developed myocyte hypertrophy by 1 month; B(DeltaI)/B(-) mice also reexpressed the cardiac beta-MHC. More than 60% of B(DeltaI)/B(-) mice developed overt hydrocephalus and showed more severe defects in neural cell migration and adhesion than did B(DeltaI)/B(DeltaI) mice. These data on B(DeltaI)/B(DeltaI) and B(DeltaI)/B(-) mice demonstrate a gene dosage effect of the amount of NMHC-B on the severity and time of onset of the defects in the heart and brain.

Objective: Epidemiologic and mechanistic evidence implicates a role for cytomegalovirus (CMV) in atherogenesis. Recently, we demonstrated that CMV has the capacity to causally contribute to atherogenesis; acute infection of rats with rat CMV (RCMV) 1 day after carotid artery injury increased neointimal accumulation. Importantly, in the injured vessel infectious virus could not be detected and viral genome was present only transiently, suggesting that additional mechanisms play a role in the virus-induced exacerbation of the vascular injury response other than the changes caused by direct infection of vessel wall cells. The present investigation was designed to determine whether chronic persistent RCMV infection, more relevant to the clinical situation, also exacerbates the response to injury and, if so, whether similar mechanisms are operative., Methods: Sixty 3-week-old male Spraque-Dawley rats received an i.p. injection of either 10(6) TCID50 RCMV (Priscott strain) or normal saline. The left carotid artery was balloon-injured 3 months after infection. Rats were killed 6 weeks later. This model produces persistent infection, as demonstrated by presence of infectious virus in the salivary glands at time of sacrifice., Results: The neointima to media (N/M) ratio of the injured vessel was 41% greater in the RCMV-infected than in control rats (1.40 +/- 0.48 vs. 0.99 +/- 0.45; P = 0.003). The aorta never contained infectious RCMV, and exhibited RCMV DNA, detected by PCR, only transiently. The persistent infection of non-vascular tissues was associated with increased serum levels of IL-2, IL-4 and IFN-gamma., Conclusions: CMV infection of young rats causes persistent infection of non-vascular tissues and increased cytokine levels. The neointimal response to subsequent vascular injury is increased, despite absence of virus from the vessel wall. These findings, as in acute infection following vascular injury, suggest that inflammatory and immune responses to chronic persistent CMV infection contribute to an exaggerated response to vascular injury.

Immunohistochemical and confocal microscopic studies were made on lung tissue from 10 women with lymphangioleiomyomatosis (LAM) to evaluate the distribution of estrogen receptors (ER) and progesterone receptors (PR) in the abnormal smooth muscle cells (LAM cells) that characterize this disorder. PR and ER were localized mainly in the nuclei of large, epithelioid LAM cells, in five patients in whom tissues were obtained before treatment. However, the reaction for PR and ER was essentially negative in similarly processed tissues from five patients studied after receiving hormonal therapy (progesterone and tamoxifen). In the untreated group, staining for ER and PR colocalized with that for HMB-45, but not with that for membrane type-1 matrix metalloproteinase (MT-1-MMP), which we have shown to be localized in proliferating LAM cells. These observations demonstrate that PR and ER are selectively expressed in a subpopulation of LAM cells that are larger in size, have a limited ability to proliferate, and do not produce MT-1-MMP, the enzyme that activates MMP-2 (which is secreted by LAM cells and is capable of lysing elastin and collagens). ER and PR in LAM cells appear to be downregulated by hormonal therapy.

The c-Cbl protooncogene product is a prominent substrate of protein tyrosine kinases and is rapidly tyrosine-phosphorylated upon stimulation of a wide variety of cell-surface receptors. We have identified a novel c-Cbl-interacting protein termed CIN85 with a molecular mass of 85 kDa which shows similarity to adaptor proteins, CMS and CD2AP. CIN85 mRNA is expressed ubiquitously in normal human tissues and cancer cell lines analyzed. CIN85 was basally associated with c-Cbl. For interaction of CIN85 with c-Cbl, the second SH3 domain of CIN85 was shown to serve as a central player. The CIN85-c-Cbl association was enhanced shortly after stimulation of 293 cells with epidermal growth factor (EGF) and gradually diminished to a basal level, which correlated with a tyrosine phosphorylation level of c-Cbl. Our results suggest that CIN85 may play a specific role in the EGF receptor-mediated signaling cascade via its interaction with c-Cbl., (Copyright 2000 Academic Press.)

Background: Matrix metalloproteinases (MMPs) have been shown to be involved in the pathogenesis of the destructive pulmonary lesions in patients with lymphangioleiomyomatosis (LAM); in the present report, the activation of these enzymes is examined., Objective: To evaluate the role of MMPs and their activating enzymes, immunohistochemical and confocalmicroscopic techniques were used to localize alpha-smooth muscle actin (alpha-SMA), HMB-45, proliferating cell nuclear antigen (PCNA), MMP-2, membrane-type 1 MMP (MT1-MMP), MT2-MMP, and MT3-MMP in lung tissues from 10 women with LAM. Tissue samples were obtained from 5 patients before treatment and in 5 patients after hormone treatment (progesterone and/or tamoxifen citrate)., Results: Staining for alpha-SMA and MMP-2 was present in all the abnormal smooth muscle cells (LAM cells) in both groups. The percentages of PCNA-, MMP-2-, or MT1-MMP-positive LAM cells were much higher in the untreated group than in the treated group, whereas the percentages of HMB-45-reactive LAM cells were similar in both groups. The reactions for MT1-MMP and PCNA were preferentially localized in small spindle-shaped LAM cells; the reaction for HMB-45 was found in large epithelioid LAM cells. Many of the PCNA-positive cells were also positive for MT1-MMP. Staining for MT2-MMP and MT3-MMP was negative., Conclusions: This study demonstrates an association between cellular proliferation and the presence of MT1-MMP in LAM cells. The activation of MMP-2 by MT1-MMP may play an important role in the destruction of lung tissue in this disorder.

Background: Previous studies suggest that infection may play a role in restenosis and atherogenesis; cytomegalovirus (CMV) is one of the implicated pathogens. To determine a potential causal role of CMV in these disease processes, we assessed whether CMV infection increases the neointimal response to injury of the rat carotid artery., Methods and Results: Carotid injury was performed on 60 rats; immediately thereafter, 30 rats were infected with rat CMV, and the other 30 were mock-infected. Six weeks later, rats were euthanized, and the salivary glands, spleen, and carotid arteries were harvested. CMV infection was associated with significant exacerbation of the neointimal response to injury (neointimal to medial ratio 0.81+/-0. 59 versus 0.31+/-0.38 in CMV-infected versus control rats; P<0.0001). This occurred despite absence of infectious virus from vascular tissues and detection of CMV DNA by polymerase chain reaction in the injured artery only at day 3 after infection. Persistent distant infection, associated with systemic cytokine response, was evidenced by isolation of infectious virus from homogenates of both salivary glands and spleen and by higher serum levels of interleukin (IL)-2 and IL-4 (but not interferon-gamma and tumor necrosis factor-alpha) in infected versus noninfected rats., Conclusions: CMV infection of immunocompetent adult rats increases the neointimal response to vascular injury, suggesting that CMV may play a causal role in atherosclerosis/restenosis. Importantly, this CMV-induced response occurs even without the presence of virus in the vascular wall, suggesting that inflammatory and immune responses to infection of nonvascular tissues may contribute to the vascular response to injury.