Clinical features of cryptococcosis in patients with different immune statuses: a multicenter study in Jiangsu Province-China.

BACKGROUND: Current guidelines support different management of cryptococcosis between severely immunodeficient and immunocompetent populations. However, few studies have focused on cryptococcosis patients with mild-to-moderate immunodeficiency. We performed this study to determine the clinical features of pulmonary (PC) and extrapulmonary cryptococcosis (EPC) and compared them among populations with different immune statuses to support appropriate clinical management of this public health threat. METHODS: All cases were reported by 14 tertiary teaching hospitals in Jiangsu Province, China from January 2013 to December 2018. The trends in incidence, demographic data, medical history, clinical symptoms, laboratory test indicators, imaging characteristics and diagnostic method of these patients were then stratified by immune status, namely immunocompetent (IC, patients with no recognized underlying disease or those with an underlying disease that does not influence immunity, such as hypertension), mild-to-moderate immunodeficiency (MID, patients with diabetes mellitus, end-stage liver or kidney disease, autoimmune diseases treated with low-dose glucocorticoid therapy, and cancer treated with chemotherapy) and severe immunodeficiency (SID, patients with acquired immunodeficiency syndrome, haematologic malignancies, solid organ transplantation or haematologic stem cell transplantation, idiopathic CD4 lymphocytosis, agranulocytosis, aggressive glucocorticoid or immunosuppressive therapy and other conditions or treatments that result in severe immunosuppression). RESULTS: The clinical data of 255 cryptococcosis patients were collected. In total, 66.3% of patients (169) were IC, 16.9% (43) had MID, and 16.9% (43) had SID. 10.1% of the patients (17) with IC were EPC, 18.6% of the patients (8) with MID were EPC, and 74.4% of patients (32) were EPC (IC/MID vs. SID, p < 0.001). Fever was more common in the SID group than in the IC and MID groups (69.8% vs. 14.8% vs. 37.2%, p < 0.001). Of chest CT scan, most lesions were distributed under the pleura (72.7%), presenting as nodules/lumps (90.3%) or consolidations (10.7%). Pleural effusion was more common in SID group compared to IC group (33.3% vs. 2.4%, p < 0.001). Positivity rate on the serum capsular polysaccharide antigen detection (CrAg) test was higher in the SID group than in the other two groups [100.0% vs. 84.4% (MID) vs. 78.2% (IC), p = 0.013]. Positivity rate on the serum CrAg test was also higher in cryptococcal meningitis patients than in PC patients (100.0% vs. 79.5%, p = 0.015). CONCLUSIONS: The clinical presentation of MID patients is intermediate between SID and IC patients and is similar to that of IC patients. The serum CrAg test is more sensitive for the identification of SID or EPC patients.

Identification, functional characterization, and estrogen regulation on gonadotropin-releasing hormone in the spotted scat, Scatophagus argus.

Gonadotropin-releasing hormone (GnRH) is a key neuropeptide of the reproductive system. However, little is known about the role of GnRH in the spotted scat (Scatophagus argus). Here, three GnRH subtypes (cGnRH-II, sGnRH, and sbGnRH) were identified in the spotted scat. cGnRH-II and sGnRH were only expressed in the brains and gonads of both male and female fish, exhibiting a tissue-specific expression pattern, while sbGnRH was expressed at different transcription levels in all examined tissues. During ovarian maturation, hypothalamus-associated sbGnRH was upregulated, while the expression of sGnRH was variable and cGnRH-II first increased and then decreased. In vivo experiments showed that sbGnRH significantly promoted the expression of fsh and lh genes in a dose-dependent manner and exhibited a desensitization effect on lh expression at high concentrations. For sGnRH and cGnRH-II, only high concentrations could induce fsh and lh expression. Furthermore, treatment with highly concentrated sbGnRH peptide also induced fsh and lh expression, whereas the sGnRH and cGnRH-II peptides only induced fsh expression in vitro. 17ß-Estradiol (E2) significantly inhibited the expression of sbGnRH mRNA in a dose-dependent manner and did not impact sGnRH and cGnRH-II mRNA levels in vivo or in vitro. The inhibitory effect of E2 on sbGnRH expression was attenuated by the estrogen receptor (ER) broad-spectrum antagonist (fulvestrant) and the ERα-specific antagonist (methyl-piperidinopyrazole), respectively, implying that the feedback regulation on sbGnRH is mediated via ERα. This study provides a theoretical basis for the reproductive endocrinology of the spotted scat by studying GnRH.

Co-Occurring Alterations of ERBB2 Exon 20 Insertion in Non-Small Cell Lung Cancer (NSCLC) and the Potential Indicator of Response to Afatinib.

Background: Human epidermal growth factor receptor 2 (ERBB2, HER-2) exon 20 insertion (ERBB2ex20ins) remains a refractory oncogenic driver in lung cancer. So far there is limited data showing the co-occurring mutation background of ERBB2ex20ins in Chinese lung cancer and its relationship with response to afatinib. Patients and Methods: A total of 112 Chinese patients with ERBB2ex20ins identified by next-generation sequencing from 17 hospitals were enrolled. The clinical outcomes of 18 patients receiving afatinib treatment were collected. Results: Among the 112 patients, insertion-site subtypes comprised of A775ins (71%; 79/112), G776indel (17%; 19/112), and P780ins (12%; 14/112). There were 66.1% (74/112) of patients carrying TP53 co-mutation and FOXA1 was the most prevalent co-amplified gene (5.5%, 3/55). The co-occurring genomic feature was similar among three insertional-site subtypes and had an overall strong concordance with the western population from the MSKCC cohort (R 2 = 0.74, P < 0.01). For the prognosis, patients with co-occurring mutation in cell-cycle pathway especially TP53 showed shorter OS than patients without [median OS: 14.5 m (95% CI:12.7-16.3 m) vs. 30.3 m (95% CI: not reached), p = 0.04], while the OS was comparable among three subtypes. For the response to afatinib, ERBB2ex20ins as a subclonal variant was an independent factor relating to shorter PFS [median PFS: 1.2 m (95% CI: 0.8-1.6 m) vs. 4.3 m (95% CI: 3.3-5.3 m), p < 0.05]. Conclusion: Our data revealed co-occurring TP53 represent an unfavorable prognosis of patients with ERBB2ex20ins, emphasizing the more valuable role of the co-mutation patterns than insertion-site subtypes in predicting prognosis of this group of patients. Moreover, the clonality status of ERBB2ex20ins was identified as a potential indicator for response to afatinib.

Gnrh3 Regulates PGC Proliferation and Sex Differentiation in Developing Zebrafish.

Gonadotropin-releasing hormone (Gnrh) plays important roles in reproduction by stimulating luteinizing hormone release, and subsequently ovulation and sperm release, ultimately controlling reproduction in many species. Here we report on a new role for this decapeptide. Surprisingly, Gnrh3-null zebrafish generated by CRISPR/Cas9 exhibited a male-biased sex ratio. After the dome stage, the number of primordial germ cells (PGCs) in gnrh3-/- fish was lower than that in wild-type, an effect that was partially rescued by gnrh3 overexpression. A terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) analysis revealed no detectable apoptosis of PGCs in gnrh3-/- embryos. Proliferating PGCs could be detected in wild-type embryos, while there was no detectable signal in gnrh3-/- embryos. Compared with wild type, the phosphorylation of AKT was not significantly different in gnrh3-/- embryos, but the phosphorylation of ERK1/2 decreased significantly. Treatment with a Gnrh analog (Alarelin) induced ERK1/2 phosphorylation and increased PGC numbers in both wild-type and gnrh3-/- embryos, and this was blocked by the MEK inhibitor PD0325901. The relative expression of sox9a, amh, and cyp11b were significantly upregulated, while cyp19a1a was significantly downregulated at 18 days post-fertilization in gnrh3-/- zebrafish. Taken together, these results indicate that Gnrh3 plays an important role in early sex differentiation by regulating the proliferation of PGCs through a MAPK-dependent path.

Fungal ß-Glucan Activates the NLRP3 Inflammasome in Human Bronchial Epithelial Cells Through ROS Production.

The nucleotide-binding domain and leucine-rich repeat protein 3 (NLRP3) inflammasome has developed as an important bridge between innate immune and infection recently, and has the ability to drive proteolytic procaspase-1 into bioactive caspase-1, then responsible for proteolytic processing of inflammatory cytokines IL-1ß and IL-18. Fungal ß-glucan, a major component of fungal cell wall, triggers inflammatory response in multiple immune cells, but rarely described in epithelial cells. Also, the relationship between fungal ß-glucan and NLRP3 inflammasome is not clear yet. In this study, we first identified that curdlan, a large particulate ß-glucan, could activate the NLRP3 inflammasome in LPS-primed human bronchial epithelial cells (HBECs). RT-PCR and Western Blot showed that curdlan upregulate the mRNA as well as intracellular protein expression of NLRP3 and IL-1ß in HBECs, along with the activity of caspase-1, and the level of mature IL-1ß in cell supernatants was higher by ELISA detection. Further studies demonstrated that the activation of NLRP3 inflammasome could be attenuated by NAC, an inhibitor of ROS. Thus, it indicated curdlan activate NLRP3 inflammasome through a pathway requiring ROS production in HBECs. These findings may provide a new therapeutic target, NLRP3 inflammasome, in invasive pulmonary fungal infections.

Cloning, expression and functional characterization on vitellogenesis of estrogen receptors in Scatophagus argus.

Estrogen receptors (Er) play a critical role in vitellogenesis. Three ers (erα, erß1 and erß2) and vitellogenins (vtg-A, vtg-B and vtg-C) subtypes were isolated in various fish species, while the contribution of each Er to the regulation of vtgs expression was not analyzed in detail. Here, erα, erß1 and erß2 were cloned and all were found to be expressed in female liver in Scatophagus argus. During proteic vitellogenesis stage, erα was simultaneously up-regulated, while erß1 and erß2 were not, with three vtgs in female liver. The effects of 17ß-estradiol (E2) alone or combined with Er antagonists on ers, vtgs mRNA expressions and Vtg protein content in incubated male liver were examined by real-time PCR and enzyme-linked immunosorbent assay (ELISA), respectively. The expressions of erα, erß1, vtgs mRNA and Vtg protein increased significantly after 24h incubation with E2 (0.1, 1 and 10µM), while Er nonselective antagonist ICI 182 780 (0.01, 0.1 and 1µM) significantly attenuated the up-regulation effects of E2 on ers, vtgs mRNA and Vtg protein in a dose-dependent manner. Erα selective antagonist Methyl-piperidinopyrazole (MPP) (0.01, 0.1 and 1µM) significantly attenuated the up-regulation effects of E2 on erα, vtg-B, vtg-C mRNA and Vtg protein, while promoted the expression of erß1 and vtg-A. Erß selective antagonist Cyclofenil (0.01, 0.1 and 1µM) attenuated the up-regulation effects of E2 on erß1, erß2, vtg-A, vtg-C mRNA and Vtg protein while promoted the expression of erα and vtg-B. Our results suggest that the regulation of Ers on different vtgs was divergent in S. argus.

Upregulated expression of ILF2 in non-small cell lung cancer is associated with tumor cell proliferation and poor prognosis.

ILF2 (NF45) is a sequence-specific DNA binding protein that is involved in mitosis control, transcriptional regulation, DNA breaks repair, microRNA processing and viral replication. In the present study, we aim to investigate the potential role of ILF2 in the progression of non-small cell lung cancer (NSCLC). Western blot analysis indicated that ILF2 was up-regulated in NSCLC tissues, compared with adjacent non-tumorous ones. Furthermore, immunohistochemistry analysis showed that the expression of ILF2 was correlated with histological differentiation, clinical stage and Ki-67 expression in NSCLC specimens. In addition, using Kaplan-Meier survival analysis, we found that high expression of ILF2 predicted poor outcome in NSCLC patients. Furthermore, we showed that up-regulated expression of ILF2 might play a regulatory role in the proliferation of NSCLC cells using serum starvation and release assay. Moreover, knockdown of ILF2 inhibited cell proliferation and cell cycle progress of NSCLC cells. In conclusion, our results indicated that ILF2 was involved in the pathogenesis of NSCLC and might be a potential target for NSCLC therapy.

Involvement of p29/SYF2/fSAP29/NTC31 in the progression of NSCLC via modulating cell proliferation.

p29, also known as SYF2/fSAP29/NTC31, is a protein associated with chromatin and involved in DNA damage response, cell cycle arrest and pre-mRNA splicing. In p29-depleted cells, DNA replication was reduced and cell population in G1 phase increased. In this study, we investigated the potential role of p29 in the regulation of non-small cell lung cancer (NSCLC) progression. Western blot and immunohistochemistry staining showed that p29 was up-regulated in clinical NSCLC tissues compared with adjacent non-cancerous tissues, and the expression of p29 had a positive correlation with clinical stage and histological differentiation, as well as expression of Ki-67, a proliferating marker. Kaplan-Meier analysis indicated that patients with high level of p29 expression had poor overall survival. In addition, small interfering RNA of p29 was performed, and the effects on NSCLC growth were examined. Interference of p29 blocked S phase entry, inhibited proliferation of A549 cells and up-regulated level of p21 expression. Taken together, these results suggested that p29 might contribute to the progression of NSCLC by enhancing cell proliferation, implicating that targeting p29 might provide beneficial effects on the clinical therapy of NSCLC.

IL-22 Up-Regulates ß-Defensin-2 Expression in Human Alveolar Epithelium via STAT3 but Not NF-κB Signaling Pathway.

Human ß-defensin-2(HBD-2) is one of the two major vertebrate antimicrobial peptide families (α and ß), which is highly expressed by proinflammatory induction in the lung and exhibit broad-spectrum antimicrobial activity. We observed that IL-22 receptors high expressed on the membrane of A549 cells; HBD-2 mRNA was expressed in a time- and concentration-dependent manners in A549 cells when treated with IL-22; further studies demonstrated that HBD-2 expression was attenuated by AG490, but to JSH-23, inhibitors of p-STAT3 DNA binding and NF-κB/p65 subunit nuclear translocation, respectively. These results support that IL-22-mediated signalling pathway of HBD-2 gene expression involved STAT3 but not NF-κB in human alveolar epithelium. These findings provide a new insight into how IL-22 may play an important link between innate and adaptive immunity, thereby anti-infection locally in the alveolar epithelium.

Transformer 2ß (Tra2ß/SFRS10) positively regulates the progression of NSCLC via promoting cell proliferation.

Transformer 2ß (Tra2ß), a member of the serine/arginine-rich-like protein family, is an important RNA-binding protein involved in alternative splice. Deregulation of Tra2ß has been observed in several cancers. However, the detailed role of Tra2ß in non-small cell lung cancer (NSCLC) has not been elucidated. In this study, the contribution of Tra2ß to NSCLC development was investigated. On histological level, the expression of Tra2ß was determined by Western and immunohistochemistry assays. It demonstrated that Tra2ß was expressed higher in NSCLC tumor tissues compared with adjacent non-tumor tissues. In addition to confirm the association of Tra2ß expression with histological differentiation and clinical stage (p < 0.05), we also confirmed significant positive correlation between the expression level of Tra2ß and that of Ki67 (p < 0.05, r = 0.446) by Spearman rank correlation test. Moreover, high expression of Tra2ß predicted poor prognosis by Kaplan-Meier survival analysis. And Tra2ß among with other clinicopathologic variables was an independent prognostic indicator for patients' overall survival by multivariate analysis. On cellular level, Tra2ß expression was demonstrated to promote proliferation of NSCLC cells through a series of assays, including serum starvation and release assay, Western blot assay and flow cytometry analysis. Moreover, knockdown of Tra2ß was confirmed to inhibit proliferation and to induce apoptosis of NSCLC cells through flow cytometry analysis, western analysis, cell counting kit-8 assay and Tunnel assay. Our results indicated that Tra2ß was involved in the tumorigenesis of NSCLC and might be a potential therapeutic target of NSCLC.

[Clinical characters of rapidly progressive crytogenic organizing pneumonia].

OBJECTIVE: To explore the clinical features of rapidly progressive crytogenic organizing pneumonia (COP). METHODS: Nine cases of rapidly progressive COP with acute respiratory failure confirmed by lung biopsy at First Affiliated Hospital, Nanjing Medical University from October 2003 to March 2014 were retrospectively reviewed. RESULTS: There were 5 males and 4 females with an age range of (52.1 ± 11.4) years. The onset of illness was 7-20 days. The major manifestations included cough (n = 9), dyspnea (n = 9), fever (n = 7), hypodynamia (n = 6), weight loss (n = 3), night sweat (n = 2), hemoptysis (n = 1), cyanosis (n = 9), "velcro" crackles (n = 6) and moist rales (n = 3). Blood tests showed elevated levels of erythrocyte sedimentation rate (n = 8), C-reactive protein (n = 8) and serum ferritin (n = 6). The results of blood gas analysis indicated acute respiratory failure in all 9 patients (PaO2<60 mmHg, 1 mmHg = 0.133 kPa). Chest computed tomography showed bilateral and multiple distribution, patchy areas of alveolar consolidation with air bronchograms (n = 5), diffuse ground-glass opacity (n = 4), tractional bronchiectasis (n = 2), pleural effusion (n = 2) and spontaneous pneumothorax (n = 1). Six cases were cured by conventional dose corticosteroid and another 3 by large-dose methylprednisolone (240 mg/d). Two cases relapsed and then recovered after a second corticosteroid therapy. CONCLUSIONS: The clinical manifestations of rapidly progressive COP show severe inflammatory reactions with a fast progression. Chest computed tomography depicts extensive lesions along with pleural effusion and spontaneous pneumothorax. Conventional dose of corticosteroid is the first choice therapy for rapidly progressive COP. Large-dose methylprednisolone is needed when conventional dose of corticosteroid proves ineffective.

Respiratory syncytial virus infection differentiates airway dysfunction in the central and peripheral airways in OVA-sensitized mice.

Much evidence suggests that respiratory syncytial virus (RSV) infection prolongs airway hyperresponsiveness (AHR) and exacerbates asthma by enhancing airway inflammation. However, the characteristic of airway inflammation and kinetics of airway dysfunction occurred in the central and peripheral airways were not fully delineated. The objective of this study was to investigate the effect of RSV on the allergic airway inflammation in different size airways and to elucidate its possible mechanism. Using a murine model of prior ovalbumin (OVA) sensitization and subsequent RSV challenge, lung resistance (R(L)), and dynamic compliance (Cdyn) was conducted by barometric whole-body plethysmography. Histological examinations were carried out. Differential cells count in bronchoalveolar lavage (BAL) fluid, serum anti-OVA IgE, and IgG1 were measured. Cytokine mRNA expression in lung tissue were determined. RSV triggered a significant increase in R(L) and reduction in Cdyn, as well as greatly prolonged the recovery of Cdyn more than that of R(L) in OVA-sensitized mice. Also, RSV resulted in more severe peripheral airway inflammation which exhibit as globe cell hyperplasia and CD8+ T cell infiltration. Furthermore, the number of lymphocytes, neutrophils and macrophages in BAL fluid, serum anti-OVA IgE and IgG1 were remarkably increased. Additionally, mice increased relative expression of cytokines IL-4, IL-13, and IFN-γ, but not IL-5, IL-17, and IL-17F. These findings demonstrated that RSV could selectively affect pathologic processes that contribute to altered airway function in the central and peripheral airways in OVA-sensitized mice. These processes may be involved in goblet cell hyperplasia and CD8+ T cell infiltration in peripheral airways.

Unusual life-threatening Rosai-Dorfman disease of the trachea: role of NF-κB.

Rosai-Dorfman disease (RDD) is a rare non-neoplastic histioproliferative disorder characterised by painless lymphadenopathy, low fever, high erythrocyte sedimentation rate, leucocytosis and hypergammaglobulinaemia. Overactivity of nuclear factor κB (NF-κB) is linked with inflammatory, cancerous and autoimmune diseases. The first case is described of an unusual life-threatening RDD of the trachea with no lymphadenopathy at risk of suffocation in a 39-year-old Chinese woman. A diagnosis of RDD was made following CT scans, thoracotomy and histological examination. Gel shift assay revealed an essential role for NF-κB overactivity in RDD. The patient remains well with no evidence of progression without treatment. Histological confirmation should be sought in all cases as the clinical manifestation of RDD is similar to asthma or lung carcinoma.

Effects of adenoviral gene transfer of mutated IkappaBalpha, a novel inhibitor of NF-kappaB, on human monocyte-derived dendritic cells.

AIM: To investigate the effects of adenoviral gene transfer of IkappaBalpha mutant (IkappaBalphaM), a novel inhibitor of nuclear factor kappaB (NF-kappaB), on apoptosis, phenotype and function of human monocyte-derived dendritic cells (DC). METHODS: Monocytes, cocultured with granulocyte/macrophage colony-stimulating factor (GM-CSF; 900 ng/mL) and interleukin (IL)-4 (300 ng/mL) for 5 d, followed by stimulation with lipopolysaccharide (LPS; 100 ng/mL) for 2 d differentiated into mature DC. Monocytes were either left untransfected or were transfected with AdIkappaBalphaM or AdLacZ. The transcription and expression of the IkappaBalphaM gene, and the inhibitory effect of IkappaBalphaM on tumor necrosis factor (TNF)-alpha-induced NF-kappaB activation in mature DC were detected by polymerase chain reaction (PCR), reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis, and electrophoretic mobility shift assays, respectively. The phenotype, apoptosis, IL-12 secretion level of DC, and ability to stimulate the proliferation of T cells were determined by flow cytometry, enzyme-linked immunosorbent assay and mixed leukocyte reaction. RESULTS: PCR and RT-PCR were used to detect a unique 801 bp band in AdIkappaBalphaM-transfected mature DC, and also a dose- and time-dependent expression of the IkappaBalphaM gene, which peaked at a multiplicity of infection of 100 pfu/cell and at 48 h. Furthermore, AdIkappaBalphaM significantly suppressed the TNF-alpha-induced NF-kappaB activation, augmented apoptosis, downregulated CD80, CD83, and CD86 surface molecules, IL-12 secretion levels and the ability to stimulate the proliferation of T cells in mature DC. CONCLUSION: AdIkappaBalphaM effectively transfected and potently inhibited NF-kappaB activation in monocyte-derived mature DC. Overexpression of the IkappaBalphaM gene in mature DC may contribute to T-cell immunosuppression through induction of DC apoptosis and downregulation of B7 molecules, providing a potential strategy for future DC-based immunotherapy of asthma.

Retinoic acid-inducible gene-I is induced by interferon-gamma and regulates the expression of interferon-gamma stimulated gene 15 in MCF-7 cells.

Retinoic acid-inducible gene-I (RIG-I) is a member of the DExH box family proteins, which have diverse roles in regulation of gene expression and cellular functions. We found RIG-I mRNA and protein were expressed in MCF-7 human breast cancer cells stimulated with interferon-gamma (IFN-gamma). This effect of IFN-gamma was observed in concentration- and time-dependent manners, and IFN-gamma also induced promoter activity of RIG-I. Transfection of GFP-RIG-I cDNA into MCF-7 cells resulted in the expression of RIG-I protein in cytoplasm. Overexpression of RIG-I induced the upregulation of IFN-gamma stimulated gene 15, which has the potential to amplify the immunomodulatory effects. We conclude that IFN-gamma induces the expression of RIG-I, which may play a role in the immunological effects of IFN-gamma.

15-Deoxy-D12,14-prostaglandin J2 inhibits CX3CL1/fractalkine expression in human endothelial cells.

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma)is a member of nuclear hormone receptor superfamily, and is knownto play a role in various biological processes including inflammatoryresponses and adipocyte differentiation. CX3CL1/fractalkineis a potent agonist for chemotaxis and adhesion of monocytes and lymphocytes. Endothelial cells produce fractalkine when stimulated with cytokinessuch as interleukin-1 (IL-1), tumour necrosis factor-alpha andinterferon-gamma (IFN-gamma). We herein report that 15-deoxy-n12,14 -prostaglandinJ2 (15d-PGJ2), a PPAR-gamma agonist,inhibits the expression of fractalkine induced by IFN-gamma orIL-1beta in human endothelial cells. Agonist for PPAR-alpha (WY14643)or PPAR-gamma (ciglitazone) did not inhibit the cytokine-inducedfractalkine expression, and the effect of 15d-PGJ2 maybe independent of PPAR. 15-Deoxy-D12,14 prostaglandinJ2 also inhibited the adhesion of blood mononuclear cellsto endothelial monolayers treated with IFN-gamma or IL-1beta. The data suggest that 15d-PGJ2 regulates inflammatoryreactions, at least in part, through the inhibition of fractalkineexpression and leucocyte traffic through the endothelium.

Interferon-gamma stimulates the expression of galectin-9 in cultured human endothelial cells.

Galectin-9 is a member of the galectin family and has been identified as an eosinophil chemoattractant produced by activated T lymphocytes. Vascular endothelial cells play an important role in the initial step of eosinophil recruitment and activation in immune and inflammatory responses. We have addressed the stimulation of galectin-9 expression in endothelial cells. Galectin-9 was detected in membrane and cytosolic fractions of human umbilical vein endothelial cells stimulated with interferon-gamma (IFN-gamma). IFN-gamma also enhanced the adhesion of human eosinophilic leukemia-1 cells to endothelial monolayers, and it was inhibited by the presence of lactose. Interleukin-4, which induces eotaxin expression, did not affect the expression of galectin-9. The in situ endothelium from patients with inflammatory diseases was found to express galectin-9. IFN-gamma-induced production of galectin-9 by endothelial cells may play an important role in immune responses by regulating interactions between the vascular wall and eosinophils.

Platelet-activating factor enhances the expression of vascular endothelial growth factor in normal human astrocytes.

Vascular endothelial growth factor (VEGF) is a potent and specific mitogen for vascular endothelial cells. To examine whether platelet-activating factor (PAF) induces the expression of VEGF in human astrocytes, we stimulated cultured normal astrocytes with PAF and performed semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) or real-time quantitative PCR for VEGF mRNA and enzyme-linked immunosorbent assay for VEGF protein. PAF increased the expression of VEGF in astrocytes in time- and dose-dependent manners. After 24-h stimulation, 10 nM PAF increased the levels of VEGF protein in astrocyte-conditioned medium by 1.3-fold. When the cells were subjected to hypoxia, the PAF-induced production of VEGF was enhanced by 6.7-fold as compared to the unstimulated cells incubated under normoxia. Dexamethasone was found to inhibit the enhanced VEGF production in response to the stimulation with PAF under hypoxia. We conclude that PAF induces VEGF gene expression in human astrocytes, and the PAF-induced increase in the expression of VEGF may modulate nervous tissue injury due to hypoxia.

Proteasome inhibitor MG-132 enhances the expression of interleukin-6 in human umbilical vein endothelial cells: Involvement of MAP/ERK kinase.

Interleukin-6 (IL-6) is a multifunctional cytokine that plays an important role in inflammatory reactions. We have addressed the possible regulation of IL-6 expression by the ubiquitin-protease system in human umbilical vein endothelial cells. Cultured endothelial cells were treated with MG-132, a protease inhibitor, and the levels of IL-6 mRNA and protein were measured by reverse transcription-PCR and ELISA. MG-132 increased the expression of IL-6 mRNA and protein;and this effect was abolished by the pretreatment of the cells with U0126, an inhibitor of MAP or ERK kinases (MEK 1/2). MG-132 treatment was also found to enhance the level of phosphorylated MEK 1/2. Treatment of the cells with actinomycin D inhibited IL-6 expression in response to MG-132, suggesting the transcriptional upregulation of IL-6 under proteasomal inhibition. We conclude that a protease inhibitor MG-132 upregulates IL-6 expression in vascular endothelial cells, at least in part, through the activation of MEK 1/2.