Samantha Wei-Man Lun

Cytokine response patterns in severe pandemic 2009 H1N1 and seasonal influenza among hospitalized adults.

Background Studying cytokine/chemokine responses in severe influenza infections caused by different virus subtypes may improve understanding on pathogenesis. Methods Adults hospitalized for laboratory-confirmed seasonal and pandemic 2009 A/H1N1 (pH1N1) influenza were studied. Plasma concentrations of 13 cytokines/chemokines were measured at presentation and then serially, using cytometric-bead-array with flow-cytometry and ELISA. PBMCs from influenza patients were studied for cytokine/chemokine expression using ex-vivo culture (Whole Blood Assay,±PHA/LPS stimulation). Clinical variables were prospectively recorded and analyzed. Results 63 pH1N1 and 53 seasonal influenza patients were studied. pH1N1 patients were younger (mean±S.D. 42.8±19.2 vs 70.5±16.7 years), and fewer had comorbidities. Respiratory/cardiovascular complications were common in both groups (71.4% vs 81.1%), although severe pneumonia with hypoxemia (54.0% vs 28.3%) and ICU admissions (25.4% vs 1.9%) were more frequent with pH1N1. Hyperactivation of the proinflammatory cytokines IL-6, CXCL8/IL-8, CCL2/MCP-1 and sTNFR-1 was found in pH1N1 pneumonia (2–15 times normal) and in complicated seasonal influenza, but not in milder pH1N1 infections. The adaptive-immunity (Th1/Th17)-related CXCL10/IP-10, CXCL9/MIG and IL-17A however, were markedly suppressed in severe pH1N1 pneumonia (2–27 times lower than seasonal influenza; P−values<0.01). This pattern was further confirmed with serial measurements. Hypercytokinemia tended to be sustained in pH1N1 pneumonia, associated with a slower viral clearance [PCR-negativity: day 3–4, 55% vs 85%; day 6–7, 67% vs 100%]. Elevated proinflammatory cytokines, particularly IL-6, predicted ICU admission (adjusted OR 12.6, 95%CI 2.6–61.5, per log10unit increase; P = 0.002), and correlated with fever, tachypnoea, deoxygenation, and length-of-stay (Spearmans rho, P-values<0.01) in influenza infections. PBMCs in seasonal influenza patients were activated and expressed cytokines ex vivo (e.g. IL-6, CXCL8/IL-8, CCL2/MCP-1, CXCL10/IP-10, CXCL9/MIG); their ‘responsiveness’ to stimuli was shown to change dynamically during the illness course. Conclusions A hyperactivated proinflammatory, but suppressed adaptive-immunity (Th1/Th17)-related cytokine response pattern was found in severe pH1N1 pneumonia, different from seasonal influenza. Cytokine/immune-dysregulation may be important in its pathogenesis.

Hypercytokinemia and Hyperactivation of Phospho-p38 Mitogen-Activated Protein Kinase in Severe Human Influenza A Virus Infection

BACKGROUND We postulate that hypercytokinemia plays a role in immunopathogenesis of severe human influenza. METHODS We prospectively studied 39 consecutive patients who were hospitalized with severe influenza A virus infection. On laboratory confirmation of the diagnosis, paired acute-phase (obtained at hospital admission) and convalescent-phase (obtained >10 days after hospital admission) plasma samples were collected for assay of 11 cytokines and chemokines (interleukin [IL] 1 beta; IL-6; IL-10; IL-12p70; tumor necrosis factor alpha; IL-8; monokine induced by interferon [IFN]-gamma; IFN-inducible protein 10; monocyte chemoattractant protein 1; regulated upon activation, normal T cell-expressed and secreted; and IFN-gamma) using cytometric bead-array analysis and enzyme-linked immunosorbent assay. Simultaneously, virus concentration in the acute-phase nasopharyngeal aspirate was determined using real-time quantitative reverse-transcriptase polymerase chain reaction. Intracellular signaling molecules regulating lymphocyte activation, phospho-p38 mitogen-activated protein kinase and phospho-extracellular signal-regulated protein kinase in CD4+ and CD8+ T lymphocytes were studied in the acute-phase samples using flow cytometric analysis and were compared with results for samples from healthy control subjects. RESULTS Statistically significant increases in plasma IL-6 (3.7-fold increase), IL-8 (2.6-fold increase), IFN-induced protein 10 (4.9-fold increase), and monokine induced by IFN-gamma (2.3-fold increase) concentrations were detected during acute illness (P < .01 for all, by Wilcoxon signed-rank test); the highest concentrations were observed on symptom days 3 and 4. Corresponding plasma cytokine and chemokine concentrations and nasopharyngeal viral loads showed statistically significant correlations (rho = 0.41, 0.49, 0.54, and 0.46, respectively; P < or = .01). Phospho-p38 mitogen-activated protein kinase expression in CD4+ lymphocytes was increased, correlating with cytokine concentrations (e.g., for IFN-induced protein 10, rho = 0.78; P < .01); phospho-extracellular signal-regulated protein kinase was suppressed. Advanced age and comorbidity were associated with aberrant IL-6, IL-8, and monokine induced by IFN-gamma responses (P < .05, by Mann-Whitney U test). An elevated IL-6 concentration was independently associated with prolonged hospitalization (hospitalization for >5 days; P = .02), adjusted for age, comorbidity, and virus load. CONCLUSIONS Hypercytokinemia (of proinflammatory and T helper 1 cytokines) is detected in severe influenza, correlating with clinical illness and virus concentration. Hyperactivation of phospho-p38 mitogen-activated protein kinase (in T helper cells) is possibly involved. Early viral suppression may attenuate these potentially deleterious cytokine responses.

Increased expression of plasma and cell surface co‐stimulatory molecules CTLA‐4, CD28 and CD86 in adult patients with allergic asthma

The co‐stimulatory interactions of the B7 family molecules CD80 and CD86 on antigen‐presenting cells, together with their T cell counter receptors CD28 and cytotoxic T lymphocyte‐associated antigen‐4 (CTLA‐4), modulate T lymphocyte‐mediated immune responses in a reciprocal manner. To investigate whether there is altered expression and the clinical significance of soluble co‐stimulatory molecules in asthmatic patients, plasma concentrations of sCTLA‐4, sCD28, sCD80 and sCD86 in 51 adult allergic asthmatic adults with or without steroid treatment, and 35 sex‐ and age‐matched control subjects were measured by enzyme‐linked immunosorbent assay (ELISA). Cell surface expression of CTLA‐4 and CD28 on peripheral blood mononuclear cells (PBMC) were analysed by flow cytometry. Results showed that the plasma sCTLA‐4 concentration was significantly higher in all asthmatic patients while sCD28 and sCD86 concentrations were significantly higher in steroid and non‐steroid treated asthmatic patients, respectively, compared with control subjects (all P < 0·01). Significantly increased cell surface expression of CD28 but not CTLA‐4 on PBMC was found in asthmatic patients compared with controls (P < 0·05). The plasma concentration and cell surface expression of CTLA‐4 were found to exhibit positive and significant correlations with those of CD28 (both P < 0·05). Serum total IgE concentration correlated positively and significantly with sCTLA‐4 and sCD28 concentrations in allergic asthmatic patients (both P < 0·05). The increased expression of these soluble co‐stimulatory molecules may reflect the dysregulation of T cell activation, thereby contributing to the immunopathogenesis of allergic asthma.

CD44+ Cancer Stem-Like Cells in EBV-Associated Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma (NPC) is a unique EBV-associated epithelial malignancy, showing highly invasive and metastatic phenotype. Despite increasing evidence demonstrating the critical role of cancer stem-like cells (CSCs) in the maintenance and progression of tumors in a variety of malignancies, the existence and properties of CSC in EBV-associated NPC are largely unknown. Our study aims to elucidate the presence and role of CSCs in the pathogenesis of this malignant disease. Sphere-forming cells were isolated from an EBV-positive NPC cell line C666-1 and its tumor-initiating properties were confirmed by in vitro and in vivo assays. In these spheroids, up-regulation of multiple stem cell markers were found. By flow cytometry, we demonstrated that both CD44 and SOX2 were overexpressed in a majority of sphere-forming C666-1 cells. The CD44+SOX2+ cells was detected in a minor population in EBV-positive xenografts and primary tumors and considered as potential CSC in NPC. Notably, the isolated CD44+ NPC cells were resistant to chemotherapeutic agents and with higher spheroid formation efficiency, showing CSC properties. On the other hand, microarray analysis has revealed a number of differentially expressed genes involved in transcription regulation (e.g. FOXN4, GLI1), immune response (CCR7, IL8) and transmembrane transport (e.g. ABCC3, ABCC11) in the spheroids. Among these genes, increased expression of CCR7 in CD44+ CSCs was confirmed in NPC xenografts and primary tumors. Importantly, blocking of CCR7 abolished the sphere-forming ability of C666-1 in vitro. Expression of CCR7 was associated with recurrent disease and distant metastasis. The current study defined the specific properties of a CSC subpopulation in EBV-associated NPC. Our findings provided new insights into developing effective therapies targeting on CSCs, thereby potentiating treatment efficacy for NPC patients.

Expression and Functional Analysis of Toll-Like Receptors of Peripheral Blood Cells in Asthmatic Patients: Implication for Immunopathological Mechanism in Asthma

BackgroundWe investigated the expression profile of toll-like receptors (TLRs) and TLR ligand-activated production profile of asthma-related inflammatory cytokines in asthmatic patients. The expression of TLR1–8 on monocytes, CD4+ T helper lymphocytes, CD8+ T cytotoxic lymphocytes, CD19+ B lymphocytes, and dendritic cells, and ex vivo production of cytokines from peripheral blood mononuclear cells activated by TLR ligands were measured by flow cytometry.DiscussionEx vivo productions of TNF-α, IL-10, and IL-1β by TLR4 and TLR5 ligand LPS and flagellin were significantly lower in asthmatic patients (all P < 0.05). Expression of TLR4 and TLR5 was also found to be significantly lower in asthmatic patients when compared to that of control subjects (all P < 0.05). Therefore, the decreased activation of TLR4 and TLR5 in asthmatic patients might contribute to the immunopathological mechanisms of asthma by reducing the release of Th1 and anti-inflammatory cytokines.

Inhibition of NOTCH3 signalling significantly enhances sensitivity to cisplatin in EBV-associated nasopharyngeal carcinoma†

Nasopharyngeal carcinoma (NPC) is an EBV‐associated epithelial malignancy which is prevalent in south‐east Asia and southern China. Despite the multiple genetic and epigenetic changes reported, the contribution of dysregulated signalling pathways to this distinct type of head and neck cancer is not well understood. Here we demonstrate the up‐regulation of NOTCH ligands (JAG1 or DLL4) and effector (HEY1) in the majority of EBV‐positive tumour lines and primary tumours. Among the NOTCH receptors, NOTCH3 was over‐expressed in all EBV‐positive tumour lines and 92.5% of primary tumours. Aberrant activation of NOTCH3 signalling was consistently detected in all these samples. These findings imply that NOTCH3 may play an crucial role in the development of NPC. By NOTCH3 specific siRNA, NOTCH3 signalling was suppressed and thereby significant growth inhibition and apoptosis induction occurred in NPC cells. Down‐regulation of a number of targets involved in cell proliferation, eg CCND1, C‐MYC,NFKB1, and survival, eg BCL2, BCL‐XL, SURVIVIN, was confirmed in the NOTCH3 knockdown NPC cells. Importantly, NOTCH3 knockdown highly enhanced the sensitivity of NPC cells to cisplatin treatment. Furthermore, we revealed that the ability of NPC cells to form spheroids in vitro and tumours in nude mice was also significantly decreased after knockdown of NICD3 expression. These findings indicate that activation of NOTCH3 pathway is a critical oncogenic event in NPC tumourigenesis. Targeting NOTCH3 signalling may serve as a potential therapeutic approach for treating patients suffering from EBV‐associated NPC. Copyright

Activation of Peripheral Th17 Lymphocytes in Patients with Asthma

A recently identified interleukin (IL)-17-producing T-helper (Th) lymphocyte subset, which comprises Th17 cells producing hallmark cytokines IL-17A, IL-17F and IL-22, is involved in chronic inflammatory diseases. Elevated gene and protein expressions of IL-17 are manifested in allergic asthma. We further characterized the activation of Th17 cells in asthmatic patients. Peripheral blood mononuclear cells (PBMC) were purified from 31 asthmatic patients and 20 sex- and age-matched control subjects. The number of IL-17A secreting cells in peripheral blood was enumerated by enzyme-linked immunosorbent spot assay. Cell surface expression of Th17-related chemokine receptor CCR6, and plasma level of IL-17A, IL-17F and IL-22, and ex vivo production of IL-17A and IL-22 were measured by flow cytometry and enzyme-linked immunosorbent assay, respectively. The number of peripheral Th17 lymphocytes, expression of CCR6 on Th cells, and ex vivo IL-23, anti-CD3 and anti-CD28 induced production of IL-22 by PBMC were significantly elevated in asthmatic patients compared with control subjects (all p < 0.01). This clinical study further confirmed increased number of peripheral Th17 lymphocytes and cell surface expression of CCR6 receptors on Th cells in asthmatic patients. Pro-inflammatory cytokine IL-23 can exacerbate disease severity by activating pathogenic Th17 lymphocytes to release downstream inflammatory cytokine IL-22 in asthma.

Increased expression of plasma and CD4+ T lymphocyte costimulatory molecule CD26 in adult patients with allergic asthma.

CD26, which is a costimulatory molecule and peptidase, is responsible for the degradation of interferon (IFN)-γ-induced chemokines. To elucidate the immunopathological role of CD26 in allergic asthma, we investigated plasma soluble CD26 (sCD26) concentration and its cell surface expression on lymphocytes, monocytes, CD4+ T helper, CD8+ T suppressor plus cytotoxic T, invariant natural killer T (iNKT), and CD19+ B lymphocytes in allergic asthmatic patients. Plasma sCD26 was significantly elevated in asthmatic patients regardless of inhaled corticosteroid treatment (all P < 0.05). Cell surface expression of CD26 was significantly up-regulated on lymphocytes, especially on CD4+ and iNKT lymphocytes (all P < 0.05), but not on other cell types. Significant positive correlations were found between sCD26 and the percentage of eosinophils, Th2-related chemokines CCL5 and CCL22, and costimulatory molecule sCTLA-4 (all P < 0.05). In conclusion, the aberrant expression of CD26 may contribute to the inflammatory process and Th2 predominance in the immunopathogenesis of allergic asthma.

miR-31 is consistently inactivated in EBV-associated nasopharyngeal carcinoma and contributes to its tumorigenesis.

BackgroundAs a distinctive type of head and neck cancers, nasopharyngeal carcinoma (NPC) is genesis from the clonal Epstein-Barr virus (EBV)-infected nasopharyngeal epithelial cells accumulated with multiple genetic lesions. Among the recurrent genetic alterations defined, loss of 9p21.3 is the most frequent early event in the tumorigenesis of EBV-associated NPC. In addition to the reported CDKN2A/p16, herein, we elucidated the role of a miRNA, miR-31 within this 9p21.3 region as NPC-associated tumor suppressor.MethodsThe expression and promoter methylation of miR-31 were assessed in a panel of NPC tumor lines and primary tumors. Its in vitro and in vivo tumor suppression function was investigated through the ectopic expression of miR-31 in NPC cells. We also determined the miR-31 targeted genes and its involvement in the growth in NPC.ResultsDownregulation of miR-31 expression was detected in almost all NPC cell line, patient-derived xenografts (PDXs) and primary tumors. Both homozygous deletion and promoter hypermethylation were shown to be major mechanisms for miR-31 silencing in this cancer. Strikingly, loss of miR-31 was also obviously observed in the dysplastic lesions of nasopharynx. Restoration of miR-31 in C666-1 cells inhibited the cell proliferation, colony-forming and migratory capacities. Dramatic reduction of in v itro anchorage-independent growth and in vivo tumorigenic potential were demonstrated in the stable clones expressing miR-31. Furthermore, we proved that miR-31 suppressed the NPC cell growth via targeting FIH1 and MCM2.ConclusionsThe findings provide strong evidence to support miR-31 as a new NPC-associated tumor suppressor on 9p21.3 region. The inactivation of miR-31 may contribute to the early development of NPC.

Signalling mechanisms regulating the activation of human eosinophils by mast-cell-derived chymase: implications for mast cell-eosinophil interaction in allergic inflammation.

Allergic diseases such as asthma and allergic dermatitis are associated with the degranulation of mast cells. Chymase, a mast‐cell‐specific protease, is the major component in mast cell granules that can induce eosinophil infiltration into inflammatory sites. We examined the immunopathological mechanisms for the activation of eosinophils by chymase in allergic inflammation. Cytokines were measured by cytometric bead array Flex Sets multiplex assay using flow cytometry and enzyme‐linked immunosorbent assay. Adhesion molecules, migration and intracellular signalling pathways were assessed by flow cytometry, Boyden chamber assay and Western blot, respectively. Chymase suppressed the apoptosis of eosinophils and induce the release of the cytokine interleukin‐6 (IL‐6) and chemokines CXCL8, CCL2 and CXCL1 by eosinophils dose‐dependently. It also up‐regulated the surface expression of adhesion molecule CD18 and stimulated the chemokinetic migration of eosinophils. The expressions of adhesion molecules, cytokines and chemokines, and chemokinetic migration were differentially regulated by the activation of extracellular signal‐regulated kinase, p38 mitogen‐activated protein kinase, Akt, Janus‐activated kinase and nuclear factor‐κB pathways. Chymase therefore plays a pivotal immunological role in the interaction between mast cells and eosinophils in allergic diseases such as allergic dermatitis by inducing adhesion molecule‐mediated chemokinetic migration and inflammatory cytokines and chemokines of eosinophils, through multiple intracellular signalling molecules and transcription factor. Our results therefore provide a further biochemical basis for the pathogenesis of allergic inflammation consequent on the interaction between mast cells and eosinophils, and give insight for the development of new therapies.