Rong-Fu Chen

Different antigens trigger different Th1/Th2 reactions in neonatal mononuclear cells (MNCs) relating to T-bet/GATA-3 expression

Neonates are known to have poor cellular immunity, especially poor Th1 response. We investigated how neonatal mononuclear cells raised different Th1/Th2 reactions in response to different antigens. Employing Dermatophagoides pteronyssinus (Der p) extract and varicella zoster virus (VZV) as antigens, we assessed Th1/Th2 reactions as demonstrated by IL‐4/IFNγ production and mRNA expression, and transcriptional factors T‐bet/GATA‐3 mRNA expression in mononuclear cells from human umbilical cord blood (CBMC). Results showed that VZV induced a dramatic increase of IFNγ production by adult peripheral blood mononuclear cells (PBMC), whereas VZV did not drive CBMC to release significant IFNγ production (1614.7±362.0 vs. 49.0±29.3,p<0.005). However, Der p induced higher IFNγ production by CBMC than VZV (298.1±171.8 vs. 49.0±29.3, P=0.047). In contrast, VZV did not induce significant IL‐4 production either by CBMC or by PBMC. Der p induced a comparative IL‐4 production by CBMC and PBMC (2.58±0.84 vs. 2.04±0.37, p>0.05). A real‐time RT‐PCR analysis of IL‐4 and IFNγ mRNA expression showed that VZV induced a significantly higher IFNγ, but not IL‐4, mRNA expression in PBMC than CBMC. Der p did not induce significant difference of IFNγ or IL‐4 mRNA expression in PBMC and CBMC. VZV enhanced Th1‐related transcription factor T‐bet mRNA expression, in association with later down‐regulation of Th2‐related GATA‐3 mRNA expression in PBMC. However, VZV did not up‐regulate T‐bet or down‐regulate GATA‐3 expression significantly in CBMC. In contrast, Der p induced an early GATA‐3 expression and later T‐bet expression in CBMC. These results suggest that different antigens trigger various Th1/Th2 reactions in PBMC and CBMC resulting from kinetic changes of T‐bet/GATA‐3 expression.

MicroRNA‐21 expression in neonatal blood associated with antenatal immunoglobulin E production and development of allergic rhinitis

Background The prevalence of allergic diseases has increased in the past decades. It is unknown whether expression of certain microRNAs (miRNAs) in neonatal leucocytes is correlated to IgE production and/or allergic diseases.

Combination of CTLA-4 and TGFβ1 gene polymorphisms associated with dengue hemorrhagic fever and virus load in a dengue-2 outbreak

The pathogenesis of dengue hemorrhagic fever (DHF) has been considered to be massive immune activation of T cells. Abnormal expression of the immune regulatory molecules, CTLA-4 and TGFbeta1, leads to disturbances of regulatory T cell immune response. We investigate the contribution of CTLA-4 and TGFbeta1 in DHF by analyzing them for association with virus load in blood and polymorphisms of CTLA-4 +49A/G, and TGFbeta1 -509C/T in a DEN-2 outbreak. The increased frequency of the TGFbeta1 -509 CC genotype in patients with DHF was compared to those with dengue fever (OR=1.9, p=0.034). Moreover, the presence of the CTLA-4 +49 G allele and TGFbeta1 -509 CC genotype increased the susceptibility to risk of DHF (OR=2.1, p=0.028) and significantly higher virus load (p=0.013). This finding suggests that a combination of CTLA-4 and TGFbeta1 polymorphisms is associated with the susceptibility of DHF and higher virus load.

Interaction of maternal atopy, CTLA‐4 gene polymorphism and gender on antenatal immunoglobulin E production

Background Genetic heritability and maternal atopy have been correlated to antenatal IgE production, but very few studies have studied gene–maternal atopy interaction on antenatal IgE production. This study investigated the interaction of CTLA‐4 polymorphism with prenatal factors on the elevation of cord blood IgE (CBIgE).

IL-12-independent Th1 polarization in human mononuclear cells infected with varicella-zoster virus.

T helper type 1 (Th1) cells perform a critical role in fighting intracellular organisms, and interleukin‐12 (IL‐12) is known to promote a Thl response. This study was conducted to identify whether an IL‐12‐independent Th1 reaction is induced by the varicella‐zoster virus (VZV) in human beings. It was found that different intracellular microorganisms could induce IFNγ but not IL‐12 production. Induction of IFNγ production by VZV was associated with IFNα production and phosphorylation of both the signal transducer and activator of transcription‐1 (STAT‐1) and STAT‐4 in lymphocytes. In contrast, Bacillus Calmette‐Guerin (BCG) induced IL‐12 production in association with STAT‐4 but not STAT‐1 activation. Anti‐IFNα but not anti‐IL‐12 antibodies blocked the VZV‐induced Th1 polarization. A patient with an IL‐12 receptor β1 chain deficiency showed a normal VZV‐ but not a normal BCG‐induced Th1 reaction, further supporting the concept of an IFNα‐mediated, IL‐12‐independent Th1 reaction in response to certain intracellular infections. Identification of the early Th1 polarization induced by IFNα versus IL‐12 in response to specific viruses may enable the development of better therapeutic strategies tailored to different infections.

Augmented TLR2 Expression on Monocytes in both Human Kawasaki Disease and a Mouse Model of Coronary Arteritis

Background Kawasaki disease (KD) of unknown immunopathogenesis is an acute febrile systemic vasculitis and the leading cause of acquired heart diseases in childhood. To search for a better strategy for the prevention and treatment of KD, this study compared and validated human KD immunopathogenesis in a mouse model of Lactobacillus casei cell wall extract (LCWE)-induced coronary arteritis. Methods Recruited subjects fulfilled the criteria of KD and were admitted for intravenous gamma globulin (IVIG) treatment at the Kaohsiung Chang Gung Memorial Hospital from 2001 to 2009. Blood samples from KD patients were collected before and after IVIG treatment, and cardiovascular abnormalities were examined by transthoracic echocardiography. Wild-type male BALB/c mice (4-week-old) were intraperitoneally injected with LCWE (1 mg/mL) to induce coronary arteritis. The induced immune response in mice was examined on days 1, 3, 7, and 14 post injections, and histopathology studies were performed on days 7 and 14. Results Both human KD patients and LCWE-treated mice developed coronary arteritis, myocarditis, valvulitis, and pericarditis, as well as elevated plasma levels of interleukin (IL)-2, IL-6, IL-10, monocyte chemoattractant protein (MCP)-1, and tumor necrosis factor (TNF)-α in acute phase. Most of these proinflammatory cytokines declined to normal levels in mice, whereas normal levels were achieved in patients only after IVIG treatment, with a few exceptions. Toll-like receptor (TLR)-2, but not TLR4 surface enhancement on circulating CD14+ monocytes, was augmented in KD patients before IVIG treatment and in LCWE-treated mice, which declined in patients after IVIG treatment. Conclusion This result suggests that that not only TLR2 augmentation on CD14+ monocytes might be an inflammatory marker for both human KD patients and LCWE-induced CAL mouse model but also this model is feasible for studying therapeutic strategies of coronary arteritis in human KD by modulating TLR2-mediated immune activation on CD14+ monocytes.

Different implications of paternal and maternal atopy for perinatal IgE production and asthma development.

Asthma is a hereditary disease associated with IgE-mediated reaction. Whether maternal atopy and paternal atopy have different impacts on perinatal IgE production and asthma development remains unclear. This paper reviews and summarizes the effects of maternal and paternal atopy on the developmental aspects of IgE production and asthma. Maternal atopy affects both pre- and postnatal IgE production, whereas paternal atopy mainly affects the latter. Maternally transmitted genes GSTP1 and FceRI-beta are associated with lung function and allergic sensitization, respectively. In IgE production and asthma development, the maternal influence on gene-environment interaction is greater than paternal influence. Maternal, paternal, and/or postnatal environmental modulation of allergic responses have been linked to epigenetic mechanisms, which may be good targets for early prevention of asthma.

Use of proteomic differential displays to assess functional discrepancies and adjustments of human bone marrow- and Wharton jelly-derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) from bone marrow are suitable for the reconstruction of connective tissues and even brain tissue but have limitations in terms of cell expansion and fully specific differentiation. In our current study, we have attempted to adjust and improve the cell expansion and differentiation properties of human MSCs from different tissues. MSCs from normal bone marrow and Wharton jelly were subjected to proteomic differential displays, followed by functional adjustments based on these displays. Bone marrow MSCs expressed more transgelin-2 and differentiated more rapidly into bone nodules but showed a slower growth rate. A knockdown of transgelin-2 expression by specific small interfering RNA (siRNA) significantly increased the growth rate of these cells, the G1/S phase cell cycle transition, and the interaction of cyclin D1 with cdk2. Wharton jelly MSCs expressed the chaperone protein HSP90β at higher levels and differentiated slowly toward an osteogenic lineage. However, the knockdown of HSP90β expression significantly increased bone nodule formation, inhibited cell growth, decreased the number of cells in the G1/S phase of the cell cycle, and decreased the interaction of cyclin D1 with cdk2 and of cyclin E with cdk2. These results were validated by the in vivo repair of segmental bone defects in a mouse model with severe combined immunodeficiency. We thus demonstrate an improvement in the cell expansion and tissue regeneration properties of human MSCs through specific adjustments.

A Model to Study Antioxidant Regulation of Endotoxemia-Modulated Neonatal Granulopoiesis and Granulocyte Apoptosis

Neonates with septicemia tend to develop granulocytopenia, which may, in part, be due to septic mediators such as oxygen free radicals and tumor necrosis factor alpha (TNF-α). Granulocytopenia may be caused by a decrease in granulocyte growth and/or an increase in granulocyte destruction. In the present study, we investigated antioxidant regulation of endotoxin-modulated neonatal granulopoiesis and granulocyte apoptosis. Using human umbilical cord blood (HUCB), we found that simulating endotoxemia in vitro elicited significant superoxide production within a few minutes. Endotoxin exposure suppressed colony-forming unit–granulocyte and monocyte formation in a dose-dependent fashion. Addition of antioxidants such as N-acetyl-cysteine could reverse the endotoxin suppression of colony-forming unit–granulocyte and monocyte formation (13 ± 5 versus 75 ± 5 colony-forming units/mL). Spontaneous in vitro granulocyte apoptosis in 6 h, as reflected by phosphatidylserine expression on the cell surface, was higher in granulocytes from HUCB than in those from adult blood (10.8 ± 1.0%versus 5.6 ± 1.2%). The addition of endotoxin or IL-8 to the cells in the in vitro model did not promote granulocyte apoptosis, but TNF-α, a major mediator of the effects of endotoxin, significantly induced granulocyte apoptosis in HUCB (control versus TNF-α: 8.9 ± 1.2%versus 35.9 ± 2.9%). Addition of the antioxidant N-acetyl-cysteine effectively blocked TNF-α-induced granulocyte apoptosis as demonstrated by DNA fragmentation. Results from these studies indicate that oxygen radicals are directly involved in endotoxin suppression of granulopoiesis, and indirectly promote granulocyte apoptosis, presumably through TNF-α-mediated action. Thus, under certain conditions, modulation of oxygen radical production in the blood may benefit neonates with granulocytopenia.

Sialic acid involved in hypermucoviscosity phenotype of Klebsiella pneumoniae and associated with resistance to neutrophil phagocytosis

Klebsiella pneumoniae (KP) with the hypermucoviscosity (HV) phenotype has abundant capsular polysaccharides (CPS) and usually causes an invasive syndrome. Sialic acid (Sia), a component of CPS in KP strains with the HV phenotype, may be anti-phagocytic. Sia-binding immunoglobulin-like lectin-9 (Siglec-9) act as an MHC class-I receptor on neutrophils that recognizes Sia and sends a signal to dampen inflammatory response. Three clinical KP strains with KP-M1 (HV-positive; capsular serotype K1), KP-14 (HV-negative; capsular serotype non-K1/K2), and DT-X (HV-negative; capsular serotype K1) were studied. We assessed total Sia in CPS extracts using enzymatic methods and phagocytosis by neutrophils of neuraminidase-treated bacteria using flow cytometry. Neutrophil killing was evaluated in the presence and absence of antibodies against Siglec-9. The concentration of Sia was significantly higher in the CPS extract of KP-M1 (56.75 ± 6.75 μmole/109 cfu) than in the CPS extract of KP-14 (0.02 ± 0.01 μmole/109 cfu) and DT-X (a negligible value). The KP-M1 (compared with the KP-14 and DT-X) was more resistant to neutrophil phagocytosis. Both the HV phenotype and resistance to phagocytosis of KP-M1 were significantly decreased after Sia removal with neuraminidase treatment. Fluorescence microscopy with an antibody against human Siglec-9 showed attachment of KP-M1 (but were absent of KP-14 and DT-X) to the surface of neutrophils and colocalization with human Siglec-9. Engagement of Siglec-9 via Sia enhanced neutrophils killing of KP-M1 by ex vivo human neutrophils bactericidal activity assay. The result showed that Sia might be a constituent of KP-M1 CPS responsible for HV, thereby contributing to anti-phagocytic activity of this pathogen.