Effector mechanisms of sunitinib-induced G1 cell cycle arrest, differentiation, and apoptosis in human acute myeloid leukaemia HL60 and KG-1 cells.

Acute myeloid leukaemia (AML) is a heterogeneous disease with dismal outcome. Sunitinib is an orally active inhibitor of multiple tyrosine kinase receptors approved for renal cell carcinoma and gastrointestinal stromal tumour that has also been studied for AML in several clinical trials. However, the precise mechanism of sunitinib action against AML remains unclear and requires further investigation. For this purpose, this study was conducted using human AML cell lines (HL60 and KG-1) and AML patients' mononucleated cells. Sunitinib induced G1 phase arrest associated with decreased cyclin D1, cyclin D3, and cyclin-dependent kinase (Cdk)2 and increased p27(Kip1), pRb1, and p130/Rb2 expression and phosphorylated activation of protein kinase C alpha and beta (PKCα/ß). Selective PKCα/ß inhibitor treatment abolished sunitinib-elicited AML differentiation, suggesting that PKCα/ß may underlie sunitinib-induced monocytic differentiation. Furthermore, sunitinib increased pro-apoptotic molecule expression (Bax, Bak, PUMA, Fas, FasL, DR4, and DR5) and decreased anti-apoptotic molecule expression (Bcl-2 and Mcl-1), resulting in caspase-2, caspase-3, caspase-8, and caspase-9 activation and both death receptor and mitochondria-dependent apoptosis. Taken together, these findings provide evidence that sunitinib targets AML cells through both differentiation and apoptosis pathways. More clinical studies are urgently needed to demonstrate its optimal clinical applications in AML.

Sensitization to Per a 2 of the American cockroach correlates with more clinical severity among airway allergic patients in Taiwan.

BACKGROUND: In Taiwan, 57.5% of asthmatic patients are allergic to cockroaches, which are a major indoor allergen for immunoglobulin E (IgE)-mediated respiratory diseases. OBJECTIVE: To determine whether sensitization to different cockroach allergenic components correlates with different clinical manifestations and severities. METHODS: The complementary DNAs (cDNAs) encoding for Per a 1 through 7 and Per a 9 were generated by reverse transcription polymerase chain reaction and cloned into the Escherichia coli expression system. Sixty-four subjects were divided into 3 groups based on the clinical severity of their allergic reaction: those with persistent asthma and rhinitis (AS), those with allergic rhinitis only (AR), and the nonallergic controls (NA). Serum levels of interleukin-8 (IL-8), monocyte chemotactic protein-1 (MCP-1), chemokine (C-C motif) ligand 20 (CCL-20), and granulocyte macrophage colony-stimulating factor (GM-CSF) were measured, and the binding frequencies to each recombinant allergen were examined. RESULTS: Serum levels of IL-8, MCP-1, and CCL-20 were significantly higher in the AS group than in the AR and NA groups. The numbers of IgE-binding allergens did not correlate with the clinical severity of airway allergy to cockroaches. However, 81% in the AS group had IgE-binding activity to Per a 2, which was significantly higher than that of the AR group (45%, P < .05). In contrast, 80% of AR patients had IgE-binding activity to Per a 9 compared with only 28.5% of AS patients (P < .01). CONCLUSION: Allergens from American cockroaches do not have equal importance in terms of pathogenicity. Sensitization to Per a 2 correlates with more severe airway allergy and elevated proinflammatory chemokines. This may help in selecting target allergens for component resolved diagnosis and immunotherapeutic agents.

Herbal Supplement Ameliorates Cardiac Hypertrophy in Rats with CCl(4)-Induced Liver Cirrhosis.

We used the carbon tetrachloride (CCl(4)) induced liver cirrhosis model to test the molecular mechanism of action involved in cirrhosis-associated cardiac hypertrophy and the effectiveness of Ocimum gratissimum extract (OGE) and silymarin against cardiac hypertrophy. We treated male wistar rats with CCl(4) and either OGE (0.02 g/kg B.W. or 0.04 g/kg B.W.) or silymarin (0.2 g/kg B.W.). Cardiac eccentric hypertrophy was induced by CCl(4) along with cirrhosis and increased expression of cardiac hypertrophy related genes NFAT, TAGA4, and NBP, and the interleukin-6 (IL-6) signaling pathway related genes MEK5, ERK5, JAK, and STAT3. OGE or silymarin co-treatment attenuated CCl(4)-induced cardiac abnormalities, and lowered expression of genes which were elevated by this hepatotoxin. Our results suggest that the IL-6 signaling pathway may be related to CCl(4)-induced cardiac hypertrophy. OGE and silymarin were able to lower liver fibrosis, which reduces the chance of cardiac hypertrophy perhaps by lowering the expressions of IL-6 signaling pathway related genes. We conclude that treatment of cirrhosis using herbal supplements is a viable option for protecting cardiac tissues against cirrhosis-related cardiac hypertrophy.

Specific IgE and IgG responses and cytokine profile in subjects with allergic reactions to biting midge Forcipomyia taiwana.

BACKGROUND: Forcipomyia taiwana is a tiny blood-sucking midge whose habitat covers large parts of Taiwan and southern China. Female midges bite during the day, causing intense pruritus and swelling in allergic individuals. In this study, we investigated the immune responses of different allergic reactions to midge bites. METHODS: F. taiwana (midge)-specific IgE, -IgG and -IgG subclasses were examined by ELISA in 62 human subjects. Peripheral blood mononuclear cells (PBMC) from 6 subjects with solely delayed reactions (SDR) to midge bites and 6 nonallergic controls (NAC) were cultured with midge extract at various time points and assayed. Proliferation of PBMC was measured by MTT assay. Expression of cytokine mRNA was measured by real-time PCR and protein levels by cytometric bead immunoassay or ELISA. Protease activity in midge extract was determined by the Azocoll method. RESULTS: Midge-specific IgE among subjects with an immediate reaction were significantly elevated compared to SDR and NAC subjects. There were no differences in the level of midge-specific-IgG, -IgG(1), -IgG(2), -IgG(3) and -IgG(4) among subjects with different biting reactions. Midge extract elicited significantly more PBMC proliferation, higher expression of IFN-gamma, IL-10, IL-6 and TNF-alpha in SDR subjects than in NAC. Protease activity was detected in midge extract. Protease inhibitors E64 and pepstatin suppressed midge-extract-induced IL-8 production. CONCLUSIONS: Our results suggest that an immediate reaction to midge bites is IgE-mediated. IFN-gamma, IL-6 and TNF-alpha are involved in delayed reactions to midge bites. A protease-activated pathway may also be involved in the intense, itchy reactions to midge bites.

Pregnancy-induced hemophagocytic lymphohistiocytosis combined with autoimmune hemolytic anemia.

Hemophagocytic lymphohistiocytosis (HLH), presenting with fever, cytopenia, liver dysfunction, hepatosplenomegaly, hypertriglyceridemia, and hyperferritinemia, is associated with various etiologies, including infections, collagen vascular diseases, and malignancies. The present report describes a 28-year-old woman who developed HLH combined with autoimmune hemolytic anemia (AIHA) at 23 weeks of gestation. Without response to corticosteroid, the patient completely recovered from both HLH and AIHA after termination of the pregnancy. Pregnancy-induced immune dysregulation and cytokine overproduction in genetically susceptible women may play critical roles in HLH. The differential diagnosis of pregnant women with fever and cytopenia should include HLH. Pregnancy termination should be considered when pregnancy-induced HLH is refractory to medical treatment.

Functional characterization of hepatitis B virus X protein based on the inhibition of tumorigenesis in nude mice injected with CCL13-HBx cells.

OBJECTIVE: This study aimed to determine the effects of HBx on the inhibition of tumorigenesis in nude mice injected with CCL13-HBx cells. Therefore, the characteristics of the induced tumors and the phenomenon of apoptosis were assessed. METHODS: The induced tumors were identified using the specific marker of hepatocellular carcinoma (HCC), anti-alpha-fetoprotein (AFP), and their characteristics were pathologically examined. Apoptosis was detected by DNA fragmentation, and the expression of the proapoptotic proteins p53, Bax, Bad, caspase-3, and caspase-8 and the anti-apoptotic protein Bcl-2 was detected by Western blotting. To identify possible molecules involved in the inhibition of tumorigenesis, extracts of the induced tumors were separated by 2D-PAGE, and the proteins were identified by MS. RESULTS: The tumors of the nude mice injected with CCL13 and CCL13-HBx cells were identified as HCCs. Moreover, HBx was found to suppress tumor growth via apoptosis in the nude mice injected with CCL13-HBx cells. The MS findings revealed that phosphorylated myosin light chain was a candidate molecule involved in the inhibition of tumorigenesis. CONCLUSION: HBx suppressed tumorigenesis in the nude mice injected with CCL13-HBx cells, which proved to be a good animal model for the in vivo study of the effects of HBx on tumorigenesis.

HBx inhibits the growth of CCL13-HBX-stable cells via the GSK-3beta/beta-catenin cascade.

OBJECTIVE: The hepatitis B virus X protein (HBx) plays critical roles in cell survival via modulation of signaling pathways. In our previous studies, we reported that HBx inhibited the growth of CCL13-HBx-stable cells (Chang-HBx cells) in vitro and tumor formation in vivo in CCL13-HBx-cell-injected nude mice; however, this inhibition mechanism is unclear. METHODS: To investigate the role of HBx in Wnt-3/beta-catenin signaling pathways, we focused on the key molecules GSK-3beta and beta-catenin, and analyzed by Western blotting and immunofluorescence staining. RESULTS: Results indicated that following HBx induction, GSK-3beta activity was up-regulated, the expression and accumulation of beta-catenin in the nucleus were decreased, and cell proliferation was suppressed. Inhibition of GSK-3beta activity by pharmacological inhibitors rescued the expression and accumulation of beta-catenin in the nucleus and facilitated cell proliferation and growth following HBx induction. The localization of beta-catenin, which is involved in cell proliferation, and mediated by GSK-3beta activation was also demonstrated. CONCLUSION: Our findings suggest that HBx negatively regulated proliferation of CCL13-HBx-stable cells via the GSK-3beta/beta-catenin cascade.

Effects of hepatitis B virus X protein (HBx) on cell-growth inhibition in a CCL13-HBx stable cell line.

OBJECTIVE: The known function of hepatitis B virus X protein (HBx) is to determine the fate of cells by modulating various signaling pathways. In our previous study, we demonstrated that HBx inhibits tumor formation in nude mice injected with CCL13-HBx stable cell lines; however, the mechanism underlying this inhibition is unclear. METHODS: To investigate the possible mechanisms underlying HBx involvement in CCL13-HBx cells, gene profiles were initially analyzed by DNA microarray technology and subsequently confirmed by performing semiquantitative RT-PCR and Western blotting. Furthermore, the phenomenon of cell death via apoptosis was detected via DNA fragmentation, TUNEL staining, caspase-3 activity assay, and propidium iodide (PI) staining. RESULTS: The results indicated that HBx induction downregulated Wnt-3 and beta-catenin that are involved in cell proliferation. Moreover, HBx induction repressed cell growth and downregulated the expressions of cyclin D1, CDK4, cyclin E, CDK2, and cyclin B1. Furthermore, HBx induction triggered cell death via apoptosis, as determined by DNA fragmentation, TUNEL staining, caspase-3 activity assay, and PI staining. CONCLUSION: Most importantly, our results indicated that HBx induction in the CCL13-HBx stable cell line downregulated Wnt-3/beta-catenin expression and suppressed cell growth by repressing cell proliferation or triggering cell apoptosis.

Primary splenic lymphoma associated with hemophagocytic lymphohistiocytosis complicated with splenic rupture.

Primary splenic lymphoma (PSL) is a rare disease with ambiguous definition, comprising less than 1% of non-Hodgkin's lymphoma. Even rarer is PSL combined with hemophagocytic lymphohistiocytosis (HLH), which has presentations of fever, cytopenia, hepatosplenomegaly, hyperferritinemia, and phagocytosis of hematopoietic cells in the reticuloendothelial system. We report the case of a 77-year-old man who presented with HLH initially. Refusing diagnostic splenectomy, he received chemotherapy. Spontaneous splenic rupture occurred after chemotherapy. In the following emergency operation, PSL was diagnosed. He received another 5 courses of chemotherapy with the R-CNOP regimen (rituximab, cyclophosphamide, mitoxantrone, vincristine, prednisolone). Now he has no residual or relapsed disease. Diagnostic splenectomy for adult HLH patients without definite etiologies may play an important role.

Ovatodiolide isolated from Anisomeles indica induces cell cycle G2/M arrest and apoptosis via a ROS-dependent ATM/ATR signaling pathways.

Ovatodiolide was isolated from the traditional Chinese medicinal herb Anisomeles indica, possesses anti-bacterial and anti-inflammatory properties; however, the anti-cancer activity and its mechanisms have been limitedly reported. This study aimed to examine the effect and molecular action of ovatodiolide in lung cancer cells. Cell cycle distribution and reactive oxygen species (ROS) generation were measured by flow cytometry. Apoptosis was detected by propidium iodide/annexin V staining and TUNEL assay. DNA damage was investigated by comet assay and γ-H2AX staining. Caspase activity was determined using caspase fluorometric kits. Moreover, protein levels were examined by western blot. Ovatodiolide provoked reactive oxygen species generation and DNA damage, as well as inhibited cell growth and induced apoptosis in human lung cancer A549 and H1299 cell lines. DNA damage-related molecules, ATM/ATR and CHK1/CHK2 were activated by ovatodiolide. Moreover, ovatodiolide-mediated G2/M arrest was associated with the decrease of Cyclin B1 and CDC25C levels, and increase of p21WAF1/CIP1 expression. Additionally, ovatodiolide-triggered apoptosis was through both intrinsic and extrinsic pathways characterized by the elevating PUMA, Bax, and DR5 proteins, decreasing Bcl-2 and Mcl-1, and activating caspase-8, caspase-9 and caspase-3. Caffeine, an ATM/ATR inhibitor, rescued ovatodiolide-mediated cell cycle arrest and apoptosis, but not reactive oxygen species generation. Nevertheless, antioxidant N-acetyl-cysteine completely blocked ovatodiolide-mediated molecular events, G2/M arrest, and apoptosis. These observations suggest that ovatodiolide stimulates reactive oxygen species generation, causes oxidative stress and DNA damage; subsequently, provokes DNA damage signaling pathways, eventually leads to block cell cycle at G2/M phase and trigger apoptosis in lung cancer A549 and H1299 cells.

Molecular cloning of Indian jujube (Zizyphus mauritiana) allergen Ziz m 1 with sequence similarity to plant class III chitinases.

Indian jujube (Zizyphus mauritiana) is a sweet fruit that is abundantly cultivated in Taiwan. We have previously identified 42 and 30 kDa allergens that are cross-reactive with latex allergen from crude Indian jujube extract. This study aimed to clone the 30 kDa Ziz m 1 Z. mauritiana allergen. The Ziz m 1 encoding cDNA was isolated from a ZAPII cDNA library constructed from Z. mauritiana mRNA, sequenced and expressed in Pichia pastoris. The protein predicted from the cDNA sequence has 330 amino acids, the first 25 of which constituted a putative signal peptide. The deduced molecular mass of the mature protein is 33.86 kDa, while its isoelectric point is estimated at 4.36. The recombinant Ziz m 1 showed chitinase activity, possessed IgE binding capacity, and had IgE cross-reactivity with the latex allergen. Moreover, anti-recombinant Ziz m 1 antibody-based ELISA was able to detect commercial skin testing latex reagent, laboratory prepared latex and Indian jujube extracts. Recombinant Ziz m 1 showed 87.5% skin reactivities on eight latex- and Indian jujube-sensitive subjects. Although no sequence similarity was found to other known allergens, Ziz m 1 was found to have amino acid sequence identity (39-45.3%) to many plant chitinases including chitinase (45.2%) of Hevea brasiliensis (hevamine), class III chitinases of Vigna angularis (45.3%), Capsicum annuum (44.7%) and Oryza sativa (41.2%). A conserved domain search revealed that Ziz m 1 belongs to the family 18 glycosyl hydrolases. The recombinant allergen may therefore be of value for diagnosis and therapeutic purposes, and the further characterization of Indian jujube allergen may help to elucidate the mechanism underlying latex-fruit syndrome.

Altered expression profile of superoxide dismutase isoforms in nasal polyps from nonallergic patients.

OBJECTIVE/HYPOTHESIS: Nasal polyposis (NP) is a chronic inflammatory disease of the upper respiratory tract. The pathophysiology is unknown but has been shown to be multifactorial. Free radical-mediated damage has been implicated in the pathogenesis of NP. Superoxide dismutases (SODs) are the first and the most important line of antioxidant enzyme defense against reactive oxygen species. Moreover, isozymes of the SOD family are critical for modulating the activity of nitric oxide, a gaseous free radical that is believed to play roles in the physiology and pathology of respiratory tracts. However, the expression profile of SOD isoforms in NP remains unclear. We aimed to investigate the expression profile of the SOD isoforms in nasal polyps from nonallergic patients. STUDY DESIGN: Prospective study. METHODS: Nasal polyp tissues were obtained from eight nonallergic patients who underwent elective polypectomy; mucosal specimens from the middle turbinates were acquired from eight subjects without NP as control tissues. The expression profile of SOD isoenzymes, SOD1, SOD2, and SOD3, in the nasal tissues was determined by reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and Western blotting (WB). RESULTS: NP in all eight of the NP patients manifested as severe or recurrent sinonasal polyposis clinically. The expression pattern of SOD isoenzymes evaluated by RT-PCR analysis indicated that the mean levels of SOD1 mRNA and, to a greater extent, SOD3 mRNA were higher in polyp tissues than in control tissues. There was no significant difference in the expression levels of SOD2 mRNA between the two groups. The data from ELISA and WB analysis showed that there were increased expressions of SOD1 and SOD3 protein in polyp tissues compared with the control tissues, but there was no difference in the expression of SOD2 protein between the two groups. The results from RT-PCR, ELISA, and WB were paralleled and revealed that the expressions of SOD1 and, to a greater extent, SOD3 were higher in polyp tissues than in the control group. CONCLUSIONS: The expressions of SOD3 and SOD1 were higher in polyp tissues. These results are consistent with previously reported data and support the hypothesis that there is increased oxidative stress in NP. Our data also suggest that the SODs might be important in the pathogenesis of NP; however, the roles these SOD isoforms, especially SOD3, play in both normal nasal mucosa and NP require further clarification.

Increased prevalence of interleukin-1 receptor antagonist gene polymorphism in patients with chronic rhinosinusitis.

OBJECTIVE: To assess the association of the interleukin (IL)-1beta and the IL-1 receptor antagonist (IL-1Ra) gene polymorphisms with chronic rhinosinusitis (CRS). DESIGN: Genotyping of the 2 IL-1beta gene (IL1B) polymorphisms (promoter and exon) and the IL-1Ra gene (IL1RN) polymorphism (intron 2) was performed using polymerase chain reaction and restriction length fragment polymorphism analyses. SETTING: Prospective study, tertiary medical center. PATIENTS: The study population comprised 88 consecutive adult Taiwan-Chinese patients who met stringent criteria for CRS and received endoscopic sinus surgery and 103 healthy volunteers of the same ethnicity and similar age range. Of the 88 patients, 61 had CRS with nasal polyps, while the other 27 had CRS without nasal polyps. RESULTS: There were significant differences in the distribution of the IL1RN polymorphism between the control subjects and patients with CRS (P<.05). The II allele of IL1RN occurred more frequently in the CRS patient group, and the odds ratio for subjects with I/II genotype was 3.39 (95% confidence interval, 1.25-9.18). In the case of CRS without nasal polyps, the odds ratio for subjects with I/II genotype was further increased to 4.75 (1.39-16.25). There was no association between the other 2 polymorphisms of IL1B and CRS. CONCLUSION: Increased prevalence of IL1RN polymorphism in patients with CRS suggests that this polymorphism, or a polymorphism in linkage disequilibrium with it, may be involved in the development of CRS.

Lactate dehydrogenase, not vascular endothelial growth factor or basic fibroblast growth factor, positively correlates to bone marrow vascularity in acute myeloid leukemia.

BACKGROUND: Angiogenesis has been extensively studied in acute myeloid leukemia (AML). Lactate dehydrogenase (LDH), a common biochemical marker for tumor burden and anaerobic glycolysis, is a poor prognostic factor for AML. Regulated by hypoxia-induced factor, both vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are responsive to cancer-related angiogenesis. To study the roles of serum LDH, VEGF and bFGF in AML angiogenesis, we investigated bone marrow vascularity in untreated AML patients, and analyzed its relationship to serum LDH, VEGF and bFGF levels. METHODS: Eighteen (11 males, 7 females; mean age, 57.7 years) de novo, untreated AML patients were enrolled. Bone marrow vascularity was evaluated by staining bone marrow core biopsy tissue with endothelial cell marker CD31 or CD34. Serum LDH was determined with the Wroblewski-La Due method. Serum VEGF and bFGF were determined with enzyme-linked immunoassay. The relationship of LDH, VEGF and bFGF level to bone marrow vessel numbers was examined by linear regression. RESULTS: Log LDH significantly correlated to AML bone marrow vascularity (r = 0.61; p = 0.007). VEGF and bFGF concentrations did not correlate with AML angiogenesis. CONCLUSION: These results suggest that serum LDH, but not VEGF and bFGF concentrations, can be used as a simple parameter for predicting vessel formation in AML bone marrow.

Potent Antiarthritic Properties of Phloretin in Murine Collagen-Induced Arthritis.

In the exploration of potential therapeutic agents for rheumatoid arthritis (RA), DBA/1J mice are used as the RA model of collagen-induced arthritis (CIA). Phloretin, a flavonoid compound extracted from Prunus mandshurica, has been found to exhibit anti-inflammatory activity, making it a potential candidate for treatment of RA. The objective of this study was to evaluate the therapeutic effects of phloretin on CIA mice. CIA mice were dosed daily with phloretin at either 50 or 100 mg/kg among two treatment groups. CIA treated mice showed mitigation of clinical symptoms of RA in addition to reduced inflammation of hind-limbs compared to mice who did not receive phloretin. Histological analysis showed that phloretin suppressed the severity of RA and effectively mitigated joint inflammation and cartilage- and bone-destruction via reducing proinflammatory cytokine productions (TNF-α, IL-6, IL-1ß, and IL-17). This was at least partially mediated by causing inadequate splenocyte activation and proliferation. Moreover, phloretin-treated CIA mice showed decreased oxidative stress and diminished levels of malondialdehyde (MDA) and hydrogen peroxide (H2O2) in paw tissues as well as reduced productivity of anti-collagen antibodies in serum. We have concluded that phloretin could be a potent and effective antiarthritis agent, demonstrating anti-inflammatory, antioxidative, and immunomodulatory effects in CIA mice.

The prevalence of HPV-18 and variants of E6 gene isolated from cervical cancer patients in Taiwan.

BACKGROUND: Cervical cancer is the second most common cancer in women worldwide. It has been considered that human papillomavirus (HPV) is associated with cervical cancer. Currently, more than 80 different serotypes of HPV have been characterized and they are divided into low- and high-risk groups. The most common types that lead to cervical cancer are HPV-16 and -18. The viral oncogenes E6 and E7 are associated with the development of cervical cancer. In previous study, the variants of HPV-16 E6 gene have been reported. It suggests that variants may influence the morbidity of carcinogenesis, but the variant study on HPV-18 remains unknown. OBJECTIVES: To identify the variants of integrated HPV-18 E6 gene in the prevalent infection of HPV-18 of cervical cancer patients. STUDY DESIGN: 25 cervical cancer patients were clinically identified and the biopsies were obtained. The infectious HPV types were identified by PCR and Southern blotting analysis. The DNA fragments of the integrated HPV-18 E6 were amplified by PCR and cloned. The nucleotide sequences were obtained by sequencing. RESULTS: The prevalence of HPV infection in our 25 cases was HPV-18 (100%) and 7 out of these 25 cases (28%) were co-infected with HPV-16. The most dominant mutation among 25 tested patients was a silence mutation C183G of the E6 coding region. CONCLUSIONS: The prevalent HPV infectious serotype is HPV-18, which differs from the worldwide prevalent type. The identified HPV-18 E6 variants had a unique silence mutation located on C183G in E6 coding region.

IgE-Binding Epitope Mapping and Tissue Localization of the Major American Cockroach Allergen Per a 2.

PURPOSE: Cockroaches are the second leading allergen in Taiwan. Sensitization to Per a 2, the major American cockroach allergen, correlates with clinical severity among patients with airway allergy, but there is limited information on IgE epitopes and tissue localization of Per a 2. This study aimed to identify Per a 2 linear IgE-binding epitopes and its distribution in the body of a cockroach. METHODS: The cDNA of Per a 2 was used as a template and combined with oligonucleotide primers specific to the target areas with appropriate restriction enzyme sites. Eleven overlapping fragments of Per a 2 covering the whole allergen molecule, except 20 residues of signal peptide, were generated by PCR. Mature Per a 2 and overlapping deletion mutants were affinity-purified and assayed for IgE reactivity by immunoblotting. Three synthetic peptides comprising the B cell epitopes were evaluated by direct binding ELISA. Rabbit anti-Per a 2 antibody was used for immunohistochemistry. RESULTS: Human linear IgE-binding epitopes of Per a 2 were located at the amino acid sequences 57-86, 200-211, and 299-309. There was positive IgE binding to 10 tested Per a 2-allergic sera in 3 synthetic peptides, but none in the controls. Immunostaining revealed that Per a 2 was localized partly in the mouth and midgut of the cockroach, with the most intense staining observed in the hindgut, suggesting that the Per a 2 allergen might be excreted through the feces. CONCLUSIONS: Information on the IgE-binding epitope of Per a 2 may be used for designing more specific diagnostic and therapeutic approaches to cockroach allergy.

Polygonum cuspidatum and its active components inhibit replication of the influenza virus through toll-like receptor 9-induced interferon beta expression.

Influenza virus infection is a global public health issue. The effectiveness of antiviral therapies for influenza has been limited by the emergence of drug-resistant viral strains. Therefore, there is an urgent need to identify novel antiviral therapies. Here we tested the effects of 300 traditional Chinese medicines on the replication of various influenza virus strains in a lung cell line, A549, using an influenza-specific luciferase reporter assay. Of the traditional medicines tested, Polygonum cuspidatum (PC) and its active components, resveratrol and emodin, were found to attenuate influenza viral replication in A549 cells. Furthermore, they preferentially inhibited the replication of influenza A virus, including clinical strains isolated in 2009 and 2011 in Taiwan and the laboratory strain A/WSN/33 (H1N1). In addition to inhibiting the expression of hemagglutinin and neuraminidase, PC, emodin, and resveratrol also increased the expression of interferon beta (IFN-ß) through Toll-like receptor 9 (TLR9). Moreover, the anti-viral activity of IFN-ß or resveratrol was reduced when the A549 cells were treated with neutralizing anti-IFN-ß antibodies or a TLR9 inhibitor, suggesting that IFN-ß likely acts synergistically with resveratrol to inhibit H1N1 replication. This potential antiviral mechanism, involving direct inhibition of virus replication and simultaneous activation of the host immune response, has not been previously described for a single antiviral molecule. In conclusion, our data support the use of PC, resveratrol or emodin for inhibiting influenza virus replication directly and via TLR-9-induced IFN-ß production.

Inhibition of tumorigenicity of the hepatitis B virus X gene in Chang liver cell line.

The hepatitis B virus X gene, which encodes the HBx protein, has multiple functions and is involved in hepatocarcinogenesis. However, the exact role of HBx in hepatocarcinogenesis is still controversial. We have established an inducible (tet-off system) HBx-expressing cell line, Chang-HBx. Compared with the original of Chang liver cell line (ATCC CCL13), Chang-HBx grows faster in serum-containing medium but slower in serum-free medium. Chang-HBx colony formation in soft agar shows an anchorage-demanding character and its tumorigenicity potential in BALB/c nude mice were substantially inhibited. HBx also causes the induction of G1 phase arrest of cell growth in early infection of HBV and therefore plays a negative role in tumorigenicity. An excellent mice animal model for producing hepatoma was also provided in this study.

Dominant mutations of hepatitis B virus variants in hepatoma accumulate in B-cell and T-cell epitopes of the HBx antigen.

Hepatitis B virus (HBV) X gene, encoding a pleotropic transactivator of HBx protein, has been associated with the development of hepatocellular carcinoma (HCC). Molecular information on liver-derived HBV variants isolated from HCC among Taiwanese population was studied. Amplification of the HBV X genes of 20 HCC patients in high stringency with HBV specific primers was observed. The resulting amplified HBV X genes were purified and individually-cloned into pUC-T vector. Sequences of the eight liver-derived X gene were aligned and compared with the wild type, the ayw HBV serotype. Results indicate that the HBx protein of variants were found predominantly within the regions of amino acid positions 26-45 in N-terminus, and positions 87, 88, 116, 118, 119, 127 and 144. Sequences from six out of the eight variants were found to be identical. These accumulated sequence mutations among the eight HBx variants were found to coincide within the B-cell epitopes (positions 29-48), particularly in the HBx proline and serine rich (PSR) domain, and the T-cell epitopes regions (positions 116-127). These frequent mutations of HBV variants, rather than subtype-specific polymorphic sites, may be involved in immunoevasion.