Parvapan Bhattarakosol

H5N1 influenza A virus and infected human plasma.

To the Editor: Since January 2004, a total of 22 persons have been confirmed infected with avian influenza A virus (H5N1) in Thailand; 14 of these patients died. Three waves of outbreaks occurred during the past 2 years. The last patient of the third wave was a 5-year-old boy whose symptoms developed on November 28, 2005; he was hospitalized on December 5 and died 2 days later. The child resided in the Ongkharak District, Nakhon Nayok Province, ≈70 km northeast of Bangkok. Villagers informed the Department of Livestock after the patients illness was diagnosed. Five dead chickens had been reported in this area from November 28 to December 1, 2005. Samples from these chickens could not be obtained, thus, no H5N1 testing was performed. The boy had fever, headache, and productive cough for 7 days before he was admitted to the Her Royal Highness Princess Maha Chakri Sirindhorn Medical Center. Clinical examination and chest radiograph showed evidence of lobar pneumonia. He was treated with antimicrobial drugs (midecamycin and penicillin G) and supportive care, including oxygen therapy. On December 7, the patients condition worsened, and severe pneumonia with adult respiratory distress syndrome developed. Laboratory tests showed leukopenia (2,300 cells/mm3), acidosis, and low blood oxygen saturation by cutaneous pulse oximetry (81.6%). Oseltamivir was administered after his parents informed hospital staff about the boys contact with the dead chicken. However, the boy died the same day; no autopsy was performed. On December 9, the cause of death was declared by the Ministry of Public Health to be H5N1 influenza virus. A blood sample was collected from the patient on December 7; anticoagulation was accomplished with ethylenediaminetetraacetic acid (EDTA) for repeated biochemistry analysis and complete blood count. The plasma from the EDTA blood sample was separated 2 days later and stored at –20°C for 12 days. The sample was subsequently given to the Center of Excellence in Viral Hepatitis, Faculty of Medicine, Chulalongkorn University, for molecular diagnosis and then stored at –70°C, where specific precautions implemented for handling highly infectious disease specimens such as H5N1 influenza virus were observed. Plasma was examined by multiplex reverse transcription–polymerase chain reaction (RT-PCR) (1) and multiplex real-time RT-PCR (2), both of which showed positive results for H5N1 virus. The virus titer obtained from the plasma was 3.08 × 103 copies/mL. The plasma specimen was processed for virus isolation by embryonated egg injection, according to the standard protocol described by Harmon (3). Briefly, 100 μL 1:2 diluted plasma was injected into the allantoic cavity of a 9-day-old embryonated egg and incubated at 37°C. The infected embryo died within 48 hours, and the allantoic fluid was shown to contain 2,048 hemagglutinin (HA) units; also, subtype H5N1 was confirmed (1,2). Whole genome sequencing was performed and submitted to the GenBank database under the strain A/Thailand/NK165/05 accession no. DQ 372591-8. The phylogenetic trees of the HA and neuraminidase (NA) genes were constructed by using MEGA 3 (4) for comparison with H5N1 viruses isolated from humans, tigers, and chickens from previous outbreaks in 2004 and 2005 (Figure). The sequence analyses of the viruses showed that the HA cleavage site contained SPQREKRRKKR, which differed from the 2004 H5N1 virus by an arginine-to-lysine substitution at position 341. That finding had also been observed in wild bird species during earlier outbreaks in Thailand in 2004 (5). Similar to the 2004–2005 H5N1 isolates from Thailand, a 20–amino acid deletion at the NA stalk region was observed. Moreover, the amino acid residues (E119, H274, R292, and N294) of the NA active site were conserved, which suggests that the virus was sensitive to oseltamivir. In addition, a single amino acid substitution from glutamic acid to lysine at position 627 of PB2 showed increased virus replication efficiency in mammals (6). Figure Phylogenetic analysis of the hemagglutinin and neuraminidase genes of H5N1 from study patient compared with sequences from previous outbreaks (2004–2005). Observing live influenza virus in human serum or plasma is unusual. However, in 1963, low quantities of virus were isolated from blood of a patient on day 4 of illness (7), and in 1970, the virus was cultivated from blood specimens from 2 patients (8). Recently, a fatal case of avian influenza A (H5N1) in a Vietnamese child was reported. The diagnosis was determined by isolating the virus from cerebrospinal fluid, fecal, throat, and serum specimens (9); viral RNA was found in 6 of 7 serum specimens 4–9 days after the onset of illness (10). In this case, the H5N1 virus could be isolated from plasma on day 10 after symptoms developed. This case showed the virus in the patients blood, which raises concern about transmission among humans. Because probable H5N1 avian influenza transmission among humans has been reported (11), this case should be a reminder of the necessity to carefully handle and transport serum or plasma samples suspected to be infected with H5N1 avian influenza. Because viable virus has been detected in blood samples, handling, transportation, and testing of blood samples should be performed in a biosafety (category III) containment laboratory to prevent the spread of the virus to healthcare and laboratory workers. We express our thanks to the Thailand Research Fund (Senior Research Scholar), Royal Golden Jubilee PhD Program and Center of Excellence in Viral Hepatitis Research, and Prasert Auewarakul for their generous support of our study.

Antitumor and Antiangiogenic Activities of Curcumin in Cervical Cancer Xenografts in Nude Mice

To evaluate the effects of curcumin (CUR) on tumor progression and angiogenesis in cervical cancer- (CaSki-) implanted nude mice and on the angiogenic biomarkers: vascular endothelial growth factor (VEGF), cyclooxygenase-2 (COX-2), and epidermal growth factor receptor (EGFR). CaSki cells were subcutaneously injected in nude mice to establish subcutaneous tumors. One month after injection, mice were orally administered vehicle or 500, 1,000, and 1,500 mg/kg of CUR daily × 30 consecutive days. Tumor volume was measured every 3-4 days. At the end of the study, tumor microvasculature was observed under confocal microscope, and immunohistochemical analyses were performed to detect CD31, VEGF, COX-2, and EGFR. CUR at the doses of 1,000 and 1,500 mg/kg showed significant tumor growth retardation (21.03% and 35.57%) versus CaSki + vehicle group. The microvascular density (MVD) in CaSki + vehicle group was significantly increased versus Control + vehicle group and significantly reduced by CUR (1,000 and 1,500 mg/kg). VEGF, COX-2, and EGFR expressions were upregulated in CaSki + vehicle group and attenuated significantly by CUR (1,000 and 1,500 mg/kg). In conclusion, high dose CUR inhibited tumor growth and angiogenesis in CaSki-implanted mice probably mediated by the downregulation of VEGF, COX-2 and EGFR. CUR may have a role in treating human cervical cancer and should be explored further.

Molecular Mechanisms of Curcumin on Diabetes-Induced Endothelial Dysfunctions: Txnip, ICAM-1, and NOX2 Expressions

We aim to investigate the effects of curcumin on preventing diabetes-induced vascular inflammation in association with its actions on Txnip, ICAM-1, and NOX2 enzyme expressions. Male Wistar rats were divided into four groups: control (CON), diabetic (DM; streptozotocin (STZ), i.v. 55 mg/kg BW), control-treated with curcumin (CONCUR; 300 mg/kg BW), and diabetes treated with curcumin (DMCUR; 300 mg/kg BW). 12th week after STZ injection, iris blood perfusion, leukocyte adhesion, Txnip, p47phox, and malondialdehyde (MDA) levels were determined by using laser Doppler, intravital fluorescent confocal microscopy, Western Blot analysis, and TBAR assay, respectively. The iris blood perfusion of DM and DMCUR was decreased significantly compared to CON and CONCUR (P < 0.001). Plasma glucose and HbA1c of DM and DMCUR were increased significantly compared to CON and CONCUR (P < 0.001). Leukocyte adhesion, ICAM-1, p47phox expression, and MDA levels in DM were increased significantly compared to CON, CONCUR, and DMCUR (P < 0.05). Txnip expression in DM and DMCUR was significantly higher than CON and CONCUR (P < 0.05). From Pearsons analysis, the correlation between the plasma MDA level and the endothelial functions was significant. It suggested that curcumin could ameliorate diabetic vascular inflammation by decreasing ROS overproduction, reducing leukocyte-endothelium interaction, and inhibiting ICAM-1 and NOX2 expression.

Molecular and demographic analysis of respiratory syncytial virus infection in patients admitted to King Chulalongkorn Memorial Hospital, Thailand, 2007.

Please cite this paper as: Boonyasuppayakorn et al. (2010) Molecular and demographic analysis of respiratory syncytial virus infection in King Chulalongkorn Memorial Hospital admitted patients, Thailand, 2007. Influenza and Other Respiratory Viruses 4(5), 313–323.

Original Article: Molecular and demographic analysis of respiratory syncytial virus infection in patients admitted to King Chulalongkorn Memorial Hospital, Thailand, 2007

Please cite this paper as: Boonyasuppayakorn et al. (2010) Molecular and demographic analysis of respiratory syncytial virus infection in King Chulalongkorn Memorial Hospital admitted patients, Thailand, 2007. Influenza and Other Respiratory Viruses 4(5), 313–323.

Intra- and Intergenotypic Variations among Human Cytomegalovirus gB Genotypes

Objectives: To study genotypic variations among human cytomegalovirus (HCMV) gB genotypes in clinical samples of Thai patients. Methods: gB genotyping of 31 HCMV-DNA-positive clinical samples were determined by PCR-RFLP, gene cloning and DNA sequencing methods. Results: Eight gB1, 7 gB2, 7 gB3, 6 gB untype (UT1 and UT2) and 3 mixed gB genotypes were first identified by PCR-RFLP. All 3 mixed gB genotype samples and 1 gB2 sample were cloned and confirmed gB genotype by PCR-RFLP. Altogether, 57 strains (27 unique types and 30 transformants) were further analyzed by DNA sequencing method. Discordant results between PCR-RFLP and DNA sequencing methods were demonstrated in 5 samples and 1 clone. The gB UT1 and UT2 were all classified as gB1 except 1 sample of UT1 (9D), suggesting a new variant. Each genotype had a similarity of more than 97%, whereas strains of different genotypes had 77.71–92.75% homology. The most divergent type was gB3. Conclusions: Intra- and intergenotypic variations among strains were demonstrated either in individual or distinct patients. Intragenotypic variation in a person occurs possibly due mainly to a point mutation mechanism rather than reinfection of a new gB genotype.

Effects of Acanthus ebracteatus Vahl on tumor angiogenesis and on tumor growth in nude mice implanted with cervical cancer.

Purpose The aim of this study was to examine the effects of the crude extract of Acanthus ebracteatus Vahl (AE) on tumor growth and angiogenesis by utilizing a tumor model in which nude mice were implanted with cervical cancer cells containing human papillomavirus 16 DNA (HPV-16 DNA). Materials and methods The growth-inhibitory effect of AE was investigated in four different cell types: CaSki (HPV-16 positive), HeLa (HPV-18 positive), hepatocellular carcinoma cells (HepG2), and human dermal fibroblast cells (HDFs). The cell viabilities and IC50 values of AE were determined in cells incubated with AE for different lengths of time. To conduct studies in vivo, female BALB/c nude mice (aged 6–7 weeks, weighing 20–25 g) were used. A cervical cancer-derived cell line (CaSki) with integrated HPV-16 DNA was injected subcutaneously (1 × 107 cells/200 μL) in the middle dorsum of each animal (HPV group). One week after injection, mice were fed orally with AE crude extract at either 300 or 3000 mg/kg body weight/day for 14 or 28 days (HPV-AE groups). Tumor microvasculature and capillary vascularity were determined using laser scanning confocal microscopy. Tumor tissue was collected from each mouse to evaluate tumor histology and vascular endothelial growth factor (VEGF) immunostaining. Results The time-response curves of AE and the dose-dependent effect of AE on growth inhibition were determined. After a 48-hour incubation period, the IC50 of AE in CaSki was discovered to be significantly different from that of HDFs (P < 0.05). A microvascular network was observed around the tumor area in the HPV group on days 21 and 35. Tumor capillary vascularity in the HPV group was significantly increased compared with the control group (P < 0.001). High-dose treatment of AE extract (HPV-3000AE group) significantly attenuated the increase in VEGF expression and tumor angiogenesis in mice that received either the 14- or 28-day treatment period (P < 0.001). Conclusion Our novel findings demonstrated that AE crude extract could inhibit cervical cancer growth, VEGF expression, and angiogenesis in a CaSki-cell transplant model in mice.

Neuraminidase Activity and Resistance of 2009 Pandemic H1N1 Influenza Virus to Antiviral Activity in Bronchoalveolar Fluid

ABSTRACT Human bronchoalveolar fluid is known to have anti-influenza activity. It is believed to be a frontline innate defense against the virus. Several antiviral factors, including surfactant protein D, are believed to contribute to the activity. The 2009 pandemic H1N1 influenza virus was previously shown to be less sensitive to surfactant protein D. Nevertheless, whether different influenza virus strains have different sensitivities to the overall anti-influenza activity of human bronchoalveolar fluid was not known. We compared the sensitivities of 2009 pandemic H1N1, seasonal H1N1, and seasonal H3N2 influenza virus strains to inhibition by human bronchoalveolar lavage (BAL) fluid. The pandemic and seasonal H1N1 strains showed lower sensitivity to human BAL fluid than the H3N2 strains. The BAL fluid anti-influenza activity could be enhanced by oseltamivir, indicating that the viral neuraminidase (NA) activity could provide resistance to the antiviral defense. In accordance with this finding, the BAL fluid anti-influenza activity was found to be sensitive to sialidase. The oseltamivir resistance mutation H275Y rendered the pandemic H1N1 virus but not the seasonal H1N1 virus more sensitive to BAL fluid. Since only the seasonal H1N1 but not the pandemic H1N1 had compensatory mutations that allowed oseltamivir-resistant strains to maintain NA enzymatic activity and transmission fitness, the resistance to BAL fluid of the drug-resistant seasonal H1N1 virus might play a role in viral fitness. IMPORTANCE Human airway secretion contains anti-influenza activity. Different influenza strains may vary in their susceptibilities to this antiviral activity. Here we show that the 2009 pandemic and seasonal H1N1 influenza viruses were less sensitive to human bronchoalveolar lavage (BAL) fluid than H3N2 seasonal influenza virus. The resistance to the pulmonary innate antiviral activity of the pandemic virus was determined by its neuraminidase (NA) gene, and it was shown that the NA inhibitor resistance mutation H275Y abolished this resistance of the pandemic H1N1 but not the seasonal H1N1 virus, which had compensatory mutations that maintained the fitness of drug-resistant strains. Therefore, the innate respiratory tract defense may be a barrier against NA inhibitor-resistant mutants, and evasion of this defense may play a role in the emergence and spread of drug-resistant strains.

Effects of Tetrahydrocurcumin on Tumor Growth and Cellular Signaling in Cervical Cancer Xenografts in Nude Mice.

Tetrahydrocurcumin (THC) is a stable metabolite of curcumin (CUR) in physiological systems. The mechanism underlying the anticancer effect of THC is not completely understood. In the present study, we investigated the effects of THC on tumor growth and cellular signaling in cervical cancer xenografts in nude mice. Cervical cancer cells (CaSki) were subcutaneously injected in nude mice to establish tumors. One month after the injection, mice were orally administered vehicle or 100, 300, and 500 mg/kg of THC daily for 30 consecutive days. Relative tumor volume (RTV) was measured every 3-4 days. COX-2, EGFR, p-ERK1&2, p-AKT, and Ki-67 expressions were measured by immunohistochemistry whereas cell apoptosis was detected by TUNELS method. THC treatments at the doses of 100, 300, and 500 mg/kg statistically retarded the RTV by 70.40%, 76.41%, and 77.93%, respectively. The CaSki + vehicle group also showed significantly increased COX-2, EGFR, p-ERK1&2, and p-AKT; however they were attenuated by all treatments with THC. Ki-67 overexpression and a decreasing of cell apoptosis were found in CaSki + vehicle group, but these findings were reversed after the THC treatments.

Effects of Tetrahydrocurcumin on Hypoxia-Inducible Factor-1α and Vascular Endothelial Growth Factor Expression in Cervical Cancer Cell-Induced Angiogenesis in Nude Mice

Tetrahydrocurcumin (THC), one of the important in vivo metabolites of curcumin, inhibits tumor angiogenesis. Its effects on angiogenesis in cervical cancer- (CaSki-) implanted nude mice and its mechanisms on hypoxia-inducible factor-1α and vascular endothelial growth factor expression were investigated. Female BALB/c nude mice were divided into control (CON) and CaSki-implanted groups (CaSki group). One month after the injection with cervical cancer cells, mice were orally administered vehicle or 100, 300, and 500 mg/kg of THC daily for 30 consecutive days. The microvascular density (MVD) was evaluated using the CD31 expression. VEGF, VEGFR-2, and HIF-1α expression were also detected by immunohistochemistry. The MVD in CaSki + vehicle group was significantly increased compared to the CON + vehicle group. Interestingly, when treated with THC at all doses, the CaSki group showed a significant smaller number of the MVD. The CaSki + vehicle group also showed significantly increased VEGF, VEGFR-2, and HIF-1α expressions, but they were downregulated when mice were treated with THC at all doses. THC demonstrated an inhibitory effect against tumor angiogenesis in CaSki-implanted nude mice model. This effect is likely to be mediated by the downregulation of HIF-1-α, VEGF expression, and its receptor. THC could be developed into a promising agent for cancer therapy in the future.