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1.
Prep Biochem Biotechnol ; 53(6): 672-682, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36244016

RESUMO

Lipopolysaccharide (LPS), as a stubborn contamination, should be monitored and kept in an acceptable level during the pharmaceutical production process. Recombinant hepatitis B surface antigen (r-HBsAg) is one of the recombinant biological products, which is probable to suffer from extrinsic endotoxin due to its long and complex production process. This research aims to assess the potential interaction between LPS and r-HBsAg by recruiting immunoaffinity chromatography (IAC) as a novel tool to quantify the interaction. Molecular modeling was performed on the HBsAg molecule to theoretically predict its potential binding and interaction sites. Then dynamic light scattering (DLS) analysis was implemented on HBsAg, LPS, and mixtures of them to reveal the interaction. The virus-like particle (VLP) structure of HBsAg and the ribbon-like structure of LPS were visualized by transmission electron microscopy (TEM). Finally, the interaction was quantified by applying various LPS/HBsAg ratios ranging from 1.67 to 120 EU/dose in the IAC. Consequently, the LPS/HBsAg ratios in the eluate were measured from 1.67 to a maximum of 92.5 EU/dose. The results indicated that 77 to 100% of total LPS interacted with HBsAg by an inverse relationship to the incubated LPS concentration. The findings implied that the introduced procedure is remarkably practical in the quantification of LPS interaction with a target recombinant protein.


Assuntos
Cromatografia de Afinidade , Antígenos de Superfície da Hepatite B , Lipopolissacarídeos , Proteínas Recombinantes , Lipopolissacarídeos/análise , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/ultraestrutura , Antígenos de Superfície da Hepatite B/química , Antígenos de Superfície da Hepatite B/genética , Antígenos de Superfície da Hepatite B/isolamento & purificação , Antígenos de Superfície da Hepatite B/ultraestrutura , Microscopia Eletrônica de Transmissão , Vacinas contra Hepatite B/química , Vacinas contra Hepatite B/genética , Vacinas contra Hepatite B/isolamento & purificação , Modelos Químicos , Sequência de Aminoácidos , Difusão Dinâmica da Luz , Cromatografia de Afinidade/métodos
2.
Hepatology ; 75(3): 673-689, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34537985

RESUMO

BACKGROUND AND AIMS: The global burden of viral hepatitis B is substantial, and monitoring infections across the care cascade is important for elimination efforts. There is little information on care disparities by immigration status, and we aimed to quantify disease burden among immigrant subgroups. APPROACH AND RESULTS: In this population-based, retrospective cohort study, we used linked laboratory and health administrative records to describe the HBV care cascade in five distinct stages: (1) lifetime prevalence; (2) diagnosis; (3) engagement with care; (4) treatment initiation; and (5) treatment continuation. Infections were identified based on at least one reactive antigen or nucleic acid test, and lifetime prevalence was estimated as the sum of diagnosed and estimated undiagnosed cases. Care cascades were compared between long-term residents and immigrant groups, including subgroups born in hepatitis B endemic countries. Stratified analyses and multivariable Poisson regression were used to identify drivers for cascade progression. Between January 1997 and December 2014, 2,014,470 persons were included, 50,475 with infections, of whom 30,118 were engaged with care, 11,450 initiated treatment, and 6554 continued treatment >1 year. Lifetime prevalence was estimated as 163,309 (1.34%) overall, 115,722 (3.42%) among all immigrants, and 50,876 (9.37%) among those from highly endemic countries. Compared to long-term residents, immigrants were more likely to be diagnosed (adjusted rate ratio [aRR], 4.55; 95% CI, 4.46, 4.63), engaged with care (aRR, 1.07; 95% CI, 1.04, 1.09), and initiate treatment (aRR, 1.09; 95% CI, 1.03, 1.16). CONCLUSIONS: In conclusion, immigrants fared well compared to long-term residents along the care cascade, having higher rates of diagnosis and slightly better measures in subsequent cascade stages, although intensified screening efforts and better strategies to facilitate linkage to care are still needed.


Assuntos
Continuidade da Assistência ao Paciente/organização & administração , Emigrantes e Imigrantes/estatística & dados numéricos , Antígenos de Superfície da Hepatite B/isolamento & purificação , Antígenos E da Hepatite B/isolamento & purificação , Hepatite B , Programas de Rastreamento , Conduta do Tratamento Medicamentoso/estatística & dados numéricos , Estudos de Coortes , Monitoramento Epidemiológico , Feminino , Necessidades e Demandas de Serviços de Saúde , Hepatite B/diagnóstico , Hepatite B/epidemiologia , Hepatite B/terapia , Humanos , Masculino , Programas de Rastreamento/métodos , Programas de Rastreamento/estatística & dados numéricos , Pessoa de Meia-Idade , Ontário/epidemiologia , Prevalência , Estudos Retrospectivos
3.
Minim Invasive Ther Allied Technol ; 31(5): 728-736, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33853487

RESUMO

INTRODUCTION: Evidence on the biological danger associated with surgical plume is lacking. We examined whether surgical plume, generated by the energy devices ultrasonically activated scalpel (US) or electrocautery (EC) contains virus-related substances. MATERIAL AND METHODS: Experiment 1, ex-vivo model: Tumor mass of a hepatocellular carcinoma line was prepared in a Nod/SCID mouse. Surgical plume generated on the mass by US or EC was collected and detection of HBs gene fragment and antigens (HBsAg or AFP) was conducted. Experiment 2, clinical specimen: Detection of HBV-DNA and HBsAg was conducted following the collection of surgical plume generated from clinically obtained liver specimens from six HBV-associated hepatocellular carcinoma patients. RESULTS: Experiment 1: HBs gene fragment was detected in the solutions regardless of the device used. HBsAg was detected in US and EC solutions and AFP was also detected in a US solution. Experiment 2: HBV-DNA was detected in both devices, in all three cases whose preoperative serum HBV-DNA was positive. In the other serum-negative cases, HBV-DNA was not detected. While serum HBsAg was positive in five of six cases, it was not detected in any solution. CONCLUSIONS: DNA fragments or antigens of virus can exist in the surgical plume generated by EC or US.


Assuntos
Aerossóis , Carcinoma Hepatocelular , Vírus da Hepatite B , Neoplasias Hepáticas , Aerossóis/análise , Animais , Carcinoma Hepatocelular/cirurgia , DNA Viral/isolamento & purificação , Eletrocoagulação , Antígenos de Superfície da Hepatite B/isolamento & purificação , Vírus da Hepatite B/isolamento & purificação , Humanos , Neoplasias Hepáticas/cirurgia , Camundongos , Camundongos SCID , Procedimentos Cirúrgicos Ultrassônicos , alfa-Fetoproteínas
4.
Hepatology ; 74(4): 1750-1765, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-33961298

RESUMO

BACKGROUND AND AIMS: We compared risk of acute liver injury and mortality in patients with COVID-19 and current, past, and no HBV infection. APPROACH AND RESULTS: This was a territory-wide retrospective cohort study in Hong Kong. Patients with COVID-19 between January 23, 2020, and January 1, 2021, were identified. Patients with hepatitis C or no HBsAg results were excluded. The primary outcome was mortality. Acute liver injury was defined as alanine aminotransferase or aspartate aminotransferase ≥2 × upper limit of normal (ULN; i.e., 80 U/L), with total bilirubin ≥2 × ULN (i.e., 2.2 mg/dL) and/or international normalized ratio ≥1.7. Of 5,639 patients included, 353 (6.3%) and 359 (6.4%) had current and past HBV infection, respectively. Compared to patients without known HBV exposure, current HBV-infected patients were older and more likely to have cirrhosis. Past HBV-infected patients were the oldest, and more had diabetes and cardiovascular disease. At a median follow-up of 14 (9-20) days, 138 (2.4%) patients died; acute liver injury occurred in 58 (1.2%), 8 (2.3%), and 11 (3.1%) patients with no, current, and past HBV infection, respectively. Acute liver injury (adjusted HR [aHR], 2.45; 95% CI, 1.52-3.96; P < 0.001), but not current (aHR, 1.29; 95% CI, 0.61-2.70; P = 0.507) or past (aHR, 0.90; 95% CI, 0.56-1.46; P = 0.681) HBV infection, was associated with mortality. Use of corticosteroid, antifungal, ribavirin, or lopinavir-ritonavir (adjusted OR [aOR], 2.55-5.63), but not current (aOR, 1.93; 95% CI, 0.88-4.24; P = 0.102) or past (aOR, 1.25; 95% CI, 0.62-2.55; P = 0.533) HBV infection, was associated with acute liver injury. CONCLUSION: Current or past HBV infections were not associated with more liver injury and mortality in COVID-19.


Assuntos
Lesão Pulmonar Aguda/epidemiologia , COVID-19/mortalidade , Hepatite B Crônica/epidemiologia , Lesão Pulmonar Aguda/sangue , Lesão Pulmonar Aguda/diagnóstico , Lesão Pulmonar Aguda/virologia , Adulto , Fatores Etários , Idoso , Alanina Transaminase , Aspartato Aminotransferases , COVID-19/complicações , COVID-19/diagnóstico , COVID-19/virologia , Feminino , Antígenos de Superfície da Hepatite B/isolamento & purificação , Vírus da Hepatite B/imunologia , Vírus da Hepatite B/isolamento & purificação , Hepatite B Crônica/complicações , Hepatite B Crônica/diagnóstico , Hepatite B Crônica/virologia , Hong Kong/epidemiologia , Humanos , Masculino , Anamnese/estatística & dados numéricos , Pessoa de Meia-Idade , Estudos Retrospectivos , Medição de Risco/estatística & dados numéricos , Fatores de Risco
5.
Mol Biol Rep ; 48(1): 139-146, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33400073

RESUMO

Aptamers as potential alternatives for antibodies could be employed against hepatitis B surface antigen (HBsAg), the great hallmark and first serological marker in HBV, for further theragnostic applications. Therefore, isolation HBsAg specific aptamer was performed in this study with a modified Cell-SELEX method. HEK293T overexpressing HBsAg and HEK293T as target and control cells respectively, were incubated with single-stranded rounds of DNA library during six SELEX and Counter SELEX rounds. Here, we introduced the new modified Cell-SELEX using deoxyribonuclease I digestion to separate single stranded DNA aptamers against the HBsAg. Characterization and evaluation of selected sequences were performed using flow cytometry analysis. The results led to isolation of 15 different ssDNA clones in six rounds of selection which were categorized to four clusters based on common structural motifs. The evaluation of SELEX progress showed growth in aptamer affinity with increasing in the cycle number. Taken together, the application of modified cell-SELEX demonstrated the isolation of HBsAg-specific ssDNA aptamers with proper affinity. Modified cell-SELEX as an efficient method can shorten the selection procedure and increase the success rate while the benefits of cell-based SELEX will be retained. Selected aptamers could be applied in purification columns, diagnostic kits, and drug delivery system against HBV-related liver cancer.


Assuntos
Aptâmeros de Nucleotídeos/farmacologia , Antígenos de Superfície da Hepatite B/isolamento & purificação , Hepatite B/genética , Neoplasias Hepáticas/tratamento farmacológico , Técnica de Seleção de Aptâmeros , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/farmacologia , Desoxirribonuclease I/genética , Sistemas de Liberação de Medicamentos , Citometria de Fluxo , Células HEK293 , Hepatite B/imunologia , Hepatite B/virologia , Antígenos de Superfície da Hepatite B/genética , Antígenos de Superfície da Hepatite B/imunologia , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/patologia
6.
Methods Mol Biol ; 2183: 83-94, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32959242

RESUMO

The small (S) envelope protein of the Hepatitis B Virus (HBV), HBV-S, has the unique ability to self-assemble into highly immunogenic subviral particles (SVPs), in the absence of other viral factors, in eukaryotic cells, including those of nonhepatic origin. This feature is currently exploited for generation of SVPs exposing heterologous epitopes on their surface that can be used as vaccine candidates to target various diseases. Here, we describe a simple and robust method for production of such chimeric HBV-S protein-based SVPs in transiently transfected HEK293T cells and purification from cell supernatants by ultracentrifugation on sucrose cushion and sucrose step gradients. The SVPs obtained by this methodology have been successfully used in immunogenicity studies in animal models.


Assuntos
Técnicas de Cultura Celular por Lotes , Antígenos de Superfície da Hepatite B/biossíntese , Proteínas Recombinantes de Fusão , Animais , Técnicas de Cultura de Células , Expressão Gênica , Células HEK293 , Antígenos de Superfície da Hepatite B/genética , Antígenos de Superfície da Hepatite B/imunologia , Antígenos de Superfície da Hepatite B/isolamento & purificação , Vacinas contra Hepatite B/biossíntese , Vacinas contra Hepatite B/imunologia , Humanos , Transfecção
7.
Am J Trop Med Hyg ; 104(3): 1101-1105, 2020 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-33350368

RESUMO

In Pakistan, viral hepatitis is a serious public health problem affecting millions of people. Both hepatitis B and hepatitis C infections are spreading rapidly in all provinces of Pakistan, including Sindh, because of lack of knowledge about routes of transmission, low literacy rate, reuse of syringes, piercing, and other factors. However, information about the prevalence and risk factors is inadequate. So, a general population-based study was conducted to determine the prevalence rate and risk factors of hepatitis B and hepatitis C in Nawabshah. Healthy individuals were screened for hepatitis B and hepatitis C using an immunochromatographic rapid test followed by confirmation through ELISA and PCR. Information about sociodemographic and risk factors was obtained through a pretested questionnaire. Descriptive frequencies, odds ratio, and CI were calculated using SPSS software version 23 (IBM Corp, Armonk, NY). In total, 523 participants were screened for hepatitis B and hepatitis C, among whom 232 were females and 291 were males. The overall prevalence of hepatitis C and hepatitis B was 14.3% and 6.7%, respectively. In a bivariate analysis, hepatitis B infection was significantly associated with risk factors such as hospitalization, blood transfusion, needle injury, multiple sex partners, reused syringe, dental extraction, surgery, injectable drug abuse, and shaving at barbershops. Hepatitis C infection was associated with factors including surgery, needle injury, blood transfusion, reused syringes, dental extraction, and shaving at barbershops. The increasing prevalence of hepatitis B surface antigen and hepatitis C virus in Nawabshah is a public health concern. There is dire need to implement preventive measures.


Assuntos
Hepatite B/epidemiologia , Hepatite C/epidemiologia , Adulto , Feminino , Hepacivirus/isolamento & purificação , Antígenos de Superfície da Hepatite B/sangue , Antígenos de Superfície da Hepatite B/isolamento & purificação , Humanos , Masculino , Pessoa de Meia-Idade , Paquistão/epidemiologia , Prevalência , Fatores de Risco
8.
STAR Protoc ; 1(3): 100129, 2020 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-33377023

RESUMO

The isolation of human antibodies with naturally paired heavy and light chains is crucial for understanding the human antibody immune response. Here, we present a protocol for antibody cloning from the sorted single human memory B cells recognizing hepatitis B virus (HBV) S antigen (HBsAg). A two-fluorescent-dye labeling strategy against HBsAg allows for an improved sorting specificity, while non-relevant protein staining allows for the exclusion of non-specific B cells. This protocol could also be widely adapted for other antigens. For complete details on the use and execution of this protocol, please refer to Wang et al. (2020).


Assuntos
Citometria de Fluxo/métodos , Células B de Memória/citologia , Linfócitos B/imunologia , Clonagem Molecular/métodos , Hepatite B/imunologia , Anticorpos Anti-Hepatite B/genética , Antígenos de Superfície da Hepatite B/imunologia , Antígenos de Superfície da Hepatite B/isolamento & purificação , Antígenos de Superfície da Hepatite B/metabolismo , Humanos , Imunoglobulinas/metabolismo , Leucócitos Mononucleares/citologia
9.
Cancer Control ; 27(1): 1073274820976594, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33297765

RESUMO

Hepatitis B reactivation (HBVr) in cancer patients is a well-established complication due to chemotherapy-induced immunosuppression. Studies have reported HBVr associated with immunosuppressive medications, such as rituximab, methotrexate, and high dose steroids. There are different risks for different types of chemotherapy with rituximab carrying one of the highest risks for hepatitis B reactivation. Tyrosine kinase inhibitors (TKIs) are the standard of care in patients with chronic myeloid leukemia (CML). The risk of HBVr in chronic myeloid leukemia has been reported in many studies, but to this date, there are no clear guidelines or recommendations regarding screening and monitoring of HBV in CML patients receiving TKIs. We conducted this review to identify the risk of HBVr in patients with CML who are treated with tyrosine kinase inhibitors. We recommend testing for HBV status in patients who are to be treated with TKIs and to consider giving prophylaxis in those who are positive for HBsAg at baseline. More studies are needed to assess the risk of reactivation in patients with Hepatitis B core antibody positive receiving TKIs. Currently, monitoring such patients for reactivation may be the best strategy.


Assuntos
Vírus da Hepatite B/isolamento & purificação , Hepatite B Crônica/diagnóstico , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Inibidores de Proteínas Quinases/efeitos adversos , Ativação Viral/imunologia , Antibioticoprofilaxia/métodos , Antibioticoprofilaxia/normas , Antivirais/uso terapêutico , Monitoramento de Medicamentos/normas , Antígenos de Superfície da Hepatite B/imunologia , Antígenos de Superfície da Hepatite B/isolamento & purificação , Vírus da Hepatite B/fisiologia , Hepatite B Crônica/imunologia , Hepatite B Crônica/prevenção & controle , Hepatite B Crônica/virologia , Humanos , Hospedeiro Imunocomprometido , Leucemia Mielogênica Crônica BCR-ABL Positiva/imunologia , Programas de Rastreamento/normas , Programas de Rastreamento/estatística & dados numéricos , Guias de Prática Clínica como Assunto , Ativação Viral/efeitos dos fármacos
10.
World J Gastroenterol ; 26(20): 2645-2656, 2020 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-32523317

RESUMO

BACKGROUND: Recent evidence has indicated the role of B cells and B cell-activating factor (BAFF) in the development of hepatocellular carcinoma (HCC). AIM: To characterize circulating BAFF receptor expression and B cell subpopulations in patients with hepatitis B virus (HBV)-related HCC. METHODS: Peripheral blood samples collected from 41 patients with chronic HBV infection (25 patients without HCC and 16 patients with HCC) and 9 healthy controls were assessed for BAFF receptors [BAFF-R(B cell-activating factor receptor), transmembrane activator and cyclophilin ligand interactor, B-cell maturation antigen] and B cell subpopulations by multicolor flow cytometry. RESULTS: The frequency of BAFF-R expressing B cells to total B cells was significantly lower in patients with HCC (3.39% ± 2.12%) compared with the non-HCC group (5.37% ± 1.90%) and healthy controls (6.23% ± 2.32%), whereas there was no difference in transmembrane activator and cyclophilin ligand interactor and B-cell maturation antigen. The frequencies of CD27+IgD+ memory B cells, CD27+IgD- class-switched memory B cells and plasmablasts were significantly lower in the patients with HCC compared to patients without HCC (1.23 ± 1.17 vs 3.09 ± 1.55, P = 0.001, 0.60 ± 0.44 vs 1.69 ± 0.86, P < 0.0001 and 0.16 ± 0.12 vs 0.37 ± 0.30, P = 0.014, respectively). However, the ratio of naïve and transitional B cell did not differ significantly between the three groups. In addition, decreased BAFF-R expression on B cells was significantly correlated with large tumor size and advanced tumor stage. CONCLUSION: Our data demonstrated BAFF-R expression was reduced in B cells that involved with the frequencies of B cells maturation in patients with HCC. The depletion of BAFF-R might play an important role in the development of HCC in patients with chronic HBV infection.


Assuntos
Receptor do Fator Ativador de Células B/metabolismo , Linfócitos B/imunologia , Carcinoma Hepatocelular/imunologia , Hepatite B Crônica/patologia , Neoplasias Hepáticas/imunologia , Adulto , Idoso , Linfócitos B/metabolismo , Biópsia , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , Feminino , Voluntários Saudáveis , Antígenos de Superfície da Hepatite B/isolamento & purificação , Vírus da Hepatite B/imunologia , Vírus da Hepatite B/isolamento & purificação , Hepatite B Crônica/diagnóstico , Hepatite B Crônica/imunologia , Hepatite B Crônica/virologia , Humanos , Fígado/imunologia , Fígado/patologia , Fígado/virologia , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Adulto Jovem
11.
J Infect Dis ; 221(2): 243-250, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31504660

RESUMO

BACKGROUND: With the nation's focus on the opioid crisis, methamphetamine has made a comeback, potentially increasing risk for hepatitis B. We examined factors associated with hepatitis B virus (HBV) exposure among people who reported ever using methamphetamine in a nationally representative survey. METHODS: We used the National Health and Nutrition Examination Survey to examine factors associated with HBV exposure among participants who reported ever using methamphetamine using bivariate and multivariable logistic regression. RESULTS: Overall, 847 participants met the study inclusion criteria. In multivariable logistic regression, female sex (adjusted odds ratio, 3.83; 95% confidence interval, 1.65-8.90), living below the poverty threshold (3.17; 1.39-7.21), injection drug use (4.89; 1.95-12.26), active hepatitis C virus infection (3.39; 1.10-12.26), and identifying as men who have sex with men (28.21; 5.19-153.38) were significantly associated with HBV exposure. CONCLUSIONS: The odds of HBV exposure for female participants who reported using methamphetamine were 4 times than that for male participants. Poverty, injection drug use, and hepatitis C virus infection were also associated. As methamphetamine use increases, it is critical to identify those at risk of acquiring HBV infections in order to target testing and vaccination.


Assuntos
Estimulantes do Sistema Nervoso Central/administração & dosagem , Hepatite B/transmissão , Metanfetamina/administração & dosagem , Adolescente , Adulto , Idoso , Estudos Transversais , Feminino , Inquéritos Epidemiológicos , Hepatite B/epidemiologia , Antígenos de Superfície da Hepatite B/isolamento & purificação , Humanos , Drogas Ilícitas , Masculino , Pessoa de Meia-Idade , Medição de Risco , Adulto Jovem
13.
Zhonghua Liu Xing Bing Xue Za Zhi ; 40(7): 854-858, 2019 Jul 10.
Artigo em Chinês | MEDLINE | ID: mdl-31357811

RESUMO

Hepatitis B virus (HBV) infection is one of the important public health problems. In order to eliminate the threat of viral hepatitis to public health, World Health Organization has proposed to achieve the goal of reducing the HBsAg positive rate in children to 0.1% by 2030. There are regional differences in HBsAg positive rates among pregnant women worldwide, from 0.6% in areas with low prevalence to 20% in areas with high prevalence. The positive rate of HBsAg in pregnant women in China is higher than global average. In recent years, several studies have found that HBV infection in pregnancy not only increase the risk of mother-to-child transmission, but also adversely affect the health of pregnant women and their children. Therefore, this paper summarizes the progress in research both at home and abroad in terms of HBsAg positive rate in pregnant women and the influence of pregnancy complicated with HBV infection on pregnant women and perinatal infants in order to explore the effect of pregnancy complicated with HBV infection on maternal and infant safety.


Assuntos
Hepatite B , Complicações Infecciosas na Gravidez , China , Feminino , Antígenos de Superfície da Hepatite B/isolamento & purificação , Humanos , Lactente , Transmissão Vertical de Doenças Infecciosas , Gravidez , Risco
14.
Indian J Med Res ; 149(3): 389-395, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-31249205

RESUMO

Background & objectives: : Nucleic acid amplification test (NAT) in blood donor screening not only detects window period (WP) donors but also those with chronic occult infections which are negative by routine serological screening. This study was conducted to determine the time trend of NAT positivity and seroprevalence of transfusion-transmitted infections (TTIs) through a period of six years and evaluate the strength of NAT as a supplementary test in identifying the cryptic carriers in blood donor population. Methods: : A total of 1,01,411 blood donations were screened between January 2011 and December 2016 by the ELISA and individual donor (ID) NAT Procleix Ultrio Plus Assay. Additional molecular and serological assays were done on the NAT yield samples to differentiate the type of cryptic carriers. Results: : NAT yields comprised 0.05 per cent (50/101411) of the total samples tested with a yield rate of 1/2028. Hepatitis B virus (HBV) contributed to 80 per cent of the total NAT yields and the rest 20 per cent due to hepatitis C virus (HCV). Majority of HBV NAT yields (75%) were from chronic occult donors and 25 per cent were WP donors. Both HBV and HCV NAT yields had a wide range of viral count. There was no HIV NAT yield. A significant decline in the prevalence rate of TTIs through the study period of six years was observed. Interpretation & conclusions: : The cryptic infections found in blood donors increase the risk of TTIs. Blood screening by both serology and NAT can reduce this threat.


Assuntos
Antígenos de Superfície da Hepatite B/sangue , Técnicas de Amplificação de Ácido Nucleico/métodos , Estudos Soroepidemiológicos , Reação Transfusional/sangue , Adulto , Povo Asiático , Doadores de Sangue , Segurança do Sangue , DNA Viral/genética , DNA Viral/isolamento & purificação , Feminino , Hepacivirus/genética , Hepacivirus/isolamento & purificação , Hepacivirus/patogenicidade , Hepatite B/sangue , Hepatite B/epidemiologia , Hepatite B/virologia , Antígenos de Superfície da Hepatite B/isolamento & purificação , Vírus da Hepatite B/genética , Vírus da Hepatite B/isolamento & purificação , Vírus da Hepatite B/patogenicidade , Hepatite C/sangue , Hepatite C/epidemiologia , Hepatite C/virologia , Humanos , Índia/epidemiologia , Masculino , Reação Transfusional/epidemiologia , Reação Transfusional/genética , Reação Transfusional/virologia
15.
Prep Biochem Biotechnol ; 49(8): 813-821, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31169457

RESUMO

Separation of biomass from culture media by centrifugation and then washing the biomass are mandatory steps in the fermentation process of recombinant Pichia pastoris expressed HBsAg intracellularly. Biomass has to be washed many times to eliminate the culture media residues thoroughly. In this study, we tried to develop the hydrocyclone as an alternative method for separation of biomass from fermentation culture, an attractive replacement for centrifugation processes. The advantages of using hydrocyclone in biomass separation could be summarized in its suitability for continuous separation and its low risk of contamination. To evaluate the performance of hydrocyclone, concentration ratio in underflow to feed stream, capacity, and centrifugal force by considering three parameters of pressure drop, concentration, and the type of hydrocyclone were investigated. Using three level factorial design a concentration ratio equation was developed, with the correlation coefficient R2 = 0.977 ensured the good fitness of the predicted data with the experimental results. In optimal conditions, maximum concentration ratio was 1.246, for flow rate 13.5 LPM and C-force equal to 1276.11 at maximum pressure drop (3 bar) and minimum concentration (0.5% w/w) in hydrocyclone 1. Herein, two different hydrocyclones with the cylindrical diameters of 19 mm and 21 mm were used for separating the yeast cells.


Assuntos
Centrifugação/instrumentação , Meios de Cultura/química , Antígenos de Superfície da Hepatite B/isolamento & purificação , Pichia/química , Técnicas de Cultura Celular por Lotes/instrumentação , Biomassa , Desenho de Equipamento , Fermentação , Pressão , Proteínas Recombinantes/isolamento & purificação
16.
Anal Chim Acta ; 1073: 30-38, 2019 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-31146833

RESUMO

Highly N-doped SiC was presented as an optimal electrode for electrochemical immunoassays with a far higher sensitivity than chemiluminescence detection. As the first step, the electrochemical properties of highly N-doped SiC, such as the double-layer capacitance (Cdl), rate constant for electron transfer (kapp) and ideal polarizable potential range (electrochemical window) were analyzed and compared with those of Au, Pt, and graphite electrodes. The highly N-doped SiC electrode was used for the quantification of oxidized 3,3',5,5'-tetramethylbenzidine (TMB) which was widely used as chromogenic substrate for commercialized immunoassay kits. In order to enhance the sensitivity for the quantification of the oxidized TMB the chronoamperometry was applied to avoid the background current of i-V measurement. Finally, the chronoamperometry based on the highly N-doped SiC electrode was applied to commercial immunoassay kits for the medical diagnosis of the human immunodeficiency virus (HIV) and the human hepatitis B surface antigen (hHBsAg). The chronoamperometric measurement based on the highly N-doped SiC electrode was proved to detect at far lower limits in comparison with the conventional optical density measurement as well as the chemiluminescence assay based on luminol as a chemiluminescent probe.


Assuntos
Benzidinas/análise , Técnicas Biossensoriais , Compostos Inorgânicos de Carbono/química , Técnicas Eletroquímicas , HIV/isolamento & purificação , Antígenos de Superfície da Hepatite B/isolamento & purificação , Imunoensaio , Compostos de Silício/química , Eletrodos , Humanos
17.
Artigo em Inglês | MEDLINE | ID: mdl-30877983

RESUMO

Production of recombinant HBsAg as a main component of the hepatitis B vaccine has already been established in commercial scale. So far, many studies have been performed to optimize the production process of this recombinant vaccine. However, still aggregation and dissociation of rHBsAg virus-like particles (VLPs) are major challenges in downstream processing of this biomedicine. The structural diversity of rHBsAg is dependent on many factors including cell types, molecular characteristics of the expressed recombinant rHBsAg, buffer composition as well as operation condition and specific characteristics of each downstream processing unit. Hence, it is not relatively easy to implement a single strategy to prevent aggregation formation in already established rHBsAg production processes. In this study, we examined the efficacy of weak anion exchange chromatography (IEC)- packed with DEAE Sepharose Fast Flow medium- on isolation of rHBsAg VLPs from aggregated structures. For this purpose, the influence of ionic strength of elution buffer as a key factor was investigated in isolation and recovery of rHBsAg VLPs. The elution buffer with electrical conductivity between 27 and 31 mS/cm showed the best results for removing aggregated rHBsAg based on SEC-HPLC analysis. The results showed that in the selected conductivity range, about 79% of rHBsAg was recovered with purity above 95%. The percentage of rHBsAg VLPs in the recovered sample was between 94% and 97.5% indicating that we could obtain highly homogeneous rHBsAg within the acceptable quality level. The TEM, SDS-PAGE and western blot analysis were also in agreement with our quantitative measurements.


Assuntos
Cromatografia por Troca Iônica/métodos , Antígenos de Superfície da Hepatite B/isolamento & purificação , Vacinas contra Hepatite B/química , Proteínas Recombinantes/isolamento & purificação , Vacinas Sintéticas/química , Animais , Células CHO , Cromatografia em Gel , Cricetinae , Cricetulus , Escherichia coli , Antígenos de Superfície da Hepatite B/química , Pichia , Estabilidade Proteica , Proteínas Recombinantes/química , Vírion/isolamento & purificação
18.
PLoS One ; 14(2): e0212800, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30794666

RESUMO

This study describes the comparative expression and purification of hepatitis B surface antigen (HBsAg) particles produced upon infection of human primary hepatocytes and human hepatoma cell lines (HuH-7 and HepG2) with recombinant vaccinia viruses. The highest levels of HBsAg expression were found in HuH-7 hepatoma cells following infection with recombinant vaccinia viruses, which contain the S gene under control of a 7.5 k-promoter. Four different methods for purification of the HBsAg particles were examined: isopycnic ultracentrifugation, sucrose cushion sedimentation, isocratic column gel filtration, and binding to anti-HBs-coated microparticles. The highest degree of purity of HBsAg particles was reached by the method based on anti-HBs-coated microparticles. The resulting product was >98% pure. Biochemical analysis and characterization of purified HBsAg particles were performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), western blotting, and electron microscopy. The HBsAg, purified from human hepatoma cell lines and from human primary hepatocytes, consisted of both the non-glycosylated (p25) and the glycosylated (gp27) form and assembled into typical 22-nm particles, and thus may be of great interest and importance for research, diagnostics, and medical treatments.


Assuntos
Carcinoma Hepatocelular/metabolismo , Antígenos de Superfície da Hepatite B , Vírus da Hepatite B/genética , Hepatócitos/metabolismo , Neoplasias Hepáticas/metabolismo , Vacinas de Partículas Semelhantes a Vírus , Vaccinia virus/genética , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Homólogo 5 da Proteína Cromobox , Glicosilação , Antígenos de Superfície da Hepatite B/biossíntese , Antígenos de Superfície da Hepatite B/química , Antígenos de Superfície da Hepatite B/genética , Antígenos de Superfície da Hepatite B/isolamento & purificação , Vírus da Hepatite B/metabolismo , Humanos , Neoplasias Hepáticas/genética , Tamanho da Partícula , Vacinas de Partículas Semelhantes a Vírus/química , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas de Partículas Semelhantes a Vírus/isolamento & purificação , Vacinas de Partículas Semelhantes a Vírus/metabolismo , Vaccinia virus/metabolismo
19.
Prep Biochem Biotechnol ; 49(2): 158-166, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30636487

RESUMO

In purification process of recombinant hepatitis B surface antigen (rHBsAg), immunoaffinity chromatography (IAF) is one of the most important and effective steps in rHBsAg purification. However, the buffer composition and the interaction of ligands-rHBsAg often lead to disassembly, deformation, and clumping of a portion of these virus-like particles (VLPs). Besides, the expensive media, variable biospecific ligand density and the possibility of product contamination are other reported drawbacks of using IAF which makes the production process of rHBsAg more challenging. This study investigated the possibility of substituting IAF with purification methods of size-exclusion chromatography (SEC) and ultracentrifugation. In the SEC, the efficacy of rHBsAg purification was examined by four different media in which Toyopearl HW 65S resin demonstrated the best results. By integrating Toyopearl HW 65S resin - with a bed height of 51 cm - and ultracentrifugation process at 47,000 rpm for 48 hr, 95% of protein impurities were removed. Compared to the IAF in rHBsAg production, the purified sample contained a higher percentage of multimeric rHBsAg particles without any noticeable monomer and aggregate forms. The result of this study indicates that the proposed integrated system could be an efficient mild purification alternative for conventional IAF.


Assuntos
Cromatografia em Gel/métodos , Antígenos de Superfície da Hepatite B/isolamento & purificação , Ultracentrifugação/métodos , Cromatografia de Afinidade , Hepatite B/virologia , Vírus da Hepatite B/química , Humanos , Pichia/química , Proteínas Recombinantes/isolamento & purificação
20.
Public Health ; 168: 36-42, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30685596

RESUMO

OBJECTIVE: The objective of the study is to evaluate the pattern and predictors of medical care received by hepatitis B virus (HBV) carriers during pregnancy and after delivery in Hong Kong. STUDY DESIGN: The study is a retrospective analysis. METHODS: Pregnant HBV carriers and their infants were followed up for 9-12 months after delivery. Face-to-face interviews were conducted to investigate what medical care they received for HBV before, during and after pregnancy. RESULTS: Data were available for 412 HBV carriers. A total of 375 (91.0%) women were known HBV carriers before pregnancy. Routine antenatal screening picked out the remaining 37 (9.0%) HBV carriers; these women were younger, more likely to be smokers and had a lower level of education (P < 0.05) than known HBV carriers. In total, 356 of 412 (86.4%) HBV carriers did not receive any medical care for HBV during pregnancy. Known HBV carrier status, history of medical check-up and the use of antiviral treatment before pregnancy were significant predictors for HBV medical care during pregnancy (P < 0.05). The results show that 217 of 412 (52.6%) HBV carriers did not receive medical care for HBV after delivery. HBV medical care before pregnancy, use of antiviral treatment before pregnancy and a higher level of education were significant predictors for postpartum HBV medical care (P < 0.05). Multivariate analysis showed that HBV medical care before pregnancy (odds ratio [OR], 7.73; 95% confidence interval [CI], 3.21-18.65; P < 0.001) and the use of antiviral treatment (OR, 5.02; 95% CI, 1.41-17.81; P = 0.013) were associated with medical care during pregnancy. Medical care before pregnancy was also associated with postpartum HBV medical care (OR, 5.05; 95% CI, 3.29-7.51; P < 0.001). CONCLUSIONS: A significant proportion of HBV carriers did not receive HBV-related medical check-ups during and after pregnancy in Hong Kong despite the majority being aware of their carrier status. Medical care before pregnancy predicted antenatal and postpartum HBV medical care.


Assuntos
Antivirais/uso terapêutico , Portador Sadio/tratamento farmacológico , Hepatite B/tratamento farmacológico , Transmissão Vertical de Doenças Infecciosas/prevenção & controle , Cuidado Pós-Natal/estatística & dados numéricos , Complicações Infecciosas na Gravidez/tratamento farmacológico , Cuidado Pré-Natal/estatística & dados numéricos , Adulto , Feminino , Antígenos de Superfície da Hepatite B/isolamento & purificação , Hong Kong , Humanos , Lactente , Gravidez , Estudos Retrospectivos
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