Search results for: polyclonal antibody

Authors: Evgeny A. Ermakov, Lyudmila P. Smirnova, Valentina N. Buneva

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Schizophrenia associated with dysregulation of neurotransmitter processes in the central nervous system and disturbances in the humoral immune system resulting in the formation of antibodies (Abs) to the various components of the nervous tissue. Abs to different neuronal receptors and DNA were detected in the blood of patients with schizophrenia. Abs hydrolyzing DNA were detected in pool of polyclonal autoantibodies in autoimmune and infectious diseases, such catalytic Abs were named abzymes. It is believed that DNA-hydrolyzing abzymes are cytotoxic, cause nuclear DNA fragmentation and induce cell death by apoptosis. Abzymes with DNAase activity are interesting because of the mechanism of formation and the possibility of use as diagnostic markers. Therefore, in our work we have set following goals: to determine the level anti-DNA Abs in the serum of patients with schizophrenia and to study DNA-hydrolyzing activity of IgG of patients with schizophrenia. Materials and methods: In our study there were included 41 patients with a verified diagnosis of paranoid or simple schizophrenia and 24 healthy donors. Electrophoretically and immunologically homogeneous IgGs were obtained by sequential affinity chromatography of the serum proteins on protein G-Sepharose and gel filtration. The levels of anti-DNA Abs were determined using ELISA. DNA-hydrolyzing activity was detected as the level of supercoiled pBluescript DNA transition in circular and linear forms, the hydrolysis products were analyzed by agarose electrophoresis followed by ethidium bromide stain. To correspond the registered catalytic activity directly to the antibodies we carried out a number of strict criteria: electrophoretic homogeneity of the antibodies, gel filtration (acid shock analysis) and in situ activity. Statistical analysis was performed in ‘Statistica 9.0’ using the non-parametric Mann-Whitney test. Results: The sera of approximately 30% of schizophrenia patients displayed a higher level of Abs interacting with single-stranded (ssDNA) and double-stranded DNA (dsDNA) compared with healthy donors. The average level of Abs interacting with ssDNA was only 1.1-fold lower than that for interacting with dsDNA. IgG of patient with schizophrenia were shown to possess DNA hydrolyzing activity. Using affinity chromatography, electrophoretic analysis of isolated IgG homogeneity, gel filtration in acid shock conditions and in situ DNAse activity analysis we proved that the observed activity is intrinsic property of studied antibodies. We have shown that the relative DNAase activity of IgG in patients with schizophrenia averaged 55.4±32.5%, IgG of healthy donors showed much lower activity (average of 9.1±6.5%). It should be noted that DNAase activity of IgG in patients with schizophrenia with a negative symptoms was significantly higher (73.3±23.8%), than in patients with positive symptoms (43.3±33.1%). Conclusion: Anti-DNA Abs of patients with schizophrenia not only bind DNA, but quite efficiently hydrolyze the substrate. The data show a correlation with the level of DNase activity and leading symptoms of patients with schizophrenia.

Keywords: anti-DNA antibodies, abzymes, DNA hydrolysis, schizophrenia

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Authors: Lavinia Ruta, Ileana Farcasanu

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The yeast surface display (YSD) technique enables the expression of proteins on yeast cell surfaces, facilitating the identification and isolation of proteins with targeted binding properties, such as nanobodies. Nanobodies, derived from camelid species, are single-domain antibody fragments renowned for their high affinity and specificity towards target proteins, making them valuable in research and potentially in therapeutics. Their advantages include a compact size (~15 kDa), robust stability, and the ability to target challenging epitopes. The project endeavors to establish and validate a platform for producing Green Fluorescent Protein (GFP) and mCherry nanobodies using the yeast surface display method. mCherry, a prevalent red fluorescent protein sourced from coral species, is commonly utilized as a genetic marker in biological studies due to its vibrant red fluorescence. The GFP-nanobody, a single variable domain of heavy-chain antibodies (VHH), exhibits specific binding to GFP, offering a potent means for isolating and engineering fluorescent protein fusions across various biological research domains. Both GFP and mCherry nanobodies find specific utility in cellular imaging and protein analysis applications.

Keywords: YSD, nanobodies, GFP, Saccharomyces cerevisiae

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Authors: Abdolreza Esmaeilzadeh, Maryam Erfanmanesh, Sousan Ghasemi, Farzaneh Mohammadi

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Background: Hepatitis C Virus (HCV), a public health problem, is an enveloped, single-stranded RNA virus and a member of the Hepacivirus genus of the Flaviviridae family. Liver cancer, cirrhosis, and end-stage liver are the outcomes of chronic infection with HCV. HCV isolates show significant heterogeneity in genetics around the world. Therefore, determining HCV genotypes is a vital step in determining prognosis and planning therapeutic strategies. Materials and Methods: Serum samples of 136 patients were collected and analyzed for anti-HCV antibodies using ELISA (The enzyme-linked immunosorbent assay) method. Then, positive samples were exposed to RT-PCR, which was performed under standard condition. Afterwards, they investigated for genotyping using allele-specific PCR (AS-PCR), and HCV genotype 2.0 line probe assay (LiPA). Results: Samples indicated 216 bp bands on 2% agarose gel. Analyses of the results demonstrated that the most dominant subtype was 3a with frequency of 38.26% in Zanjan Province followed by subtypes of 1b, 1a, 2, and 4 with frequencies of 25.73%, 22.05%, 5.14%, and 4.41%, respectively. The frequency of unknown HCV genotypes was 4.41%. Conclusions: According to the results, it was found that HCV high prevalent genotype in Zanjan is subtype 3a. Analysis of the results provides identification of certain HCV genotypes, and these valuable findings could affect the type and duration of the treatment.

Keywords: anti-HCV antibody, Hepatitis C Virus (HCV), genotype, RT-PCR, AS-PCR

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Authors: Natalia Caldeira De Carvalho, Tassia Batista Pessato, Luis Gustavo R. Fernandes, Ricardo L. Zollner, Flavia Maria Netto

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Protein hydrolysates are ingredients of enteral diets and hypoallergenic formulas. Enzymatic hydrolysis is the most commonly used method for reducing the antigenicity of milk protein. The antigenicity and physicochemical characteristics of the protein hydrolysates depend on the reaction parameters. Among them, pH has been pointed out as of the major importance. Hydrolysis reaction in laboratory scale is commonly carried out under controlled pH (pH-stat). However, from the industrial point of view, controlling pH during hydrolysis reaction may be infeasible. This study evaluated the impact of pH control on the physicochemical properties and antigenicity of the hydrolysates of whey proteins with Alcalase. Whey protein isolate (WPI) solutions containing 3 and 7 % protein (w/v) were hydrolyzed with Alcalase 50 and 100 U g-1 protein at 60°C for 180 min. The reactions were carried out under controlled and uncontrolled pH conditions. Hydrolyses performed under controlled pH (pH-stat) were initially adjusted and maintained at pH 8.5. Hydrolyses carried out without pH control were initially adjusted to pH 8.5. Degree of hydrolysis (DH) was determined by OPA method, peptides profile was evaluated by HPLC-RP, and molecular mass distribution by SDS-PAGE/Tricine. The residual α-lactalbumin (α-La) and β-lactoglobulin (β-Lg) concentrations were determined using commercial ELISA kits. The specific IgE and IgG binding capacity of hydrolysates was evaluated by ELISA technique, using polyclonal antibodies obtained by immunization of female BALB/c mice with α-La, β-Lg and BSA. In hydrolysis under uncontrolled pH, the pH dropped from 8.5 to 7.0 during the first 15 min, remaining constant throughout the process. No significant difference was observed between the DH of the hydrolysates obtained under controlled and uncontrolled pH conditions. Although all hydrolysates showed hydrophilic character and low molecular mass peptides, hydrolysates obtained with and without pH control exhibited different chromatographic profiles. Hydrolysis under uncontrolled pH released, predominantly, peptides between 3.5 and 6.5 kDa, while hydrolysis under controlled pH released peptides smaller than 3.5 kDa. Hydrolysis with Alcalase under all conditions studied decreased by 99.9% the α-La and β-Lg concentrations in the hydrolysates detected by commercial kits. In general, β-Lg concentrations detected in the hydrolysates obtained under uncontrolled pH were significantly higher (p<0.05) than those detected in hydrolysates produced with pH control. The anti-α-La and anti-β-Lg IgE and IgG responses to all hydrolysates decreased significantly compared to WPI. Levels of specific IgE and IgG to the hydrolysates were below 25 and 12 ng ml-1, respectively. Despite the differences in peptide composition and α-La and β-Lg concentrations, no significant difference was found between IgE and IgG binding capacity of hydrolysates obtained with or without pH control. These results highlight the impact of pH on the hydrolysates characteristics and their concentrations of antigenic protein. Divergence between the antigen detection by commercial ELISA kits and specific IgE and IgG binding response was found in this study. This result shows that lower protein detection does not imply in lower protein antigenicity. Thus, the use of commercial kits for allergen contamination analysis should be cautious.

Keywords: allergy, enzymatic hydrolysis, milk protein, pH conditions, physicochemical characteristics

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Authors: Itzel Amaro-Estrada, Eduardo Vergara-Rivera, Virginia Juarez-Flores, Mayra Cobaxin-Cardenas, Rosa Estela Quiroz, Jesus F. Preciado, Sergio Rodriguez-Camarillo

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Bovine anaplasmosis is an infectious, tick-borne disease caused mainly by Anaplasma marginale; typical signs include anemia, fever, abortion, weight loss, decreased milk production, jaundice, and potentially death. Sick bovine can recover when antibiotics are administered; however, it usually remains as carrier for life, being a risk of infection for susceptible cattle. Anaplasma marginale is an obligate intracellular Gram-negative bacterium with genetic composition highly diverse among geographical isolates. There are currently no vaccines fully effective against bovine anaplasmosis; therefore, the economic losses due to disease are present. Vaccine formulation became a hard task for several pathogens as Anaplasma marginale, but peptide-based vaccines are an interesting proposal way to induce specific responses. Phage-displayed peptide libraries have been proved one of the most powerful technologies for identifying specific ligands. Screening of these peptides libraries is also a tool for studying interactions between proteins or peptides. Thus, it has allowed the identification of ligands recognized by polyclonal antiserums, and it has been successful for the identification of relevant epitopes in chronic diseases and toxicological conditions. Protective immune response to bovine anaplasmosis includes high levels of immunoglobulins subclass G2 (IgG2) but not subclass IgG1. Therefore, IgG2 from the serum of protected bovine can be useful to identify ligands, which can be part of an immunogen for cattle. In this work, phage display random peptide library Ph.D. ™ -12 was incubating with IgG2 or blood sera of immunized bovines against A. marginale as targets. After three rounds of biopanning, several candidates were selected for additional analysis. Subsequently, their reactivity with sera immunized against A. marginale, as well as with positive and negative sera to A. marginale was evaluated by immunoassays. A collection of recognized peptides tested by ELISA was generated. More than three hundred phage-peptides were separately evaluated against molecules which were used during panning. At least ten different peptides sequences were determined from their nucleotide composition. In this approach, three phage-peptides were selected by their binding and affinity properties. In the case of the development of vaccines or diagnostic reagents, it is important to evaluate the immunogenic and antigenic properties of the peptides. Immunogenic in vitro and in vivo behavior of peptides will be assayed as synthetic and as phage-peptide for to determinate their vaccine potential. Acknowledgment: This work was supported by grant SEP-CONACYT 252577 given to I. Amaro-Estrada.

Keywords: bovine anaplasmosis, peptides, phage display, veterinary vaccines

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Authors: Nasib Zaman, Maneesha Kour, Muhammad Rizwan, Fazal Akbar

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Abstract: Chikungunya fever is a re-emerging rapidly spreading mosquito-borne disease cause by Aedes albopictus and Aedes aegypti mosquito vectors. Currently, it is affecting millions of people globally. Objective: This study's main objective was to find the incidence of chikungunya fever in the Swat region and the factors associated with the spread of this infection. Method: This study was carried out in different areas of Swat. Blood samples and data were collected from selected patients, and a questionnaire was filled for each patient. 3-5ml of the specimen was taken from the patient's vein and serum, or plasma was separated by centrifugation. Chikungunya tests were performed for IgG and IgM antibodies. The data was analyzed by SPSS and Graph Paid Prism 5. Results: A total of 169 patients were included in this study, out of which 103 (60.9%) having age less than 30 years were positive for chikungunya infection and 66 (39.1%) having more than 30 years were negative for this infection. Only 1 (0.6%) were positive for both IgG and IgM antibody. About 15 (8.9%) patients have diagnosed with positive IgG antibodies, and 25 (26.6%) patients were positive for IgM positive antibodies. The infection rate was significantly higher in males compared to females 71 (59.6%) vs. 14 (38%) P value=0.088, OR=1.7. Conclusion: This study concludes clinical knowledge and awareness that are necessary for a diagnosis of chikungunya infection properly. Therefore it is important to educate people for the eradication of this infection. Recommendation: This study also recommends investigating the other risk factors associated with this infection.

Keywords: Chikungunya, risk factor, Incidence, antibodies, mosquito

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Authors: Vishal Kumar Singh

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Bioprocess development is a time-constrained activity aimed at harnessing the full potential of culture performance in an ambience that is not natural to cells. Even with the use of chemically defined media and feeds, a significant amount of time is devoted in identifying the apt operating parameters. In addition, the scale-up of these processes is often accompanied by loss of antibody titer and product quality, which further delays the commercialization of the drug product. In such a scenario, the investigation of this disparity of culture performance is done by further experimentation at a smaller scale that is representative of at-scale production bioreactors. These scale-down model developments are also time-intensive. In this study, a computation fluid dynamics-based multi-objective scaling approach has been illustrated to speed up the process transfer. For the implementation of this approach, a transient multiphase water-air system has been studied in Ansys CFX to visualize the air bubble distribution and volumetric mass transfer coefficient (kLa) profiles, followed by the design of experiment based parametric optimization approach to define the operational space. The proposed approach is completely in silico and requires minimum experimentation, thereby rendering a high throughput to the overall process development.

Keywords: bioprocess development, scale up, scale down, computation fluid dynamics, multi-objective, Ansys CFX, design of experiment

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Authors: Muhamad Azuddin Hassan, Siti Shafura Karim, Ambri Mohamed, Iskandar Yahya

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Human serum albumin plays a significant part in the physiological functions of the human body system (HSA).HSA level monitoring is critical for early detection of HSA-related illnesses. The goal of this study is to show that a field effect transistor (FET)-based immunosensor can assess HSA using high aspect ratio carbon nanotubes network (CNT) as a transducer. The CNT network were deposited using air brush technique, and the FET device was made using a shadow mask process. Field emission scanning electron microscopy and a current-voltage measurement system were used to examine the morphology and electrical properties of the CNT network, respectively. X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy were used to confirm the surface alteration of the CNT. The detection process is based on covalent binding interactions between an antibody and an HSA target, which resulted in a change in the manufactured biosensor's drain current (Id).In a linear range between 1 ng/ml and 10zg/ml, the biosensor has a high sensitivity of 0.826 mA (g/ml)-1 and a LOD value of 1.9zg/ml.HSA was also identified in a genuine serum despite interference from other biomolecules, demonstrating the CNT-FET immunosensor's ability to quantify HSA in a complex biological environment.

Keywords: carbon nanotubes network, biosensor, human serum albumin

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Authors: Ting Zou, Chengfei Zhang

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Objectives: The purpose of this study was to investigate the role of Semaphorin 4D (Sema4D)/Plexin-B1 signaling on osteo/odontogenic differentiation of human dental pulp stem cells (DPSCs) and uncover its molecular mechanism. Methods: DPSCs were cultured in osteo/odontogenic medium. After treatment with Sema4D (10μg/mL), osteo/odontogenic differentiation and mineralization was evaluated by measuring alkaline phosphatase (ALP) activity and alizarin red S staining respectively. The expression of osteo/odontogenic genes (ALP, Col1A1, BSP, and Runx2) was determined by real-time polymerase chain reaction. p-Plexin-B1, Plexin-B1, Col1A1, RhoA, and ErbB2 were analyzed by western. Results: ALP activity and mineralization formation of DPSCs were significantly decreased after treatment with Sema4D (P<0.05). Sema4D significantly down-regulated osteo/odontogenic-related genes expression (ALP, Col1A1, BSP, and Runx2). p-Plexin-B1, Plexin-B1 and RhoA protein expression levels increased after stimulated with Sema4D, while the expression of Col1A1 decreased. Pretreatment with Plexin-B1 antibody blocked Sema4D induced p-Plexin-B1 expression. Conclusion: Sema4D suppressed osteo/odontogenic differentiation of DPSCs via RhoA-mediated pathways.

Keywords: Sema4D/Plexin-B1, dental pulp stem cells, osteo/odontogenic differentiation, alkaline phosphatase (ALP)

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Authors: Sina Mahdavi

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Background and Objective: Multiple sclerosis (MS) is the most common inflammatory autoimmune disease of the central nervous system (CNS) that affects the myelination process in the CNS. Complex interactions of various "environmental or infectious" factors may act as triggers in autoimmunity and disease progression. The association between viral infections, especially the John Cunningham virus (JCV) and MS is one potential cause that is not well understood. This study aims to summarize the available data on JCV infection in MS disease progression. Materials and Methods: For this study, the keywords "Multiple sclerosis", " John Cunningham virus ", and "central nervous system" in the databases PubMed, Google Scholar, Sid, and MagIran between 2019 and 2022 were searched, and 12 articles were chosen, studied, and analyzed. Results: MS patients are candidates for natalizumab therapy, which inhibits lymphocyte migration and increases the risk of progressive multifocal leukoencephalopathy (PML), a rare lytic infection of glial cells caused by JCV. Oligodendrocytes may be the target of JCV infection in the central nervous system (CNS). Conclusion: There is a high expression of JCV during the natalizumab treatment period for MS patients, suggesting that the virus may play a role in the development of MS by inducing an inflammatory state. Therefore, it is necessary to evaluate anti-JCV antibody serum as an important risk factor for the development of PML before deciding on the treatment course for these patients.

Keywords: multiple sclerosis, John Cunningham virus, central nervous system, autoimmunity

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Authors: Saeed Ahmed, Muhammad Imran, Yasir Allah Ditta, Shahid Mehmood, Zahid Rasool

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A study was conducted to investigate the effect of different levels of microencapsulated butyric (MEB) on growth performance, gut health and immunity in commercial broiler chickens. In total, 336 day-old Hubbard classic broilers chicks were randomly assigned to 4 dietary treatments (Control, 0.25, 0.35 and 0.45g/kg of butyric acid) under completely randomized design. Each treatment was replicated 3 times with 28 birds in each replicate. Feed intake, body weight gain, feed conversion ratio, intestinal morphology, apparent ileal digestibility of protein and immunity parameters were evaluated. At the end of the experiment (35-d) 3 birds/replicate in each group were randomly selected and slaughtered to collect blood, duodenal samples and ileal digesta. The data were analyzed by using ANOVA technique. The results indicated improved body weight gain (P = 0.0222), feed conversion ratio (P = 0.0056), duodenal villus height (P = 0.0512), AID (P = 0.0098) antibody titer against Newcastle disease improved (P = 0.0326). Treatments remained unresponsive with respect to feed intake (P = 0.9685).

Keywords: butyric acid, broilers, gut health, ileal digestibility

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Authors: Giang Tran Thi Huong, Hieu Dong Van, Tung Dao Duy, Saadanov Iskender, Isakeev Mairambek, Tsutomu Omatsu, Yukie Katayama, Tetsuya Mizutani, Yuki Ozeki, Yohei Takeda, Haruko Ogawa, Kunitoshi Imai

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Newcastle disease virus (NDV) is a contagious viral disease of the poultry industry and other birds throughout the world. At present, very little is known about molecular epidemiological data regarding the causes of ND outbreak in commercial poultry farms in Kyrgyzstan. In the current study, the NDV isolated from the one out of three samples from the unvaccinated flock was confirmed as NDV. Phylogenetic analysis indicated that this NDV strain is clustered in the Class II subgenotype VIId, and closely related to the Chinese NDV isolate. Phylogenetic analyses revealed that the isolated NDV strain has an origin different from the 4 NDV strains previously identified in Kyrgyzstan. According to the mean death time (MDT: 61.1 h) and a multibasic amino acid (aa) sequence at the F0 proteolytic cleavage site (¹¹²R-R-Q-K-R-F¹¹⁷), the NDV isolate was determined as mesogenic strain. Several mutations in the neutralizing epitopes (notably, ³⁴⁷E→K) and the global head were observed in the hemagglutinin-neuraminidase (HN) protein of the current isolate. The present study represents the molecular characterization of the coding gene region of NDV in Kyrgyzstan. Additionally, further study will be investigated on the antigenic characterization using monoclonal antibody.

Keywords: Kyrgyzstan, Newcastle disease, genotype, genome characterization

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Authors: Waode Nurmayani, Nikmatul Riswanda

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Broilers are chickens which are kept with the most efficient time and hoped get a good body weight. All things are done, for example with the improvement of feed and use antibiotics. Feed cost is the most cost to be spent. Nearly 80% of the cost is spent just for buy feed. Earthworm (Lumbricus rubellus) is a good choice to reduce the cost of feed protein source. The Earthworm has a high crude protein content of about 48.5%-61.9%, rich with proline amino acid about 15% of the 62 amino acids. Not only about protein, this earthworm also has a role in disease prevention. Prevention of disease in livestock usual with use feed supplement. Earthworm (Lumbricus rubellus) is one of the natural materials used as feed. In addition, several types of earthworms that have been known to contain active substances about antibacterial pathogens namely Lumbricus rubellus. The earthworm could be used as an antibiotic because it contain the antibody of Lumbricine active substance. So that, this animal feed from Lumbricus rubellus could improve the performance of broilers. Bioactive of anti-bacterial is called Lumbricine able to inhibit the growth of pathogenic bacteria in the intestinal wall so that the population of pathogenic bacteria is reduced. The method of write in this scientific writing is divided into 3 techniques, namely data completion, data analysis, and thinking pan from various literature about earthworm (Lumbricus rubellus) as broiler feed. It is expected that innovation of feed material of earthworm (Lumbricus rubellus) could reduce the cost of protein feed and the use of chemical antibiotics.

Keywords: earthworm, broiler, protein, antibiotic

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Authors: Saekil Yun, Sib Sankar Giri, Jin Woo Jun, Hyoun Joong Kim, Sang Guen Kim, Sang Wha Kim, Jung Woo Kang, Se Jin Han, Se Chang Park

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In aquaculture, vaccination is important to control and prevent diseases. In the study, we utilized poly D,L-lactide-co-glycolic acid (PLGA) microparticles (MPs) for encapsulating formalin-killed Aeromonas hydrophila cells. To assess the innate and adaptive immune responses, carps and loaches were used for the experiments. Fish were divided into three groups (A, B, C). Total antigen of 0.1 ml vaccine was adjusted by 2 x 108 CFU and injected via intraperitoneal route. Group A was vaccinated with 0.1 ml of PLGA vaccine, group B was with 0.1 ml of FKC vaccine and group C was with 0.1 ml of sterile PBS. All three groups were challenged with A. hydrophila and challenge dose was lethal dose (LD50). Loaches and carp were then challenged with A. hydrophila at 12 and 20 weeks post vaccination (wpv), and 10 and 14 wpv, respectively, and relative survival rates were calculated. For both fish species, the curve of antibody titer over time was shallower in the PLGA group than the FKC group and the PLGA groups demonstrated higher survival rates at all time-points. In the groups of PLGA-MP, relative mRNA levels of IL-1β, TNF-α, lysozyme C and IgM were significantly upregulated than FKC treated groups. Biodegradable PLGA microparticle vaccine could induce longer immune responses than original FKC vaccines to protect from A. hydrophila infection.

Keywords: PLGA, microparticles, Aeromonas hydrophila, vaccine

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Authors: Jihene Bettaieb, Kaouther Ayouni, Ghassen Kharroubi, Rym Mallekh, Walid Hammemi, Afif Ben Salah, Henda Triki

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In Tunisia, the hepatitis A virus (HAV) represents a public health concern. Due to the progress in sanitation and socio-economic conditions, the epidemiology of HAV has shown dynamic changes over the past years. This study aimed to investigate the current seroprevalence of HAV antibodies (anti-HAV) among the residents of Thala, a rural setting in central-west Tunisia, to determine the age-specific seroprevalence for HAV infection and co-infection with hepatitis C and B virus. A total of 1379 subjects (mean age: 25.0 ± 17.3 years, 555 males/ 824 females) were recruited between January and June 2014. The study population included 95 individuals previously known as hepatitis C positive. Serum samples were collected and screened for the detection of IgG anti-HAV, HBsAg, and HBcAb by the Elisa Test. The overall anti- HAV seroprevalence was about 84.7%. There was no statistically significant difference between males and females. On the 1379 tested individual, 219 were positive for HBcAb, and 67 were positive for HBsAg. IgG anti- HAV were positive in 80.6% of HBsAg-positive patients (54 out of 67), 81.3% of HBcAb-positive patients (178 out of 219), and in 95.8% of HCV-positive patients (91 out of 95). HBV infection and HCV infection were statistically associated with a greater risk of positive anti-HAV antibody (p < 0.001). Our study revealed that Thala represents an intermediate endemicity level and that the introduction of vaccination against HAV in this region is recommended, especially for the hepatitis B or C infected person seronegative for HAV.

Keywords: coinfection, hepatitis A, seroprevalence, Tunisia

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Authors: Mohammad Hossein Modarressi, Mozhgan Mondeali, Khabat Barkhordari, Ali Etemadi

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The COVID-19 pandemic, caused by SARS-CoV-2, has led to significant concerns worldwide due to its catastrophic effects on public health. The SARS-CoV-2 infection is initiated with the binding of the receptor-binding domain (RBD) in its spike protein to the ACE2 receptor in the host cell membrane. Due to the error-prone entity of the viral RNA-dependent polymerase complex, the virus genome, including the coding region for the RBD, acquires new mutations, leading to the appearance of multiple variants. These variants can potentially impact transmission, virulence, antigenicity and evasive immune properties. S477N mutation located in the RBD has been observed in the SARS-CoV-2 omicron (B.1.1. 529) variant. In this study, we investigated the consequences of S477N mutation at the molecular level using computational approaches such as molecular dynamics simulation, protein-protein interaction analysis, immunoinformatics and free energy computation. We showed that displacement of Ser with Asn increases the stability of the spike protein and its affinity to ACE2 and thus increases the transmission potential of the virus. This mutation changes the folding and secondary structure of the spike protein. Also, it reduces antibody neutralization, raising concern about re-infection, vaccine breakthrough and therapeutic values.

Keywords: S477N, COVID-19, molecular dynamic, SARS-COV2 mutations

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Authors: Lynn Lehmann, Dinorah Leyva, Ana Lazic, Stefan Duhr, Philipp Baaske

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Characterization of biomolecular interactions, such as protein-protein, protein-nucleic acid or protein-small molecule, provides critical insights into cellular processes and is essential for the development of drug diagnostics and therapeutics. Here we present a novel, label-free, and tether-free technology to analyze picomolar to millimolar affinities of biomolecular interactions by Microscale Thermophoresis (MST). The entropy of the hydration shell surrounding molecules determines thermophoretic movement. MST exploits this principle by measuring interactions using optically generated temperature gradients. MST detects changes in the size, charge and hydration shell of molecules and measures biomolecule interactions under close-to-native conditions: immobilization-free and in bioliquids of choice, including cell lysates and blood serum. Thus, MST measures interactions under close-to-native conditions, and without laborious sample purification. We demonstrate how MST determines the picomolar affinities of antibody::antigen interactions, and protein::protein interactions measured from directly from cell lysates. MST assays are highly adaptable to fit to the diverse requirements of different and complex biomolecules. NanoTemper´s unique technology is ideal for studies requiring flexibility and sensitivity at the experimental scale, making MST suitable for basic research investigations and pharmaceutical applications.

Keywords: biochemistry, biophysics, molecular interactions, quantitative techniques

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Authors: Y. Suchitra Devi, L. Hemchandra Singh

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Background: Because of its geographical location, Manipur lies in the conventional goiter endemic belt. During the post salt iodization period, endemic goiter was prevalent in the valley districts of Manipur without iodine deficiency. Objectives: The present study aim at the prevalence of goiter among school children (6-12 years) and women of reproductive age group (above 20 years) of Andro Assembly Constituency, Imphal- East, Manipur, India. Method: A total of 3992 individuals were clinically examined for thyroid enlargement. Hormones like TSH, FT₄, FT₃, and Anti-TPO, Anti-Tg were tested, UIC, USCN, testing of iodine in water and salt. Result: Total goiter prevalence was found to be 13.98%, median urinary iodine level was 166.0 µg/l, mean urinary thiocyanate concentration was 0.726 ± 0.408, mean water iodine concentration was 3.843 ± 2.291, and all the salt samples were above 15ppm. 6 out of 41 children and 93 out of 176 women were auto antibody positive. 41 children and 176 women were tested for TSH, FT₄, and FT₃, which shows disturbance in hormone level. Conclusion: The present study showed that the region is mildly goiter endemic without biochemical iodine deficiency.

Keywords: goiter, TSH, FT₄, FT₃, anti-TPO, anti-Tg, UIC, USCN, school children and women of reproductive age

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Authors: Fantu Lombamo Untiso, Sylvia Murphy, Emily Pieracci

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Background: Rabies is a highly fatal viral disease of all warm-blooded animals including human globally. However, effective rabies control program still remains to be a reality and needs to be strengthened. Objective: Reviewing of recorded data and analyzing it to generate information on the status of rabies in Addis Ababa in the year 2012/13. Methods: A retrospective data were used from the Ethiopian Public Health Institute rabies case record book registered in the year 2012/13. Results: Among 1357 suspected rabid animals clinically examined; only 8.84% were positive for rabies. Out of 216 animal brains investigated in the laboratory with Fluorescent Antibody Technique, 55.5% were confirmed rabies positive. Among the laboratory confirmed positive rabies cases, high percentage of the animals came from Yeka (20%) and lower number from Kirkos subcity (3.3%). Out of 1149 humans who came to the institute seeking anti-rabies post-exposure prophylaxis, 85.65% and 7.87% of them were exposed to suspected dogs and cats respectively. 3 human deaths due to rabies were reported in the year after exposure to dog bite of unknown vaccination status. Conclusion: The principal vector of rabies in Addis Ababa is dog. Effective rabies management and control based on confirmed cases and mass-immunization and control of stray dog populations is recommended.

Keywords: Addis Ababa, exposure, rabies, surveillance

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Authors: A. Zarei, M. Porkhalili, B. Gholamhosseini

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In this experiment, sixty Hy-Line (W-36) laying hens were selected in 40weeks of age. Experimental diets were consumed for 12 weeks duration by them. The experimental design was completely randomized block included four treatments and each of them with five replications and three sample in each replicate. Treatments were as follow: Basal diet+probiotic, basal diet + prebiotic and basal diet+probiotic+ prebiotic. Performance traits were measured such as: hen production, egg weight, feed intake, feed conversion ratio ,shell thickness, shell strength, shell weight, hough unit, yolk color, and yolk cholesterol. Blood parameters like; Ca, cholesterol, triglyceride, VLDL and antibody titer and so morphological of intestine were determined. At the end of experimental period, after sampling from end of cecum, bacterial colony count was measured. Results showed; shell weight was significantly greater than other treatments in probiotic treatment.Yolk weight in prebiotic treatment was significantly greater than other treatments. The ratio of height of villi to dept of crypt cells in duodenum, jejunum, ileum and secum in prebiotic treatment were significantly greater. Results from the other traits were not significant between treatments, however there were totally good results in other traits with simultaneous usage of probiotic and prebiotic.

Keywords: probiotic, prebiotic, laying hens, performance, blood parameters, intestine morphology

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Authors: Husham Bayazed

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A growing number of studies indicate that atherosclerotic coronary artery disease (CAD) has a complex pathogenesis that extends beyond cholesterol intimal infiltration. The atherosclerosis process may involve an immune micro-environmental condition driven by local activation of the adaptive and innate immunity arrays, resulting in the formation of atherosclerotic plaques. Therefore, despite the wide usage of lipid-lowering agents, these devastating coronary diseases are not averted either at primary or secondary prevention levels. Many trials have recently shown an interest in the immune targeting of the inflammatory process of atherosclerotic plaques, with the promised improvement in atherosclerotic cardiovascular disease outcomes. This recently includes the immune-modulatory drug “Canakinumab” as an anti-interleukin-1 beta monoclonal antibody in addition to "Colchicine,” which's established as a broad-effect drug in the management of other inflammatory conditions. Recent trials and studies highlight the importance of inflammation and immune reactions in the pathogenesis of atherosclerosis and plaque formation. This provides an insight to discuss and extend the therapies from old lipid-lowering drugs (statins) to anti-inflammatory drugs (colchicine) and new targeted immune-modulatory therapies like inhibitors of IL-1 beta (canakinumab) currently under investigation.

Keywords: atherosclerotic plagues, immune microenvironment, lipid-lowering agents, and immunomodulatory drugs

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Authors: Aleksandra Twarda, Dobrosława Krzemień, Grzegorz Dubin, Tad A. Holak

Abstract:

Stabilin-2 belongs to the group of scavenger receptors and plays a crucial role in clearance of more than 10 ligands from the bloodstream, including hyaluronic acid, products of degradation of extracellular matrix and metabolic products. The Link domain, a defining feature of stabilin-2, has a sequence similar to Link domains in other hyaluronic acid receptors, such as CD44 or TSG-6, and is responsible for most of ligands binding. Present knowledge of signal transduction by stabilin-2, as well as ligands’ recognition and binding mechanism, is limited. Until now, no experimental structures have been solved for any segments of stabilin-2. It has recently been demonstrated that the stabilin-2 knock-out or blocking of the receptor by an antibody effectively opposes cancer metastasis by elevating the level of circulating hyaluronic acid. Moreover, loss of expression of stabilin-2 in a peri-tumourous liver correlates with increased survival. Solving of the crystal structure of stabilin-2 and elucidation of the binding mechanism of hyaluronic acid could enable the precise characterization of the interactions in the binding site. These results may allow for designing specific small-molecule inhibitors of stabilin-2 that could be used in cancer therapy. To carry out screening for crystallization of stabilin-2, we cloned constructs of the Link domain of various lengths with or without surrounding domains. The folding properties of the constructs were checked by nuclear magnetic resonance (NMR). It is planned to show the binding of hyaluronic acid to the Link domain using several biochemical methods, i.a. NMR, isothermal titration calorimetry and fluorescence polarization assay.

Keywords: stabilin-2, Link domain, X-ray crystallography, NMR, hyaluronic acid, cancer

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Authors: Arati Inamdar, Siddharth Bhattacharyya, Kester Haye

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Collision tumors are rare entities characterized by neoplasms of two different cell populations with distinct separating boundaries. Such tumors could be benign, malignant, or a combination of both. The exact mechanism of origin for collision tumors is predicted to be tumor heterogeneity or concurrent occurrence of neoplasm in the same organ. We present two cases of plasmacytoma presenting as a collision tumor, one with a tumor of hematological origin and another with a non-hematological origin, namely Chronic Lymphocytic Leukemia and Adenocarcinoma of the colon, respectively. The immunohistochemical stains and flowcytometry analysis performed on the specimens aided incorrect diagnosis. Interestingly, neoplastic cells of plasmacytoma in the first case demonstrated strong cytokeratin along with weak Epithelial Specific Antigen/ Epithelial cell adhesion molecule Monoclonal Antibody (MOC31) positivity, indicating that the tumor may influence the microenvironment of the tumor in the vicinity. Furthermore, the next-generation sequencing studies performed on the specimen with plasmacytoma and chronic lymphocytic lymphoma demonstrated BReast CAncer gene (BRCA2) and Tumor Necrosis Factor Alpha Induced Protein 3 (TNFAIP3) as a disease associated variants suggestive of risk for multiple tumors including collision tumors. Our reports highlight the unique collision tumors involving plasmacytoma, which have never been reported previously, as well as provide necessary insights about the underline genetic aberrations and tumor heterogeneity through sequencing studies and allow clonality assessment for subsequent tumors.

Keywords: BRCA2, collision tumor, chronic lymphocytic leukemia, plasmacytoma

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Authors: Dong-ping Yuan, Lin Gu, Jun Long, Jie Chen, Ni Jie, Ying-Li Shi

Abstract:

Endometriosis is a common disease in women of reproductive age, whose classic characteristic is mononuclear cell infiltration into lesions. Shikonin is an anti-inflammatory phytocompound from Lithospermum erythrorhizon, whose potential therapeutic effects for the endometriosis remain unclear. The working hypothesis was that shikonin can inhibit the development of endometriosis by the inhibition of chemotactic effect. Shikonin significantly inhibited the growth of human endometrial tissue implanted into mice (P<0.05). No observable adverse effects were found. The mouse regulated upon activation normal T-cell expressed and secreted (mRANTES) level in peritoneal fluid of animal endometriosis model was higher than that in normal SCID mice (P<0.05), and decreased dramatically after shikonin treatment in a dose-dependent manner (P<0.05). Peritoneal fluid from NOD/SCID mice treated with shikonin inhibited monocytes chemotaxis, which could be abolished by mRANTES antibody. In vitro, shikonin significantly inhibited RANTES expression of U937 cells cultured alone or co-cultured with human methothelail cells and endometrial stromal cells, and inhibited RANTES-induced chemotaxis of U937 cells (P<0.05). The present results suggest that shikonin can inhibit the development of endometriosis by mechanisms that at least include the inhibition of RANTES expression and decreased migration of mononuclear cells to lesions. Shikonin may be a useful and safe new approach for treating endometriosis.

Keywords: endometriosis, shikonin, RANTES chemotaxis

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Authors: Wanwisa Deenin, Abdulhadee Yakoh, Chahya Kreangkaiwal, Orawon Chailapakul, Kanitha Patarakul, Sudkate Chaiyo

Abstract:

LipL32 is an outer membrane protein present only on pathogenic Leptospira species, which are the causative agent of leptospirosis. Leptospirosis symptoms are often misdiagnosed with other febrile illnesses as the clinical manifestations are non-specific. Therefore, an accurate diagnostic tool for leptospirosis is indeed critical for proper and prompt treatment. Typical diagnosis via serological assays is generally performed to assess the antibodies produced against Leptospira. However, their delayed antibody response and complicated procedure are undoubtedly limited the practical utilization especially in primary care setting. Here, we demonstrate for the first time an early-stage detection of LipL32 by an integrated lateral-flow immunoassay with electrochemical readout (eLFIA). A ferrocene trace tag was monitored via differential pulse voltammetry operated on a smartphone-based device, thus allowing for on-field testing. Superior performance in terms of the lowest detectable limit of detection (LOD) of 8.53 pg/mL and broad linear dynamic range (5 orders of magnitude) among other sensors available thus far was established. Additionally, the developed test strip provided a straightforward yet sensitive approach for diagnosis of leptospirosis using the collected human sera from patients, in which the results were comparable to the real-time polymerase chain reaction technique.

Keywords: leptospirosis, electrochemical detection, lateral flow immunosensor, point-of-care testing, early-stage detection

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Authors: Zaw Z Aung

Abstract:

Background: High-risk groups for hepatitis B infection (HBV) are people who injected drugs (PWID), men who have sex with men (MSM), people living with HIV (PLHIV) and persons with hepatitis C (HCV), etc. HBV/HCV coinfected patients are at increased risk of cirrhosis, hepatic decompensation and hepatocellular carcinoma. To the best of author’s knowledge, there is currently no data for hepatitis B vaccine utilization in HCV positive patients and their antibody response. Methodology: From February 2018 to May 2018, consented participants at or above 18 years who came to the clinic in Mandalay were tested with the anti-HCV rapid test. Those who tested HCV positive (n=168) were further tested with hepatitis B profile and asked about their previous hepatitis B vaccination history and risk factors. Results: Out of 168 HCV positive participants, three were excluded for active HBV infections. The remaining 165 were categorized into previously vaccinated 64% (n=106) and unvaccinated 36% (n=59) There were three characteristics groups- PWID monoinfected (n=77), General Population (GP) monoinfected (n=22) and HIV/HCV coinfected participants (n=66). Unvaccinated participants were highest in HIV/HCV, with 68%(n=45) followed by GP (23%, n=5) and PWID (12%, n=9). Among previously vaccinated participants, the highest percentage was PWID (88%, n=68), the second highest was GP (77%, n=17) and lowest in HIV/HCV patients (32%, n=21). 63 participants completed third doses of vaccination (PWID=36, GP=13, HIV/HCV=14). 53% of participants who completed 3 dose of hepatitis B were non-responders (n=34): HIV/HCV (86%, n=12), PWID (44%, n=16), and GP (46%, n=6) Conclusion: Even in the presence of effective and safe hepatitis B vaccine, uptake is low among high risk groups especially PLHIV that needs to be improved. Integration or collaboration of hepatitis B vaccination program, HIV/AIDS and hepatitis C treatment centers is desirable. About half of vaccinated participants were non-responders so that optimal doses, schedule and follow-up testing need to be addressed carefully for those groups.

Keywords: Hepatitis B vaccine, Hepatitis C, HIV, Myanmar

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Authors: Recep Keşli, Onur Türkyılmaz, Hayriye Tokay, Kasım Demir

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Objective: Coeliac disease (CD) is a primary small intestine disorder caused by high sensitivity to gluten which is present in the crops, characterized by inflammation in the small intestine mucosa. The goal of this study was to determine and to compare the sensitivity and specificity values of anti-endomysial IgA (EMA IgA) (IFA) and anti-tissue transglutaminase IgA (anti-tTG IgA) (ELISA) antibodies in the diagnosis of patients suspected with the CD. Methods: One thousand two hundred seventy three patients, who have applied to gastroenterology and pediatric disease polyclinics of Afyon Kocatepe University ANS Research and Practice Hospital were included into the study between 23.09.2010 and 30.05.2016. Sera samples were investigated by immunofluorescence method for EMA positiveness (Euroimmun, Luebeck, Germany). In order to determine quantitative value of Anti-tTG IgA (EIA) (Orgentec Mainz, Germany) fully automated ELISA device (Alisei, Seac, Firenze, Italy) were used. Results: Out of 1273 patients, 160 were diagnosed with coeliac disease according to ESPGHAN 2012 diagnosis criteria. Out of 160 CD patients, 120 were female, 40 were male. The EMA specificity and sensitivity were calculated as 98% and 80% respectively. Specificity and sensitivity of Anti-tTG IgA were determined as 99% and 96% respectively. Conclusion: The specificity of EMA for CD was excellent because all EMA-positive patients (n = 144) were diagnosed with CD. The presence of human anti-tTG IgA was found as a reliable marker for diagnosis and follow-up the CD. Diagnosis of CD should be established on both the clinical and serologic profiles together.

Keywords: anti-endomysial antibody, anti-tTG IgA, coeliac disease, immunofluorescence assay (IFA)

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Authors: Mir Mohammad Reza Hosseini

Abstract:

In new years immune checkpoint inhibitors have gathered care as being one of the greatest talented kinds of immunotherapy on the prospect. There has been a specific emphasis on the immune checkpoint molecules, cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed cell death protein 1 (PD-1). In 2011, ipilimumab, the primary antibody obstructive an immune checkpoint (CTLA4) was authorized. It is now documented that recognized tumors have many devices of overpowering the antitumor immune response, counting manufacture of repressive cytokines, staffing of immunosuppressive immune cells, and upregulation of coinhibitory receptors recognized as immune checkpoints. This was fast followed by the growth of monoclonal antibodies directing PD1 (pembrolizumab and nivolumab) and PDL1 (atezolizumab and durvalumab). Anti-PD1/PDL1 antibodies have developed some of the greatest extensively set anticancer therapies. We also compare and difference their present place in cancer therapy and designs of immune-related toxicities and deliberate the role of dual immune checkpoint inhibition and plans for the organization of immune-related opposing proceedings. In this review, the employed code and present growth of numerous immune checkpoint inhibitors are abridged, while the communicating device and new development of Immune checkpoint inhibitors in cancer therapy-based synergistic therapies with additional immunotherapy, chemotherapy, phototherapy, and radiotherapy in important and clinical educations in the historical 5 years are portrayed and tinted. Lastly, we disapprovingly measure these methods and effort to find their fortes and faintness based on pre-clinical and clinical information.

Keywords: checkpoint, cancer therapy, PD-1, PDL-1, CTLA4, immunosuppressive

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Authors: Lucian Mocan, Flaviu Tabaran, Teodora Mocan, Cristian Matea, Cornel Iancu

Abstract:

We present method of Gold Nanoparticle enhanced laser thermal ablation of HepG2 cells (Human hepatocellular liver carcinoma cell line), based on a simple gold nanoparticle carrier system, such as serum albumin (BSA), and demonstrate its selective therapeutic efficacy. Hyperspectral, contrast phase, and confocal microscopy combined immunochemical staining were used to demonstrate the selective internalization of HSA-GNPs via Gp60 receptors and the caveolin-mediated endocytosis inside HepG2 cells. We examined the ability of laser-activated carbon nanotubes to induce Hsp70 expression using confocal microscopy. Hep G2 cells heat-shocked (laser activated BSA-GNPs) to 42°C demonstrated an up-regulation of Hsp70 compared with control cells (BSA-GNPs treated cells without laser), which showed no detectable constitutive expression of Hsp70. We observed a time-dependent induction in Hsp70 expression in Hep G2 treated with BSA-GNPs and LASER irradiated. The post-irradiation apoptotic rate of HepG2 cells treated with HSA-GNPs ranged from 88.24% (for 50 mg/L) at 60 seconds, while at 30 minute the rate increased to 92.34% (50 mg/L). These unique results may represent a major step in liver cancer treatment using nanolocalized thermal ablation by laser heating.

Keywords: gold nanoparticles, liver cancer, albumin, laser irradiation

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Authors: Atefeh Esmailnejad, Mohammad Abbaszadeh Hasiri

Abstract:

Coxiella burnetii is a gram-negative obligatory intracellular bacterium that causes Q fever, a significant zoonotic disease. Sheep, cattle, and goats are the most commonly reported reservoirs for the bacteria, but infected cats and dogs have also been implicated in the transmission of the disease to human. The aim of present study was to investigate the presence of antibodies against Coxiella burnetii among companion dogs in Fars province, South of Iran. A total of 181 blood samples were collected from asymptomatic dogs, mostly referred to Veterinary Hospital of Shiraz University for regular vaccination. The IgG antibody detection against Coxiella burnetii was made by indirect Enzyme-linked Immunosorbent Assay (ELISA), employing phase I and II Coxiella burnetii antigens. A logistic regression model was developed to analyze multiple risk factors associated with seropositivity. An overall seropositivity of 7.7% (n=14) was observed. Prevalence was significantly higher in adult dogs above five years (18.18 %) compared with dogs between 1 and five years (7.86 %) and less than one year (6.17%) (P=0.043). Prevalence was also higher in male dogs (11.21 %) than in female (2.7 %) (P=0.035). There were no significant differences in the prevalence of positive cases and breed, type of housing, type of food and exposure to other farm animals (P>0.05). The results of this study showed the presence of Coxiella burnetii infection among the companion dogs population in Fars province. To our knowledge, this is the first study regarding Q fever in dogs carried out in Iran. In areas like Iran, where human cases of Q fever are not common or remain unreported, the public health implications of Q fever seroprevalence in dogs are quite significant.

Keywords: Coxiella burnetii, dog, Iran, Q fever

Procedia PDF Downloads 280