Search results for: excretory-secretory antigens
Commenced in January 2007
Frequency: Monthly
Edition: International
Paper Count: 91

Search results for: excretory-secretory antigens

31 Results of the Safety Evaluation of Cancer Vaccines Dealing with Novel Targets for Cancer Immunotherapy

Authors: Axel Mancebo, Ana M. Bada, Angel Casacó, Bárbara González, Avelina León, María E. Arteaga, Consuelo González, Belinda Sánchez, Adriana Carr, Nuris Ledón, Arianna Iglesias

Abstract:

Despite the many preventive and therapeutic modalities aimed at curing cancer, it remains as a serious world health problem. Promising recent developments suggest that cancer immunotherapy may be the next great hope for cancer treatment. EGFRs are receptor tyrosine kinases and it is considered an important therapeutic target related with tumor progression, and several types of molecular therapies, including monoclonal antibodies, small molecules, and vaccines, have been developed to target the HER family of receptors. On the other hand, gangliosides are membrane glycosphingolipids that contain two variants of sialic acid, the N-acetylated (NeuAc) and the N-glycolylated (NeuGc) variant. The high expression of this antigen-specific molecule has been associated with malignant tumor progression and immunosuppressive mechanisms, so ganglioside could be considered as the target for cancer immunotherapy. We have been working for several years in the safety evaluation of cancer vaccines targeting these two systems, the EGF receptor and ganglioside. We presented in this work results of repeated dose toxicity studies performed in Sprague Dawley rats and Cynomolgus monkeys, including clinical observations, body weight and rectal temperature measuring, clinical pathology analysis, gross necropsy and histological examination in rodent studies, and immunological evaluation. Immunizations were capable of inducing mainly inflammatory effects at the injection site, with findings largely attributable to the adjuvants used and probably enhanced by the immunological properties of the antigens. In general, these vaccines were shown to be well tolerated, and these studies in relevant species allow treating cancer patients with tumors during long periods with relative weight safety margin.

Keywords: cancer vaccines, safety, toxicology, rats, non human primates

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30 Seroprevalence of Hepatitis B and C among Healthcare Workers in Dutse Metropolis, Jigawa State, Nigeria

Authors: N. M. Sani, I. Bitrus, A. M. Sarki, N. S. Mujahid

Abstract:

Hepatitis is one of the neglected infectious diseases in sub Saharan Africa, and most of the available data is based on blood donors. Health care workers (HCWs) often get infected as a result of their close contact with patients. A cross-sectional study was conducted to determine the prevalence of hepatitis B and C among this group of professionals with a view to improving the quality of care to their patients. Hepatitis B and C infections pose a major public health problem worldwide. While infection is highest in the developing world particularly Asia and sub-Saharan Africa, healthcare workers are at higher risk of acquiring blood-borne viral infections, particularly Hepatitis B and C which are mostly asymptomatic. This study was aimed at determining the prevalence of Hepatitis B and C infections and associated risk factors among health care workers in Dutse Metropolis, Jigawa State - Nigeria. A standard rapid immuno-chromatographic technique i.e. rapid ELISA was used to screen all sera for Hepatitis B surface antigen (HBsAg) and Hepatitis C viral antibody (HCVAb) respectively. Strips containing coated antibodies and antigens to HBV and HCV respectively were removed from the foil. Strips were labeled according to samples. Using a separate disposable pipette, 2 drops of the sample (plasma) were added into each test strip and allowed to run across the absorbent pad. Results were read after 15 minutes. The prevalence of HBV and HCV infection in 100 healthcare workers was determined by testing the plasma collected from the clients during their normal checkup using HBsAg and HCVAb test strips. Results were subjected to statistical analysis using chi-square test. The prevalence of HBV among HCWs was 19 out of 100 (19.0%) and that of HCV was 5 out of 100 (5.0%) where in both cases, higher prevalence was observed among female nurses. It was also observed that all HCV positive cases were recorded among nurses only. The study revealed that nurses are at greater risk of contracting HBV and HCV due to their frequent contact with patients. It is therefore recommended that effective vaccination and other infection control measures be encouraged among healthcare workers.

Keywords: prevalence, hepatitis, viruses, healthcare workers, infection

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29 Immune Modulation and Cytomegalovirus Reactivation in Sepsis-Induced Immunosuppression

Authors: G. Lambe, D. Mansukhani, A. Shetty, S. Khodaiji, C. Rodrigues, F. Kapadia

Abstract:

Introduction: Sepsis is known to cause impairment of both innate and adaptive immunity and involves an early uncontrolled inflammatory response, followed by a protracting immunosuppression phase, which includes decreased expression of cell receptors, T cell anergy and exhaustion, impaired cytokine production, which may cause high risk for secondary infections due to reduced response to antigens. Although human cytomegalovirus (CMV) is widely recognized as a serious viral pathogen in sepsis and immunocompromised patients, the incidence of CMV reactivation in patients with sepsis lacking strong evidence of immunosuppression is not well defined. Therefore, it is important to determine an association between CMV reactivation and sepsis-induced immunosuppression. Aim: To determine the association between incidence of CMV reactivation and immune modulation in sepsis-induced immunosuppression with time. Material and Methods: Ten CMV-seropositive adult patients with severe sepsis were included in this study. Blood samples were collected on Day 0, and further weekly up to 21 days. CMV load was quantified by real-time PCR using plasma. The expression of immunosuppression markers, namely, HLA-DR, PD-1, and regulatory T cells, were determined by flow cytometry using whole blood. Results: At Day 0, no CMV reactivation was observed in 6/10 patients. In these patients, the median length for reactivation was 14 days (range, 7-14 days). The remaining four patients, at Day 0, had a mean viral load of 1802+2599 copies/ml, which increased with time. At Day 21, the mean viral load for all 10 patients was 60949+179700 copies/ml, indicating that viremia increased with the length of stay in the hospital. HLA-DR expression on monocytes significantly increased from Day 0 to Day 7 (p = 0.001), following which no significant change was observed until Day 21, for all patients except 3. In these three patients, HLA-DR expression on monocytes showed a decrease at elevated viral load (>5000 copies/ml), indicating immune suppression. However, the other markers, PD-1 and regulatory T cells, did not show any significant changes. Conclusion: These preliminary findings suggest that CMV reactivation can occur in patients with severe sepsis. In fact, the viral load continued to increase with the length of stay in the hospital. Immune suppression, indicated by decreased expression of HLA-DR alone, was observed in three patients with elevated viral load.

Keywords: CMV reactivation, immune suppression, sepsis immune modulation, CMV viral load

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28 Studies of Single Nucleotide Polymorphism of Proteosomal Gene Complex and Their Association with HBV Infection Risk in India

Authors: Jasbir Singh, Devender Kumar, Davender Redhu, Surender Kumar, Vandana Bhardwaj

Abstract:

Single Nucleotide polymorphism (SNP) of proteosomal gene complex is involved in the pathogenesis of hepatitis B Virus (HBV) infection. Some of such proteosomal gene complex are large multifunctional proteins (LMP) and antigen associated transporters that help in antigen presentation. Both are involved in intracellular processing and presentation of viral antigens in association with Major Histocompatability Complex (MHC) Class I molecules. A total of hundred each of hepatitis B virus infected and control samples from northern India were studied. Genomic DNA was extracted from all studied samples and PCR-RFLP method was used for genotyping at different positions of LMP genes. Genotypes at a given position were inferred from the pattern of bands and genotype frequencies and haplotype frequencies were also calculated. Homozygous SNP {A>C} was observed at codon 145 of LMP7 gene and having a protective role against HBV as there was statistically significant high distribution of this SNP among controls than cases. Heterozygous SNP {A>C} was observed at codon 145 of LMP7 gene and made individuals more susceptible to HBV infection as there was statistically significant high distribution of this SNP among cases than control. SNP {T>C} was observed at codon 60 of LMP2 gene but statistically significant differences were not observed among controls and cases. For codon 145 of LMP7 and codon 60 of LMP2 genes, four haplotypes were constructed. Haplotype I (LMP2 ‘C’ and LMP7 ‘A’) made individuals carrying it more susceptible to HBV infection as there was statistically significant high distribution of this haplotype among cases than control. Haplotype II (LMP2 ‘C’ and LMP7 ‘C’) made individuals carrying it more immune to HBV infection as there was statistically significant high distribution of this haplotype among control than cases. Thus it can be concluded that homozygous SNP {A>C} at codon 145 of LMP7 and Haplotype II (LMP2 ‘C’ and LMP7 ‘C’) has a protective role against HBV infection whereas heterozygous SNP {A>C} at codon 145 of LMP7 and Haplotype I (LMP2 ‘C’ and LMP7 ‘A’) made individuals more susceptible to HBV infection.

Keywords: Hepatitis B Virus, single nucleotide polymorphism, low molecular weight proteins, transporters associated with antigen presentation

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27 Lung Icams and Vcam-1 in Innate and Adaptive Immunity to Influenza Infections: Implications for Vaccination Strategies

Authors: S. Kozlovski, S.W. Feigelson, R. Alon

Abstract:

The b2 integrin ligands ICAM-1 ICAM-2 and the endothelial VLA-4 integrin ligand VCAM-1 are constitutively expressed on different lung vessels and on high endothelial venules (HEVs), the main portal for lymphocyte entry from the blood into lung draining lymph nodes. ICAMs are also ubiquitously expressed by many antigen-presenting leukocytes and have been traditionally suggested as critical for the various antigen-specific immune synapses generated by these distinct leukocytes and specific naïve and effector T cells. Loss of both ICAM-1 and ICAM-2 on the lung vasculature reduces the ability to patrol monocytes and Tregs to patrol the lung vasculature at a steady state. Our new findings suggest, however, that in terms of innate leukocyte trafficking into the lung lamina propria, both constitutively expressed and virus-induced vascular VCAM-1 can functionally compensate for the loss of these ICAMs. In a mouse model for influenza infection, neutrophil and NK cell recruitment and clearance of influenza remained normal in mice deficient in both ICAMs. Strikingly, mice deficient in both ICAMs also mounted normal influenza-specific CD8 proliferation and differentiation. In addition, these mice normally combated secondary influenza infection, indicating that the presence of ICAMs on conventional dendritic cells (cDCs) that present viral antigens are not required for immune synapse formation between these APCs and naïve CD8 T cells as previously suggested. Furthermore, long-lasting humoral responses critical for protection from a secondary homosubtypic influenza infection were also normal in mice deficient in both ICAM-1 and ICAM-2. Collectively, our results suggest that the expression of ICAM-1 and ICAM-2 on lung endothelial and epithelial cells, as well as on DCs and B cells, is not critical for the generation of innate or adaptive anti-viral immunity in the lungs. Our findings also suggest that endothelial VCAM-1 can substitute for the functions of vascular ICAMs in leukocyte trafficking into various lung compartments.

Keywords: emigration, ICAM-1, lymph nodes, VCAM-1

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26 Evaluation of Existence of Antithyroid Antibodies, Anti-Thyroid Peroxidase and Anti-Thyroglobulin in Patients with Hepatitis C Viral Infections

Authors: Junaid Mahmood Alam, Sana Anwar, Sarah Sughra Asghar

Abstract:

Chronic hepatitis or Hepatitis C viral (HCV) infection has been identified as one of the factors that could elicit autoimmune disease resulting in the development of auto-antibodies. Furthermore, HCV is implicated in contravening of forbearance to antigens, therefore, inciting auto-reactivity. In this regard, several near and past studies noted the prevalence of thyroid dysfunction and production of anti-thyroid antibodies (ATAb) such as anti-thyroid peroxidase (AntiTPO) and anti-thyroglobulin (AntiTG) in patients with HCV. Likewise, one of the etiologies of augmentation of thyroid disease is basically interferon therapy for HCV infections, for which a number of autoimmune diseases have been noted including Grave’s disease, Hishimoto thyroiditis. A prospectively case-control study was therefore carried out at department of clinical biochemistry lab services and chemical pathology in collaboration with department of clinical microbiology, at Liaquat National Hospital and Medical College, Karachi Pakistan for the period January 2015 to December 2017. Two control groups were inducted for comparison purpose, control group 1 = without HCV infection and with thyroid disorders (n = 20), control group 2 = with HCV infection and without thyroid disorders (n = 20), whereas HCV infected were n = 40 where more than half were noted to be positive for either of HCV IgG and Ag. In HCV group, patients with existing sub-clinical hypothyroidism and clinical hyperthyroidism were less than 5%. Analysis showed the presence of AntiTG in 12 HCV patients (30%), AntiTPO in 15 (37.5%) and both AntiTG and antiTPO in 10 patients (25%). Only 3 patients were found with the history of anti-thyroid auto-antibodies (7.5%) and one with parents and relatives with auto-immune disorders (2.5%). Patients that remained untreated were 12 (30%), under treatment 18 (45%) and with complete-course of treatment 10 (25%). As per review of the literature, meta-analysis of evident data and cross-sectional studies of selective cohorts (as studied in presented research), thyroid connection is designated as one of the most recurrent endocrine ailment associated with chronic HCV infection. Moreover, it also represents an extrahepatic disease in the continuum of HCV syndrome. In conclusion, HCV patients were more likely to encompass thyroid disorders especially related to development of either of ATAb or both antiTG and AntiTPO.

Keywords: Hepatitis C viral (HCV) infection, anti-thyroid antibodies, anti-thyroid peroxidase antibodies, anti-thyroglobulin antibodies

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25 Evaluation of Antibody Titer Produced in Layer Chicken after Vaccination with an Experimental Ornitobacterium rhinotracheal Vaccine

Authors: Mohammad Javad Mehrabanpour, Mohammad Hosein Hosseini, Ali Shirazi, Dorsa Mehrabanpour

Abstract:

Respiratory infections are the most important diseases that affect poultry. Ornithobacterium rhinotracheale is a bacterium that causes respiratory infections including alveolar inflation and pneumonia in birds. The aim of this study was to evaluated antibody titer against Ornitobacterium rhinotracheal in layer chicken sera after vaccination with an experimental ORT vaccine that produced in Razi Vaccine and Serum Research Institute. Cultured bacteria were inactivated by formalin, and controlled tests were conducted on it. The obtained antigens were formulated using Montanide oil and were homogenized using homogenizer. Eighty SPF chickens were kept until the age of 14 days under existing standards for temperature, humidity, and light. At the age of 14 days, chickens were divided into 3 groups. The first group included 50 chickens injected with prepared ORT vaccine, the second group, as control group, included 15 chickens injected with sterile PBS to get stress of infection and the third group included 15 chickens with no injection performed to them. All 3 groups were kept in separate cages at same room. Blood samples were regularly taken from the chickens every week for serum separation and evaluation of antibody titer. During the fifth week post vaccination, booster vaccine was injected into the chickens of vaccinated group. The chickens were inspected every day in terms of mortality as well as any injection site reactions. Three weeks after the booster injection, blood samples were taken from all chickens of all groups, and sera were isolated. The sera of immunized (vaccinated) SPF chickens with ORT vaccine as well as that of SPF chickens in the control groups were reviewed according to the recommendations of ELISA kit manufacturer to examine the chicken’s humeral immune response to the studied vaccine. Potency, stability and sterility tests were also performed on the above mentioned vaccine. Results obtained indicate high antibody titer in sera of chickens vaccinated with experimental ORT vaccine as compared with the control groups that emphasize the ability of experimentally prepared ORT vaccine to stimulate humoral immune response of chicken. After the second injection, antibody titer increased and remained almost stable up to 9 weeks after the injection. ORT vaccine can cause potency in chickens and can protect them against disease.

Keywords: antibody, layer chicken, Ornithobactrium rhinotracitis, vaccine

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24 Cytology Is a Promising Tool for the Diagnosis of High-Grade Serous Ovarian Carcinoma from Ascites

Authors: Miceska Simona, Škof Erik, Frković Grazio Snježana, Jeričević Anja, Smrkolj Špela, Cvjetićanin Branko, Novaković Srdjan, Grčar Kuzmanov Biljana, Kloboves-Prevodnik Veronika

Abstract:

Objectives: High-grade serous ovarian cancer (HGSOC) is characterized by the dissemination of the tumor cells (TC) in the peritoneal cavity forming malignant ascites at the time of diagnosis or recurrence. Still, cytology itself has been underutilized as a modality for the diagnosis of HGSOC from ascites, and histological examination from the tumor tissue is yet the only validated method used. The objective of this study was to evaluate the reliability of cytology in the diagnosis of HGSOC in relation to the histopathological examination. Methods: The study included 42 patients with histologically confirmed HGSOC, accompanied by malignant ascites. To confirm the malignancy of the TC in the ascites and to define their immunophenotype, immunohistochemical reaction (IHC) of the following antigens: Calretinin, MOC, WT1, PAX8, p53, p16 & Ki-67 was evaluated on ascites cytospins and tissue blocks. For complete cytological determination of HGSOC, BRCA 1/2 gene mutation was determined from ascites, tissue block, and blood. BRCA1/2 mutation from blood was performed to define the type of mutation, somatic vs germline. Results: Among 42 patients, the immunophenotype of HGSOC from ascites was confirmed in 36 cases (86%). For more profound analysis, the patients were divided in 3 groups regarding the number of TC present in the ascites: patients with less than 10% TC, 10% TC, and more than 10% TC. From all included patients, in the group with less than 10% TC, there were 10 cases, and only 5 of them(50%) showed HGSOC phenotype; 12 cases had equally 10% of TC, and 11 cases (92%) showed HGSOC phenotype; 20 cases had more than 10% TC and all of them (100%) confirmed the HGSOC immunophenotype from ascites. Only 33 patients were eligible for further BRCA1/2 analysis. Eleven BRCA1/2 mutations were detected from thetissue block: 6 germline and 5 somatic. In 2 cases with less than 10% TC, BRCA1/2 mutation was not detected; 4 cases had 10% TC, and 2 of them (50%) confirmed the mutation; 4 cases had more than 10% TC, and all showed 100% reliability with the tumor tissue. Conclusions: Cytology is a highly reliable method for determining the immunophenotype of HGSOC and BRCA1/2 mutation if more than 10% of tumor cells are present in the ascites. This may present an additional non-invasive clinical approach for fast and effective diagnose in the future, especially in inoperable conditions or relapses.

Keywords: cytology, ascites, high-grade serous ovarian cancer, immunophenotype, BRCA1/2

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23 Normal Hematopoietic Stem Cell and the Toxic Effect of Parthenolide

Authors: Alsulami H., Alghamdi N., Alasker A., Almohen N., Shome D.

Abstract:

Most conventional chemotherapeutic agents which are used for the treatment of cancers not only eradicate cancer cells but also affect normal hematopoietic Stem cells (HSCs) that leads to severe pancytopenia during treatment. Therefore, a need exists for novel approaches to treat cancer without or with minimum effect on normal HSCs. Parthenolide (PTL), a herbal product occurring naturally in the plant Feverfew, is a potential new chemotherapeutic agent for the treatment of many cancers such as acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL). In this study we investigated the effect of different PTL concentrations on the viability of normal HSCs and also on the ability of these cells to form colonies after they have been treated with PTL in vitro. Methods: In this study, 24 samples of bone marrow and cord blood were collected with consent, and mononuclear cells were separated using density gradient separation. These cells were then exposed to various concentrations of PTL for 24 hours. Cell viability after culture was determined using 7ADD in a flow cytometry test. Additionally, the impact of PTL on hematopoietic stem cells (HSCs) was evaluated using a colony forming unit assay (CFU). Furthermore, the levels of NFҝB expression were assessed by using a PE-labelled anti-pNFκBP65 antibody. Results: this study showed that there was no statistically significant difference in the percentage of cell death between untreated and PTL treated cells with 5 μM PTL (p = 0.7), 10 μM PTL (p = 0.4) and 25 μM (p = 0.09) respectively. However, at higher doses, PTL caused significant increase in the percentage of cell death. These results were significant when compared to untreated control (p < 0.001). The response of cord blood cells (n=4) on the other hand was slightly different from that for bone marrow cells in that the percentage of cell death was significant at 100 μM PTL. Therefore, cord blood cells seemed more resistant than bone marrow cells. Discussion &Conclusion: At concentrations ≤25 μM PTL has a minimum or no effect on HSCs in vitro. Cord blood HSCs are more resistant to PTL compared to bone marrow HSCs. This could be due to the higher percentage of T-lymphocytes, which are resistant to PTL, in CB samples (85% in CB vs. 56% in BM. Additionally, CB samples contained a higher proportion of CD34+ cells, with 14.5% of brightly CD34+ cells compared to only 1% in normal BM. These bright CD34+ cells in CB were mostly negative for early-stage stem cell maturation antigens, making them young and resilient to oxidative stress and high concentrations of PTL.

Keywords: stem cell, parthenolide, NFKB, CLL

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22 Proteomic Analysis of Cytoplasmic Antigen from Brucella canis to Characterize Immunogenic Proteins Responded with Naturally Infected Dogs

Authors: J. J. Lee, S. R. Sung, E. J. Yum, S. C. Kim, B. H. Hyun, M. Her, H. S. Lee

Abstract:

Canine brucellosis is a critical problem in dogs leading to reproductive diseases which are mainly caused by Brucella canis. There are, nonetheless, not clear symptoms so that it may go unnoticed in most of the cases. Serodiagnosis for canine brucellosis has not been confirmed. Moreover, it has substantial difficulties due to broad cross-reactivity between the rough cell wall antigens of B. canis and heterospecific antibodies present in normal, uninfected dogs. Thus, this study was conducted to characterize the immunogenic proteins in cytoplasmic antigen (CPAg) of B. canis, which defined the antigenic sensitivity of the humoral antibody responses to B. canis-infected dogs. In analysis of B. canis CPAg, first, we extracted and purified the cytoplasmic proteins from cultured B. canis by hot-saline inactivation, ultrafiltration, sonication, and ultracentrifugation step by step according to the sonicated antigen extract method. For characterization of this antigen, we checked the sort and range of each protein on SDS-PAGE and verified the immunogenic proteins leading to reaction with antisera of B. canis-infected dogs. Selected immunodominant proteins were identified using MALDI-MS/MS. As a result, in an immunoproteomic assay, several polypeptides in CPAg on one or two-dimensional electrophoresis (DE) were specifically reacted to antisera from B. canis-infected dogs but not from non-infected dogs. The polypeptides with approximate 150, 80, 60, 52, 33, 26, 17, 15, 13, 11 kDa on 1-DE were dominantly recognized by antisera from B. canis-infected dogs. In the immunoblot profiles on 2-DE, ten immunodominant proteins in CPAg were detected with antisera of infected dogs between pI 3.5-6.5 at approximate 35 to 10 KDa, without any nonspecific reaction with sera in non-infected dogs. Ten immunodominant proteins identified by MALDI-MS/MS were identified as superoxide dismutase, bacteroferritin, amino acid ABC transporter substrate-binding protein, extracellular solute-binding protein family3, transaldolase, 26kDa periplasmic immunogenic protein, Rhizopine-binding protein, enoyl-CoA hydratase, arginase and type1 glyceraldehyde-3-phosphate dehydrogenase. Most of these proteins were determined by their cytoplasmic or periplasmic localization with metabolism and transporter functions. Consequently, this study discovered and identified the prominent immunogenic proteins in B. canis CPAg, highlighting that those antigenic proteins may accomplish a specific serodiagnosis for canine brucellosis. Furthermore, we will evaluate those immunodominant proteins for applying to the advanced diagnostic methods with high specificity and accuracy.

Keywords: Brucella canis, Canine brucellosis, cytoplasmic antigen, immunogenic proteins

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21 Immunocytochemical Stability of Antigens in Cytological Samples Stored in In-house Liquid-Based Medium

Authors: Anamarija Kuhar, Veronika Kloboves Prevodnik, Nataša Nolde, Ulrika Klopčič

Abstract:

The decision for immunocytochemistry (ICC) is usually made in the basis of the findings in Giemsa- and/or Papanicolaou- smears. More demanding diagnostic cases require preparation of additional cytological preparations. Therefore, it is convenient to suspend cytological samples in a liquid based medium (LBM) that preserve antigen and morphological properties. However, the duration of these properties being preserved in the medium is usually unknown. Eventually, cell morphology becomes impaired and altered, as well as antigen properties may be lost or become diffused. In this study, the influence of cytological sample storage length in in-house liquid based medium on antigen properties and cell morphology is evaluated. The question is how long the cytological samples in this medium can be stored so that the results of immunocytochemical reactions are still reliable and can be safely used in routine cytopathological diagnostics. The stability of 6 ICC markers that are most frequently used in everyday routine work were tested; Cytokeratin AE1/AE3, Calretinin, Epithelial specific antigen Ep-CAM (MOC-31), CD 45, Oestrogen receptor (ER), and Melanoma triple cocktail were tested on methanol fixed cytospins prepared from fresh fine needle aspiration biopsies, effusion samples, and disintegrated lymph nodes suspended in in-house cell medium. Cytospins were prepared on the day of the sampling as well as on the second, fourth, fifth, and eight day after sample collection. Next, they were fixed in methanol and immunocytochemically stained. Finally, the percentage of positive stained cells, reaction intensity, counterstaining, and cell morphology were assessed using two assessment methods: the internal assessment and the UK NEQAS ICC scheme assessment. Results show that the antigen properties for Cytokeratin AE1/AE3, MOC-31, CD 45, ER, and Melanoma triple cocktail were preserved even after 8 days of storage in in-house LBM, while the antigen properties for Calretinin remained unchanged only for 4 days. The key parameters for assessing detection of antigen are the proportion of cells with a positive reaction and intensity of staining. Well preserved cell morphology is highly important for reliable interpretation of ICC reaction. Therefore, it would be valuable to perform a similar analysis for other ICC markers to determine the duration in which the antigen and morphological properties are preserved in LBM.

Keywords: cytology samples, cytospins, immunocytochemistry, liquid-based cytology

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20 Morphological and Molecular Evaluation of Dengue Virus Serotype 3 Infection in BALB/c Mice Lungs

Authors: Gabriela C. Caldas, Fernanda C. Jacome, Arthur da C. Rasinhas, Ortrud M. Barth, Flavia B. dos Santos, Priscila C. G. Nunes, Yuli R. M. de Souza, Pedro Paulo de A. Manso, Marcelo P. Machado, Debora F. Barreto-Vieira

Abstract:

The establishment of animal models for studies of DENV infections has been challenging, since circulating epidemic viruses do not naturally infect nonhuman species. Such studies are of great relevance to the various areas of dengue research, including immunopathogenesis, drug development and vaccines. In this scenario, the main objective of this study is to verify possible morphological changes, as well as the presence of antigens and viral RNA in lung samples from BALB/c mice experimentally infected with an epidemic and non-neuroadapted DENV-3 strain. Male BALB/c mice, 2 months old, were inoculated with DENV-3 by intravenous route. After 72 hours of infection, the animals were euthanized and the lungs were collected. Part of the samples was processed by standard technique for analysis by light and transmission electronic microscopies and another part was processed for real-time PCR analysis. Morphological analyzes of lungs from uninfected mice showed preserved tissue areas. In mice infected with DENV-3, the analyzes revealed interalveolar septum thickening with presence of inflammatory infiltrate, foci of alveolar atelectasis and hyperventilation, bleeding foci in the interalveolar septum and bronchioles, peripheral capillary congestion, accumulation of fluid in the blood capillary, signs of interstitial cell necrosis presence of platelets and mononuclear inflammatory cells circulating in the capillaries and/or adhered to the endothelium. In addition, activation of endothelial cells, platelets, mononuclear inflammatory cell and neutrophil-type polymorphonuclear inflammatory cell evidenced by the emission of cytoplasmic membrane prolongation was observed. DEN-like particles were seen in the cytoplasm of endothelial cells. The viral genome was recovered from 3 in 12 lung samples. These results demonstrate that the BALB / c mouse represents a suitable model for the study of the histopathological changes induced by DENV infection in the lung, with tissue alterations similar to those observed in human cases of DEN.

Keywords: BALB/c mice, dengue, histopathology, lung, ultrastructure

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19 A Matched Case-Control Study to Asses the Association of Chikunguynya Severity among Blood Groups and Other Determinants in Tesseney, Gash Barka Zone, Eritrea

Authors: Ghirmay Teklemicheal, Samsom Mehari, Sara Tesfay

Abstract:

Objectives: A total of 1074 suspected chikungunya cases were reported in Tesseney Province, Gash Barka region, Eritrea, during an outbreak. This study was aimed to assess the possible association of chikungunya severity among ABO blood groups and other potential determinants. Methods: A sex-matched and age-matched case-control study was conducted during the outbreak. For each case, one control subject had been selected from the mild Chikungunya cases. Along the same line of argument, a second control subject had also been designated through which neighborhood of cases were analyzed, scrutinized, and appeared to the scheme of comparison. Time is always the most sacrosanct element in pursuance of any study. According to the temporal calculation, this study was pursued from October 15, 2018, to November 15, 2018. Coming to the methodological dependability, calculating odds ratios (ORs) and conditional (fixed-effect) logistic regression methods were being applied. As a consequence of this, the data was analyzed and construed on the basis of the aforementioned methodological systems. Results: In this outbreak, 137 severe suspected chikungunya cases and 137 mild chikungunya suspected patients, and 137 controls free of chikungunya from the neighborhood of cases were analyzed. Non-O individuals compared to those with O blood group indicated as significant with a p-value of 0.002. Separate blood group comparison among A and O blood groups reflected as significant with a p-value of 0.002. However, there was no significant difference in the severity of chikungunya among B, AB, and O blood groups with a p-value of 0.113 and 0.708, respectively, and a strong association of chikungunya severity was found with hypertension and diabetes (p-value of < 0.0001); whereas, there was no association between chikungunya severity and asthma with a p-value of 0.695 and also no association with pregnancy (p-value =0.881), ventilator (p-value =0.181), air conditioner (p-value = 0.247), and didn’t use latrine and pit latrine (p-value = 0.318), among individuals using septic and pit latrine (p-value = 0.567) and also among individuals using flush and pit latrine (p-value = 0.194). Conclusions: Non- O blood groups were found to be at risk more than their counterpart O blood group individuals with severe form of chikungunya disease. By the same token, individuals with chronic disease were more prone to severe forms of the disease in comparison with individuals without chronic disease. Prioritization is recommended for patients with chronic diseases and non-O blood group since they are found to be susceptible to severe chikungunya disease. Identification of human cell surface receptor(s) for CHIKV is quite necessary for further understanding of its pathophysiology in humans. Therefore, molecular and functional studies will necessarily be helpful in disclosing the association of blood group antigens and CHIKV infections.

Keywords: Chikungunya, Chikungunya virus, disease outbreaks, case-control studies, Eritrea

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18 Designing of Multi-Epitope Peptide Vaccines for Fasciolosis (Fasciola gigantica) using Immune Epitope and Analysis Resource (IEDB) Server

Authors: Supanan Chansap, Werachon Cheukamud, Pornanan Kueakhai, Narin Changklungmoa

Abstract:

Fasciola species (Fasciola spp.) is caused fasciolosis in ruminants such as cattle, sheep, and buffalo. Fasciola gigantica (F.gigantica) commonly infects tropical regions. Fasciola hepatica (F.hepatica) in temperate regions. Liver fluke infection affects livestock economically, for example, reduced milk and meat production, weight loss, sterile animals. Currently, Triclabendazole is used to treat liver flukes. However, liver flukes have also been found to be resistant to drugs in countries. Therefore, vaccination is an attractive alternative to prevent liver fluke infection. Peptide vaccines are new vaccine technologies that mimic epitope antigens that trigger an immune response. An interesting antigen used in vaccine production is catepsin L, a family of proteins that play an important role in the life of the parasite in the host. This study aims to identify immunogenic regions of protein and construct a multi-epidetope vaccine using an immunoinformatic tool. Fasciola gigantica Cathepsin L1 (FgCatL1), Fasciola gigantica Cathepsin L1G (FgCatL1G), and Fasciola gigantica Cathepsin L1H (FgCatL1H) were predicted B-cell and Helper T lymphocytes (HTL) by Immune Epitope and Analysis Resource (IEDB) servers. Both B-cell and HTL epitopes aligned with cathepsin L of the host and Fasciola hepatica (F. hepatica). Epitope groups were selected from non-conserved regions and overlapping sequences with F. hepatica. All overlapping epitopes were linked with the GPGPG and KK linker. GPGPG linker was linked between B-cell epitope. KK linker was linked between HTL epitope and B-cell and HTL epitope. The antigenic scores of multi-epitope peptide vaccine was 0.7824. multi-epitope peptide vaccine was non-allergen, non-toxic, and good soluble. Multi-epitope peptide vaccine was predicted tertiary structure and refinement model by I-Tasser and GalaxyRefine server, respectively. The result of refine structure model was good quality that was generated by Ramachandran plot analysis. Discontinuous and linear B-cell epitopes were predicted by ElliPro server. Multi-epitope peptide vaccine model was two and seven of discontinuous and linear B-cell epitopes, respectively. Furthermore, multi-epitope peptide vaccine was docked with Toll-like receptor 2 (TLR-2). The lowest energy ranged from -901.3 kJ/mol. In summary, multi-epitope peptide vaccine was antigenicity and probably immune response. Therefore, multi-epitope peptide vaccine could be used to prevent F. gigantica infections in the future.

Keywords: fasciola gigantica, Immunoinformatic tools, multi-epitope, Vaccine

Procedia PDF Downloads 55
17 Data Quality and Associated Factors on Regular Immunization Programme at Ararso District: Somali Region- Ethiopia

Authors: Eyob Seife, Molla Alemayaehu, Tesfalem Teshome, Bereket Seyoum, Behailu Getachew

Abstract:

Globally, immunization averts between 2 and 3 million deaths yearly, but Vaccine-Preventable Diseases still account for more in Sub-Saharan African countries and takes the majority of under-five deaths yearly, which indicates the need for consistent and on-time information to have evidence-based decision so as to save lives of these vulnerable groups. However, ensuring data of sufficient quality and promoting an information-use culture at the point of collection remains critical and challenging, especially in remote areas where the Ararso district is selected based on a hypothesis of there is a difference in reported and recounted immunization data consistency. Data quality is dependent on different factors where organizational, behavioral, technical and contextual factors are the mentioned ones. A cross-sectional quantitative study was conducted on September 2022 in the Ararso district. The study used the world health organization (WHO) recommended data quality self-assessment (DQS) tools. Immunization tally sheets, registers and reporting documents were reviewed at 4 health facilities (1 health center and 3 health posts) of primary health care units for one fiscal year (12 months) to determine the accuracy ratio, availability and timeliness of reports. The data was collected by trained DQS assessors to explore the quality of monitoring systems at health posts, health centers, and at the district health office. A quality index (QI), availability and timeliness of reports were assessed. Accuracy ratios formulated were: the first and third doses of pentavalent vaccines, fully immunized (FI), TT2+ and the first dose of measles-containing vaccines (MCV). In this study, facility-level results showed poor timeliness at all levels and both over-reporting and under-reporting were observed at all levels when computing the accuracy ratio of registration to health post reports found at health centers for almost all antigens verified. A quality index (QI) of all facilities also showed poor results. Most of the verified immunization data accuracy ratios were found to be relatively better than that of quality index and timeliness of reports. So attention should be given to improving the capacity of staff, timeliness of reports and quality of monitoring system components, namely recording, reporting, archiving, data analysis and using information for decisions at all levels, especially in remote and areas.

Keywords: accuracy ratio, ararso district, quality of monitoring system, regular immunization program, timeliness of reports, Somali region-Ethiopia

Procedia PDF Downloads 45
16 Application of Mesenchymal Stem Cells in Diabetic Therapy

Authors: K. J. Keerthi, Vasundhara Kamineni, A. Ravi Shanker, T. Rammurthy, A. Vijaya Lakshmi, Q. Hasan

Abstract:

Pancreatic β-cells are the predominant insulin-producing cell types within the Islets of Langerhans and insulin is the primary hormone which regulates carbohydrate and fat metabolism. Apoptosis of β-cells or insufficient insulin production leads to Diabetes Mellitus (DM). Current therapy for diabetes includes either medical management or insulin replacement and regular monitoring. Replacement of β- cells is an attractive treatment option for both Type-1 and Type-2 DM in view of the recent paper which indicates that β-cells apoptosis is the common underlying cause for both the Types of DM. With the development of Edmonton protocol, pancreatic β-cells allo-transplantation became possible, but this is still not considered as standard of care due to subsequent requirement of lifelong immunosuppression and the scarcity of suitable healthy organs to retrieve pancreatic β-cell. Fetal pancreatic cells from abortuses were developed as a possible therapeutic option for Diabetes, however, this posed several ethical issues. Hence, in the present study Mesenchymal stem cells (MSCs) were differentiated into insulin producing cells which were isolated from Human Umbilical cord (HUC) tissue. MSCs have already made their mark in the growing field of regenerative medicine, and their therapeutic worth has already been validated for a number of conditions. HUC samples were collected with prior informed consent as approved by the Institutional ethical committee. HUC (n=26) were processed using a combination of both mechanical and enzymatic (collagenase-II, 100 U/ml, Gibco ) methods to obtain MSCs which were cultured in-vitro in L-DMEM (Low glucose Dulbecco's Modified Eagle's Medium, Sigma, 4.5 mM glucose/L), 10% FBS in 5% CO2 incubator at 37°C. After reaching 80-90% confluency, MSCs were characterized with Flowcytometry and Immunocytochemistry for specific cell surface antigens. Cells expressed CD90+, CD73+, CD105+, CD34-, CD45-, HLA-DR-/Low and Vimentin+. These cells were differentiated to β-cells by using H-DMEM (High glucose Dulbecco's Modified Eagle's Medium,25 mM glucose/L, Gibco), β-Mercaptoethanol (0.1mM, Hi-Media), basic Fibroblast growth factor (10 µg /L,Gibco), and Nicotinamide (10 mmol/L, Hi-Media). Pancreatic β-cells were confirmed by positive Dithizone staining and were found to be functionally active as they released 8 IU/ml insulin on glucose stimulation. Isolating MSCs from usually discarded, abundantly available HUC tissue, expanding and differentiating to β-cells may be the most feasible cell therapy option for the millions of people suffering from DM globally.

Keywords: diabetes mellitus, human umbilical cord, mesenchymal stem cells, differentiation

Procedia PDF Downloads 240
15 Efficacy of Pooled Sera in Comparison with Commercially Acquired Quality Control Sample for Internal Quality Control at the Nkwen District Hospital Laboratory

Authors: Diom Loreen Ndum, Omarine Njimanted

Abstract:

With increasing automation in clinical laboratories, the requirements for quality control materials have greatly increased in order to monitor daily performance. The constant use of commercial control material is not economically feasible for many developing countries because of non-availability or the high-cost of the materials. Therefore, preparation and use of in-house quality control serum will be a very cost-effective measure with respect to laboratory needs.The objective of this study was to determine the efficacy of in-house prepared pooled sera with respect to commercially acquired control sample for routine internal quality control at the Nkwen District Hospital Laboratory. This was an analytical study, serum was taken from leftover serum samples of 5 healthy adult blood donors at the blood bank of Nkwen District Hospital, which had been screened negative for human immunodeficiency virus (HIV), hepatitis C virus (HCV) and Hepatitis B antigens (HBsAg), and were pooled together in a sterile container. From the pooled sera, sixty aliquots of 150µL each were prepared. Forty aliquots of 150µL each of commercially acquired samples were prepared after reconstitution and stored in a deep freezer at − 20°C until it was required for analysis. This study started from the 9th June to 12th August 2022. Every day, alongside with commercial control sample, one aliquot of pooled sera was removed from the deep freezer and allowed to thaw before analyzed for the following parameters: blood urea, serum creatinine, aspartate aminotransferase (AST), alanine aminotransferase (ALT), potassium and sodium. After getting the first 20 values for each parameter of pooled sera, the mean, standard deviation and coefficient of variation were calculated, and a Levey-Jennings (L-J) chart established. The mean and standard deviation for commercially acquired control sample was provided by the manufacturer. The following results were observed; pooled sera had lesser standard deviation for creatinine, urea and AST than commercially acquired control samples. There was statistically significant difference (p<0.05) between the mean values of creatinine, urea and AST for in-house quality control when compared with commercial control. The coefficient of variation for the parameters for both commercial control and in-house control samples were less than 30%, which is an acceptable difference. The L-J charts revealed shifts and trends (warning signs), so troubleshooting and corrective measures were taken. In conclusion, in-house quality control sample prepared from pooled serum can be a good control sample for routine internal quality control.

Keywords: internal quality control, levey-jennings chart, pooled sera, shifts, trends, westgard rules

Procedia PDF Downloads 54
14 Nano-Immunoassay for Diagnosis of Active Schistosomal Infection

Authors: Manal M. Kame, Hanan G. El-Baz, Zeinab A.Demerdash, Engy M. Abd El-Moneem, Mohamed A. Hendawy, Ibrahim R. Bayoumi

Abstract:

There is a constant need to improve the performance of current diagnostic assays of schistosomiasis as well as develop innovative testing strategies to meet new testing challenges. This study aims at increasing the diagnostic efficiency of monoclonal antibody (MAb)-based antigen detection assays through gold nanoparticles conjugated with specific anti-Schistosoma mansoni monoclonal antibodies. In this study, several hybidoma cell lines secreting MAbs against adult worm tegumental Schistosoma antigen (AWTA) were produced at Immunology Department of Theodor Bilharz Research Institute and preserved in liquid nitrogen. One MAb (6D/6F) was chosen for this study due to its high reactivity to schistosome antigens with highest optical density (OD) values. Gold nanoparticles (AuNPs) were functionalized and conjugated with MAb (6D/6F). The study was conducted on serum samples of 116 subjects: 71 patients with S. mansoni eggs in their stool samples group (gp 1), 25 with other parasites (gp2) and 20 negative healthy controls (gp3). Patients in gp1 were further subdivided according to egg count in their stool samples into Light infection {≤ 50 egg per gram(epg) (n= 17)}, moderate {51-100 epg (n= 33)} and severe infection {>100 epg(n= 21)}. Sandwich ELISA was performed using (AuNPs -MAb) for detection of circulating schistosomal antigen (CSA) levels in serum samples of all groups and the results were compared with that after using MAb/ sandwich ELISA system. Results Gold- MAb/ ELISA system reached a lower detection limit of 10 ng/ml compared to 85 ng/ml on using MAb/ ELISA and the optimal concentrations of AuNPs -MAb were found to be 12 folds less than that of MAb/ ELISA system for detection of CSA. The sensitivity and specificity of sandwich ELISA for detection of CSA levels using AuNPs -MAb were 100% & 97.8 % respectively compared to 87.3% &93.38% respectively on using MAb/ ELISA system. It was found that CSA was detected in 9 out of 71 S.mansoni infected patients on using AuNPs - MAb/ ELISA system and was not detected by MAb/ ELISA system. All those patients (9) was found to have an egg count below 50 epg feces (patients with light infections). ROC curve analyses revealed that sandwich ELISA using gold-MAb was an excellent diagnostic investigator that could differentiate Schistosoma patients from healthy controls, on the other hand it revealed that sandwich ELISA using MAb was not accurate enough as it could not recognize nine out of 71 patients with light infections. Conclusion Our data demonstrated that: Loading gold nanoparticles with MAb (6D/6F) increases the sensitivity and specificity of sandwich ELISA for detection of CSA, thus active (early) and light infections could be easily detected. Moreover this binding will decrease the amount of MAb consumed in the assay and lower the coast. The significant positive correlation that was detected between ova count (intensity of infection) and OD reading in sandwich ELISA using gold- MAb enables its use to detect the severity of infections and follow up patients after treatment for monitoring of cure.

Keywords: Schistosomiasis, nanoparticles, gold, monoclonal antibodies, ELISA

Procedia PDF Downloads 351
13 Anisakidosis in Turkey: Serological Survey and Risk for Humans

Authors: E. Akdur Öztürk, F. İrvasa Bilgiç, A. Ludovisi , O. Gülbahar, D. Dirim Erdoğan, M. Korkmaz, M. Á. Gómez Morales

Abstract:

Anisakidosis is a zoonotic human fish-borne parasitic disease caused by accidental ingestion of anisakid third-stage larvae (L3) of members of the Anisakidae family present in infected marine fish or cephalopods. Infection with anisakid larvae can lead to gastric, intestinal, extra-gastrointestinal and gastroallergic forms of the disease. Anisakid parasites have been reported in almost all seas, particularly in the Mediterranean Sea. There is a remarkably high level of risk exposure to these zoonotic parasites as they are present in economically and ecologically important fish of Europe. Anisakid L3 larvae have been also detected in several fish species from the Aegean Sea. Turkey is a peninsular country surrounded by Black, Aegean and the Mediterranean Sea. In this country, fishing habit and fishery product consumption are highly common. In recent years, there was also an increase in the consumption of raw fish due to the increasing interest in the cuisine of the Far East countries. In different regions of Turkey, A. simplex (inMerluccius Merluccius Scomber japonicus, Trachurus mediterraneus, Sardina pilchardus, Engraulis encrasicolus, etc.), Anisakis spp., Contraceucum spp., Pseudoterronova spp. and, C. aduncum were identified as well. Although it is accepted both the presence of anisakid parasites in fish and fishery products in Turkey and the presence of Turkish people with allergic manifestations after fish consumption, there are no reports of human anisakiasis in this country. Given the high prevalence of anisakid parasites in the country, the absence of reports is likely not due to the absence of clinical cases rather to the unavailability of diagnostic tools and the low awareness of the presence of this infection. The aim of the study was to set up an IgE-Western Blot (WB) based test to detect the anisakidosis sensitization among Turkish people with a history of allergic manifestation related to fish consumption. To this end, crude worm antigens (CWA) and allergen enriched fraction (50-66% ) were prepared from L3 of A. simplex (s.l.) collected from Lepidopus caudatus fished in the Mediterranean Sea. These proteins were electrophoretically separated and transferred into the nitrocellulose membranes. By WB, specific proteins recognized by positive control serum samples from sensitized patients were visualized on nitrocellulose membranes by a colorimetric reaction. The CWA and 50–66% fraction showed specific bands, mainly due to Ani s 1 (20-22 kD) and Ani s 4 (9-10 kD). So far, a total of 7 serum samples from people with allergic manifestation and positive skin prick test (SPT) after fish consumption, have been tested and all of them resulted negative by WB, indicating the lack of sensitization to anisakids. This preliminary study allowed to set up a specific test and evidence the lack of correlation between both tests, SPT and WB. However, the sample size should be increased to estimate the anisakidosis burden in Turkish people.

Keywords: anisakidosis, fish parasite, serodiagnosis, Turkey

Procedia PDF Downloads 111
12 Data Quality on Regular Childhood Immunization Programme at Degehabur District: Somali Region, Ethiopia

Authors: Eyob Seife

Abstract:

Immunization is a life-saving intervention which prevents needless suffering through sickness, disability, and death. Emphasis on data quality and use will become even stronger with the development of the immunization agenda 2030 (IA2030). Quality of data is a key factor in generating reliable health information that enables monitoring progress, financial planning, vaccine forecasting capacities, and making decisions for continuous improvement of the national immunization program. However, ensuring data of sufficient quality and promoting an information-use culture at the point of the collection remains critical and challenging, especially in hard-to-reach and pastoralist areas where Degehabur district is selected based on a hypothesis of ‘there is no difference in reported and recounted immunization data consistency. Data quality is dependent on different factors where organizational, behavioral, technical, and contextual factors are the mentioned ones. A cross-sectional quantitative study was conducted on September 2022 in the Degehabur district. The study used the world health organization (WHO) recommended data quality self-assessment (DQS) tools. Immunization tally sheets, registers, and reporting documents were reviewed at 5 health facilities (2 health centers and 3 health posts) of primary health care units for one fiscal year (12 months) to determine the accuracy ratio. The data was collected by trained DQS assessors to explore the quality of monitoring systems at health posts, health centers, and the district health office. A quality index (QI) was assessed, and the accuracy ratio formulated were: the first and third doses of pentavalent vaccines, fully immunized (FI), and the first dose of measles-containing vaccines (MCV). In this study, facility-level results showed both over-reporting and under-reporting were observed at health posts when computing the accuracy ratio of the tally sheet to health post reports found at health centers for almost all antigens verified where pentavalent 1 was 88.3%, 60.4%, and 125.6% for Health posts A, B, and C respectively. For first-dose measles-containing vaccines (MCV), similarly, the accuracy ratio was found to be 126.6%, 42.6%, and 140.9% for Health posts A, B, and C, respectively. The accuracy ratio for fully immunized children also showed 0% for health posts A and B and 100% for health post-C. A relatively better accuracy ratio was seen at health centers where the first pentavalent dose was 97.4% and 103.3% for health centers A and B, while a first dose of measles-containing vaccines (MCV) was 89.2% and 100.9% for health centers A and B, respectively. A quality index (QI) of all facilities also showed results between the maximum of 33.33% and a minimum of 0%. Most of the verified immunization data accuracy ratios were found to be relatively better at the health center level. However, the quality of the monitoring system is poor at all levels, besides poor data accuracy at all health posts. So attention should be given to improving the capacity of staff and quality of monitoring system components, namely recording, reporting, archiving, data analysis, and using information for decision at all levels, especially in pastoralist areas where such kinds of study findings need to be improved beside to improving the data quality at root and health posts level.

Keywords: accuracy ratio, Degehabur District, regular childhood immunization program, quality of monitoring system, Somali Region-Ethiopia

Procedia PDF Downloads 80
11 Defense Priming from Egg to Larvae in Litopenaeus vannamei with Non-Pathogenic and Pathogenic Bacteria Strains

Authors: Angelica Alvarez-Lee, Sergio Martinez-Diaz, Jose Luis Garcia-Corona, Humberto Lanz-Mendoza

Abstract:

World aquaculture is always looking for improvements to achieve productions with high yields avoiding the infection by pathogenic agents. The best way to achieve this is to know the biological model to create alternative treatments that could be applied in the hatcheries, which results in greater economic gains and improvements in human public health. In the last decade, immunomodulation in shrimp culture with probiotics, organic acids and different carbon sources has gained great interest, mainly in larval and juvenile stages. Immune priming is associated with a strong protective effect against a later pathogen challenge. This work provides another perspective about immunostimulation from spawning until hatching. The stimulation happens during development embryos and generates resistance to infection by pathogenic bacteria. Massive spawnings of white shrimp L. vannamei were obtained and placed in experimental units with 700 mL of sterile seawater at 30 °C, salinity of 28 ppm and continuous aeration at a density of 8 embryos.mL⁻¹. The immunostimulating effect of three death strains of non-pathogenic bacterial (Escherichia coli, Staphylococcus aureus and Bacillus subtilis) and a pathogenic strain for white shrimp (Vibrio parahaemolyticus) was evaluated. The strains killed by heat were adjusted to O.D. 0.5, at A 600 nm, and directly added to the seawater of each unit at a ratio of 1/100 (v/v). A control group of embryos without inoculum of dead bacteria was kept under the same physicochemical conditions as the rest of the treatments throughout the experiment and used as reference. The duration of the stimulus was 12 hours, then, the larvae that hatched were collected, counted and transferred to a new experimental unit (same physicochemical conditions but at a salinity of 28 ppm) to carry out a challenge of infection against the pathogen V. parahaemolyticus, adding directly to seawater an amount 1/100 (v/v) of the live strain adjusted to an OD 0.5; at A 600 nm. Subsequently, 24 hrs after infection, nauplii survival was evaluated. The results of this work shows that, after 24 hrs, the hatching rates of immunostimulated shrimp embryos with the dead strains of B. subtillis and V. parahaemolyticus are significantly higher compared to the rest of the treatments and the control. Furthermore, survival of L. vanammei after a challenge of infection of 24 hrs against the live strain of V. parahaemolyticus is greater (P < 0.05) in the larvae immunostimulated during the embryonic development with the dead strains B. subtillis and V. parahaemolyticus, followed by those that were treated with E. coli. In summary superficial antigens can stimulate the development cells to promote hatching and can have normal development in agreeing with the optical observations, plus exist a differential response effect between each treatment post-infection. This research provides evidence of the immunostimulant effect of death pathogenic and non-pathogenic bacterial strains in the rate of hatching and oversight of shrimp L. vannamei during embryonic and larval development. This research continues evaluating the effect of these death strains on the expression of genes related to the defense priming in larvae of L. vannamei that come from massive spawning in hatcheries before and after the infection challenge against V. parahaemolyticus.

Keywords: immunostimulation, L. vannamei, hatching, survival

Procedia PDF Downloads 119
10 Development of Biosensor Chip for Detection of Specific Antibodies to HSV-1

Authors: Zatovska T. V., Nesterova N. V., Baranova G. V., Zagorodnya S. D.

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In recent years, biosensor technologies based on the phenomenon of surface plasmon resonance (SPR) are becoming increasingly used in biology and medicine. Their application facilitates exploration in real time progress of binding of biomolecules and identification of agents that specifically interact with biologically active substances immobilized on the biosensor surface (biochips). Special attention is paid to the use of Biosensor analysis in determining the antibody-antigen interaction in the diagnostics of diseases caused by viruses and bacteria. According to WHO, the diseases that are caused by the herpes simplex virus (HSV), take second place (15.8%) after influenza as a cause of death from viral infections. Current diagnostics of HSV infection include PCR and ELISA assays. The latter allows determination the degree of immune response to viral infection and respective stages of its progress. In this regard, the searches for new and available diagnostic methods are very important. This work was aimed to develop Biosensor chip for detection of specific antibodies to HSV-1 in the human blood serum. The proteins of HSV1 (strain US) were used as antigens. The viral particles were accumulated in cell culture MDBK and purified by differential centrifugation in cesium chloride density gradient. Analysis of the HSV1 proteins was performed by polyacrylamide gel electrophoresis and ELISA. The protein concentration was measured using De Novix DS-11 spectrophotometer. The device for detection of antigen-antibody interactions was an optoelectronic two-channel spectrometer ‘Plasmon-6’, using the SPR phenomenon in the Krechman optical configuration. It was developed at the Lashkarev Institute of Semiconductor Physics of NASU. The used carrier was a glass plate covered with 45 nm gold film. Screening of human blood serums was performed using the test system ‘HSV-1 IgG ELISA’ (GenWay, USA). Development of Biosensor chip included optimization of conditions of viral antigen sorption and analysis steps. For immobilization of viral proteins 0.2% solution of Dextran 17, 200 (Sigma, USA) was used. Sorption of antigen took place at 4-8°C within 18-24 hours. After washing of chip, three times with citrate buffer (pH 5,0) 1% solution of BSA was applied to block the sites not occupied by viral antigen. It was found direct dependence between the amount of immobilized HSV1 antigen and SPR response. Using obtained biochips, panels of 25 positive and 10 negative for the content of antibodies to HSV-1 human sera were analyzed. The average value of SPR response was 185 a.s. for negative sera and from 312 to. 1264 a.s. for positive sera. It was shown that SPR data were agreed with ELISA results in 96% of samples proving the great potential of SPR in such researches. It was investigated the possibility of biochip regeneration and it was shown that application of 10 mM NaOH solution leads to rupture of intermolecular bonds. This allows reuse the chip several times. Thus, in this study biosensor chip for detection of specific antibodies to HSV1 was successfully developed expanding a range of diagnostic methods for this pathogen.

Keywords: biochip, herpes virus, SPR

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9 Immunoliposome-Mediated Drug Delivery to Plasmodium-Infected and Non-Infected Red Blood Cells as a Dual Therapeutic/Prophylactic Antimalarial Strategy

Authors: Ernest Moles, Patricia Urbán, María Belén Jiménez-Díaz, Sara Viera-Morilla, Iñigo Angulo-Barturen, Maria Antònia Busquets, Xavier Fernàndez-Busquets

Abstract:

Bearing in mind the absence of an effective vaccine against malaria and its severe clinical manifestations causing nearly half a million deaths every year, this disease represents nowadays a major threat to life. Besides, the basic rationale followed by currently marketed antimalarial approaches is based on the administration of drugs on their own, promoting the emergence of drug-resistant parasites owing to the limitation in delivering drug payloads into the parasitized erythrocyte high enough to kill the intracellular pathogen while minimizing the risk of causing toxic side effects to the patient. Such dichotomy has been successfully addressed through the specific delivery of immunoliposome (iLP)-encapsulated antimalarials to Plasmodium falciparum-infected red blood cells (pRBCs). Unfortunately, this strategy has not progressed towards clinical applications, whereas in vitro assays rarely reach drug efficacy improvements above 10-fold. Here, we show that encapsulation efficiencies reaching >96% can be achieved for the weakly basic drugs chloroquine (CQ) and primaquine using the pH gradient active loading method in liposomes composed of neutrally charged, saturated phospholipids. Targeting antibodies are best conjugated through their primary amino groups, adjusting chemical crosslinker concentration to retain significant antigen recognition. Antigens from non-parasitized RBCs have also been considered as targets for the intracellular delivery of drugs not affecting the erythrocytic metabolism. Using this strategy, we have obtained unprecedented nanocarrier targeting to early intraerythrocytic stages of the malaria parasite for which there is a lack of specific extracellular molecular tags. Polyethylene glycol-coated liposomes conjugated with monoclonal antibodies specific for the erythrocyte surface protein glycophorin A (anti-GPA iLP) were capable of targeting 100% RBCs and pRBCs at the low concentration of 0.5 μM total lipid in the culture, with >95% of added iLPs retained into the cells. When exposed for only 15 min to P. falciparum in vitro cultures synchronized at early stages, free CQ had no significant effect over parasite viability up to 200 nM drug, whereas iLP-encapsulated 50 nM CQ completely arrested its growth. Furthermore, when assayed in vivo in P. falciparum-infected humanized mice, anti-GPA iLPs cleared the pathogen below detectable levels at a CQ dose of 0.5 mg/kg. In comparison, free CQ administered at 1.75 mg/kg was, at most, 40-fold less efficient. Our data suggest that this significant improvement in drug antimalarial efficacy is in part due to a prophylactic effect of CQ found by the pathogen in its host cell right at the very moment of invasion.

Keywords: immunoliposomal nanoparticles, malaria, prophylactic-therapeutic polyvalent activity, targeted drug delivery

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8 The Porcine Reproductive and Respiratory Syndrome Virus Genotype 2 (PRRSV-2)-derived Oncolytic Protein Reprograms Tumor-Associated Macrophages

Authors: Farrah Putri Salmanida, Mei-Li Wu, Rika Wahyuningtyas, Wen-Bin Chung, Hso-Chi Chaung, Ko-Tung Chang

Abstract:

Within the field of immunotherapy, oncolytic virotherapy (OVT) employs dual approaches that directly eliminate tumor cells while preserving healthy ones and indirectly reprogram the tumor microenvironment (TME) to elicit antitumor responses. Within the TME, tumor associated macrophages (TAMs) manifest characteristics akin to those of anti-inflammatory M2 macrophages, thus earning the designation of M2-like TAMs. In prior research, two antigens denoted as A1 (g6Ld10T) and A3 (ORF6L5), derived from a complete sequence of ORF5 with partial sequence of ORF6 in Porcine Reproductive and Respiratory Syndrome Virus Genotype 2 (PRRSV-2), demonstrated the capacity to repolarize M2-type porcine alveolar macrophages (PAMs) into M1 phenotypes. In this study, we sought for utilizing OVT strategies by introducing A1 or A3 on TAMs to endow them with the anti-tumor traits of M1 macrophages while retaining their capacity to target cancer cells. Upon exposing human THP-1-derived M2 macrophages to a cross-species test with 2 µg/ml of either A1 or A3 for 24 hours, real time PCR revealed that A3, but not A1, treated cells exhibited upregulated gene expressions of M1 markers (CCR7, IL-1ß, CCL2, Cox2, CD80). These cells reacted to virus-derived antigen, as evidenced by increased expression of pattern-recognition receptors TLR3, TLR7, and TLR9, subsequently providing feedback in the form of type I interferon responses like IFNAR1, IFN-ß, IRF3, IRF7, OAS1, Mx1, and ISG15. Through an MTT assay, only after 15 µg/ml of A3 treatment could the cell viability decrease, with a predicted IC50 of 16.96 µg/ml. Interestingly, A3 caused dose-dependent toxicity to a rat C6 glial cancer cell line even at doses as low as 2.5 µg/ml and reached its IC50 at 9.419 µg/ml. Using Annexin V/7AAD staining and PCR test, we deduced that a significant proportion of C6 cells were undergoing the early apoptosis phase predominantly through the intrinsic apoptosis cascade involving Bcl-2 family proteins. Following this stage, we conducted a test on A3’s repolarization ability, which revealed a significant rise in M1 gene expression markers, such as TNF, CD80, and IL-1ß, in M2-like TAMs generated in vitro from murine RAW264.7 macrophages grown with conditioned medium of 4T1 breast cancer cells. This was corroborated by the results of transcriptome analysis, which revealed that the primary subset among the top 10 to top 30 significantly upregulated differentially expressed genes (DEGs) dominantly consisted of M1 macrophages profiles, including Ccl3, Ccl4, Csf3, TNF, Bcl6b, Stc1, and Dusp2. Our findings unveiled the remarkable potential of the PRRSV-derived antigen A3 to repolarize macrophages while also being capable of selectively inducing apoptosis in cancerous cells. While further in vivo study is needed for A3, it holds promise as an adjuvant by its dual effects in cancer therapy modalities.

Keywords: cancer cell apoptosis, interferon responses, macrophage repolarization, recombinant protein

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7 Transformers in Gene Expression-Based Classification

Authors: Babak Forouraghi

Abstract:

A genetic circuit is a collection of interacting genes and proteins that enable individual cells to implement and perform vital biological functions such as cell division, growth, death, and signaling. In cell engineering, synthetic gene circuits are engineered networks of genes specifically designed to implement functionalities that are not evolved by nature. These engineered networks enable scientists to tackle complex problems such as engineering cells to produce therapeutics within the patient's body, altering T cells to target cancer-related antigens for treatment, improving antibody production using engineered cells, tissue engineering, and production of genetically modified plants and livestock. Construction of computational models to realize genetic circuits is an especially challenging task since it requires the discovery of flow of genetic information in complex biological systems. Building synthetic biological models is also a time-consuming process with relatively low prediction accuracy for highly complex genetic circuits. The primary goal of this study was to investigate the utility of a pre-trained bidirectional encoder transformer that can accurately predict gene expressions in genetic circuit designs. The main reason behind using transformers is their innate ability (attention mechanism) to take account of the semantic context present in long DNA chains that are heavily dependent on spatial representation of their constituent genes. Previous approaches to gene circuit design, such as CNN and RNN architectures, are unable to capture semantic dependencies in long contexts as required in most real-world applications of synthetic biology. For instance, RNN models (LSTM, GRU), although able to learn long-term dependencies, greatly suffer from vanishing gradient and low-efficiency problem when they sequentially process past states and compresses contextual information into a bottleneck with long input sequences. In other words, these architectures are not equipped with the necessary attention mechanisms to follow a long chain of genes with thousands of tokens. To address the above-mentioned limitations of previous approaches, a transformer model was built in this work as a variation to the existing DNA Bidirectional Encoder Representations from Transformers (DNABERT) model. It is shown that the proposed transformer is capable of capturing contextual information from long input sequences with attention mechanism. In a previous work on genetic circuit design, the traditional approaches to classification and regression, such as Random Forrest, Support Vector Machine, and Artificial Neural Networks, were able to achieve reasonably high R2 accuracy levels of 0.95 to 0.97. However, the transformer model utilized in this work with its attention-based mechanism, was able to achieve a perfect accuracy level of 100%. Further, it is demonstrated that the efficiency of the transformer-based gene expression classifier is not dependent on presence of large amounts of training examples, which may be difficult to compile in many real-world gene circuit designs.

Keywords: transformers, generative ai, gene expression design, classification

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6 Transcriptional Differences in B cell Subpopulations over the Course of Preclinical Autoimmunity Development

Authors: Aleksandra Bylinska, Samantha Slight-Webb, Kevin Thomas, Miles Smith, Susan Macwana, Nicolas Dominguez, Eliza Chakravarty, Joan T. Merrill, Judith A. James, Joel M. Guthridge

Abstract:

Background: Systemic Lupus Erythematosus (SLE) is an interferon-related autoimmune disease characterized by B cell dysfunction. One of the main hallmarks is a loss of tolerance to self-antigens leading to increased levels of autoantibodies against nuclear components (ANAs). However, up to 20% of healthy ANA+ individuals will not develop clinical illness. SLE is more prevalent among women and minority populations (African, Asian American and Hispanics). Moreover, African Americans have a stronger interferon (IFN) signature and develop more severe symptoms. The exact mechanisms involved in ethnicity-dependent B cell dysregulation and the progression of autoimmune disease from ANA+ healthy individuals to clinical disease remains unclear. Methods: Peripheral blood mononuclear cells (PBMCs) from African (AA) and European American (EA) ANA- (n=12), ANA+ (n=12) and SLE (n=12) individuals were assessed by multimodal scRNA-Seq/CITE-Seq methods to examine differential gene signatures in specific B cell subsets. Library preparation was done with a 10X Genomics Chromium according to established protocols and sequenced on Illumina NextSeq. The data were further analyzed for distinct cluster identification and differential gene signatures in the Seurat package in R and pathways analysis was performed using Ingenuity Pathways Analysis (IPA). Results: Comparing all subjects, 14 distinct B cell clusters were identified using a community detection algorithm and visualized with Uniform Manifold Approximation Projection (UMAP). The proportion of each of those clusters varied by disease status and ethnicity. Transitional B cells trended higher in ANA+ healthy individuals, especially in AA. Ribonucleoprotein high population (HNRNPH1 elevated, heterogeneous nuclear ribonucleoprotein, RNP-Hi) of proliferating Naïve B cells were more prevalent in SLE patients, specifically in EA. Interferon-induced protein high population (IFIT-Hi) of Naive B cells are increased in EA ANA- individuals. The proportion of memory B cells and plasma cells clusters tend to be expanded in SLE patients. As anticipated, we observed a higher signature of cytokine-related pathways, especially interferon, in SLE individuals. Pathway analysis among AA individuals revealed an NRF2-mediated Oxidative Stress response signature in the transitional B cell cluster, not seen in EA individuals. TNFR1/2 and Sirtuin Signaling pathway genes were higher in AA IFIT-Hi Naive B cells, whereas they were not detected in EA individuals. Interferon signaling was observed in B cells in both ethnicities. Oxidative phosphorylation was found in age-related B cells (ABCs) for both ethnicities, whereas Death Receptor Signaling was found only in EA patients in these cells. Interferon-related transcription factors were elevated in ABCs and IFIT-Hi Naive B cells in SLE subjects of both ethnicities. Conclusions: ANA+ healthy individuals have altered gene expression pathways in B cells that might drive apoptosis and subsequent clinical autoimmune pathogenesis. Increases in certain regulatory pathways may delay progression to SLE. Further, AA individuals have more elevated activation pathways that may make them more susceptible to SLE.

Keywords:

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5 On the Utility of Bidirectional Transformers in Gene Expression-Based Classification

Authors: Babak Forouraghi

Abstract:

A genetic circuit is a collection of interacting genes and proteins that enable individual cells to implement and perform vital biological functions such as cell division, growth, death, and signaling. In cell engineering, synthetic gene circuits are engineered networks of genes specifically designed to implement functionalities that are not evolved by nature. These engineered networks enable scientists to tackle complex problems such as engineering cells to produce therapeutics within the patient's body, altering T cells to target cancer-related antigens for treatment, improving antibody production using engineered cells, tissue engineering, and production of genetically modified plants and livestock. Construction of computational models to realize genetic circuits is an especially challenging task since it requires the discovery of the flow of genetic information in complex biological systems. Building synthetic biological models is also a time-consuming process with relatively low prediction accuracy for highly complex genetic circuits. The primary goal of this study was to investigate the utility of a pre-trained bidirectional encoder transformer that can accurately predict gene expressions in genetic circuit designs. The main reason behind using transformers is their innate ability (attention mechanism) to take account of the semantic context present in long DNA chains that are heavily dependent on the spatial representation of their constituent genes. Previous approaches to gene circuit design, such as CNN and RNN architectures, are unable to capture semantic dependencies in long contexts, as required in most real-world applications of synthetic biology. For instance, RNN models (LSTM, GRU), although able to learn long-term dependencies, greatly suffer from vanishing gradient and low-efficiency problem when they sequentially process past states and compresses contextual information into a bottleneck with long input sequences. In other words, these architectures are not equipped with the necessary attention mechanisms to follow a long chain of genes with thousands of tokens. To address the above-mentioned limitations, a transformer model was built in this work as a variation to the existing DNA Bidirectional Encoder Representations from Transformers (DNABERT) model. It is shown that the proposed transformer is capable of capturing contextual information from long input sequences with an attention mechanism. In previous works on genetic circuit design, the traditional approaches to classification and regression, such as Random Forrest, Support Vector Machine, and Artificial Neural Networks, were able to achieve reasonably high R2 accuracy levels of 0.95 to 0.97. However, the transformer model utilized in this work, with its attention-based mechanism, was able to achieve a perfect accuracy level of 100%. Further, it is demonstrated that the efficiency of the transformer-based gene expression classifier is not dependent on the presence of large amounts of training examples, which may be difficult to compile in many real-world gene circuit designs.

Keywords: machine learning, classification and regression, gene circuit design, bidirectional transformers

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4 In vivo Evaluation of LAB Probiotic Potential with the Zebrafish Animal Model

Authors: Iñaki Iturria, Pasquale Russo, Montserrat Nacher-Vázquez, Giuseppe Spano, Paloma López, Miguel Angel Pardo

Abstract:

Introduction: It is known that some Lactic Acid Bacteria (LAB) present an interesting probiotic effect. Probiotic bacteria stimulate host resistance to microbial pathogens and thereby aid in immune response, and modulate the host's immune responses to antigens with a potential to down-regulate hypersensitivity reactions. Therefore, probiotic therapy is valuable against intestinal infections and may be beneficial in the treatment of Inflammatory Bowel Disease (IBD). Several in vitro tests are available to evaluate the probiotic potential of a LAB strain. However, an in vivo model is required to understand the interaction between the host immune system and the bacteria. During the last few years, zebrafish (Danio rerio) has gained interest as a promising vertebrate model in this field. This organism has been extensively used to study the interaction between the host and the microbiota, as well as the host immune response under several microbial infections. In this work, we report on the use of the zebrafish model to investigate in vivo the colonizing ability and the immunomodulatory effect of probiotic LAB. Methods: Lactobacillus strains belonging to different LAB species were fluorescently tagged and used to colonize germ-free zebrafish larvae gastrointestinal tract (GIT). Some of the strains had a well-documented probiotic effect (L. acidophilus LA5); while others presented an exopolysaccharide (EPS) producing phenotype, thus allowing evaluating the influence of EPS in the colonization and immunomodulatory effect. Bacteria colonization was monitored for 72 h by direct observation in real time using fluorescent microscopy. CFU count per larva was also evaluated at different times. The immunomodulatory effect was assessed analysing the differential expression of several innate immune system genes (MyD88, NF-κB, Tlr4, Il1β and Il10) by qRT- PCR. The anti-inflammatory effect was evaluated using a chemical enterocolitis zebrafish model. The protective effect against a pathogen was also studied. To that end, a challenge test was developed using a fluorescently tagged pathogen (Vibrio anguillarum-GFP+). The progression of the infection was monitored up to 3 days using a fluorescent stereomicroscope. Mortality rates and CFU counts were also registered. Results and conclusions: Larvae exposed to EPS-producing bacteria showed a higher fluorescence and CFU count than those colonized with no-EPS phenotype LAB. In the same way, qRT-PCR results revealed an immunomodulatory effect on the host after the administration of the strains with probiotic activity. A downregulation of proinflammatory cytoquines as well as other cellular mediators of inflammation was observed. The anti-inflammatory effect was found to be particularly marked following exposure to LA% strain, as well as EPS producing strains. Furthermore, the challenge test revealed a protective effect of probiotic administration. As a matter of fact, larvae fed with probiotics showed a decrease in the mortality rate ranging from 20 to 35%. Discussion: In this work, we developed a promising model, based on the use of gnotobiotic zebrafish coupled with a bacterial fluorescent tagging in order to evaluate the probiotic potential of different LAB strains. We have successfully used this system to monitor in real time the colonization and persistence of exogenous LAB within the gut of zebrafish larvae, to evaluate their immunomodulatory effect and for in vivo competition assays. This approach could bring further insights into the complex microbial-host interactions at intestinal level.

Keywords: gnotobiotic, immune system, lactic acid bacteria, probiotics, zebrafish

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3 The Role of Cholesterol Oxidase of Mycobacterium tuberculosis in the Down-Regulation of TLR2-Signaling Pathway in Human Macrophages during Infection Process

Authors: Michal Kielbik, Izabela Szulc-Kielbik, Anna Brzostek, Jaroslaw Dziadek, Magdalena Klink

Abstract:

The goal of many research groups in the world is to find new components that are important for survival of mycobacteria in the host cells. Mycobacterium tuberculosis (Mtb) possesses a number of enzymes degrading cholesterol that are considered to be an important factor for its survival and persistence in host macrophages. One of them - cholesterol oxidase (ChoD), although not being essential for cholesterol degradation, is discussed as a virulence compound, however its involvement in macrophages’ response to Mtb is still not sufficiently determined. The recognition of tubercle bacilli antigens by pathogen recognition receptors is crucial for the initiation of the host innate immune response. An important receptor that has been implicated in the recognition and/or uptake of Mtb is Toll-like receptor type 2 (TLR2). Engagement of TLR2 results in the activation and phosphorylation of intracellular signaling proteins including IRAK-1 and -4, TRAF-6, which in turn leads to the activation of target kinases and transcription factors responsible for bactericidal and pro-inflammatory response of macrophages. The aim of these studies was a detailed clarification of the role of Mtb cholesterol oxidase as a virulence factor affecting the TLR2 signaling pathway in human macrophages. As human macrophages the THP-1 differentiated cells were applied. The virulent wild-type Mtb strain (H37Rv), its mutant lacking a functional copy of gene encoding cholesterol oxidase (∆choD), as well as complimented strain (∆choD–choD) were used. We tested the impact of Mtb strains on the expression of TLR2-depended signaling proteins (mRNA level, cytosolic level and phosphorylation status). The cytokine and bactericidal response of THP-1 derived macrophages infected with Mtb strains in relation to TLR2 signaling pathway dependence was also determined. We found that during the 24-hours of infection process the wild-type and complemented Mtb significantly reduced the cytosolic level and phosphorylation status of IRAK-4 and TRAF-6 proteins in macrophages, that was not observed in the case of ΔchoD mutant. Decreasement of TLR2-dependent signaling proteins, induced by wild-type Mtb, was not dependent on the activity of proteasome. Blocking of TLR2 expression, before infection, effectively prevented the induced by wild-type strain reduction of cytosolic level and phosphorylation of IRAK-4. None of the strains affected the surface expression of TLR2. The mRNA level of IRAK-4 and TRAF-6 genes were significantly increased in macrophages 24 hours post-infection with either of tested strains. However, the impact of wild-type Mtb strain on both examined genes was significantly stronger than its ΔchoD mutant. We also found that wild-type strain stimulated macrophages to release high amount of immunosuppressive IL-10, accompanied by low amount of pro-inflammatory IL-8 and bactericidal nitric oxide in comparison to mutant lacking cholesterol oxidase. The influence of wild-type Mtb on this type of macrophages' response strongly dependent on fully active IRAK-1 and IRAK-4 signaling proteins. In conclusion, Mtb using cholesterol oxidase causes the over-activation of TLR2 signaling proteins leading to the reduction of their cytosolic level and activity resulting in the modulation of macrophages response to allow its intracellular survival. Supported by grant: 2014/15/B/NZ6/01565, National Science Center, Poland

Keywords: Mycobacterium tuberculosis, cholesterol oxidase, macrophages, TLR2-dependent signaling pathway

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2 Targeting Basic Leucine Zipper Transcription Factor ATF-Like Mediated Immune Cells Regulation to Reduce Crohn’s Disease Fistula Incidence

Authors: Mohammadjavad Sotoudeheian, Soroush Nematollahi

Abstract:

Crohn’s disease (CD) is a chronic gastrointestinal segment inflammation encompassing immune dysregulation in a genetically susceptible individual in response to the environmental triggers and interaction between the microbiome and immune system. Uncontrolled inflammation leads to long-term complications, including fibrotic strictures and enteric fistulae. Increased production of Th1 and Th17-cell cytokines and defects in T-regulatory cells have been associated with CD. Th17-cells are essential for protection against extracellular pathogens, but their atypical activity can cause autoimmunity. Intrinsic defects in the control of programmed cell death in the mucosal T-cell compartment are strongly implicated in the pathogenesis of CD. The apoptosis defect in mucosal T-cells in CD has been endorsed as an imbalance of the Bcl-2 and the Bax. The immune system encounters foreign antigens through microbial colonization of mucosal surfaces or infections. In addition, FOSL downregulated IL-26 expression, a cytokine that marks inflammatory Th17-populations in patients suffering from CD. Furthermore, the expression of IL-23 is associated with the transcription factor primary leucine zipper transcription factor ATF-like (Batf). Batf-deficiency demonstrated the crucial role of Batf in colitis development. Batf and IL-23 mediate their effects by inducing IL-6 production. Strong association of IL-23R, Stat3, and Stat4 with IBD susceptibility point to a critical involvement of T-cells. IL-23R levels in transfer fistula were dependent on the AP-1 transcription factor JunB that additionally controlled levels of RORγt by facilitating DNA binding of Batf. T lymphocytes lacking JunB failed to induce IL-23- and Th17-mediated experimental colitis highlighting the relevance of JunB for the IL-23/ Th17 pathway. The absence of T-bet causes unrestrained Th17-cell differentiation. T-cells are central parts of immune-mediated colon fistula. Especially Th17-cells were highly prevalent in inflamed IBD tissues, as RORγt is effective in preventing colitis. Intraepithelial lymphocytes (IEL) contain unique T-cell subsets, including cells expressing RORγt. Increased activated Th17 and decreased T-regulatory cells in inflamed intestinal tissues had been seen. T-cells differentiate in response to many cytokines, including IL-1β, IL-6, IL-23, and TGF-β, into Th17-cells, a process which is critically dependent on the Batf. IL-23 promotes Th17-cell in the colon. Batf manages the generation of IL-23 induced IL-23R+ Th17-cells. Batf is necessary for TGF-β/IL-6-induced Th17-polarization. Batf-expressing T-cells are the core of T-cell-mediated colitis. The human-specific parts of three AP-1 transcription factors, FOSL1, FOSL2, and BATF, are essential during the early stages of Th17 differentiation. BATF supports the Th17 lineage. FOSL1, FOSL2, and BATF make possession of regulatory loci of genes in the Th17 lineage cascade. The AP1 transcription factor Batf is identified to control intestinal inflammation and seems to regulate pathways within lymphocytes, which could theoretically control the expression of several genes. It shows central regulatory properties over Th17-cell development and is intensely upregulated within IBD-affected tissues. Here, we demonstrated that targeting Batf in IBD appears as a therapeutic approach that reduces colitogenic T-cell activities during fistula formation while aiming to affect inflammation in the gut epithelial cells.

Keywords: immune system, Crohn’s Disease, BATF, T helper cells, Bcl, interleukin, FOSL

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