Search results for: lymphocytes

Authors: Malinee Pongsavee

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Sodium formate is the chemical substance used for food additive. Catalase is the important antioxidative enzyme in protecting the cell from oxidative damage by reactive oxygen species (ROS). The resultant level of oxidative stress in sodium formatetreated lymphocytes was investigated. The sodium formate concentrations of 0.05, 0.1, 0.2, 0.4 and 0.6 mg/mL were treated in human lymphocytes for 12 hours. After 12 treated hours, catalase activity change was measured in sodium formate-treated lymphocytes. The results showed that the sodium formate concentrations of 0.4 and 0.6 mg/mL significantly decreased catalase activities in lymphocytes (P < 0.05). The change of catalase activity in sodium formate-treated lymphocytes may be the oxidative damage marker for detect sodium formate exposure in human.

Keywords: sodium formate, catalase activity, oxidative damage marker, toxicity

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Authors: Olena Grygorieva

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Nowadays mechanisms of thymocytes emigration from the thymus to the periphery are investigated actively. We have proposed a hypothesis of thymocytes’ migration from the thymus through lymphatic vessels during periodical short-term local edema. By morphological, hystochemical methods we have examined quantity of lymphocytes, epitelioreticulocytes, mast cells, blood and lymphatic vessels in morpho-functional areas of rats’ thymuses during the first week after birth in 4 hours interval. In newborn and beginning from 8 hour after birth every 12 hours specific density of the thymus, absolute quantity of microcirculatory vessels, especially of lymphatic ones, lymphcyte-epithelial index, quantity of mast cells and their degranulative forms increase. Structure of extracellular matrix, intrathymical microenvironment and lymphocytes’ adhesive properties change. Absolute quantity of small lymphocytes in thymic cortex changes wavy. All these changes are straightly expressed from 0 till 2, from 12 till 16, from 108 till 120 hours of postnatal life. During this periods paravasal lymphatic vessels are stuffed with lymphocytes, i.e. discrete migration of lymphocytes from the thymus occurs. After rapid edema reduction, quantity of lymphatic vessels decrease, they become empty. Therefore, in the thymus of newborn periodical short-term local edema is observed, on its top discrete migration of lymphocytes from the thymus occurs.

Keywords: lymphocytes, lymphatic vessels, mast cells, thymus

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Authors: Małgorzata Tomczyńska, Joanna Saluk-Bijak

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Background: Graves’ disease is a frequent organ-specific autoimmune thyroid disease, which characterized by the presence of different kind autoantibodies, that, in most cases, act as agonists of the thyrotropin receptor, leading to hyperthyroidism. Role of platelets and lymphocytes can be modulated in the pathophysiology of thyroid autoimmune diseases. Interference in the physiology of platelets can lead to enhanced activity of these cells. Activated platelets can bind to circulating lymphocytes and to affect lymphocyte adhesion. Platelets and lymphocytes can regulate mutual functions. Therefore, the activation of T lymphocytes, as well as blood platelets, is associated with the development of inflammation and oxidative stress within the target tissue. The present study was performed to investigate a platelet-lymphocyte relation by assessing the degree of their mutual aggregation in whole blood of patients with Graves’ disease. Also, the purpose of this study was to examine the impact of platelet interaction on lymphocyte migration capacity. Methods: 30 patients with Graves’ disease were recruited in the study. The matched 30 healthy subjects were served as the control group. Immunophenotyping of lymphocytes was carried out by flow cytometry method. A CytoSelect™ Cell Migration Assay Kit was used to evaluate lymphocyte migration and adhesion to blood platelets. Visual assessment of lymphocyte-platelet aggregate morphology was done using confocal microscope after magnetic cell isolation by Miltenyi Biotec. Results: The migration and functional responses of lymphocytes to blood platelets were greater in the group of Graves’ disease patients compared with healthy controls. The group of Graves’ disease patients exhibited a reduced T lymphocyte and a higher B cell count compared with controls. Based on microscopic analysis, more platelet-lymphocyte aggregates were found in patients than in control. Conclusions: Studies have shown that in Graves' disease, lymphocytes show increased platelet affinity, more strongly migrating toward them, and forming mutual cellular conglomerates. This may be due to the increased activation of blood platelets in this disease.

Keywords: blood platelets, cell migration, Graves’ disease, lymphocytes, lymphocyte-platelet aggregates

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Authors: Rodrigo Fernandes da Silva, Daniela Maira Cardozo, Paulo Cesar Martins Alves, Sophie Françoise Derchain, Fernando Guimarães

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T-reg lymphocytes are important for the control of peripheral tolerance. They control the adaptive immune system and prevent autoimmunity through its suppressive action on CD4+ and CD8+ lymphocytes. The suppressive action also includes B lymphocytes, dendritic cells, monocytes/macrophages and recently, studies have shown that T-reg are also able to inhibit NK cells, therefore they exert their control of the immune response from innate to adaptive response. Most tumors express self-ligands, therefore it is believed that T-reg cells induce tolerance of the immune system, hindering the development of successful immunotherapies. T-reg cells have been linked to the suppression mechanisms of the immune response against tumors, including ovarian cancer. The goal of this study was to disclose the sub-population of the expanded CD3+ lymphocytes reported by previous studies, using the long-term culture model designed by Carlens et al 2001, to generate effector cell suspensions enriched with cytotoxic CD3-CD56+ NK cells, from PBMC of ovarian neoplasia patients. Methods and Results: Blood was collected from 12 patients with ovarian neoplasia after signed consent: 7 benign (Bng) and 5 malignant (Mlg). Mononuclear cells were separated by Ficoll-Paque gradient. Long-term culture was conducted by a 21 day culturing process with SCGM CellGro medium supplemented with anti-CD3 (10ng/ml, first 5 days), IL-2 (1000UI/ml) and FBS (10%). After 21 days of expansion, there was an increase in the population of CD3+ lymphocytes in the benign and malignant group. Within CD3+ population, there was a significant decrease in the population of CD4+ lymphocytes in the benign (median Bgn D-0=73.68%, D-21=21.05%) (p<0.05) and malignant (median Mlg D-0=64.00%, D-21=11.97%) (p < 0.01) group. Inversely, after 21 days of expansion, there was an increase in the population of CD8+ lymphocytes within the CD3+ population in the benign (median Bgn D-0=16.80%, D-21=38.56%) and malignant (median Mlg D-0=27.12%, D-21=72.58%) group. However, this increase was only significant on the malignant group (p<0.01). Within the CD3+CD4+ population, there was a significant increase (p < 0.05) in the population of T-reg lymphocytes in the benign (median Bgn D-0=9.84%, D-21=39.47%) and malignant (median Mlg D-0=3.56%, D-21=16.18%) group. Statistical analysis inter groups was performed by Kruskal-Wallis test and intra groups by Mann Whitney test. Conclusion: The CD4+ and CD8+ sub-population of CD3+ lymphocytes shifts with the culturing process. This might be due to the process of the immune system to produce a cytotoxic response. At the same time, T-reg lymphocytes increased within the CD4+ population, suggesting a modulation of the immune response towards cells of the immune system. The expansion of the T-reg population can hinder an immune response against cancer. Therefore, an immunotherapy using this expansion procedure should aim to halt the expansion of T-reg or its immunosuppresion capability.

Keywords: regulatory T cells, CD8+ T cells, CD4+ T cells, NK cell expansion

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Authors: Aisha Arshad, Samina Naz Mukry, Tahir Shamsi

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Background: Immune thrombocytopenia (ITP) is an autoimmune disorder. Besides platelets counts, immature platelets fraction (IPF) can be used as tool to predict megakaryocytic activity in ITP patients. The clinical biomarkers like Neutrophils to lymphocytes ratio (NLR) and platelet to lymphocytes ratio(PLR) predicts inflammation and can be used as prognostic markers.The present study was planned to assess the ratios in ITP and their utility in predicting prognosis after treatment. Methods: A total of 111 patients of ITP with same number of healthy individuals were included in this case control study during the period of January 2015 to December 2017.All the ITP patients were grouped according to guidelines of International working group of ITP. A 3cc blood was collected in EDTA tube and blood parameters were evaluated using Sysmex 1000 analyzer.The ratios were calculated by using absolute counts of Neutrophils,Lymphocytes and platelets.The significant (p=<0.05) difference between ITP patients and healthy control groups was determined by Kruskal wallis test, Dunn’s test and spearman’s correlation test was done using SPSS version 23. Results: The significantly raised total leucocytes counts (TLC) and IPF along with low platelets counts were observed in ITP patients as compared to healthy controls.In ITP groups,very low platelet count with median and IQR of 2(3.8)3x109/l with highest mean and IQR IPF 25.4(19.8)% was observed in newly diagnosed ITP group. The NLR was high with prognosis of disease as higher levels were observed in P-ITP. The PLR was significantly low in ND-ITP ,P-ITP, C-ITP, R-ITP and compared to controls with p=<0.001 as platelet were less in number in all ITP patients. Conclusion: The IPF can be used in evaluation of bone marrow response in ITP. The simple, reliable and calculated NLR and PLR ratios can be used in predicting prognosis and response to treatment in ITP and to some extend the severity of disease.

Keywords: neutrophils, platelets, lymphocytes, infection

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Authors: Felipe Osorio Ospina, Maria Adelaida Mejia Arango, Esteban Onésimo Vallejo Agudelo, Victoria Lucía Dávila Osorio, Natalia Vargas Grisales, Lina María Martínez Sanchez, Camilo Andrés Agudelo Vélez, Ángela Maria Londoño García, Isabel Cristina Ortiz Trujillo

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Excessive exposure to ultraviolet radiation, has shown to be a risk factor for photodamage, alteration of the immune mechanisms to recognize malignant cells and cutaneous pro-inflamatorios States and skin cancers. Objective: Identify the time of exposure to ultraviolet radiation for the production of chromosomal damage in human lymphocytes. Methodology: We conducted an in vitro study serial, in which samples were taken from heparinized blood of healthy people, who do not submit exposure to agents that could induce chromosomal alterations. The samples were cultured in RPMI-1640 medium containing 10% fetal bovine serum, penicillin and streptomycin antibiotic. Subsequently, they were grouped and exposed to ultraviolet light for 1 to 20 seconds. At the end of the treatments, cytology samples were prepared, and it was colored with Giemsa (5%). Reading was carried out in an optical microscope and 100 metaphases analysed by treatment for posting chromosomal alterations. Each treatment was conducted at three separate times and each became two replicas. Results: We only presented chromosomal alterations in lymphocytes exposed to UV for a groups 1 to 3 seconds (p<0.05). Conclusions: Exposure to ultraviolet radiation generates visible damage in chromosomes from human lymphocytes observed in light microscopy, the highest rates of injury was observed between two and three seconds, and above this value, the reduction in the number of mitotic cells was evident.

Keywords: ultraviolet rays, lymphocytes, chromosome breakpoints, photodamage

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Authors: Felipe Osorio Ospina, Maria Adelaida Mejia Arango, Esteban Onésimo Vallejo Agudelo, Victoria Lucía Dávila Osorio, Natalia Vargas Grisales, Lina María Martínez Sanchez, Camilo Andrés Agudelo Vélez, Ángela Maria Londoño García, Isabel Cristina Ortiz Trujillo

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Excessive exposure to ultraviolet radiation, has shown to be a risk factor for photodamage, alteration of the immune mechanisms to recognize malignant cells and cutaneous pro-inflamatorios states and skin cancers. Objective: To identify the time of exposure to ultraviolet radiation for the production of chromosomal damage in human lymphocytes. Methodology: We conducted an in vitro study serial, in which samples were taken from the heparinized blood of healthy people, who do not submit exposure to agents that could induce chromosomal alterations. The samples were cultured in RPMI-1640 medium containing 10% fetal bovine serum, penicillin, and streptomycin antibiotic. Subsequently, they were grouped and exposed to ultraviolet light for 1 to 20 seconds. At the end of the treatments, cytology samples were prepared, and it was colored with Giemsa (5%). Reading was carried out in an optical microscope and 100 metaphases analysed by treatment for posting chromosomal alterations. Each treatment was conducted at three separate times and each became two replicas. Results: We only presented chromosomal alterations in lymphocytes exposed to UV for groups 1 to 3 seconds (p < 0.05). Conclusions: Exposure to ultraviolet radiation generates visible damage in chromosomes from human lymphocytes observed in light microscopy, the highest rates of injury was observed between two and three seconds, and above this value, the reduction in the number of mitotic cells was evident.

Keywords: chromosome breakpoints, lymphocytes, photodamage, ultraviolet rays

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Authors: Amira E. Derbalah, Doaa M. Zaghloul

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10 clinically healthy hemal nodes were collected from male bulls aged 2-3 years. Light microscopy revealed a capsule of connective tissue consisted mainly of collagen fiber surrounding hemal node, numerous erythrocytes were found in wide subcapsular sinus under the capsule. The parenchyma of the hemal node was divided into cortex and medulla. Diffused lymphocytes, and lymphoid follicles, having germinal centers were the main components of the cortex, while in the medulla there was wide medullary sinus, diffused lymphocytes and few lymphoid nodules. The area occupied with lymph nodules was larger than that occupied with non-nodular structure of lymphoid cords and blood sinusoids. Electron microscopy revealed the cellular components of hemal node including elements of circulating erythrocytes intermingled with lymphocytes, plasma cells, mast cells, reticular cells, macrophages, megakaryocytes and endothelial cells lining the blood sinuses. The lymphocytes were somewhat triangular in shape with cytoplasmic processes extending between adjacent erythrocytes. Nuclei were triangular to oval in shape, lightly stained with clear nuclear membrane indentation and clear nucleoli. The reticular cells were elongated in shape with cytoplasmic processes extending between adjacent lymphocytes, rough endoplasmic reticulum, ribosomes and few lysosomes were seen in their cytoplasm. Nucleus was elongated in shape with less condensed chromatin. Plasma cells were oval to irregular in shape with numerous dilated rough endoplasmic reticulum containing electron lucent material occupying the whole cytoplasm and few mitochondria were found. Nuclei were centrally located and oval in shape with heterochromatin emarginated and often clumped near the nuclear membrane. Occasionally megakaryocytes and mast cells were seen among lymphocytes. Megakaryocytes had multilobulated nucleus and free ribosomes often appearing as small aggregates in their cytoplasm, while mast cell had their characteristic electron dense granule in the cytoplasm, few electron lucent granules were found also, we conclude that, the main function of the hemal node of cattle is proliferation of lymphocytes. No role for plasma cell in erythrophagocytosis could be suggested.

Keywords: cattle, electron microscopy, hemal node, histology, immune system

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Authors: Piamsiri Sawaisorn, Tienrat Tangchaikeeree, Waraporn Chan-On, Chaniya Leepiyasakulchai, Rachanee Udomsangpetch, Suradej Hongeng, Kulachart Jangpatarapongsa

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Human Vγ9Vδ2 T lymphocytes are regarded as promising effector cells for cancer immunotherapy since they have the ability to eliminate several tumor cells through non-peptide antigen recognition and non-major histocompatibility complex (MHC) restriction. An issue of recent interest is the capability to activate γδ T cells by use of a group of drugs, such as pamidronate, that cause accumulation of phosphoantigen which is recognized by γδ T cell receptors. Moreover, their antigen presenting cell-like phenotype and function have been confirmed in many clinical trials. In this study, Vγ9Vδ2 T cells derived from normal peripheral blood mononuclear cells were activated with pamidronate and the expanded Vγ9Vδ2 T cells can recognize and kill chronic myeloid leukemia (CML) cells treated with pamidronate through their cytotoxic activity. To support the strong role played by Vγ9Vδ2 T cells against cancer, we provide the evidence that Vγ9Vδ2 T cells activated with CML cell lysate antigen can efficiently express antigen presenting cell (APC) phenotype and function. In conclusion, pamidronate can be used in intentional activation of human Vγ9Vδ2 T cells and can increase the susceptibility of CML cells to cytotoxicity of Vγ9Vδ2 T cells. The activated Vγ9Vδ2 T cells by cancer cells lysate can show their APC characteristics, and so greatly increase the interest in exploring their therapeutic potential in hematologic malignancy.

Keywords: γδ T lymphocytes, antigen-presenting cells, chronic myeloid leukemia, cancer, immunotherapy

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Authors: Fatimah A. Alhomaid

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A total of 40 one day-old LSL chicks (Lohman Selected Loghorn) were used in this study. In 20 days-old chicks, the bursa was formed of mucosa, musculosa and serosa. Its lamina propria was lymphoid in nature. After hatching, the bursa continued to grow and became fully developed at the 30th day post- hatching. It appeared as a blind sac. Its lumen was occupied by 12-13 mucosal folds. Each fold was lined by tall columnar or pseudo- stratified columnar epithelium. Its core was made of lamina propria infiltrated by a large number of lymphoid follicles. Most follicles possessed an outer corona surrounding a germinal center. At the age of 6 weeks physiological regression of the bursa was observed. The lymphoid follicles were decreased in size, the lymphocytes were depleted and the interfollicular stroma became obvious, thicker and more fibrous. Fibrosis of the lymphoid follicles was frequently seen in some sections at the age of 30 weeks.

Keywords: Bursa of fabricius, lymphocytes, cloacal Bursa

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Authors: Wooseok Byon, Eunyoung Kim, Junseong Kwon, Byung Joo Song, Chan Heun Park

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Background/Purpose: Previous preclinical and clinical data suggest that increased lymphocytic infiltration would be associated with good prognosis and benefit from immunogenic chemotherapy especially in triple-negative breast cancer (TNBC). We investigated a single-center experience of TNBC and relationship with lymphocytic infiltration. Methods: From January 2004 to December 2012, at the Department of Surgery, Kangbuk Samsung Hospital, Sungkyunkwan University, School of Medicine, we retrospectively reviewed 897 breast cancer patients-clinical outcomes, clinicopathological characteristics, breast cancer subtypes. And we reviewed lymphocytic infiltration of TNBC specimens by two pathologists. Statistical analysis of risk factors associated with recurrence was performed. Results: A total of 897 patients, 76 were TNBC (8.47%). Mean age of TNBC patients were 50.95 (SD10.42) years, mean follow-up periods was 40.06 months. We reviewed 49 slides, and there were 8 recurrent breast cancer patients (16.32%), and 4 patients were expired (8.16%). There were 9 lymphocytic predominant breast cancers (LPBC)-carcinomas with either intratumoral lymphocytes in >60% of tumor cell nests. 1 patient of LPBC was recurred and 8 were not. In multivariate logistic regression, the odds ratio of lymphocytic infiltration was 0.59 (p=0.643). Conclusion: In a single-center experience of TNBC, the lymphocytic infiltration in tumor cell nest might be a good trend on the prognosis but there was not statistically significant.

Keywords: tumor-infiltrating lymphocytes, triple negative breast cancer, medical and health sciences

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Authors: Vahid Changizi, Aram Rostami, Akbar Mosavi

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Purpose of study: Critical macromolecules of cells such as DNA are in exposure to damage of free radicals that induced from interaction of ionizing radiation with biological systems. Selenium and vitamin-E are natural compound that has been shown to be a direct free radical scavenger. The aim of this study was to investigate the in vivo/in vitro radioprotective effect of selenium and vitamin-E separately and synergistically against genotoxicity induced by 6MV x-rays irradiation in cultured blood lymphocytes from 15 human volunteers. Methods: Fifteen volunteers were divided in three groups include A, B and C. These groups were given slenium(800 IU), vitamin-E(100 mg) and selenium(400 IU) + vitamin-E(50 mg), respectively. Peripheral blood samples were collected from each group before(0 hr) and 1, 2 and 3 hr after selenium and vitamin-E administration (separately and synergistically). Then the blood samples were irradiated to 200 cGy of 6 Mv x-rays. After that, lymphocyte samples were cultured with mitogenic stimulation to determine the chromosomal aberrations wih micronucleus assay in cytokinesis-blocked binucleated cells. Results: The lymphocytes in the blood samples collected at 1 hr after ingestion selenium and vitamin-E, exposed in vitro to x-rays exhibited a significant decrease in the incidence of micronuclei, compared with control group at 0 hr. The maximum protection and decrease in frequency of micronuclei(50%) was observed at 1 hr after administration of selenium and vitamin-E synergistically. Conclusion: The data suggest that ingestion of selenium and vitamin-E as a radioprotector substances before exposures may reduce genetic damage caused by x-rays irradiation.

Keywords: x-rays, selenium, vitamin-e, lymphocyte, micronuclei

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Authors: Q. B. Maria de la Luz Ortega Juárez, Saúl Rojas Hernández, Itzel Berenice Rodríguez Mera, María Maricela Carrasco Yépez, Mara Gutierrez Sánchez

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Naegleria fowleri is a free-living amoeba found mainly in temperate freshwater and is the etiologic agent of primary amebic meningoencephalitis (PAM), a fatal acute disease with a mortality rate greater than 95%. At present, there are no treatments available for MAP, and the development of effective vaccines that generate long-term immunological memory allowing protection against MAP would be of great importance. The objective of this work was to analyze the effector and memory immune response in BALB/c mice immunized with total extract of N. fowleri co-administered with cholera toxin. In this study, BALB/c mice were immunized four times intranasally with ET of N. fowleri adjuvanted with CT with or without booster at three months and were challenged or not with the lethal dose of N. fowleri, determining survival, the humoral, effector and memory response, by ELISA and flow cytometry techniques. The results obtained showed that the survival of mice immunized with booster had 60% protection compared to the group without booster, which obtained 20% protection. Evaluating the humoral response, it was found that both IgG and IgA levels were higher in sera than in nasal washes in both treatments. In the cellular response, the increase in the percentage of positive cells was found for effector T and B lymphocytes in the nasal passages (NP) in the group with boost and nasopharynx-associated lymphoid tissue (NALT) in the group without boost and lymphocytes only. B in both treatments, as well as in memory cells treatment with boost T lymphocytes in PN and NALT and without boost in cervical lymph nodes (CG) with respect to B lymphocytes, in PN, GC and NALT in treatment with boost and NALT in treatment without booster. Therefore, the involvement of the effector immune response and memory play a fundamental role for protection against N. fowleri and for the development of vaccine candidates.

Keywords: immune response, immunological memory, naegleria fowleri, primary amebic meningoencephalitis

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Authors: Tanveer Beg, Yasir H. Siddique, Gulshan Ara, Asfar S. Azmi, Mohammad Afzal

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Doxorubicin is a well-known DNA intercalating chemotherapy drug that is widely used for treatment of different cancers. Its clinical utility is limited due to the observed genotoxic side effects on healthy cells suggesting that newer combination and genoprotective regimens are urgently needed for the management of doxorubicin chemotherapy. Some dietary phytochemicals are well known for their protective mechanism of action and genistein from soy is recognized as an anti-oxidant with similar properties. Therefore, the present study investigates the effect of genistein against the genotoxic doses of doxorubicin by assessing chromosomal aberrations, sister chromatid exchanges, cell cycle kinetics, cell viability, apoptosis, and DNA damage markers in cultured human lymphocytes. Our results reveal that genistein treatment significantly suppresses genotoxic damage induced by doxorubicin. It is concluded that genistein has the potential to reduce the genotoxicity induced by anti-cancer drugs, thereby reducing the chances of developing secondary tumors during the therapy.

Keywords: apoptosis, DNA damage markers, doxorubicin, genistein, genotoxicity, human lymphocyte culture

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Authors: A. Ojha, Y. K. Gupta

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Organophosphate (OP) pesticides are among the most widely used synthetic chemicals for controlling a wide variety of pests throughout the world. Chlorpyrifos (CPF), methyl parathion (MPT), and malathion (MLT) are among the most extensively used OP pesticides in India. DNA strand breaks and DNA-protein crosslinks (DPC) are toxic lesions associated with the mechanisms of toxicity of genotoxic compounds. In the present study, we have examined the potential of CPF, MPT, and MLT individually and in combination, to cause DNA strand breakage and DPC formation. Peripheral blood lymphocytes of rat were exposed to 1/4 and 1/10 LC50 dose of CPF, MPT, and MLT for 2, 4, 8, and 12h. The DNA strand break was measured by the comet assay and expressed as DNA damage index while DPC estimation was done by fluorescence emission. There was significantly marked increase in DNA damage and DNA-protein crosslink formation in time and dose dependent manner. It was also observed that MPT caused the highest level of DNA damage as compared to other studied OP compounds. Thus, from present study, we can conclude that studied pesticides have genotoxic potential. The pesticides mixture does not potentiate the toxicity of each other. Nonetheless, additional in vivo data are required before a definitive conclusion can be drawn regarding hazard prediction to humans.

Keywords: organophosphate, pesticides, DNA damage, DNA protein crosslink, genotoxic

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Authors: Mohammad Y. Alfaifi

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Methidathion (MTD) (Trade name Supracide®) is a non-systemic organophosphorus insecticide used intensively worldwide including Saudi Arabia. However, there is a lack in published studies about it's genotoxicity. In this study we evaluated MTD toxicity in rat bone marrow cells (in vivo) and in lymphocytes (in vitro) using different doses based on LD50. MNNCE (Micronucleated normocromatic erythrocytes) and MNPCE (Micronucleated polychromatic erythrocytes), NDI (Nuclear division index) and NDCI (nuclear division cytotoxicity index), necrotic and apoptotic cells were recorded in rat's bone marrow samples. CA, MI (number of cells undergoing mitosis) necrotic, and apoptotic cells recorded in lymphocytes. Results showed that there was a slight increase in the frequency of micronucleated bone marrow cells. However, no structural chromosomal aberrations were detected in vivo or in vitro. On the other hand, the results showed significant increase in necrotic and apoptotic cells following MTD administration in a dose-dependent manner comparing to positive and negative control groups. In light of these results, MTD can be considered highly cytotoxic and moderate genotoxic, and precaution should be taken when using MTD.

Keywords: methidathion, micronucleus, NDI, NDCI, toxicity, chromosomal aberrations

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Authors: Seyyed Mohammad Amin Mousavi Sagharchi, Elham Javanroudi

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Introduction: Tuberculosis (TB) is a known disease with hidden features caused by Mycobacterium tuberculosis (MTB). This disease, which is one of the 10 deadliest in the world, has caused millions of deaths in recent decades. Furthermore, TB is responsible for infecting about 30% population of world. Like any infection, TB can activate the immune system by locating and colonization in the human body, especially in the alveoli. TB is granulomatosis, so MTB can absorb the host’s immune cells and other cells to form granuloma. Method: Different databases (e.g., PubMed) were recruited to prepare this paper and fulfill our goals to search and find effective papers and investigations. Results: Immune response to MTB is related to T cell killers and contains CD1, CD4, and CD8 T lymphocytes. CD1 lymphocytes can recognize glycolipids, which highly exist in the Mycobacterial fatty cell wall. CD4 lymphocytes and macrophages form granuloma, and it is the main line of immune response to Mycobacteria. On the other hand, CD8 cells have cytolytic function for directly killing MTB by secretion of granulysin. Other functions and secretion to the deal are interleukin-12 (IL-12) by induction of expression interferon-γ (INF-γ) for macrophages activation and creating a granuloma, and tumor necrosis factor (TNF) by promoting macrophage phagolysosomal fusion. Conclusion: Immune cells in battle with MTB are macrophages, dendritic cells (DCs), neutrophils, and natural killer (NK) cells. These immune cells can recognize the Mycobacterium by various receptors, including Toll-like receptors (TLRs), Nod-like receptors (NLRs), and C-type lectin receptors (CLRs) located in the cell surface. In human alveoli exist about 50 dendritic macrophages, which have close communication with other immune cells in the circulating system and epithelial cells to deal with Mycobacteria. Against immune cells, MTB handles some factors (e.g., cordfactor, O-Ag, lipoarabinomannan, sulfatides, and adenylate cyclase) and practical functions (e.g., inhibition of macrophages).

Keywords: mycobacterium tuberculosis, immune responses, immunological mechanisms, respiratory tuberculosis

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Authors: Khalid Ahmed Abdoun, Mohamed Abdulwahid Alsoufi, Ibrahim Abdullah Alhidary

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The intention of this study was to evaluate the effect of dietary Cr supplementation on haematological parameters in camel calves reared under hot summer conditions. Fifteen male camel calves (5 – 6 months old) were randomly allotted to three dietary treatments (n = 5) for a period of 84 days. Camel calves were fed ad libitum on basal diet without Cr supplementation (control), basal diet supplemented with 0.5 mg Cr/kg DM (Cr 0.5) or basal diet supplemented with 1.0 mg Cr/kg DM (Cr 1.0). During this, blood samples were collected every four weeks for hematological examination. The obtained results revealed that dietary Cr supplementation to camel calves reared under hot summer did not show significant effects (P> 0.05) on hematological variables. However, the neutrophil to lymphocytes ratio (N: L ratio) was significantly (P < 0.05) reduced in camel calves fed on diets supplemented with chromium. In conclusion, Chromium supplementation to the diet of camel calves did not show any significant effects on hematological variables. Whereas, the neutrophil to lymphocytes ratio (N: L ratio) was reduced in camel calves fed diets supplemented with chromium.

Keywords: camel calves, chromium, haematological, immune response

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Authors: S. I. Nwadioha, M. S. Odimayo, G. T. A. Jombo, E. O. P. Nwokedi

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Aims and Objectives: To determine the baseline CD4 positive T lymphocytes count of HIV/AIDS treatment naïve adults clients presenting for the first time treatment in Benue State University Teaching Hospital. Subjects and Methods: A total of 700 subjects age between 18 years to 70 years, were recruited for the study, comprising 600 HIV sero-positive patients and 100 healthy controls in Benue State University Teaching Hospital, Makurdi from 2013 to 2014. The CD4 counts of the subjects were evaluated using a Partec flow cytometer. Results: CD4 count of 200-299 cells/μl peaked with 25% (n=150/600)[control; 0%( n= 0/100)]. The study also showed that 44% (266/600) of HIV subjects had acquired immunodeficiency syndrome as defined by low CD4 counts below 200 cells/μl. Seventy-five per cent (n=451/600)of our patients would require to be placed on antiretroviral therapy with CD4 count of less than 350 cells/μl. At CD4 350 baseline criterion, age group 20-29 years had the highest demand 35%(160/451) for ARV followed by age groups 30-39 and 40-49 years with 28%(128/451) and 22%(98/451) respectively. Conclusion: There is a high prevalence of acquired immunodeficiency syndrome as defined by CD4 counts below 200 cells/μl, among the young active productive age group. The strict adopting of the ART WHO 2010 scale- up criteria doubles the number of the HIV clients that would qualify for ART with its attendant health benefits on the long run.

Keywords: CD4 counts, HIV patients, young age group, Nigeria

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Authors: Anuranjani Kumar, Madhu Bala

Abstract:

Ionising radiation exposure induces generation of free radicals and the oxidative DNA damage. SBL-1, a radioprotective leaf extract prepared from leaves Hippophae rhamnoides L. (Common name; Seabuckthorn), showed > 90% survival in mice population that was treated with lethal dose (10 Gy) of ⁶⁰Co gamma irradiation. In this study, early effects of pre-treatment with or without SBL-1 in blood peripheral blood lymphocytes (PBMCs) were investigated by cell viability assays (trypan blue and MTT). The quantitative in vitro study of Hoescht/PI staining was performed to check the apoptosis/necrosis in PBMCs irradiated at 2 Gy with or without pretreatment of SBL-1 (at different concentrations) up to 24 and 48h. Comet assay was performed in vivo, to detect the DNA strands breaks and its repair mechanism on peripheral blood lymphocytes at lethal dose (10 Gy). For this study, male mice (wt. 28 ± 2g) were administered radioprotective dose (30mg/kg body weight) of SBL-1, 30 min prior to irradiation. Animals were sacrificed at 24h and 48h. Blood was drawn through cardiac puncture, and blood lymphocytes were separated using histopaque column. Both neutral and alkaline comet assay were performed using standardized technique. In irradiated animals, alkaline comet assay revealed single strand breaks (SSBs) that showed significant (p < 0.05) increase in percent DNA in tail and Olive tail moment (OTM) at 24 h while at 48h the percent DNA in tail further increased significantly (p < 0.02). The double strands breaks (DSBs) increased significantly (p < 0.01) at 48 h in neutral assay, in comparison to untreated control. The animals pre-treated with SBL-1 before irradiation showed significantly (p < 0.05) less DSBs at 48 h treatment in comparison to irradiated group of animals. The SBL-1 alone treated group itself showed no toxicity. The antioxidant potential of SBL-1 were also investigated by in vitro biochemical assays such as DPPH (p < 0.05), ABTS, reducing ability (p < 0.09), hydroxyl radical scavenging (p < 0.05), ferric reducing antioxidant power (FRAP), superoxide radical scavenging activity (p < 0.05), hydrogen peroxide scavenging activity (p < 0.05) etc. SBL-1 showed strong free radical scavenging power that plays important role in the studies of radiation-induced injuries. The SBL-1 treated PBMCs showed significant (p < 0.02) viability in trypan blue assay at 24-hour incubation.

Keywords: radiation, SBL-1, SSBs, DSBs, FRAP, PBMCs

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Authors: Reza Fardid, Aliyeh Alipour

Abstract:

Introduction: Coronary heart disease (CHD) is the most common and deadliest diseases. A coronary angiography is an important tool for the diagnosis and treatment of this disease. Because angiography is performed by exposure to ionizing radiation, it can lead to harmful effects. Ionizing radiation induces double-stranded breaks in DNA, which is a potentially life-threatening injury. The purpose of the present study is an investigation of the phosphorylation of histone H2AX in the location of the double-stranded break in Peripheral blood lymphocytes as an indication of Biological effects of radiation on angiography patients. Materials and Methods: This method is based on measurement of the phosphorylation of histone (gamma-H2AX, gH2AX) level on serine 139 after formation of DNA double-strand break. 5 cc of blood from 24 patients with angiography were sampled before and after irradiation. Blood lymphocytes were removed, fixed and were stained with specific ϒH2AX antibodies. Finally, ϒH2AX signal as an indicator of the double-strand break was measured with Flow Cytometry Technique. Results and discussion: In all patients, an increase was observed in the number of breaks in double-stranded DNA after irradiation (20.15 ± 14.18) compared to before exposure (1.52 ± 0.34). Also, the mean of DNA double-strand break was showed a linear correlation with DAP. However, although induction of DNA double-strand breaks associated with radiation dose in patients, the effect of individual factors such as radiosensitivity and regenerative capacity should not be ignored. If in future we can measure DNA damage response in every patient angiography and it will be used as a biomarker patient dose, will look very impressive on the public health level. Conclusion: Using flow cytometry readings which are done automatically, it is possible to detect ϒH2AX in the number of blood cells. Therefore, the use of this technique could play a significant role in monitoring patients.

Keywords: coronary angiography, DSB of DNA, ϒH2AX, ionizing radiation

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Authors: Vaiyapuri Subbarayn Periasamy, Jegan Athinarayanan, Ali A. Alshatwi

Abstract:

The widespread use of Titanium oxide nanoparticles (TiO2-NPs), now are found with different physic-chemical properties (size, shape, chemical properties, agglomeration, etc.) in many processed foods, agricultural chemicals, biomedical products, food packaging and food contact materials, personal care products, and other consumer products used in daily life. Growing evidences have been highlighted that there are risks of physico-chemical properties dependent toxicity with special attention to “TiO2-NPs and human immune system”. Unfortunately, agglomeration and aggregation have frequently been ignored in immuno-toxicological studies, even though agglomeration and aggregation would be expected to affect nanotoxicity since it changes the size, shape, surface area, and other properties of the TiO2-NPs. In this present investigation, we assessed the immune toxic effect of TiO2-NPs on human immune cells Total WBC including Lymphocytes (T cells (CD3+), T helper cells (CD3+, CD4+), Suppressor/cytotoxic T cells (CD3+/CD8+) and NK cells (CD3-/CD16+ and CD56+), Monocytes (CD14+, CD3-) and B lymphocytes (CD19+, CD3-) in order to find the immunological response (IL1A, IL1B, IL2 IL-4, IL5 IL-6, IL-10, IL-12, IL-13, IFN-γ, TGF-β, and TNF-a) and redox gene regulation (TNF, p53, BCl-2, CAT, GSTA4, TNF, CYP1A, POR, SOD1, GSTM3, GPX1, and GSR1)-linking physicochemical properties with special reference to agglomeration of TiO2-NPs. Our findings suggest that TiO2-NPs altered cytokine production, enhanced phagocytic indexing, metabolic stress through specific immune regulatory- genes expression in different WBC subsets and may contribute to pro-inflammatory response. Although TiO2-NPs have great advantages in the personal care products, biomedical, food and agricultural products, its chronic and acute immune-toxicity still need to be assessed carefully with special reference to food and environmental safety.

Keywords: TiO2 nanoparticles, oxidative stress, cytokine, human immune cells

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Authors: Saman Khan, Imrana Naseem

Abstract:

Copper is an essential trace element required for pro-angiogenic co-factors including vascular endothelial growth factor (VEGF). Elevated levels of copper are found in various types of cancer including prostrate, colon, breast, lung and liver for angiogensis and metastasis. Therefore, targeting copper via copper-specific chelators in cancer cells can be developed as effective anticancer treatment strategy. In continuation of our pursuit to design and synthesize copper chelators, herein we opted for a reaction to incorporate di-(2-picolyl) amine in 3-(bromoacetyl) coumarin (parent backbone) for the synthesis of complex 1. We evaluated lipid peroxidation, protein carbonylation, ROS generation, DNA damage and consequent apoptosis by complex 1 in exogenously added Cu(II) in human peripheral lymphocytes (simulate malignancy condition). Results showed that Cu(II)-complex 1 interaction leads to cell proliferation inhibition, apoptosis, ROS generation and DNA damage in human lymphocytes, and these effects were abrogated by cuprous chelator neocuproine and ROS scavengers (thiourea, catalase, SOD). This indicates that complex 1 cytotoxicity is due to redox cycling of copper to generate ROS which leads to pro-oxidant cell death in cancer cells. To further confirm our hypothesis, using the rat model of diethylnitrosamine (DEN) induced hepatocellular carcinoma; we showed that complex 1 mediates DNA breakage and cell death in isolated carcinoma cells. Membrane permeant copper chelator, neocuproine, and ROS scavengers inhibited the complex 1-mediated cellular DNA degradation and apoptosis. In summary, complex 1 anticancer activity is due to its copper chelation capability. These results will provide copper chelation as an effective targeted cancer treatment strategy for selective cytotoxic action against malignant cells without affecting normal cells.

Keywords: cancer treatment, copper chelation, ROS generation, DNA damage, redox cycling, apoptosis

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Authors: Fatimah Alhomaid

Abstract:

The present study was planned to investigate the role of molecular changes and immunohistochemical in early detection of colon cancer in Saudi patients. Our results were carried out on 48 patients colon cancer. We obtained our data from laboratory in King Khalid university hospital. The specimens were taken (48) patients with colon cancer 34 male and 14 female and 2 control. The average age of varied from 37-85 years. The tumor was diagnosed as I in tow patients (male and female) and grade 2 in 42 patients (29 male and 13 female) while the grade 3 in 4 patients (all males). The specimens were processed for haematoxylin and eosin staining , immunohistochemical technique and flow cytometry analysis. Our study noted that most patients had adenocarcinoma which characterized by presence of signet-ring cells were very clear in advanced patients of adenocarcinoma. Our sections in adenocarcinoma in grade 2 and stage 3 had an increase in signet ring cells,an increase in the acini of glands and an increase in number of lymphocytes which spread to the muscularis layer. With advancing the disease, there were haemorge in blood and increase in lymphocytes and increase number of nuclei in the tubular glands. Our study was carried on 48 patients, immunohistochemical diagnosis (CK20,PCNA,P53) and the analysis of DNA content by flow cytometry technique. Our study indicated that the presence of correlation between the immunohistochemical analysis for P53 and the grades. The reaction of P53 appeared as strong in nucleus in grades &stage 3 and appeared in other sections as dark brown pigment. Our study indicated that the absence of correlation between the immunohistochemical analysis for pcan and the grades. In our sections, there were strong reactions in the more 80% of nuclei in grade 1& stage 2. Our study indicated that the presence of correlation between the immunohistochemical analysis for CK20 and the grades. Our results indicated the presence of positive reaction in cytoplasm varied from weak to moderate in grade 3 & stage 4. Concerning the Flow cytometry technique our results indicated that the presence of correlation between the DNA and different stages of colon cancer.

Keywords: DNA-CK20, PCNA, P53, colon cancer

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Authors: Fatimah A. Alhomaid

Abstract:

The present study was planned to investigate the role of molecular changes and immunohistochemical in early detection of liver cancer in Saudi patients. our results were carried out on 54 patients liver cancer. We obtained our data from laboratory in King Khalid University Hospital. The specimens were taken (54) patients with liver cancer 34 male and 14 female and 2 control. The average age of varied from 37-85 years. The tumor was diagnosed as grade I in tow patients (male and female) and grade 2 in 45 patients (28 male and 17 female) while the grade 3 in 4 patients (all males). The specimens were processed for haematoxylin and eosin staining, immunohistochemical technique and flow cytometry analysis. Our study noted that most patients had adenocarcinoma which characterized by presence of signet-ring cells were very clear in advanced patients with adenocarcinoma. Our sections in adenocarcinoma in grade 2 and stage 3 had an increase in signet ring cells,an increase in the acini of glands and an increase in number of lymphocytes which spread to the muscular layer. With advancing the disease, there were haemorrhage in blood and increase in lymphocytes and increase in the number of nuclei in the tubular glands. Our study was carried on 48 patients, immunohistochemical diagnosis (CK20, PCNA, P53) and the analysis of DNA content by flow cytometry technique. Our study indicated that the presence of correlation between the immunohistochemical analysis for P53 and the grades. The reaction of P53 appeared as strong in nucleus in grades &stage 3 and appeared in other sections as dark brown pigment. Our study indicated that the absence of correlation between the immunohistochemical analysis for PCAN and the grades. In our sections there were strong reaction in the more 80% of nuclei in grade 1& stage 2. Our study indicated that the presence of correlation between the immunohistochemical analysis for CK20 and the grades. Our results indicated the presence of positive reaction in cytoplasm varied from weak to moderate in grade 3 & stage 4. Concerning the Flow cytometry technique our results indicated that the presence of correlation between the DNA and different stages of liver cancer.

Keywords: cancer, CK20, DNA, cytometry analysis, liver, immunohistochemical, molecular changes, PCNA, p53

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Authors: Ugwuowo Leonard Chidi, Oparaji Chetachukwu Jecinta., Ogidi Chibuzor Agafenachukwu, Onuoha Rebecca Obianuju

Abstract:

This experiment was conducted to investigate the growth performance, haematology and serum biochemistry indices of broiler birds fed diets containing Neem leaf meal. A total of 96 unsexed day-old broiler birds were allocated to four treatments of T1, T2, T3 and T4 and replicated three times with eight birds per replicate in a Completely Randomized Design. The treatments were diets containing 2.0, 4.0, 6.0 and 8.0% Neem leaf meal respectively. Growth performances, packed cell volume, red blood cell count, haemoglobin, white blood cell count, lymphocytes, mean corpuscular volume, mean corpuscular haemoglobin concentration, platelet count, aspartate amino transaminase, alanine amino transaminase, alkaline phosphate, cholesterol, albumin, globulin, urea, glucose, total protein and creatinine were evaluated. Results showed that there were no significant differences (P>0.05) in all the growth performance parameters among the treatments. The results of the experiment showed that there were significant differences (P<0.05) in all the heamatological and serum biochemistry parameters at finisher phases. Mean corpuscular volume, white blood cell count, lymphocytes, red blood cell count, haemoglobin, platelet count, creatinine and triglyceride increased and were highest in treatment two while treatment four had the least values in mean corpuscular volume, urea, white blood cell, haemoglobin and triglyceride. This implies that the levels of inclusion of Neem leaf meal in this experiment did not affect the growth performance of the broiler chicks but the haematological and serum biochemistry indices were affected. Treatment two with a 4% inclusion level of Neem leaf meal has shown the capacity to replace vaccines and antibiotics in broilers due to the positive effects it had on both the haematological and serum biochemistry.

Keywords: leaf meal, broiler, Aziradicta indica, serum biochemistry, haematology

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Authors: Nurkhairul Bariyah Baharun, Afzan Adam, Reena Rahayu Md Zin

Abstract:

Tumor microenvironment (TME) in breast cancer is mainly composed of cancer cells, immune cells, and stromal cells. The interaction between cancer cells and their microenvironment plays an important role in tumor development, progression, and treatment response. The TME in breast cancer includes tumor-infiltrating lymphocytes (TILs) that are implicated in killing tumor cells. TILs can be found in tumor stroma (sTILs) and within the tumor (iTILs). TILs in triple negative breast cancer (TNBC) have been demonstrated to have prognostic and potentially predictive value. The international Immune-Oncology Biomarker Working Group (TIL-WG) had developed a guideline focus on the assessment of sTILs using hematoxylin and eosin (H&E)-stained slides. According to the guideline, the pathologists use “eye balling” method on the H&E stained- slide for sTILs assessment. This method has low precision, poor interobserver reproducibility, and is time-consuming for a comprehensive evaluation, besides only counted sTILs in their assessment. The TIL-WG has therefore recommended that any algorithm for computational assessment of TILs utilizing the guidelines provided to overcome the limitations of manual assessment, thus providing highly accurate and reliable TILs detection and classification for reproducible and quantitative measurement. This study is carried out to develop a TNBC digital whole slide image (WSI) dataset from H&E-stained slides and IHC (CD4+ and CD8+) stained slides. TNBC cases were retrieved from the database of the Department of Pathology, Hospital Canselor Tuanku Muhriz (HCTM). TNBC cases diagnosed between the year 2010 and 2021 with no history of other cancer and available block tissue were included in the study (n=58). Tissue blocks were sectioned approximately 4 µm for H&E and IHC stain. The H&E staining was performed according to a well-established protocol. Indirect IHC stain was also performed on the tissue sections using protocol from Diagnostic BioSystems PolyVue™ Plus Kit, USA. The slides were stained with rabbit monoclonal, CD8 antibody (SP16) and Rabbit monoclonal, CD4 antibody (EP204). The selected and quality-checked slides were then scanned using a high-resolution whole slide scanner (Pannoramic DESK II DW- slide scanner) to digitalize the tissue image with a pixel resolution of 20x magnification. A manual TILs (sTILs and iTILs) assessment was then carried out by the appointed pathologist (2 pathologists) for manual TILs scoring from the digital WSIs following the guideline developed by TIL-WG 2014, and the result displayed as the percentage of sTILs and iTILs per mm² stromal and tumour area on the tissue. Following this, we aimed to develop an automated digital image scoring framework that incorporates key elements of manual guidelines (including both sTILs and iTILs) using manually annotated data for robust and objective quantification of TILs in TNBC. From the study, we have developed a digital dataset of TNBC H&E and IHC (CD4+ and CD8+) stained slides. We hope that an automated based scoring method can provide quantitative and interpretable TILs scoring, which correlates with the manual pathologist-derived sTILs and iTILs scoring and thus has potential prognostic implications.

Keywords: automated quantification, digital pathology, triple negative breast cancer, tumour infiltrating lymphocytes

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Authors: Zahra Shokrolahi, Mohammad Reza Atashzar

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The goals of treating patients with breast cancer are to cure the disease, prolong survival, and improve quality of life. Immune cells in the tumor microenvironment have an important role in regulating tumor progression. The term of cellular immunotherapy refers to the administration of living cells to a patient; this type of immunotherapy can be active, such as a dendritic cell (DC) vaccine, in that the cells can stimulate an anti-tumour response in the patient, or the therapy can be passive, whereby the cells have intrinsic anti-tumour activity; this is known as adoptive cell transfer (ACT) and includes the use of autologous or allogeneic lymphocytes that may, or may not, be modified. The most important breast cancer cellular immunotherapies involving the use of T cells and natural killer (NK) cells in adoptive cell transfer, as well as dendritic cells vaccines. T cell-based therapies including tumour-infiltrating lymphocytes (TILs), engineered TCR-T cells, chimeric antigen receptor (CAR T cell), Gamma-delta (γδ) T cells, natural killer T (NKT) cells. NK cell-based therapies including lymphokine-activated killers (LAK), cytokine-induced killer (CIK) cells, CAR-NK cells. Adoptive cell therapy has some advantages and disadvantages some. TILs cell strictly directed against tumor-specific antigens but are inactive against tumor changes due to immunoediting. CIK cell have MHC-independent cytotoxic effect and also need concurrent high dose IL-2 administration. CAR T cell are MHC-independent; overcome tumor MHC molecule downregulation; potent in recognizing any cell surface antigen (protein, carbohydrate or glycolipid); applicable to a broad range of patients and T cell populations; production of large numbers of tumor-specific cells in a moderately short period of time. Meanwhile CAR T cells capable of targeting only cell surface antigens; lethal toxicity due to cytokine storm reported. Here we present the most popular cancer cellular immunotherapy approaches and discuss their clinical relevance referring to data acquired from clinical trials .To date, clinical experience and efficacy suggest that combining more than one immunotherapy interventions, in conjunction with other treatment options like chemotherapy, radiotherapy and targeted or epigenetic therapy, should guide the way to cancer cure.

Keywords: breast cancer , cell therapy , CAR T cell , CIK cells

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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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Authors: K. Rawojć, J. Miszczyk, A. Możdżeń, A. Panek, J. Swakoń, M. Rydygier

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Due to the mitotic delay, poor mitotic index and disappearance of lymphocytes from peripheral blood circulation, assessing the DNA damage after high dose exposure is less effective. Conventional chromosome aberration analysis or cytokinesis-blocked micronucleus assay do not provide an accurate dose estimation or radiosensitivity prediction in doses higher than 6.0 Gy. For this reason, there is a need to establish reliable methods allowing analysis of biological effects after exposure in high dose range i.e., during particle radiotherapy. Lately, Premature Chromosome Condensation (PCC) has become an important method in high dose biodosimetry and a promising treatment modality to cancer patients. The aim of the study was to evaluate the usefulness of drug-induced PCC scoring procedure in an experimental mode, where 100 G2/M cells were analyzed in different dose ranges. To test the consistency of obtained results, scoring was performed by 3 independent persons in the same mode and following identical scoring criteria. Whole-body exposure was simulated in an in vitro experiment by irradiating whole blood collected from healthy donors with 60 MeV protons and 250 keV X-rays, in the range of 4.0 – 20.0 Gy. Drug-induced PCC assay was performed on human peripheral blood lymphocytes (HPBL) isolated after in vitro exposure. Cells were cultured for 48 hours with PHA. Then to achieve premature condensation, calyculin A was added. After Giemsa staining, chromosome spreads were photographed and manually analyzed by scorers. The dose-effect curves were derived by counting the excess chromosome fragments. The results indicated adequate dose estimates for the whole-body exposure scenario in the high dose range for both studied types of radiation. Moreover, compared results revealed no significant differences between scores, which has an important meaning in reducing the analysis time. These investigations were conducted as a part of an extended examination of 60 MeV protons from AIC-144 isochronous cyclotron, at the Institute of Nuclear Physics in Kraków, Poland (IFJ PAN) by cytogenetic and molecular methods and were partially supported by grant DEC-2013/09/D/NZ7/00324 from the National Science Centre, Poland.

Keywords: cell response to radiation exposure, drug induced premature chromosome condensation, premature chromosome condensation procedure, proton therapy

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