Medicine
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This repository contains the published and unpublished research of the Faculty of Medicine by the staff members of the faculty
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Item Optimization of the cell culture media to obtain the most effective nutrient concentrations in the medium for the growth and maintenance of the Myeloma cells(University of Peradeniya, 2015) Munasinghe, M.M.E,; Athapaththu, A.M.M.H.; Gunathilaka, H.N.; Abeyewickreme, W.Cell culture can be described as the removal of cells, tissues or organs from an animal or a plant and their subsequent placement into an artificial environment. Basically, proper temperature, substrate for cell attachment, appropriate growth medium and correct pH and osmolality in the medium should be properly maintained in order to achieve a better growth in the cells. Typically, a culture medium is composed of a complement of amino acids, vitamins, inorganic salts, glucose, and serum as a source of growth factors, hormones, and attachment factors. The objective of this study is to optimize culture media in order to obtain the most effective nutrient concentrations in the medium for the growth/maintenance of NSO Myeloma cell. Myeloma cells for the monoclonal antibody production were prepared using Dulbecco's Modified Eagle Medium (DMEM) as the growth media for the NSO cell culture. In this study, the culture media was optimized in order to obtain the most effective concentrations in the media. Primarily, in order to culture the cells soon after thawing, 10% growth media was used and then the grown cells were transferred in to a nutrition rich media- Hypoxanthine Thymidine (HT) medium. The growth of the Primary cell culture, soon after thawing, was observed within 2 days of culturing. A 60% of the bottom of the culture flask was covered with the healthy NSO myeloma cells. The transferred cells were also grown to a rate of 60% within 2 days of transferring. The 10% growth media comprises with 422 mL of DMEM with added 4500 mg/L glucose without L-glutamine and sodium pyruvate, 50 mL of Fetal Clone Serum, IZ5 mL of 1M HEPES buffer (4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid), 5 m of 200 mM Glutamin, 5 mL of 100X Non-essential amino acids, 5 mL of 100 mM Sodium pyruvate and 0.5 mL of 55mM jj-mercaptoethanol. The HT medium comprises with 366.5 422 mL of DMEM with added 4500 mg/L glucose without L- glutamine and sodium pyruvate, 100 of mL FetalClone serum, 12.5 mL of 1M HEPES buffer, 5 mL 100X HT supplement, 5 mL of 200 mM Glutamin, 5 mL of 100X Non-essential amino acids, 5 mL of 100 mM Sodium pyruvate, 0.5 mL of 50 mg/mL Genatamicin, 0.5 mL of 55 mM p-mercaptoethanol and 25 uL of Interlukin-6. Since both the culture media showed optimum growth of the Myeloma cells, the above protocol with the provided concentrations of the nutrients could be used to maintain/ grow NSO myeloma cell line in the laboratory.Item Development of recombinant proteins as diagnostic intermediates for chikungunya infection(Sri Lanka Association for the Advancement of Science, 2010) Athapaththu, A.M.M.H.; Khanna, N.; Gunasena, S.; Abeyewickreme, W.; Hapugoda, M.D.Chikungunya is an important disease with explosive outbreaks occurring in many South East Asian countries. As the clinical symptoms associated with chikungunya viral infections are often indistinguishable from those of many other viral, bacterial and parasitic infections confirmation of chikungunya outbreaks is important for clinicians for proper management of patients and for vector control programmers. Laboratory diagnosis of chikungunya in Sri Lanka is hindered by the non-availability of reliable commercial diagnostic kits and inaccessibility to reagents. There is a need to develop an assay that can confirm chikungunya, produced at low cost and easily standardized for the use in field settings. Currently available laboratory diagnostic kits depend on ELISA based on whole viral antigens which cause biohazard risk, high production cost and cross reactivity with other organisms of the same genus/family. Therefore, a diagnostic intermediate using a single recombinant protein antigen to overcome problems associated with whole viral antigen/lysate is important. The objective of this study was to assist laboratory confirmation of outbreaks through developing competencies for a rapid laboratory diagnostic method using recombinant protein antigens for chikungunya infection. We have designed 2 novel recombinant protein antigens based on Envelope domain (E), a critical antigenic region of the major structural protein of chikungunya virus. They were expressed in Escherichia coli separately, and resultant proteins were affinity purified and obtained ~5mg and 10mg respectively and protein of >95% purity per liter of culture. These 2 proteins were evaluated as potential diagnostic intermediates in ELISA separately for the detection of anti-chikungunya Immunoglobulin M (IgM) antibody using a panel of well characterized serum samples. E1 and E2 showed 60% and 67% positivity respectively. Specificity proteins were tested using serum from healthy volunteers and infected with other viral diseases. Two proteins could detect only anti-chikungunya IgM antibodies. We demonstrated that these 2 novel recombinant protein antigens can function as diagnostic intermediates. Acknowledgements: Financial assistance from the International Centre for Genetic Engineering and Biotechnology (ICGEB CRP/ SRI08 02) and International Atomic Energy Agency (IAEA SRI TC 5-042) are gratefully acknowledgedItem Correlation of clinical presentation and laboratory confirmation of dengue patients(Sri Lanka Association for the Advancement of Science, 2010) Manamperi, N.H.; Athapaththu, A.M.M.H.; Premawansa, V.; Wellawaththage, C.; Jayarathna, T.D.S.S.; Abeyewickreme, W.; Hapugoda, M.D.Dengue is one of the most important arthropod-borne diseases in the world and it has become a very important disease in Sri Lanka, today. In Sri Lanka, diagnosis of dengue depends mainly on clinical signs and symptoms. Only a few suspected patients are confirmed by laboratory assays based on aetiological agents. The objective of this study was to determine the correlation between clinical presentation and laboratory confirmation of dengue patients. Acute serum samples (n=100) collected from patients clinically suspected of having dengue fever ("'ª-"ý¦> 5 days) warded at the North Colombo Teaching Hospital, Ragama were used for the present study. Serum samples were collected after obtaining informed written consent frompatients and samples were tested by RT-PCR which has high sensitivity (10 FFU/reaction) and specificity. Final diagnosis as dengue or non-dengue was assigned based on the results of RT-PCR assay. Differences in clinical and laboratory data were analyzed in dengue and non dengue patients. Chi-square test was used for comparison of data. The proportion of laboratory confirmed dengue patients were 56% (56/100). Mean platelet count and PCV in laboratory confirmed dengue patients were 60 269/mm 3 (range 3000-306000) and 41% (range 27-61%) and in non dengue patients were 106 318/mm 3 (range 5000-290000) and 41.6% (range 29-53%). Based on WHO criteria for diagnosis of dengue, heada (48/56 vs 41/44, ÝÖ 2 =0.7, p=0.38), retro-orbital pain (30/56 vs 14/44, ÝÖ 2 =3.8, p=0.04), limb pain (51/56 vs 30/44, ÝÖ 2 =7, p=0.00) and external bleeding (29/56 vs 4/44, ÝÖ 2 =18, p=0.00) showed significant association with dengue. Neck pain (10/56 vs 09/44, ÝÖ 2 =0.01, p=0.94), and lymphadenopathy (3/56 vs 02/44, ÝÖ 2 =0.08, p=0.78) did not show significant association with dengue. The infection was confirmed as dengue fever in 11% (6/56) and dengue hemorrhagic fever in 89% (50/56) based on WHO criteria. Surveillance based on clinical diagnosis may result in over estimation of the disease as clinical diagnosis is not specific enough. Laboratory confirmation of dengue suspected patients is important to measure the real incidence of the disease which leads implementation of control measures. Further, thisis important for efficient management of patients.Acknowledgements: Financial and technical assistance from the International Centre for Genetic Engineering and Biotechnology (ICGEB CRP/ SRI08-02) and International Atomic Energy Agency (IAEA SRI 5/042)