Browsing by Author "Galhena, B.P."

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Apoferritin and Dps as drug delivery vehicles: Some selected examples in oncology
(Elsevier Ltd, 2022) Kuruppu, A.I.; Turyanska, L.; Bradshaw, T.D.; Manickam, S.; Galhena, B.P.; Paranagama, P.; de Silva, R.
Background: The ideal nanoparticle should be able to encapsulate either pharmaceutical agents or imaging probes so that it could treat or image clinical tumours by targeting the cancer site efficiently. Further, it would be an added advantage if it demonstrates: small size, built in targeting, biocompatibility and biodegradability. Ferritin, which is an endogenous self-assembling protein, stores iron and plays a role in iron homeostasis. When iron atoms are removed apoferritin (AFt) is formed which consists of a hollow shell where it can be used to load guest molecules. Due to its unique architecture, AFt has been investigated as a versatile carrier for tumour theranostic applications. DNA-binding protein from starved cells (Dps), which also belongs to the ferritin family, is a protein found only in prokaryotes. It is used to store iron and protect chromosomes from oxidative damage; because of its architecture, Dps could also be used as a delivery vehicle. Conclusions: Both these nano particles are promising in the field of oncology, especially due to their stability, solubility and biocompatibility features. Further their exterior surface can be modified for better tumour-targeting ability. More studies, are warranted to determine the immunogenicity, biodistribution, and clearance from the body. General perspective: This review discusses a few selected examples of the remarkable in vitro and in vivo studies that have been carried out in the recent past with the use of AFt and Dps in targeting and delivery of various pharmaceutical agents, natural products and imaging probes in the field of oncology.
Biochemical and histopathological changes in Wistar Rats after consumption of boiled and un-boiled water from high and low disease prevalent areas for Chronic Kidney Disease of Unknown Etiology (CKDu) in North Central Province (NCP) and Its comparison with Low disease prevalent Colombo, Sri Lanka
(BioMed Central,, 2020) Thammitiyagodage, M.G.; de Silva, N.R.; Rathnayake, C.; Karunakaran, R.; Wgss, K.; Gunatillka, M.M.; Ekanayaka, N.; Galhena, B.P.; Thabrew, M.I.
BACKGROUND: Chronic Kidney Disease of unknown etiology (CKDu) is prevalent in North Central Province (NCP) of Sri Lanka. Consumption of un-boiled dug well water has been identified as one of the causative factors. This in-vivo study was performed to investigate some of the suspected factors associated with the pathogenesis of CKDu mediated via ground water. METHOD: Rats were given water, collected from high and low disease prevalent areas from the NCP of Sri Lanka and the results compared with those obtained from previously identified low disease prevalent area; Colombo. Blood Urea Nitrogen, creatinine, urinary microalbumin:creatinine ratio together with ALT and AST levels were analyzed and results were compared using one-way ANOVA and paired t-Test. Histopathology was analyzed using non-parametric method. RESULTS: Rats that ingested water from New Town Medirigiriya (NTM) from high disease prevalent NCP reported significantly elevated microalbumin:creatinine ratios compared to other water sources after 8 months, whilst boiled water from NTM had been able to significantly reduce it. Histopathological findings after the 14 months experimental period revealed significantly high tubular lesion index in rats that ingested water from NCP compared to Colombo. Rats that ingested water from high disease prevalent Divuldamana (DD) from NCP showed the highest kidney lesion index though the fluoride content was relatively low in this area compared to other water sources from high disease prevalent NCP. Rats that ingested boiled and un-boiled water from NTM also developed severe lesions whilst the group from Colombo reported the lowest. Low disease prevalent area from NCP, Huruluwewa (HW) also reported elevated liver enzymes and altered renal histopathology. Association of Na+:Ca2+ ratio in the disease progression was not reflected by the current study. Compared to Colombo, high fluoride, calcium and sodium contents were observed in water from high disease prevalent areas. All the water samples were negative for heavy metals. CONCLUSIONS: Though Fluoride is a known kidney toxic agent it cannot be the sole reason for CKDu in NCP, Sri Lanka. Various toxic elements present in NCP water may contribute to different grade of kidney and liver lesions in Wistar rats. KEYWORDS: BUN; CKDu; Microalbumin:creatinine; NCP.
Development of a method for the detection of glycated low density lipoprotein in serum
(University of Kelaniya, Sri Lanka, 2003) Galhena, B.P.; Thabrew, M.I.; Keerthisena, O.P.K.S.; Chandresekera, A.; IIIangasekera, U.
Abstracts available
Effects of a Decoction of Nigella Sativa, Hemidesmus lndicus and Smilax G/abra on Human Hepatoma HepG2 Cell Integrity
(University of Kelaniya, 2007) Galhena, B.P.; Thabrew, M.I.; Solomon F.D.P.; Rachel, V.A.H.
Introduction In Sri Lanka, a herbal decoction comprised of Nigella sativa (seeds), Hemidesmus indicus (root), and Smilax glahra (rhizome) has been used for many years by a particular family of indigenous medical practitioners (personal communication, Ayurvedic physician, Dr. N. Jayathilake) for the treatment of cancer, despite the lack of scientific evidence to prove or disprove its therapeutic efficacy. Recent in vivo investigations have demonstrated that the above decoction can offer significant protection against diethylnitrosamine induced hepatocarcinogenic changes in rats. Objective The aim of the present study was to investigate how changes in liver cell integrity could contribute to the anti-hcpatocarcinogenic actions of the decoction comprised of Nigella sativa, Ilemidesmus indicus and Smilax glahra. Methods and materials For experimental purpose, HepG2 cells were cultured in T25 plates (lx106 cells/ plate) in DMEM supplemented with 10% foetal bovine serum. Cells in test plates were exposed to different doses of the decoction ( 1 0~-tg/ml, 80~-tg/ml, and 160~-tg/ml) for a total of 72h. Cells in control plates were maintained in the same manner without exposure to the decoction. Cellular integrity in both test and control was assessed by (a). microscopic examination of cell morphology, (b). evaluation of lactate dehydrogenase (LDH) release from cells. cellular ATPase activity and intracellular reduced glutathione (GSH) status at the end of 6h. 24h, 48h and 72h incubation with or without the decoction respectively. Results Results demonstrate that in comparison with control cells, in the cells exposed to the decoction even at the lowest dose of 1 0~-tg/ml, there was a 12.99% increase in the leakage of cellular LDH along with a 14.79% decrease in cellular ATP'ase. Although there was a decrease in cellular GSH also, it was interesting to note that at each time point (6h, 24h, 48h and 72h), the cellular GSH level in test cells was marginally higher than the corresponding values in control cells. Reduction in cell growth was microscopically apparent even as early as 6h post-incubation. Conclusion The overall outcome of this study indicates a potent cytotoxicity towards human liver can~er cells once exposed to the decoction of Nigella sative (seeds), Hemidesmus indicus (root), and Smilax glabra (rhizome). This direct effect on cells helps confirm the results of recent in vivo investigations, which demonstrated a protection against chemically induced hepatocarcinogenesis in rat liver. The marginal increase in GSH subsequent to decoction exposure is supportive of a possible anti-oxidant role of the decoction, which is essential for protection against chemically induced cancers. Therefore, it may be concluded that cytotoxicity and antioxidant activity may be two mechanisms through which the DC mediates its anti-hepatocarcinogenic actions.
An investigation of the anti-carcinogenic mechanisms of a Sri Lankan herbalremedy and its ability to protect against radiation induced tissue injury
(University of Kelaniya, 2011) Galhena, B.P.
Decoction comprised of Nigella sativa (seeds). Hemidesmus indicus (roots) and Smilax glabra (rhizome) has traditionally been used to treat malignancies of various origins. Recent in-vivo and in-vitro studies have confirmed that the above decoction is effective against HCC, the commonest primary malignancy in the liver which accounts for highest mortality rate. However, the exact mechanism/s by which the decoction mediates its anti-hepatocarcinogenic activity is poorly understood. Use of herbs and their derivatives has also been reported to be beneficial for the patients suffering from severe adverse side effects during radiotherapy. Therefore the present study was carried out with the aims of investigating (a) the possible mechanisms by which the decoction mediates its claimed anti-hepatocarcinogenic activity, and (b) its ability to protect against radiation induced cellular damages. Anti-carcinogenic mechanisms were evaluated by assessing the effects of the decoction on (a) oxidative stress, (b) cell signaling and host immune response, (c) malignant cell proliferation, and (d) expression of key genes associated with carcinogenesis. Possible radioprotection mediated by the decoction was evaluated by examining the protection against cytogenetic damages induced by bleomycin, a known radiomimetic drug, in human peripheral blood lymphocytes in vitro. Results showed that the decoction could mediate a potent anti-oxidant effect by; (a) stimulating both enzymic (GPx, SOD, and GST activity) and non-enzymic (GSH) anti-oxidant defenses in rats bearing DEN-induced early hepatocarcinogenic changes in vivo, and (b) scavenging of nitric oxide (NO) and Diphenyl-2-picrylhyrazyl in vitro. Free radical induced activation of NF-KB plays a key role in cell proliferation. apoptosis and modulation of inflammation by activating an array of its down-stream targets. In the present study, it was observed that oral administration of the decoction could mediate a significant inhibition of hepatic NF-KB activity through hepatic IKKo/p suppression in C3H mice bearing early hepatocarcinogenic changes. Such inactivation was further confirmed by the down-regulation of two key down-stream targets of NF-KB transcription factor; TNFa and IL-6. These two cytokines actively participate in co-ordination of the host inflammatory response and are potent stimulators of NF-KB activation. Therefore, inhibition of these cytokines is considered to be vital in breaking the cytokine mediated NF-KB activation cycle, thus restricting the expression of its downstream targets associated with signal transduction, cell cycle regulation, apoptosis and inflammation. Generalized inhibition of inflammation mediated by the decoction was also evident by (a) significant reduction in carrageenan induced rat paw oedema formation, (b) human red cell membrane stabilization, and (c) inhibition of NO production by carrageenan induced rat peritoneal cell infiltrate. Reduction in CD4+ clonal expansion in response to oral administration of the decoction also supports the immunomodulatory potential of the decoction. Cellular oxidative stress induced mutations lead to expression of functionally defective genetic products. Altered and/or defective expression of onco-suppressor, p53wt and cell cycle regulator, p21 are considered to be more critical during carcinogenesis. Upregul#tion of hepatic p53%vt and p21WAri expression in C3H mice bearing early hepatocarcinogenic changes by the oral administration of the decoction observed in the present study strongly justify its claimed anti-hepatocarcinogenic activity. Human hepatoma (HepG2) cells treated with the decoction in-vitro also showed a significant; (a) morphological changes under microscope, (b) overall reduction in cell activity, (c) reduction in cell survival, and (d) cellular necrosis. Therefore, it is reasonable to hypothesize that the restriction of malignant cell survival and proliferation could be mediated by p53M and p2iWAF1 over-expression in the presence of the decoction. Claimed anti-oxidant role of the decoction may influence its observed protection against cytogenetic damages mediated by radiomimetic drug; bleomycin, in human peripheral blood lymphocytes. This protection was evident by a significant reduction in (a) chromosomal aberration, (b) formation of micronuclei, and (c) y-H2AX foci yield. Overall findings of the study suggest that the decoction mediates its anti-hepatocarcinogenic effects by modulating multiple targets that are closely associated with carcinogenesis. Further the ability of the decoction to modulate expression of key genes associated with carcinogenesis produce a new insight with the possible molecular mechanisms by which the decoction may be of benefit in the therapy of hepatocellular cancer. The decoction also has the ability to protect against radiation induced cytogenetic damages, thus provides an added advantage for cancer the patients who are treated with radiotherapy. Therefore, the present study strongly suseests for further investigation of the decoction in search for novel chemotherapeutic leads.
An investigation of the anti-carcinogenic mechanisms of Sri Lankan herbal remedy and its ability to protect against radiation induced tissue injury
(2011) Galhena, B.P.
Mutational analysis of driver and non-driver mutations of Philadelphia chromosome-negative myeloproliferative neoplasms;diagnosis and recent advances in treatment
(Science Publications, 2024) Afolabi, B.O.; Riwaz, A.; Weerasena, J.; Williams, S.; Denipitiya, T.; Somawardana, B.; Faizan, M.; Galhena, B.P.
Myeloproliferative neoplasms (MPNs) are hematological disorders affecting myeloid stem cells. They are classified as Philadelphia (Ph) chromosome positive-chronic myeloid leukemia, and Ph-negative polycythemia vera, essential thrombocythemia, primary myelofibrosis, chronic neutrophilic leukemia, chronic eosinophilic leukemia, juvenile myelomonocytic leukemia, and MPN unclassifiable. This review is mainly focused on the Ph-negative MPNs namely, PV, ET, and PMF. These affect both males and females with a slight male predominance, with patients mainly presenting in the seventh decade. Patients often present with thrombotic events resulting in complications that lower survival rates. The major driver mutations that have been identified in MPNs are JAK2 Exon 14, JAK2 Exon 12, MPL Exon 10, and CALR Exon 9. The importance of these driver mutations gives due recognition to their inclusion into the 2022 diagnostic criteria of the MPN WHO Classification. However, other non-driver mutations have also been reported, especially in triple-negative cases. These mutations lead to downstream constitutive activation of the JAK/STAT signaling pathway, as well as the MAPK, and PI3K/Akt pathways. Insights into the molecular pathogenesis of MPN and its association with JAK2, CALR, and MPL mutations have identified JAK2 as a rational therapeutic target. Thus, as an approach to MPN therapy, JAK2 inhibitors, such as ruxolitinib, have been shown to effectively inhibit JAK2, and are currently in clinical trials in combination with other drug classes. This review comprehensively examines the molecular markers of the main Ph-negative MPNs, as well as diagnosis and treatment options.
Optimization of reverse transcriptase PCR for selected hepatic cytokines in Wistar Rats
(Faculty of Graduate Studies, University of Kelaniya, 2015) Samaranayake, H.A.E.; Thammitiyagodage, M.G.; Galhena, B.P.; Chakrewarthy, S.; Wickremasinghe, A.R.
Expression patterns of hepatic cytokines elucidates the immune and pathological pathways involved in inflammatory responses. Cytokine mRNA quantification is widely used approach in this regard that involves RNA extraction, cDNA synthesis and real-time polymerase chain reaction of selected targets. In the present study, we optimized the reverse transcriptase PCR conditions for selected hepatic cytokines; TNF alpha and IL 6 in Wistar Rats. Liver tissues obtained from Wistar rats were washed with diethylpyrocarbonate (DEPC) treated water and frozen immediately in liquid nitrogen. Samples were stored at -800C. Total RNA was extracted from 0.1 g of liver tissue using Trizol@ according to the manufacturer‘s instructions. Subsequently, cDNA was synthesized from 2000ng of RNA using random primers and M-MLV reverse transcriptase enzyme. PCR for target cytokines was carried out using newly synthesized cDNA based on following PCR conditions. For TNF alpha, 5'-TTC TGT CTA CTG AAC TTG GGG GTG ATC GGT CC-3' and 5'-GTA TGA GAT AGC AAA TCG GCT GAC GGT GTG GG -3' were used as primers. PCR was optimized with initial denaturation at 940C for 1 and 30 sec followed by 35 cycles of 30 sec denaturation at 940C, 1 min annealing and 1 min extension at 720C. A temperature gradient of 530C, 550C and 570C was used for annealing step. Final extension was done at 720C for 3 min. For IL 6, 5‘-TCC TAC CCC AAC TTC CAA TGC TC-3‘ and 5‘-TTG GAT GGT CTT GGT CCT TAG CC-3‘were used as primers. PCR was optimized with initial denaturation at 940C for 1 and 30 sec followed by 35 cycles of 30 sec denaturation at 940C, 1 min annealing and 1 min extension at 720C. A temperature gradient of 570C, 590C, and 610C was used for annealing step. Final extension was done at 720C 3 min. Based on PCR products of TNF alpha and IL-6 separated by agarose gel electrophoresis, annealing temperatures for both genes were decided as 550C and 590C respectively.
Validation of fluorescence in situ hybridization (FISH) assay using an analyte-specific reagent in detecting aneuploidies of chromosomes 13, 18, 21, X, and Y in prenatal diagnosis
(LIDSEN Publishers, 2023) Ralalage, B.M.S.K.P.; Kaluarachchi, N.; Randunu, M.; Jainulabdeen, M.; Nanthakumar, R.; Viswakula, S.; Galhena, B.P.
Fluorescence In-Situ hybridization (FISH) is a sensitive and highly efficient technique commonly used in routine diagnostics. Most of these tests that use analyte-specific reagents are not approved by US Food and Drug Administration (FDA) but are developed by individual test laboratories. There is an emerging demand for prenatal diagnosis of aneuploidies by FISH. Since most of these assays are laboratory-developed tests, it is essential to validate them prior to their use in diagnosis. However, validation procedures of these assays are oversight despite the presence of several validation guidelines. To validate FISH assay using analyte-specific reagents in detecting aneuploidies of chromosomes 13, 18, 21, X, and Y as per American College of Medical Genetics (ACMG) guidelines in 2016. Analyte-specific reagents supplied by Oxford Gene Technologies were used in the validation process using blood and amniotic fluid samples obtained from healthy male and female adults and fetuses respectively. The validation process includes probe localization, evaluation of assay specificity, and establishment of lower cut-off and reportable reference ranges. Probe localization indicated a 100% specificity for all probes tested. Interphase FISH on uncultured amniotic fluid demonstrated significantly high (≥95%) overall disomic signal patterns for all autosomes and sex chromosomes tested. The reportable 95% confidence interval was 94.84, 94.84, 95.24, 94.54, and 94.54 for chromosomes 13, 18, 21, X, and Y respectively. The present study illustrates an experimental design in validating laboratory-developed FISH assay using analytespecific reagents in detecting aneuploidies of chromosomes 13, 18, 21, X, and Y as per ACMG guidelines. Test probes used in the present study are consistent with probe localization characteristics, assay specificity, and reportable reference ranges recommended by ACMG. Therefore, the FISH assay used in the present study could be recommended as a supplementary prenatal diagnostic test that can be carried out along with standard chromosomal analysis.