Immunomodulatory Effects of Rhaphidophora Korthalsii Methanol Extract on Natural Killer Cell Activation and Cytolytic Activity

Rhaphidophora korthalsii (Araceae) is a root-climber plant which has been previously identified as splenocyte immunostimulator. The purpose of this study was to examine the in vitro and in vivo immunomodulatory effect of R. korthalsii methanol extract on immune cell proliferation, cytokine expression and cytotoxicity. More specifically, immunomodulatory effects of R. korthalsii methanol extract on the stimulation of NK cells activity and cytotoxicity against HepG2 monolayer and spheroid culture were determined. Immune cells [peripheral blood mononuclear cells (PBMC) and mice splenocytes] treated with this extract resulted in stimulation of cell proliferation, cytokine expression and cytotoxicity in dose and time dependent manner. For the in vivo immunostimulatory effect study, unlike rIL-2 which degraded rapidly, the stimulatory effect from the extract managed to last until day 15. In order to understand the activation of NK cells by R. korthalsii methanol extract, NK cells were treated directly or indirectly. Both direct and indirect stimulated NK cells showed high-level expression of cell surface FasL, NKG2D, 16B4 and extra-cellular IFN-γ and TNF-α. These activations contributed to the killing of NK cells against HepG2 monolayer cells through Granzyme B mitochondria caspases dependent secretory apoptosis pathway where DNA fragmentation, phosphatidylserine (PS) externalisation, caspase 3, caspase 8, caspase 9 up-regulation and XIAP, Bid downregulation were observed. Apart from that, extract stimulated NK cells which caused cell death on the HepG2 spheroid and inhibited the HepG2 cell invasion, suggesting that R. korthalsii methanol extract was a potential agent to inhibit liver tumour metastasis. Our findings indicated a potential IL-2 free immunotherapy through direct and indirect R. korthalsii activation on NK cells which can further induce apoptosis on the HepG2 monolayer and spheroid culture

The best method for isolated total RNA from durian tissues

Plant tissues, especially durian tissues contain high content of polysaccharides, polyphenols and other secondary metabolites which can co-precipitate with RNA causing problem in further transcriptomic study. In this experiment, three basic chaotic agents, CTAB, SDS and guanidine are used in three basic protocols for RNA isolation. The effectiveness of each method was determined by spectrophotometer, denaturing agarose gels analysis and northern blot hybridization. CTAB combining with additional sodium acetate precipitation step showed highest yield and best quality of isolated RNA which was free from contaminations of polysaccharides, polyphenols and other secondary metabolites. Furthermore, the total RNA from 4-month old durian flesh of clone D24 was successfully used to construct a cDNA library. In conclusion, CTAB method is effective to isolate total RNA on various types of durian tissues for further gene expression analysis

Isolation and characterization of genotype VII Newcastle disease virus from NDV vaccinated farms in Malaysia

Molecular analysis, particularly sub-genotype classification, and study on the relationship of recent Malaysian NDVs with other isolates from around the world are lacking. Therefore, in the present study, a molecular epidemiological investigation was conducted to characterise six Newcastle disease viruses (NDV) isolated between 2014 and 2015 from vaccinated commercial poultry flocks. Partial Fusion (F) and Hemagglutinin-neuraminidase (HN) genes were amplified from IBS046/2014, IBS060/2014, IBS061/2014, IBS074/2014, IBS160/2015, and IBS162A/2015 isolates using one-step reverse transcription polymerase chain reaction (RT-PCR), sequenced and phylogenetically analysed. Sequence and phylogenetic analysis revealed that all the recently isolated strains of NDV belonged to sub-genotype VIIa and lineage 5a. Moreover, deduced amino acid sequence at the F protein cleavage site of the isolates revealed either 112RRQKRF117 or 112KRRKRF117 consistent with the motif found in velogenic pathotypes. The study concluded that the genotype VIIa was the causative agent of recent ND outbreaks in vaccinated broiler flocks from Malaysia. Interestingly, five out of the six isolates characterised in this study had a unique F0 protein cleavage site (112KRRKRF117). Further studies are required to determine the role of these motifs on the virulent potential of the isolates

The effects of conjugated linoleic acid isomers on the morphological changes in adipose tissue and adipogenic genes expressions on primary adipose tissue

Previous studies carried out in mouse 3T3-L1 cell culture have shown that conjugated linoleic acid (CLA) inhibited adipocyte differentiation. The present study was undertaken to investigate the effect of cis-9, trans-11 and trans-10, cis-12 CLA isomers on morphological changes and on mRNA expressions in the in vitro adipocyte isolated from specific pathogen-free (SPF) chicken. The adipocytes were isolated from SPF chicken and cultured in differentiation-induction medium with different concentrations of cis-9, trans-11 and trans-10, cis-12 CLA. After day 7, the adipocyte differentiations were monitored morphologically and mRNA expressions of lipoprotein lipase (LPL) and acyl-coenzyme A binding domain containing 5 (ACBD 5) were quantified by real-time polymerase chain reaction (PCR) analysis. Our data suggested that cis-9, trans-11 CLA downregulated the expression of LPL and ACBD 5 genes, which was concurrently observed with decrease in the adipocyte area size and cell number compared to the control and trans-10, cis-12 treated groups. Based on this finding, we concluded that dietary CLA modulate fat reduction in chicken via alteration of transcription of key adipogenic genes and adipose cellularity

Potential recombinant vaccine against influenza A virus based on M2e displayed on nodaviral capsid nanoparticles

Influenza A virus poses a major threat to human health, causing outbreaks from time to time. Currently available vaccines employ inactivated viruses of different strains to provide protection against influenza virus infection. However, high mutation rates of influenza virus hemagglutinin (H) and neuraminidase (N) glycoproteins give rise to vaccine escape mutants. Thus, an effective vaccine providing protection against all strains of influenza virus would be a valuable asset. The ectodomain of matrix 2 protein (M2e) was found to be highly conserved despite mutations of the H and N glycoproteins. Hence, one to five copies of M2e were fused to the carboxyl-terminal end of the recombinant nodavirus capsid protein derived from Macrobrachium rosenbergii. The chimeric proteins harboring up to five copies of M2e formed nanosized virus-like particles approximately 30 nm in diameter, which could be purified easily by immobilized metal affinity chromatography. BALB/c mice immunized subcutaneously with these chimeric proteins developed antibodies specifically against M2e, and the titer was proportional to the copy numbers of M2e displayed on the nodavirus capsid nanoparticles. The fusion proteins also induced a type 1 T helper immune response. Collectively, M2e displayed on the nodavirus capsid nanoparticles could provide an alternative solution to a possible influenza pandemic in the future

Bioactivity studies and chemical constituents of Murraya paniculata (Linn) Jack

Murraya paniculata (Linn) Jack (Orange Jasmine), known as "Kemuning Putih" in Malaysia, has been widely used as food flavor additive in cuisine by local residences. This is due to the strong fragrances of the leaves which make it suitable to be used in Indian and Malay dishes. Besides as a flavoring, leaves, branches, stem barks and roots of the plant are used in folk medicine to treat dysentery and morning sickness. Flowers of the plants are used in cosmetics. Since 1970's, flavonoids and coumarins were isolated from Murraya paniculata, but no further bioactivity has been tested from the isolated compounds. The aim of this paper is to review and update the research related to chemical constituents and bioactivities of Murraya paniculata (L) Jack

Bromelain enhances the anti-tumor effects of cisplatin on 4T1 breast tumor model in vivo

Background: This study aimed to evaluate the antitumor enhancing effect of bromelain consumption on 4T1-challenged mice treated with cisplatin. Methods: Mice challenged with 4T1 triple-negative breast cancer cells received water, bromelain, cisplatin, or bromelain + cisplatin treatment for 28 days. Tumor size was measured, and lung metastasis was evaluated by clonogenic assay. Expression of tumor inflammatory genes of the harvested tumor was quantified by polymerase chain reaction array and ELISA (enzyme-linked immunosorbent assay). Results: All treatments significantly reduced the size of tumor and lung metastasis, with combination treatment showing the best effect. Also, bromelain alone and combination treatment showed downregulation of the expression of tumor inflammatory genes (Gremlin [GREM1], interleukin 1β [IL-1β], interleukin-4 [IL-4], nuclear factor κB subunit 1 [NFκB1], and prostaglandin-endoperoxide synthase 2 [PTGS2]), tumor nitric oxide level, and serum IL-1β, and IL-4 levels. On the other hand, cisplatin treatment increased the expression of selected inflammatory markers. Conclusion: This study suggests that bromelain treatment could potentiate the antitumor effect of cisplatin on triple-negative breast cancer 4T1 cells through modulating the tumor environmental inflammation

Relationship between Virus Replication and Apoptosis Events in IgM + Cells from Chicken Spleen and Bursa of Fabricius Infected with Malaysia Strain of Very Virulent Infectious Bursal Disease Virus

Background: Infection of IBDV was reported to be endemic in worldwide including Malaysia and can be spread orally thru polluted fodder and water source, thus causing economic losses especially in commercial poultry industry. The infection resulted in depletion of B lymphocytes and subsequently destruction of the bursa which leaded to immunosuppression of the bird and it was postulated that the depletion of cells in the bursa was due to induction of apoptosis. In the current study, the infection of Malaysia isolated very virulent IBDV UPM0081 on IgM bearing B lymphocytes (IgM+ cells) from chicken spleen and bursa was compared.Materials, Methods & Results: A total of sixty eggs were obtained and raised until the age of 3 weeks old. The birds were divided into two groups (n = 30), which one of them served as control while IBDV strain UPM0081 was used to infect another group of birds at the concentration of 103 ELD50. The birds were observed and sacrificed at day 2, 4 and 5 post infections. Spleen and bursa of Fabricius were harvested and subjected to IgM+ cell enrichment using microbeads. The cell viability of enriched cells was assayed using MTT and cell cycle was analyzed using propidium iodide. Annexin V FITC and acridine orange/propidium iodide double stain assays were used to determine the event of apoptosis in the enriched IgM+ cells. Also, the IBDV viral load was also quantified by using real time PCR to evaluate the relationship between virus replication and apoptosis events in the infected chickens. Current results showed that the apoptotic events were observed to be significantly higher in IgM+ cells isolated from chicken bursa as compared to the cells isolated from spleen. The bursal B lymphocytes cell viability was observed to be decreasing following the infection of very virulent IBDV. The cells were then investigated of their apoptotic rate and data showed that increasing apoptotic cells (early and late apoptosis) were observed in AO/PI double stain as well as increment of SubG0/G1 population in the cell cycle analysis and also increment of Annexin V FITC bound cells in the apoptosis study. As for B lymphocytes from chicken spleen, the magnitude of damage caused by very virulent IBDV was not as severe as what being observed in the chicken bursa, with the cell viability drastically decreased on day 4 following IBDV infection.Discussion: IBDV caused severe destruction in bursa of Fabricius compared to spleen, in which cell death events in the former was reported to be directly caused by the virus. Apoptotic event in chicken spleen following IBDV infection was observed to be caused by oxidative stress. Thus, viral replication played a role in inducing bursal IgM+ cells death while such phenomenon was not observed in spleen isolated IgM+ cells. In summary, the cell death events of IgM+ cells in chicken spleen and bursa of Fabricius may be accounted by different factors upon infection with Malaysia strain of IBDV UPM0081. It is obvious that IgM+ cells from chicken bursa suffered from apoptotic cell death in an increasing manner considerably with time of infection and RNA load detected in the cells, which supported by previous literature that IBDV induces host cells apoptosis, with both VP2 and VP5 playing a role in binding and apoptosis. Meanwhile, the cell death events of B lymphocytes in chicken spleen was observed to be more relevant to other factors such as the oxidative stress or proinflammatory cytokines that caused by the virus infection rather than the viral RNA load

Relationship between Virus Replication and Apoptosis Events in IgM + Cells from Chicken Spleen and Bursa of Fabricius Infected with Malaysia Strain of Very Virulent Infectious Bursal Disease Virus

Background: Infection of IBDV was reported to be endemic in worldwide including Malaysia and can be spread orally thru polluted fodder and water source, thus causing economic losses especially in commercial poultry industry. The infection resulted in depletion of B lymphocytes and subsequently destruction of the bursa which leaded to immunosuppression of the bird and it was postulated that the depletion of cells in the bursa was due to induction of apoptosis. In the current study, the infection of Malaysia isolated very virulent IBDV UPM0081 on IgM bearing B lymphocytes (IgM+ cells) from chicken spleen and bursa was compared.Materials, Methods & Results: A total of sixty eggs were obtained and raised until the age of 3 weeks old. The birds were divided into two groups (n = 30), which one of them served as control while IBDV strain UPM0081 was used to infect another group of birds at the concentration of 103 ELD50. The birds were observed and sacrificed at day 2, 4 and 5 post infections. Spleen and bursa of Fabricius were harvested and subjected to IgM+ cell enrichment using microbeads. The cell viability of enriched cells was assayed using MTT and cell cycle was analyzed using propidium iodide. Annexin V FITC and acridine orange/propidium iodide double stain assays were used to determine the event of apoptosis in the enriched IgM+ cells. Also, the IBDV viral load was also quantified by using real time PCR to evaluate the relationship between virus replication and apoptosis events in the infected chickens. Current results showed that the apoptotic events were observed to be significantly higher in IgM+ cells isolated from chicken bursa as compared to the cells isolated from spleen. The bursal B lymphocytes cell viability was observed to be decreasing following the infection of very virulent IBDV. The cells were then investigated of their apoptotic rate and data showed that increasing apoptotic cells (early and late apoptosis) were observed in AO/PI double stain as well as increment of SubG0/G1 population in the cell cycle analysis and also increment of Annexin V FITC bound cells in the apoptosis study. As for B lymphocytes from chicken spleen, the magnitude of damage caused by very virulent IBDV was not as severe as what being observed in the chicken bursa, with the cell viability drastically decreased on day 4 following IBDV infection.Discussion: IBDV caused severe destruction in bursa of Fabricius compared to spleen, in which cell death events in the former was reported to be directly caused by the virus. Apoptotic event in chicken spleen following IBDV infection was observed to be caused by oxidative stress. Thus, viral replication played a role in inducing bursal IgM+ cells death while such phenomenon was not observed in spleen isolated IgM+ cells. In summary, the cell death events of IgM+ cells in chicken spleen and bursa of Fabricius may be accounted by different factors upon infection with Malaysia strain of IBDV UPM0081. It is obvious that IgM+ cells from chicken bursa suffered from apoptotic cell death in an increasing manner considerably with time of infection and RNA load detected in the cells, which supported by previous literature that IBDV induces host cells apoptosis, with both VP2 and VP5 playing a role in binding and apoptosis. Meanwhile, the cell death events of B lymphocytes in chicken spleen was observed to be more relevant to other factors such as the oxidative stress or proinflammatory cytokines that caused by the virus infection rather than the viral RNA load