Asian Pacific Journal of Tropical Biomedicine
Issue 7,2018 Table of Contents
2018(7):333-339.
DOI: 10.4103/2221-1691.237075
Abstract:
Objective: To investigate the role of toll-like receptor 2 (TLR2) in inflammatory activity of macrophage infected with the recombinant Mycobacterium bovis bacillus Calmette-Guerin (rBCG). Methods: Mouse macrophage cell line J774A.1 was infected with Mycobacterium bovis bacillus Calmette-Guerin (BCG) and rBCG cultures for 48 h in the presence or absence of 10 μg/mL of TLR2 inhibitor. Untreated macrophages were used as a negative control while lipopolysaccharide-stimulated macrophages were used as a positive control. The ability of the macrophage to engulf the BCG and rBCG in the absence or presence of TLR2 inhibitor was assessed using a phagocytic assay, while the production of inflammatory cytokines and nitric oxide by the infected macrophages was evaluated using ELISA and Griess reagent method, while the expression of the inducible nitric oxide synthase was determined using Western blot analysis. Results: The results showed that blocking TLR2 function reduced the phagocytic activity, nitric oxide production and proinflammatory cytokine secretion such as TNF-α, IL-1β and IL-12p40 as well as inducible nitric oxide synthase expression in the infected macrophages. These data showed the importance of TLR2 in the activation of macrophages following BCG and rBCG infections. Conclusions: Through exploring the immunological mechanism which underlies the protection conferred by the candidate vaccine, this study will improve our understanding of the vaccine candidate’s mechanism to protect the host from malaria infection.
Objective: To investigate the role of toll-like receptor 2 (TLR2) in inflammatory activity of macrophage infected with the recombinant Mycobacterium bovis bacillus Calmette-Guerin (rBCG). Methods: Mouse macrophage cell line J774A.1 was infected with Mycobacterium bovis bacillus Calmette-Guerin (BCG) and rBCG cultures for 48 h in the presence or absence of 10 μg/mL of TLR2 inhibitor. Untreated macrophages were used as a negative control while lipopolysaccharide-stimulated macrophages were used as a positive control. The ability of the macrophage to engulf the BCG and rBCG in the absence or presence of TLR2 inhibitor was assessed using a phagocytic assay, while the production of inflammatory cytokines and nitric oxide by the infected macrophages was evaluated using ELISA and Griess reagent method, while the expression of the inducible nitric oxide synthase was determined using Western blot analysis. Results: The results showed that blocking TLR2 function reduced the phagocytic activity, nitric oxide production and proinflammatory cytokine secretion such as TNF-α, IL-1β and IL-12p40 as well as inducible nitric oxide synthase expression in the infected macrophages. These data showed the importance of TLR2 in the activation of macrophages following BCG and rBCG infections. Conclusions: Through exploring the immunological mechanism which underlies the protection conferred by the candidate vaccine, this study will improve our understanding of the vaccine candidate’s mechanism to protect the host from malaria infection.
2018(7):340-344.
DOI: 10.4103/2221-1691.237076
Abstract:
Objective: To evaluate effects of docosahexaenoic acid-enriched phosphatidylcholine (DHAPC) on cytokine production induced by lipopolysaccharide (LPS). Methods: The culture supernatants of splenocytes exposed to DHA-PC along with LPS were harvested to determine the production of Th 1 (IFN-Y and IL-2) and Th2 [IL-4, IL-5, IL-6 and IL-12/IL-23 (p40)] cytokines. Cytokines were measured using ELISA. Results: Co-administration of DHAPC with LPS resulted in significantly lower IL-2 expression compared to that observed with administration of only LPS (P<0.01). Treatment with DHA-PC and LPS significantly increased IL-5 expression (P<0.01). Moreover, co-administration of DHA-PC with LPS significantly decreased IL-6 and IL-12/IL-23 (p40) expressions compared to that observed with administration of only LPS (P<0.01). Conclusions: Our results suggest that DHA-PC inhibits pro-inflammatory cytokines [IL-2, IL-6 and IL-12/IL-23 (p40)] expression on induction of inflammation.
Objective: To evaluate effects of docosahexaenoic acid-enriched phosphatidylcholine (DHAPC) on cytokine production induced by lipopolysaccharide (LPS). Methods: The culture supernatants of splenocytes exposed to DHA-PC along with LPS were harvested to determine the production of Th 1 (IFN-Y and IL-2) and Th2 [IL-4, IL-5, IL-6 and IL-12/IL-23 (p40)] cytokines. Cytokines were measured using ELISA. Results: Co-administration of DHAPC with LPS resulted in significantly lower IL-2 expression compared to that observed with administration of only LPS (P<0.01). Treatment with DHA-PC and LPS significantly increased IL-5 expression (P<0.01). Moreover, co-administration of DHA-PC with LPS significantly decreased IL-6 and IL-12/IL-23 (p40) expressions compared to that observed with administration of only LPS (P<0.01). Conclusions: Our results suggest that DHA-PC inhibits pro-inflammatory cytokines [IL-2, IL-6 and IL-12/IL-23 (p40)] expression on induction of inflammation.
Paulo Fernando Machado Paredes
,
Selene Maia de Morais
,
Fernando César Rodrigues Brito
,
Luiz Francisco Wemmenson Gon alves Moura
,
Patrícia de Araújo Rodrigues
,
Stephen Rathinaraj Benjamin
,
Francisco Ernani Alves Magalh es
,
Eridan Orlando Pereira Tramontina Florean
,
Maria Izabel Florindo Guedes
2018(7):345-351.
DOI: 10.4103/2221-1691.237077
Abstract:
Objective: To evaluate the chemical components of active extract from Cnidoscolus quercifolius root bark and its cytotoxic potential against several tumor strains. Methods: The high-performance liquid chromatography with diode-array detection and 1H and 13C nuclear magnetic resonance spectroscopy of the extract were used to distinguish the existence of possible functional groups in the root bark extract. The in vitro cytotoxic activity of methanol extract on human colon cancer cell lines was evaluated using OVCAR-8, SF-295, HCT116, HL-60 strains and the samples were assessed by 3-(4,5-dimethylthiazol2-yl)-2,5diphenyltetrazolium bromide method. Results: The analysis of nuclear magnetic spectra of the active chloroform fraction revealed the presence of absorptions bands correspondent to a mixture of favelines such as neofavelanone, deoxofaveline or methyl-faveline, which structures were confirmed by ultraviolet spectra upon high-performance liquid chromatography with diode-array detection analysis. The active fraction showed cytotoxic effects in the tested strains, HCT-116, SF-295, OVCAR-8 and HL-60 cells with IC50 of 72 hours ranging from 4.95 to 15.23 μg/mL. Conclusions: The results suggest that the substances present in faveleira (Cnidoscolus quercifolius) root bark extract have a cytotoxic potential against several tumor lines, showing a broader antitumour potential, and in addition no adverse to healthy cells. Therefore, the root bark extract of Cnidoscolus quercifolius has a possibility of use for anticarcinogenic therapies.
Objective: To evaluate the chemical components of active extract from Cnidoscolus quercifolius root bark and its cytotoxic potential against several tumor strains. Methods: The high-performance liquid chromatography with diode-array detection and 1H and 13C nuclear magnetic resonance spectroscopy of the extract were used to distinguish the existence of possible functional groups in the root bark extract. The in vitro cytotoxic activity of methanol extract on human colon cancer cell lines was evaluated using OVCAR-8, SF-295, HCT116, HL-60 strains and the samples were assessed by 3-(4,5-dimethylthiazol2-yl)-2,5diphenyltetrazolium bromide method. Results: The analysis of nuclear magnetic spectra of the active chloroform fraction revealed the presence of absorptions bands correspondent to a mixture of favelines such as neofavelanone, deoxofaveline or methyl-faveline, which structures were confirmed by ultraviolet spectra upon high-performance liquid chromatography with diode-array detection analysis. The active fraction showed cytotoxic effects in the tested strains, HCT-116, SF-295, OVCAR-8 and HL-60 cells with IC50 of 72 hours ranging from 4.95 to 15.23 μg/mL. Conclusions: The results suggest that the substances present in faveleira (Cnidoscolus quercifolius) root bark extract have a cytotoxic potential against several tumor lines, showing a broader antitumour potential, and in addition no adverse to healthy cells. Therefore, the root bark extract of Cnidoscolus quercifolius has a possibility of use for anticarcinogenic therapies.
2018(7):352-359.
DOI: 10.4103/2221-1691.237078
Abstract:
Objective: To evaluate the protective effect of morin against pentylenetetrazol (PTZ)-induced tonic-clonic convulsions in mice. Methods: Swiss albino mice (18-22 g) was used to induce convulsions by intraperitoneal (i.p.) administration of PTZ (90 mg/kg). Mice were either pretreated with morin (10, 20 and 40 mg/kg) or vehicle (distilled water, 10 mg/kg) 45 min before PTZ administration. Various behavioral and biochemical parameters were assessed. Results: PTZ administration resulted in significant production (P<0.001) of tonic-clonic conclusion and mortality in mice. PTZ-induced increase in the duration of convulsion, onset of convulsion and mortality was inhibited significantly by morin (20 and 40 mg/kg) administration. The PTZ-induced decrease in brain GABA, dopamine and Na+K+ATPase levels and increase in xanthine oxidase activity were inhibited significantly by morin (20 and 40 mg/kg) treatment. The increased levels of malondialdehyde and nitric oxide level were significantly decreased by morin (20 and 40 mg/kg) treatment. Also, reduced levels of superoxide dismutase and glutathione were increased significantly by morin treatment. Conclusions: Results of the present study indicate that morin showed its anti-convulsant effect via modulating the levels of brain GABA, Na+K+ATPase, and oxido-nitrosative stress. Thus, morin can be a potential candidate for further clinical evaluations as an anti-epileptic agent.
Objective: To evaluate the protective effect of morin against pentylenetetrazol (PTZ)-induced tonic-clonic convulsions in mice. Methods: Swiss albino mice (18-22 g) was used to induce convulsions by intraperitoneal (i.p.) administration of PTZ (90 mg/kg). Mice were either pretreated with morin (10, 20 and 40 mg/kg) or vehicle (distilled water, 10 mg/kg) 45 min before PTZ administration. Various behavioral and biochemical parameters were assessed. Results: PTZ administration resulted in significant production (P<0.001) of tonic-clonic conclusion and mortality in mice. PTZ-induced increase in the duration of convulsion, onset of convulsion and mortality was inhibited significantly by morin (20 and 40 mg/kg) administration. The PTZ-induced decrease in brain GABA, dopamine and Na+K+ATPase levels and increase in xanthine oxidase activity were inhibited significantly by morin (20 and 40 mg/kg) treatment. The increased levels of malondialdehyde and nitric oxide level were significantly decreased by morin (20 and 40 mg/kg) treatment. Also, reduced levels of superoxide dismutase and glutathione were increased significantly by morin treatment. Conclusions: Results of the present study indicate that morin showed its anti-convulsant effect via modulating the levels of brain GABA, Na+K+ATPase, and oxido-nitrosative stress. Thus, morin can be a potential candidate for further clinical evaluations as an anti-epileptic agent.
2018(7):360-364.
DOI: 10.4103/2221-1691.237079
Abstract:
Objective: To produce fluorescent tagged recombinant erythroferrone protein (ERFE_eGFP) for laboratory investigations. Methods: Erythroferrone (ERFE) gene was fused to green fluorescent protein (eGFP) gene and cloned in a pSecTag2Hygro plasmid. The constructed plasmid was amplified in Escherichia coli DH5α and the eGFP-fused ERFE (ERFE_eGFP) protein was expressed in human embryonic kidney (HEK293T) cell line. Results: The plasmid constructed from colony C6 contained ERFE_eGFP with the correct restriction sizes of 4.2 kb and expressed secretory ERFE_eGFP fusion protein (approximately size of 75 kDa) in HEK293T cell line. Conclusions: ERFE_eGFP recombinant protein is successfully expressed as a secretory functional protein and could be sensitively detected using fluorometry. This fusion protein might benefit future applications for localization of cellular ERFE receptors and competitive immunoassay of ERFE concentration.
Objective: To produce fluorescent tagged recombinant erythroferrone protein (ERFE_eGFP) for laboratory investigations. Methods: Erythroferrone (ERFE) gene was fused to green fluorescent protein (eGFP) gene and cloned in a pSecTag2Hygro plasmid. The constructed plasmid was amplified in Escherichia coli DH5α and the eGFP-fused ERFE (ERFE_eGFP) protein was expressed in human embryonic kidney (HEK293T) cell line. Results: The plasmid constructed from colony C6 contained ERFE_eGFP with the correct restriction sizes of 4.2 kb and expressed secretory ERFE_eGFP fusion protein (approximately size of 75 kDa) in HEK293T cell line. Conclusions: ERFE_eGFP recombinant protein is successfully expressed as a secretory functional protein and could be sensitively detected using fluorometry. This fusion protein might benefit future applications for localization of cellular ERFE receptors and competitive immunoassay of ERFE concentration.
2018(7):365-370.
DOI: 10.4103/2221-1691.237080
Abstract:
Objective: To identify the region conferring stability to pBSSB2 (a linear plasmid, pBSSB1, containing a kanamycin cassette), which is unique to Indonesian isolates of Salmonella enterica serovar Typhi. Methods: The open reading frame (ORF) 009 was identified as a toxin coding gene in the plasmid through introduction of translational termination codons in the ORF. Results: The stability function was located in a fragment that spanned nucleotides 5 766 to 6 828 in the linear plasmid genetic map. Ectopic expression of ORF009 in pBAD18 vector indicated ORF009 codes for a toxin. This fragment could stabilize plasmid pUC18 previously destabilized through mutation of the pcnB (plasmid copy number control) gene that codes for polyA polymerase. Majority of the cells expressing ORF009 were non-viable according to phase contrast microscopy. Conclusions: This study demonstrated that a linear plasmid fragment that carries a gene encoding a toxin possibly conferred stability to the parent plasmid. It was able to stabilize a multicopy plasmid of Escherichia coli.
Objective: To identify the region conferring stability to pBSSB2 (a linear plasmid, pBSSB1, containing a kanamycin cassette), which is unique to Indonesian isolates of Salmonella enterica serovar Typhi. Methods: The open reading frame (ORF) 009 was identified as a toxin coding gene in the plasmid through introduction of translational termination codons in the ORF. Results: The stability function was located in a fragment that spanned nucleotides 5 766 to 6 828 in the linear plasmid genetic map. Ectopic expression of ORF009 in pBAD18 vector indicated ORF009 codes for a toxin. This fragment could stabilize plasmid pUC18 previously destabilized through mutation of the pcnB (plasmid copy number control) gene that codes for polyA polymerase. Majority of the cells expressing ORF009 were non-viable according to phase contrast microscopy. Conclusions: This study demonstrated that a linear plasmid fragment that carries a gene encoding a toxin possibly conferred stability to the parent plasmid. It was able to stabilize a multicopy plasmid of Escherichia coli.
SS. Hasson
,
MS. Al-Shaqsi
,
JZ. Albusaidi
,
MS. Al-Balushi
,
FL. Hakkim
,
GM. Aleemallah
,
AA. Al-Jabri
2018(7):371-376.
DOI: 10.4103/2221-1691.237081
Abstract:
Objective: To investigate different types of dates and medical properties of influencing blood clotting and wound healing in an animal model. Methods: Three different cultivars of dates (Ajwa, Khalas, and Fardh) were examined in-vivo, for blood clotting and wound healing using CD1 mice of both sexes. Study of toxicity to animals was performed accordingly prior to further investigations. The ethanolic extracts were given orally to animals as a constituent in their daily water. Blood samples were obtained from the mice inferior vena cava to carry out the prothrombin time (PT) assay using the manual method and confirmed using a semi-automated machine. The bleeding time (BT) assay was performed using the cutting technique. In the wound healing analysis, a small cut (5-10 mm) in the skin overlying the thigh was conducted in all mice under anesthesia. The diameter of the cut and healing status were measured on a daily basis throughout the time of the experiment using a roller. Results: Ajwa was able to elevate both PT and BT (P<0.05), significantly in a time-dependent manner followed by Khalas date (P<0.05). The results of PT and BT of Fardh date were found to be very close to those of the control group (P<0.05). Despite its activity as an anticoagulant, Khalas date showed a potential property to enhance wound healing in contrast to other dates and the control groups in this study. Conclusions: Omani Khalas date fruit has both antithrombotic as well as wound healing properties. The results open a new gate with these fruits for exploring the potential component(s) that may play an important role in antithrombotic as well as wound healing process.
Objective: To investigate different types of dates and medical properties of influencing blood clotting and wound healing in an animal model. Methods: Three different cultivars of dates (Ajwa, Khalas, and Fardh) were examined in-vivo, for blood clotting and wound healing using CD1 mice of both sexes. Study of toxicity to animals was performed accordingly prior to further investigations. The ethanolic extracts were given orally to animals as a constituent in their daily water. Blood samples were obtained from the mice inferior vena cava to carry out the prothrombin time (PT) assay using the manual method and confirmed using a semi-automated machine. The bleeding time (BT) assay was performed using the cutting technique. In the wound healing analysis, a small cut (5-10 mm) in the skin overlying the thigh was conducted in all mice under anesthesia. The diameter of the cut and healing status were measured on a daily basis throughout the time of the experiment using a roller. Results: Ajwa was able to elevate both PT and BT (P<0.05), significantly in a time-dependent manner followed by Khalas date (P<0.05). The results of PT and BT of Fardh date were found to be very close to those of the control group (P<0.05). Despite its activity as an anticoagulant, Khalas date showed a potential property to enhance wound healing in contrast to other dates and the control groups in this study. Conclusions: Omani Khalas date fruit has both antithrombotic as well as wound healing properties. The results open a new gate with these fruits for exploring the potential component(s) that may play an important role in antithrombotic as well as wound healing process.
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