For the successful establishment of secondary tumours, blood-borne metastatic tumour cells must a... more For the successful establishment of secondary tumours, blood-borne metastatic tumour cells must adhere and spread on the vascular endothelium before they can migrate through it to form secondary growths in the tissue beneath. In this study an in vitro assay was developed to study the behavourial interactions between B16F10 cells and Bovine aortic endothelial cells. It was hypothesized that molecules synthesized by the endothelial cells may be involved in the mediation of the adhesion, spreading and migration events and hence that such molecules may possibly be involved in the process of haematogenic metastasis. Endothelial derived extracts were obtained from the cell surface and from conditioned medium. The extracts were tested for their adhesion promoting abilities in a quick dot blot adhesion assay. To verify that these molecules promoted adhesion, antibodies were raised against the extracts. Partial characterisation of the molecules was achieved using SDS-PAGE and immunoprobing. ...
American Journal of Reproductive Immunology, Dec 1, 2002
We previously immunoneutralized gonadotrophin releasing hormone (GnRH), using an analogue of GnRH... more We previously immunoneutralized gonadotrophin releasing hormone (GnRH), using an analogue of GnRH (des-1 GnRH-I), conjugated to tetanus toxoid via a carbodiimide reaction. The castration effect on the reproductive system was not consistent in all the treated animals. Therefore, we examined the possibility that conjugation to the carrier protein via the N- or C-terminal could have an effect on efficacy. GnRH analogue sequences were synthesized consisting of an additional cysteine at either terminal and specific conjugation was carried out using a bifunctional linker agent. Conjugation of the monomer through the N-terminal proved to be a highly effective means of causing immunocastration in terms of decreased gonadotrophin and testosterone concentrations and testicular size, whereas conjugation through the C-terminal proved to be ineffective. This was reflected in the ability of the antibodies to bind native GnRH, but not the levels of the anti-GnRH antibodies. Immunoneutralization efficacy was attributed to the importance of preserving the GnRH C-terminal.
Profiling a propolis sample from Papua New Guinea (PNG) using high-resolution mass spectrometry i... more Profiling a propolis sample from Papua New Guinea (PNG) using high-resolution mass spectrometry indicated that it contained several triterpenoids. Further fractionation by column chromatography and medium-pressure liquid chromatography (MPLC) followed by nuclear magnetic resonance spectroscopy (NMR) identified 12 triterpenoids. Five of these were obtained pure and the others as mixtures of two or three compounds. The compounds identified were: mangiferonic acid, ambonic acid, isomangiferolic acid, ambolic acid, 27-hydroxyisomangiferolic acid, cycloartenol, cycloeucalenol, 24-methylenecycloartenol, 20-hydroxybetulin, betulin, betulinic acid and madecassic acid. The fractions from the propolis and the purified compounds were tested in vitro against Crithidia fasciculata, Trypanosoma congolense, drug-resistant Trypanosoma congolense, Trypanosoma b. brucei and multidrug-resistant Trypanosoma b. brucei (B48). They were also assayed for their toxicity against U947 cells. The compounds and...
The formation of a protein layer “corona” on the nanoparticle surface upon entry into a biologica... more The formation of a protein layer “corona” on the nanoparticle surface upon entry into a biological environment was shown to strongly influence the interactions with cells, especially affecting the uptake of nanomedicines. In this work, we present the impact of the protein corona on the uptake of PEGylated zein micelles by cancer cells, macrophages, and dendritic cells. Zein was successfully conjugated with poly(ethylene glycol) (PEG) of varying chain lengths (5K and 10K) and assembled into micelles. Our results demonstrate that PEGylation conferred stealth effects to the zein micelles. The presence of human plasma did not impact the uptake levels of the micelles by melanoma cancer cells, regardless of the PEG chain length used. In contrast, it decreased the uptake by macrophages and dendritic cells. These results therefore make PEGylated zein micelles promising as potential drug delivery systems for cancer therapy.
A new compound (1) (named as artemceramide-B) together with eight known compounds (taraxerol (2),... more A new compound (1) (named as artemceramide-B) together with eight known compounds (taraxerol (2), taraxerol acetate (3), β-sitosterol (4), stigmasterol (5), trans-ethyl caffeate, dracunculin (7), scoparone (8) and isoscopoletin (9) were isolated from an ethanolic extract of the roots of Artemisia incisa Pamp (Asteracae). The structures of the compounds were determined through IR, 1D NMR (1 H NMR, 13 C NMR) and 2D NMR (COSY, NOESY, HSQC and HMBC) analyses. Accurate mass analyses were done with EI-MS, ESI-MS and acid methanolysis of compound 1 followed by GS-MS studies. The relative stereochemistry of artemceramide-B was determined by comparing its specific rotation and spectroscopic data with the literature. Compounds 1-9 were tested for their anti-bacterial potential against five bacteria strains; Staphylococcus epidermidis, Staphylococcus aureus, Klebsiella pneumoniae, Bacillus subtilis and Escherichia coli. Compound 1 (new) (MIC: 0.0157, 0.0313 mg/mL) and 7 (MIC: 0.0815, 1.000 mg/mL) showed excellent activities against S. epidermidis and S. aureus while compound 9 showed excellent activities (MIC: 0.0700, 1.234, 1.890 and 2.286 mg/mL) against S. epidermidis, S. aureus, K. pneumoniae and E. coli, respectively. Compound 6 (MIC: 2.000 mg/mL) was found to be active against E. coli while neither of the compounds showed potential activity against B. subtilis.
Antibodies play a crucial role in the immune response, in fighting off pathogens as well as helpi... more Antibodies play a crucial role in the immune response, in fighting off pathogens as well as helping create strong immunological memory. Antibody-dependent enhancement (ADE) occurs when non-neutralising antibodies recognise and bind to a pathogen, but are unable to prevent infection, and is widely known and is reported as occurring in infection caused by several viruses. This narrative review explores the ADE phenomenon, its occurrence in viral infections and evaluates its role in infection by SARS-CoV-2 virus, which causes coronavirus disease 2019 (COVID-19). As of yet, there is no clear evidence of ADE in SARS-CoV-2, though this area is still subject to further study.
Introduction: Serum Bactericidal Assays (SBAs) are considered as the gold standard to evaluate th... more Introduction: Serum Bactericidal Assays (SBAs) are considered as the gold standard to evaluate the immunogenicity of many vaccine formulations against infectious agents, for example Neisseria meningitidis vaccines. SBAs are also used to evaluate vaccine lots before release to the market, because it has been demonstrated that there is a correlation between bactericidal antibody titers and protection. For Laboratory and Clinical Good Practice, it is very important to have a positive control in each assay. To our knowledge, there is no commercial positive control to serve this function, therefore the purpose of this work was to evaluate a monoclonal antibody (mAb) panel against N. meningitidis strains produced at Institute Finlay of Vaccines as a reference material in the established bactericidal assay, with the advantage of high homogeneity and specificity and relative low cost of the mAbs test agents. Materials and Methods: Specificity of a panel of mAbs was evaluated by a whole cell...
Background: Staphylococcus aureus is an important human pathogen, especially causing skin and sof... more Background: Staphylococcus aureus is an important human pathogen, especially causing skin and soft tissue infections (SSTIs). Over the decades, the infections caused by antibioticresistant strains have often become life-threatening. Consequently, exploration and development of competent approaches to combat these serious circumstances are urgently required. Methods: The antibacterial activity of melittin (Mel) on S. aureus, methicillin-resistant S. aureus (MRSA) and clinical isolates of vancomycin-intermediate S. aureus (VISA) was investigated by minimum inhibitory concentration (MIC) and time-killing assays. The localization of Mel on the bacterial cell was visualized by confocal laser scanning microscopy and its effect on the membrane was indicated based on propidium iodide uptake. The non-ionic surfactant vesicle (NISV) or niosome nanocarrier was established for Mel loading (Mel-loaded NISV) by the thin-film hydration method. Physicochemical and in vitro biological properties of Mel-loaded NISVs were characterized. The cellular uptake of Mel-loaded NISVs was evaluated by holotomography analysis. In addition, an ex vivo study was conducted on a porcine ear skin model to assess the permeation ability of Mel-loaded NISVs and their potential to inhibit bacterial skin infection. Results: The effective inhibitory activity of Mel on skin pathogens was demonstrated. Among the tested strains, VISA was most susceptible to Mel. Regarding to its function, Mel targeted the bacterial cell envelope and disrupted cell membrane integrity. Mel-loaded NISVs were successfully fabricated with a nano-size of 120-200 nm and entrapment efficiency of greater than 90%. Moreover, Mel-loaded NISVs were taken up and accumulated in the intracellular space. Meanwhile, Mel was released and distributed throughout the cytosol and nucleus. Mel-loaded NISVs efficiently inhibited the growth of bacteria, particularly MRSA and VISA. Importantly, they not only penetrated epidermal and dermal skin layers, but also reduced the bacterial growth in infected skin. Conclusion: Mel-loaded NISVs have a great potential to exhibit antibacterial activity. Therapeutic application of Mel-loaded NISVs could be further developed as an alternative platform for the treatment of skin infection via dermal and transdermal delivery.
Molecular dynamics (MD) simulations can provide a detailed view of molecule behaviour at an atomi... more Molecular dynamics (MD) simulations can provide a detailed view of molecule behaviour at an atomic level, which can be useful when attempting to interpret experiments or design new systems. The decapeptide gonadotrophin-releasing hormone I (GnRH-I) is known to control fertility in mammals for both sexes. It was previously shown that inoculation with silica nanoparticles (SiNPs) coated with GnRH-I makes an effective anti-fertility vaccine due to how the peptide adsorbs to the nanoparticle and is presented to the immune system. In this paper, we develop and employ a protocol to simulate the development of a GnRH-I peptide adlayer by allowing peptides to diffuse and adsorb in a staged series of trajectories. The peptides start the simulation in an immobile state in solution above the model silica surface, and are then released sequentially. This facile approach allows the adlayer to develop in a natural manner and appears to be quite versatile. We find that the GnRH-I adlayer tends to ...
Previous research has shown that propolis has immunomodulatory activity. Propolis extracts from d... more Previous research has shown that propolis has immunomodulatory activity. Propolis extracts from different geographic origins were assessed for their anti-inflammatory activities by investigating their ability to alter the production of tumour necrosis factor-α (TNF-α) and the cytokines interleukin-1β (IL-1β), IL-6 and IL-10 in THP-1-derived macrophage cells co-stimulated with lipopolysaccharide (LPS). All the propolis extracts suppressed the TNF-α and IL-6 LPS-stimulated levels. Similar suppression effects were detected for IL-1β, but the release of this cytokine was synergised by propolis samples from Ghana and Indonesia when compared with LPS. Overall, the Cameroonian propolis extract (P-C) was the most active and this was evaluated for its effects on the metabolic profile of unstimulated macrophages or macrophages activated by LPS. The levels of 81 polar metabolites were identified by liquid chromatography (LC) coupled with mass spectrometry (MS) on a ZIC-pHILIC column. LPS alter...
The venom of Apis mellifera (honey bee) has been reported to play a role in immunotherapy, but ex... more The venom of Apis mellifera (honey bee) has been reported to play a role in immunotherapy, but existing evidence to support its immuno-modulatory claims is insufficient. Four fractions from whole bee venom (BV) were separated using medium pressure liquid chromatography. Their ability to induce the production of cytokines TNFα, IL-1β and IL-6 in phorbol-12-myristate-13-acetate (PMA)-treated U937 cells was assessed. The levels of the three cytokines produced by stimulation with the four fractions and crude BV without LPS were not significantly different from negative control values. However, co-stimulation of the cells with LPS and Fraction 4 (F-4) induced a 1.6-fold increase in TNF-α level (p < 0.05) compared to LPS alone. Likewise, LPS-induced IL-1β production was significantly synergised in the presence of F-1 (nine-fold), F-2 (six-fold), F-3 (four-fold) and F-4 (two-fold) fractions, but was only slightly enhanced with crude BV (1.5-fold) relative to LPS. Furthermore, the LPS-st...
The present study was carried out to examine the anti-inflammatory activity of the inner leaf gel... more The present study was carried out to examine the anti-inflammatory activity of the inner leaf gel component of Aloe barbadensis Miller. A simple in vitro assay was designed to determine the effect of the inner gel on bacterial-induced pro-inflammatory cytokine production, namely TNF-a and IL-1b, from peripheral blood leukocytes stimulated with Shigella flexneri or LPS. This report describes the suppression of both cytokines with a freeze-dried inner gel powder and a commercial health drink from the same source. Comparison was made with a human monocytic cell-line (THP-1 cells) and a similar trend in responses was demonstrated.
Trypanosomiasis and leishmaniasis are important parasitic diseases affecting millions of people i... more Trypanosomiasis and leishmaniasis are important parasitic diseases affecting millions of people in Africa, Asia, and South America. In a previous study, we identified several flavonoid glycosides as antiprotozoal principles from a Turkish plant. Here we surveyed a large set of flavonoid aglycones and glycosides, as well as a panel of other related compounds of phenolic and phenylpropanoid nature, for their in vitro activities against Trypanosoma brucei rhodesiense , Trypanosoma cruzi , and Leishmania donovani . The cytotoxicities of more than 100 compounds for mammalian L6 cells were also assessed and compared to their antiparasitic activities. Several compounds were investigated in vivo for their antileishmanial and antitrypanosomal efficacies in mouse models. Overall, the best in vitro trypanocidal activity for T. brucei rhodesiense was exerted by 7,8-dihydroxyflavone (50% inhibitory concentration [IC 50 ], 68 ng/ml), followed by 3-hydroxyflavone, rhamnetin, and 7,8,3′,4′-tetrahyd...
Staphylococcus protein A (SpA) is found in the cell wall of Staphylococcus aureus bacteria. Its a... more Staphylococcus protein A (SpA) is found in the cell wall of Staphylococcus aureus bacteria. Its ability to bind to the constant Fc regions of antibodies means it is useful for antibody extraction, and further integration with inorganic materials can lead to the development of diagnostics and therapeutics. We have investigated the adsorption of SpA on inorganic surface models such as experimentally relevant negatively charged silica, as well as positively charged and neutral surfaces, by use of fully atomistic molecular dynamics simulations. We have found that SpA, which is itself negatively charged at pH7, is able to adsorb on all our surface models. However, adsorption on charged surfaces is more specific in terms of protein orientation compared to a neutral Au (111) surface, while the protein structure is generally well maintained in all cases. The results indicate that SpA adsorption is optimal on the siloxide-rich silica surface, which is negative at pH7 since this keeps the Fc ...
The existence of pathogens that escape recognition by specific vaccines, the need to improve exis... more The existence of pathogens that escape recognition by specific vaccines, the need to improve existing vaccines and the increased availability of therapeutic (non-infectious disease) vaccines necessitate the rational development of novel vaccine concepts based on the induction of protective cell-mediated immune responses. For naive T-cell activation, several signals resulting from innate and adaptive interactions need to be integrated, and adjuvants may interfere with some or all of these signals. Adjuvants, for example, are used to promote the immunogenicity of antigens in vaccines, by inducing a pro-inflammatory environment that enables the recruitment and promotion of the infiltration of phagocytic cells, particularly antigen-presenting cells (APC), to the injection site. Adjuvants can enhance antigen presentation, induce cytokine expression, activate APC and modulate more downstream adaptive immune reactions (vaccine delivery systems, facilitating immune Signal 1). In addition, adjuvants can act as immunopotentiators (facilitating Signals 2 and 3) exhibiting immune stimulatory effects during antigen presentation by inducing the expression of co-stimulatory molecules on APC. Together, these signals determine the strength of activation of specific T-cells, thereby also influencing the quality of the downstream T helper cytokine profiles and the differentiation of antigen-specific T helper populations (Signal 3).
Non-ionic surfactant vesicles (NISV) are synthetic membrane vesicles formed by selfassembly of a ... more Non-ionic surfactant vesicles (NISV) are synthetic membrane vesicles formed by selfassembly of a non-ionic surfactant, often in a mixture with cholesterol and a charged chemical species. Different methods can be used to manufacture NISV, with the majority of these requiring bulk mixing of two phases. This mixing process is time-consuming and leads to the preparation of large and highly dispersed vesicles, which affects the consistency of the final product and could hinder subsequent regulatory approval. In this study, we have compared the physical characteristics of NISV prepared using two conventional methods (thin-film hydration method and heating method) with a recently introduced microfluidic method. The resulting particles from these methods were assessed for their physical characteristics and in vitro cytotoxicity. Through microfluidics, nanosized NISV were prepared in seconds, through rapid and controlled mixing of two miscible phases (lipids dissolved in alcohol and an aqueous medium) in a microchannel, without the need of a size reduction step, as required for the conventional methods. Stability studies over two months showed the particles were stable regardless of the method of preparation and there were no differences in terms of EC50 on A375 and A2780 cell lines. However, this work demonstrates the flexibility and ease of applying lab-on-chip microfluidics for the preparation of NISV that could be used to significantly improve formulation research and development, by enabling the rapid manufacture of a consistent end-product, under controlled conditions.
Honey bee venom has been established to have significant effect in immunotherapy. In the present ... more Honey bee venom has been established to have significant effect in immunotherapy. In the present study, (Z)-11-eicosenol-a major constituent of bee venom, along with its derivations methyl cis-11-eicosenoate and cis-11-eicosenoic acid, were synthesised to investigate their immune stimulatory effect and possible use as vaccine adjuvants. Stimuli that prime and activate the immune system have exerted profound effects on immune cells, particularly macrophages; however, the effectiveness of bee venom constituents as immune stimulants has not yet been established. Here, the abilities of these compounds to act as pro-inflammatory stimuli were assessed, either alone or in combination with lipopolysaccharide (LPS), by examining the secretion of tumour necrosis factor-α (TNF-α) and the cytokines interleukin-1β (IL-1β), IL-6 and IL-10 by THP-1 macrophages. The compounds clearly increased the levels of IL-1β and decreased IL-10, whereas a decrease in IL-6 levels suggested a complex mechanism o...
RNA interference (RNAi) is an effective and naturally occurring post-transcriptional gene regulat... more RNA interference (RNAi) is an effective and naturally occurring post-transcriptional gene regulatory mechanism. This mechanism involves the degradation of a target messenger RNA (mRNA) through the introduction of short interfering RNA (siRNA) that is complementary to the target mRNA. The application of siRNA-based therapeutics is limited by the development of an effective delivery system, as naked siRNA is unstable and cannot penetrate the cell membrane. In this study, we investigated the use of cationic niosomes (CN) prepared by microfluidic mixing for siRNA delivery. In an in vitro model, these vesicles were able to deliver anti-luciferase siRNA and effectively suppress luciferase expression in B16-F10 mouse melanoma cells. More importantly, in an in vivo mouse model, intratumoral administration of CN-carrying anti-luciferase siRNA led to significant suppression of luciferase expression compared with naked siRNA. Thus, we have established a novel and effective system for the deliv...
Melittin, the major active peptide of honeybee venom (BV), has potential for use in adjuvant immu... more Melittin, the major active peptide of honeybee venom (BV), has potential for use in adjuvant immunotherapy. The immune system response to different stimuli depends on the secretion of different metabolites from macrophages. One potent stimulus is lipopolysaccharide (LPS), a component isolated from gram-negative bacteria, which induces the secretion of pro-inflammatory cytokines in macrophage cell cultures. This secretion is amplified when LPS is combined with melittin. In the present study, pure melittin was isolated from whole BV by flash chromatography to obtain pure melittin. The ability of melittin to enhance the release of tumour necrosis factor-α (TNF-α), Interleukin (IL-1β, IL-6, and IL-10) cytokines from a macrophage cell line (THP-1) was then assessed. The response to melittin and LPS, applied alone or in combination, was characterised by metabolic profiling, and the metabolomics results were used to evaluate the potential of melittin as an immune adjuvant therapy. The addi...
For the successful establishment of secondary tumours, blood-borne metastatic tumour cells must a... more For the successful establishment of secondary tumours, blood-borne metastatic tumour cells must adhere and spread on the vascular endothelium before they can migrate through it to form secondary growths in the tissue beneath. In this study an in vitro assay was developed to study the behavourial interactions between B16F10 cells and Bovine aortic endothelial cells. It was hypothesized that molecules synthesized by the endothelial cells may be involved in the mediation of the adhesion, spreading and migration events and hence that such molecules may possibly be involved in the process of haematogenic metastasis. Endothelial derived extracts were obtained from the cell surface and from conditioned medium. The extracts were tested for their adhesion promoting abilities in a quick dot blot adhesion assay. To verify that these molecules promoted adhesion, antibodies were raised against the extracts. Partial characterisation of the molecules was achieved using SDS-PAGE and immunoprobing. ...
American Journal of Reproductive Immunology, Dec 1, 2002
We previously immunoneutralized gonadotrophin releasing hormone (GnRH), using an analogue of GnRH... more We previously immunoneutralized gonadotrophin releasing hormone (GnRH), using an analogue of GnRH (des-1 GnRH-I), conjugated to tetanus toxoid via a carbodiimide reaction. The castration effect on the reproductive system was not consistent in all the treated animals. Therefore, we examined the possibility that conjugation to the carrier protein via the N- or C-terminal could have an effect on efficacy. GnRH analogue sequences were synthesized consisting of an additional cysteine at either terminal and specific conjugation was carried out using a bifunctional linker agent. Conjugation of the monomer through the N-terminal proved to be a highly effective means of causing immunocastration in terms of decreased gonadotrophin and testosterone concentrations and testicular size, whereas conjugation through the C-terminal proved to be ineffective. This was reflected in the ability of the antibodies to bind native GnRH, but not the levels of the anti-GnRH antibodies. Immunoneutralization efficacy was attributed to the importance of preserving the GnRH C-terminal.
Profiling a propolis sample from Papua New Guinea (PNG) using high-resolution mass spectrometry i... more Profiling a propolis sample from Papua New Guinea (PNG) using high-resolution mass spectrometry indicated that it contained several triterpenoids. Further fractionation by column chromatography and medium-pressure liquid chromatography (MPLC) followed by nuclear magnetic resonance spectroscopy (NMR) identified 12 triterpenoids. Five of these were obtained pure and the others as mixtures of two or three compounds. The compounds identified were: mangiferonic acid, ambonic acid, isomangiferolic acid, ambolic acid, 27-hydroxyisomangiferolic acid, cycloartenol, cycloeucalenol, 24-methylenecycloartenol, 20-hydroxybetulin, betulin, betulinic acid and madecassic acid. The fractions from the propolis and the purified compounds were tested in vitro against Crithidia fasciculata, Trypanosoma congolense, drug-resistant Trypanosoma congolense, Trypanosoma b. brucei and multidrug-resistant Trypanosoma b. brucei (B48). They were also assayed for their toxicity against U947 cells. The compounds and...
The formation of a protein layer “corona” on the nanoparticle surface upon entry into a biologica... more The formation of a protein layer “corona” on the nanoparticle surface upon entry into a biological environment was shown to strongly influence the interactions with cells, especially affecting the uptake of nanomedicines. In this work, we present the impact of the protein corona on the uptake of PEGylated zein micelles by cancer cells, macrophages, and dendritic cells. Zein was successfully conjugated with poly(ethylene glycol) (PEG) of varying chain lengths (5K and 10K) and assembled into micelles. Our results demonstrate that PEGylation conferred stealth effects to the zein micelles. The presence of human plasma did not impact the uptake levels of the micelles by melanoma cancer cells, regardless of the PEG chain length used. In contrast, it decreased the uptake by macrophages and dendritic cells. These results therefore make PEGylated zein micelles promising as potential drug delivery systems for cancer therapy.
A new compound (1) (named as artemceramide-B) together with eight known compounds (taraxerol (2),... more A new compound (1) (named as artemceramide-B) together with eight known compounds (taraxerol (2), taraxerol acetate (3), β-sitosterol (4), stigmasterol (5), trans-ethyl caffeate, dracunculin (7), scoparone (8) and isoscopoletin (9) were isolated from an ethanolic extract of the roots of Artemisia incisa Pamp (Asteracae). The structures of the compounds were determined through IR, 1D NMR (1 H NMR, 13 C NMR) and 2D NMR (COSY, NOESY, HSQC and HMBC) analyses. Accurate mass analyses were done with EI-MS, ESI-MS and acid methanolysis of compound 1 followed by GS-MS studies. The relative stereochemistry of artemceramide-B was determined by comparing its specific rotation and spectroscopic data with the literature. Compounds 1-9 were tested for their anti-bacterial potential against five bacteria strains; Staphylococcus epidermidis, Staphylococcus aureus, Klebsiella pneumoniae, Bacillus subtilis and Escherichia coli. Compound 1 (new) (MIC: 0.0157, 0.0313 mg/mL) and 7 (MIC: 0.0815, 1.000 mg/mL) showed excellent activities against S. epidermidis and S. aureus while compound 9 showed excellent activities (MIC: 0.0700, 1.234, 1.890 and 2.286 mg/mL) against S. epidermidis, S. aureus, K. pneumoniae and E. coli, respectively. Compound 6 (MIC: 2.000 mg/mL) was found to be active against E. coli while neither of the compounds showed potential activity against B. subtilis.
Antibodies play a crucial role in the immune response, in fighting off pathogens as well as helpi... more Antibodies play a crucial role in the immune response, in fighting off pathogens as well as helping create strong immunological memory. Antibody-dependent enhancement (ADE) occurs when non-neutralising antibodies recognise and bind to a pathogen, but are unable to prevent infection, and is widely known and is reported as occurring in infection caused by several viruses. This narrative review explores the ADE phenomenon, its occurrence in viral infections and evaluates its role in infection by SARS-CoV-2 virus, which causes coronavirus disease 2019 (COVID-19). As of yet, there is no clear evidence of ADE in SARS-CoV-2, though this area is still subject to further study.
Introduction: Serum Bactericidal Assays (SBAs) are considered as the gold standard to evaluate th... more Introduction: Serum Bactericidal Assays (SBAs) are considered as the gold standard to evaluate the immunogenicity of many vaccine formulations against infectious agents, for example Neisseria meningitidis vaccines. SBAs are also used to evaluate vaccine lots before release to the market, because it has been demonstrated that there is a correlation between bactericidal antibody titers and protection. For Laboratory and Clinical Good Practice, it is very important to have a positive control in each assay. To our knowledge, there is no commercial positive control to serve this function, therefore the purpose of this work was to evaluate a monoclonal antibody (mAb) panel against N. meningitidis strains produced at Institute Finlay of Vaccines as a reference material in the established bactericidal assay, with the advantage of high homogeneity and specificity and relative low cost of the mAbs test agents. Materials and Methods: Specificity of a panel of mAbs was evaluated by a whole cell...
Background: Staphylococcus aureus is an important human pathogen, especially causing skin and sof... more Background: Staphylococcus aureus is an important human pathogen, especially causing skin and soft tissue infections (SSTIs). Over the decades, the infections caused by antibioticresistant strains have often become life-threatening. Consequently, exploration and development of competent approaches to combat these serious circumstances are urgently required. Methods: The antibacterial activity of melittin (Mel) on S. aureus, methicillin-resistant S. aureus (MRSA) and clinical isolates of vancomycin-intermediate S. aureus (VISA) was investigated by minimum inhibitory concentration (MIC) and time-killing assays. The localization of Mel on the bacterial cell was visualized by confocal laser scanning microscopy and its effect on the membrane was indicated based on propidium iodide uptake. The non-ionic surfactant vesicle (NISV) or niosome nanocarrier was established for Mel loading (Mel-loaded NISV) by the thin-film hydration method. Physicochemical and in vitro biological properties of Mel-loaded NISVs were characterized. The cellular uptake of Mel-loaded NISVs was evaluated by holotomography analysis. In addition, an ex vivo study was conducted on a porcine ear skin model to assess the permeation ability of Mel-loaded NISVs and their potential to inhibit bacterial skin infection. Results: The effective inhibitory activity of Mel on skin pathogens was demonstrated. Among the tested strains, VISA was most susceptible to Mel. Regarding to its function, Mel targeted the bacterial cell envelope and disrupted cell membrane integrity. Mel-loaded NISVs were successfully fabricated with a nano-size of 120-200 nm and entrapment efficiency of greater than 90%. Moreover, Mel-loaded NISVs were taken up and accumulated in the intracellular space. Meanwhile, Mel was released and distributed throughout the cytosol and nucleus. Mel-loaded NISVs efficiently inhibited the growth of bacteria, particularly MRSA and VISA. Importantly, they not only penetrated epidermal and dermal skin layers, but also reduced the bacterial growth in infected skin. Conclusion: Mel-loaded NISVs have a great potential to exhibit antibacterial activity. Therapeutic application of Mel-loaded NISVs could be further developed as an alternative platform for the treatment of skin infection via dermal and transdermal delivery.
Molecular dynamics (MD) simulations can provide a detailed view of molecule behaviour at an atomi... more Molecular dynamics (MD) simulations can provide a detailed view of molecule behaviour at an atomic level, which can be useful when attempting to interpret experiments or design new systems. The decapeptide gonadotrophin-releasing hormone I (GnRH-I) is known to control fertility in mammals for both sexes. It was previously shown that inoculation with silica nanoparticles (SiNPs) coated with GnRH-I makes an effective anti-fertility vaccine due to how the peptide adsorbs to the nanoparticle and is presented to the immune system. In this paper, we develop and employ a protocol to simulate the development of a GnRH-I peptide adlayer by allowing peptides to diffuse and adsorb in a staged series of trajectories. The peptides start the simulation in an immobile state in solution above the model silica surface, and are then released sequentially. This facile approach allows the adlayer to develop in a natural manner and appears to be quite versatile. We find that the GnRH-I adlayer tends to ...
Previous research has shown that propolis has immunomodulatory activity. Propolis extracts from d... more Previous research has shown that propolis has immunomodulatory activity. Propolis extracts from different geographic origins were assessed for their anti-inflammatory activities by investigating their ability to alter the production of tumour necrosis factor-α (TNF-α) and the cytokines interleukin-1β (IL-1β), IL-6 and IL-10 in THP-1-derived macrophage cells co-stimulated with lipopolysaccharide (LPS). All the propolis extracts suppressed the TNF-α and IL-6 LPS-stimulated levels. Similar suppression effects were detected for IL-1β, but the release of this cytokine was synergised by propolis samples from Ghana and Indonesia when compared with LPS. Overall, the Cameroonian propolis extract (P-C) was the most active and this was evaluated for its effects on the metabolic profile of unstimulated macrophages or macrophages activated by LPS. The levels of 81 polar metabolites were identified by liquid chromatography (LC) coupled with mass spectrometry (MS) on a ZIC-pHILIC column. LPS alter...
The venom of Apis mellifera (honey bee) has been reported to play a role in immunotherapy, but ex... more The venom of Apis mellifera (honey bee) has been reported to play a role in immunotherapy, but existing evidence to support its immuno-modulatory claims is insufficient. Four fractions from whole bee venom (BV) were separated using medium pressure liquid chromatography. Their ability to induce the production of cytokines TNFα, IL-1β and IL-6 in phorbol-12-myristate-13-acetate (PMA)-treated U937 cells was assessed. The levels of the three cytokines produced by stimulation with the four fractions and crude BV without LPS were not significantly different from negative control values. However, co-stimulation of the cells with LPS and Fraction 4 (F-4) induced a 1.6-fold increase in TNF-α level (p < 0.05) compared to LPS alone. Likewise, LPS-induced IL-1β production was significantly synergised in the presence of F-1 (nine-fold), F-2 (six-fold), F-3 (four-fold) and F-4 (two-fold) fractions, but was only slightly enhanced with crude BV (1.5-fold) relative to LPS. Furthermore, the LPS-st...
The present study was carried out to examine the anti-inflammatory activity of the inner leaf gel... more The present study was carried out to examine the anti-inflammatory activity of the inner leaf gel component of Aloe barbadensis Miller. A simple in vitro assay was designed to determine the effect of the inner gel on bacterial-induced pro-inflammatory cytokine production, namely TNF-a and IL-1b, from peripheral blood leukocytes stimulated with Shigella flexneri or LPS. This report describes the suppression of both cytokines with a freeze-dried inner gel powder and a commercial health drink from the same source. Comparison was made with a human monocytic cell-line (THP-1 cells) and a similar trend in responses was demonstrated.
Trypanosomiasis and leishmaniasis are important parasitic diseases affecting millions of people i... more Trypanosomiasis and leishmaniasis are important parasitic diseases affecting millions of people in Africa, Asia, and South America. In a previous study, we identified several flavonoid glycosides as antiprotozoal principles from a Turkish plant. Here we surveyed a large set of flavonoid aglycones and glycosides, as well as a panel of other related compounds of phenolic and phenylpropanoid nature, for their in vitro activities against Trypanosoma brucei rhodesiense , Trypanosoma cruzi , and Leishmania donovani . The cytotoxicities of more than 100 compounds for mammalian L6 cells were also assessed and compared to their antiparasitic activities. Several compounds were investigated in vivo for their antileishmanial and antitrypanosomal efficacies in mouse models. Overall, the best in vitro trypanocidal activity for T. brucei rhodesiense was exerted by 7,8-dihydroxyflavone (50% inhibitory concentration [IC 50 ], 68 ng/ml), followed by 3-hydroxyflavone, rhamnetin, and 7,8,3′,4′-tetrahyd...
Staphylococcus protein A (SpA) is found in the cell wall of Staphylococcus aureus bacteria. Its a... more Staphylococcus protein A (SpA) is found in the cell wall of Staphylococcus aureus bacteria. Its ability to bind to the constant Fc regions of antibodies means it is useful for antibody extraction, and further integration with inorganic materials can lead to the development of diagnostics and therapeutics. We have investigated the adsorption of SpA on inorganic surface models such as experimentally relevant negatively charged silica, as well as positively charged and neutral surfaces, by use of fully atomistic molecular dynamics simulations. We have found that SpA, which is itself negatively charged at pH7, is able to adsorb on all our surface models. However, adsorption on charged surfaces is more specific in terms of protein orientation compared to a neutral Au (111) surface, while the protein structure is generally well maintained in all cases. The results indicate that SpA adsorption is optimal on the siloxide-rich silica surface, which is negative at pH7 since this keeps the Fc ...
The existence of pathogens that escape recognition by specific vaccines, the need to improve exis... more The existence of pathogens that escape recognition by specific vaccines, the need to improve existing vaccines and the increased availability of therapeutic (non-infectious disease) vaccines necessitate the rational development of novel vaccine concepts based on the induction of protective cell-mediated immune responses. For naive T-cell activation, several signals resulting from innate and adaptive interactions need to be integrated, and adjuvants may interfere with some or all of these signals. Adjuvants, for example, are used to promote the immunogenicity of antigens in vaccines, by inducing a pro-inflammatory environment that enables the recruitment and promotion of the infiltration of phagocytic cells, particularly antigen-presenting cells (APC), to the injection site. Adjuvants can enhance antigen presentation, induce cytokine expression, activate APC and modulate more downstream adaptive immune reactions (vaccine delivery systems, facilitating immune Signal 1). In addition, adjuvants can act as immunopotentiators (facilitating Signals 2 and 3) exhibiting immune stimulatory effects during antigen presentation by inducing the expression of co-stimulatory molecules on APC. Together, these signals determine the strength of activation of specific T-cells, thereby also influencing the quality of the downstream T helper cytokine profiles and the differentiation of antigen-specific T helper populations (Signal 3).
Non-ionic surfactant vesicles (NISV) are synthetic membrane vesicles formed by selfassembly of a ... more Non-ionic surfactant vesicles (NISV) are synthetic membrane vesicles formed by selfassembly of a non-ionic surfactant, often in a mixture with cholesterol and a charged chemical species. Different methods can be used to manufacture NISV, with the majority of these requiring bulk mixing of two phases. This mixing process is time-consuming and leads to the preparation of large and highly dispersed vesicles, which affects the consistency of the final product and could hinder subsequent regulatory approval. In this study, we have compared the physical characteristics of NISV prepared using two conventional methods (thin-film hydration method and heating method) with a recently introduced microfluidic method. The resulting particles from these methods were assessed for their physical characteristics and in vitro cytotoxicity. Through microfluidics, nanosized NISV were prepared in seconds, through rapid and controlled mixing of two miscible phases (lipids dissolved in alcohol and an aqueous medium) in a microchannel, without the need of a size reduction step, as required for the conventional methods. Stability studies over two months showed the particles were stable regardless of the method of preparation and there were no differences in terms of EC50 on A375 and A2780 cell lines. However, this work demonstrates the flexibility and ease of applying lab-on-chip microfluidics for the preparation of NISV that could be used to significantly improve formulation research and development, by enabling the rapid manufacture of a consistent end-product, under controlled conditions.
Honey bee venom has been established to have significant effect in immunotherapy. In the present ... more Honey bee venom has been established to have significant effect in immunotherapy. In the present study, (Z)-11-eicosenol-a major constituent of bee venom, along with its derivations methyl cis-11-eicosenoate and cis-11-eicosenoic acid, were synthesised to investigate their immune stimulatory effect and possible use as vaccine adjuvants. Stimuli that prime and activate the immune system have exerted profound effects on immune cells, particularly macrophages; however, the effectiveness of bee venom constituents as immune stimulants has not yet been established. Here, the abilities of these compounds to act as pro-inflammatory stimuli were assessed, either alone or in combination with lipopolysaccharide (LPS), by examining the secretion of tumour necrosis factor-α (TNF-α) and the cytokines interleukin-1β (IL-1β), IL-6 and IL-10 by THP-1 macrophages. The compounds clearly increased the levels of IL-1β and decreased IL-10, whereas a decrease in IL-6 levels suggested a complex mechanism o...
RNA interference (RNAi) is an effective and naturally occurring post-transcriptional gene regulat... more RNA interference (RNAi) is an effective and naturally occurring post-transcriptional gene regulatory mechanism. This mechanism involves the degradation of a target messenger RNA (mRNA) through the introduction of short interfering RNA (siRNA) that is complementary to the target mRNA. The application of siRNA-based therapeutics is limited by the development of an effective delivery system, as naked siRNA is unstable and cannot penetrate the cell membrane. In this study, we investigated the use of cationic niosomes (CN) prepared by microfluidic mixing for siRNA delivery. In an in vitro model, these vesicles were able to deliver anti-luciferase siRNA and effectively suppress luciferase expression in B16-F10 mouse melanoma cells. More importantly, in an in vivo mouse model, intratumoral administration of CN-carrying anti-luciferase siRNA led to significant suppression of luciferase expression compared with naked siRNA. Thus, we have established a novel and effective system for the deliv...
Melittin, the major active peptide of honeybee venom (BV), has potential for use in adjuvant immu... more Melittin, the major active peptide of honeybee venom (BV), has potential for use in adjuvant immunotherapy. The immune system response to different stimuli depends on the secretion of different metabolites from macrophages. One potent stimulus is lipopolysaccharide (LPS), a component isolated from gram-negative bacteria, which induces the secretion of pro-inflammatory cytokines in macrophage cell cultures. This secretion is amplified when LPS is combined with melittin. In the present study, pure melittin was isolated from whole BV by flash chromatography to obtain pure melittin. The ability of melittin to enhance the release of tumour necrosis factor-α (TNF-α), Interleukin (IL-1β, IL-6, and IL-10) cytokines from a macrophage cell line (THP-1) was then assessed. The response to melittin and LPS, applied alone or in combination, was characterised by metabolic profiling, and the metabolomics results were used to evaluate the potential of melittin as an immune adjuvant therapy. The addi...
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Papers by Valerie Ferro