RESUMO
Optimizations of the gene expression cassette combined with the selection of an appropriate signal peptide are important factors that must be considered to enhance heterologous protein expression in Chinese Hamster Ovary (CHO) cells. In this study, we investigated the effectiveness of different signal peptides on the production of recombinant human chorionic gonadotropin (r-hCG) in CHO-K1 cells. Four optimized expression constructs containing four promising signal peptides were stably transfected into CHO-K1 cells. The generated CHO-K1 stable pool was then evaluated for r-hCG protein production. Interestingly, human serum albumin and human interleukin-2 signal peptides exhibited relatively greater extracellular secretion of the r-hCG with an average yield of (16.59 ± 0.02 µg/ml) and (14.80 ± 0.13 µg/ml) respectively compared to the native and murine IgGκ light chain signal peptides. The stably transfected CHO pool was further used as the cell substrate to develop an optimized upstream process followed by a downstream phase of the r-hCG. Finally, the biological activity of the purified r-hCG was assessed using in vitro bioassays. The combined data highlight that the choice of signal peptide can be imperative to ensure an optimal secretion of a recombinant protein in CHO cells. In addition, the stable pool technology was a viable approach for the production of biologically active r-hCG at a research scale with acceptable bioprocess performances and consistent product quality.
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BACKGROUND: Mass vaccination of dogs as important rabies reservoir is proposed to most effectively reduce and eliminate rabies also in humans. However, a minimum coverage of 70% needs to be achieved for control of the disease in zoonotic regions. In numerous developing countries, dog vaccination rate is still dangerously low because of economic constraints and due to a high turnover in dog populations. Improved vaccine production processes may help to alleviate cost and supply limitations. In this work, we studied and optimized the replication and vaccine potency of PV rabies virus strain in the muscovy-duck derived AGE1.CR and AGE1.CR.pIX suspension cell lines. RESULTS: The BHK-21-adapted PV rabies virus strain replicated efficiently in the avian cell lines without requirement for prior passaging. CR.pIX was previously shown to augment heat shock responses and supported slightly higher infectious titers compared to the parental CR cell line. Both cell lines allowed replication of rabies virus also in absence of recombinant IGF, the only complex component of the chemically defined medium that was developed for the two cell lines. After scale-up from optimization experiments in shake flask to production in 7-l bioreactors peak virus titers of 2.4 × 108 FFU/ml were obtained. The potency of inactivated rabies virus harvest according to the NIH test was 3.5 IU/ml. Perfusion with the chemically defined medium during the virus replication phase improved the potency of the vaccine twofold, and increased the number of doses 9.6 fold. CONCLUSION: This study demonstrates that a rabies vaccine for animal vaccination can be produced efficiently in the AGE1.CR.pIX suspension cell line in a scalable process in chemically defined medium.
Assuntos
Vacina Antirrábica , Raiva , Animais , Reatores Biológicos , Linhagem Celular , Cães , Patos , Raiva/prevenção & controle , Raiva/veterináriaRESUMO
Hepatitis E virus (HEV) is a nonenveloped virus causing an emerging zoonotic disease posing a severe threat to the public health in the world, especially to pregnant women. In this study, a truncated form (aa 368-606) of the open reading frame 2 of the capsid protein (tORF2-HEV), a major structural protein of HEV, was expressed in Escherichia coli. This work characterizes for the first time, the fused Glutathione-S-Transferase-tagged tORF2 (GST-tORF2) and tORF2-HEV forms in E. coli. The fusion protein was purified by affinity chromatography with a purity higher than 90% and to yield about 27% after thrombin digestion. The purified GST-tORF2 protein was then characterized by western blot, using anti-GST antibodies, and CD spectroscopy. The GST-tORF2 and tORF2-HEV proteins were shown to be efficient to develop an ELISA test to detect anti-HEV IgG in mice sera immunized with a recombinant full length ORF2 protein. Sera showed a significant increase of the absorbance signal at 450 nm, in plate wells coated with a quantity of 0.5, 1 and 2 µg of proteins. ELISA plates coated with the purified GST-tORF2 and tORF2-HEV showed similar response when compared to the HEV ELISA where total insect cell lysate, infected with the recombinant baculovirus expressing full ORF2, was used as positive control.
Assuntos
Proteínas do Capsídeo , Vírus da Hepatite E , Proteínas Recombinantes de Fusão , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/isolamento & purificação , Escherichia coli/genética , Escherichia coli/metabolismo , Vírus da Hepatite E/química , Vírus da Hepatite E/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
Rabies is a fatal disease that can be prevented by vaccination. Different approaches were investigated to develop novel human rabies vaccines with improved features compared to the current available vaccines, among them is the use of heterologous gene expression technology. Here, we describe the expression of the surface rabies virus glycoprotein (RABV-G), which is the major antigen responsible for the induction of protective immunity, in Pichia pastoris. Six transformants were selected according to their gene copy number as determined by real time qPCR. Upon induction by methanol, low level of RABV-G was secreted into the culture medium, around 60 ng/mL. To understand the effect of foreign gene dosage on cellular physiology of P. pastoris, transcriptional analysis of key genes involved in unfolded protein response (UPR) and endoplasmic reticulum associated degradation (ERAD) pathway was performed. Results showed that these pathways were highly activated; misfolded RABV-G was degraded in the cytosol via the ERAD mechanism. To study the functionality of the secreted RABV-G, in vitro competitive neutralizing assay was conducted. Data showed the secreted recombinant RABV-G had enabled a reduction of the neutralizing activity of human immune rabies serum, indicating that the secreted recombinant protein had reached its correct conformational form.
Assuntos
Expressão Gênica , Glicoproteínas de Membrana , Pichia/química , Pichia/metabolismo , Vírus da Raiva/genética , Proteínas Virais , Humanos , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Pichia/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Virais/biossíntese , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
The dimorphic yeast Yarrowia lipolytica has become an emerging cell factory for recombinant proteins production. Expression vectors involving LIP2 promoter (pLIP2) have been developed and used successfully. However, the relationship between dimorphic transition (i.e., cell morphology) and pLIP2 regulation is still unclear and must be assessed to improve process robustness. This requests to discriminate the effect of cell morphology from that of effectors, such as pH, that trigger the dimorphic transition. This was performed using gene reporter system based on ß-galactosidase activity and DsRed fluorescence, single-cell analysis by flow cytometry, and quantification of gene expression. Our results clearly pointed out that cell morphology has not effect on the regulation of pLIP2. By contrast, pH modification yielded to phenotypic heterogeneity, potentially leading to a lack of robustness of the cell population. Taken altogether, our results demonstrated that, under appropriate environmental conditions (e.g., pH being an important factor), Y. lipolytica could be considered as a robust and reliable host for recombinant protein production.
Assuntos
Yarrowia/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Concentração de Íons de Hidrogênio , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Yarrowia/crescimento & desenvolvimento , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
The use of Vero cells for rabies vaccine production was recommended from the WHO in 2005. A controlled production process is necessary to reduce the risk of contaminants in the product. One step towards this is to turn away from animal-derived components (e.g. serum, trypsin, bovine serum albumin) and face a production process in animal component-free medium. In this study, a proteomic approach was applied, using 2-D differential gel electrophoresis and mass spectrometry to compare rabies virus propagation in Vero cells under different cultivation conditions in microcarrier culture. Protein alterations were investigated for uninfected and infected Vero cells over a time span from 1 to 8 days post-infection in two different types of media (serum-free versus serum-containing media). For mock-infected cells, proteins involved in stress response, redox status, protease activity or glycolysis, and protein components in the endoplasmic reticulum were found to be differentially expressed comparing both cultivation media at all sampling points. For virus-infected cells, additionally changes in protein expression involved in general cell regulation and in calcium homeostasis were identified under both cultivation conditions. The fact that neither of these additional proteins was identified for cells during mock infection, but similar protein expression changes were found for both systems during virus propagation, indicates for a specific response of the Vero cell proteome on rabies virus infection.
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Interações Hospedeiro-Patógeno , Proteoma/análise , Vírus da Raiva/crescimento & desenvolvimento , Animais , Chlorocebus aethiops , Eletroforese em Gel Bidimensional , Espectrometria de Massas , Células VeroRESUMO
Measles vaccination remains the most efficient way to control the spread of the virus. This work focuses on the production of a measles vaccine using stirred conditions as an advanced option for process scale up. Non-porous Cytodex 1 microcarriers were used to support MRC-5 cell growth in suspension cultures. Virus replication was first optimized in spinner flasks, and the effects of various operational parameters were investigated. Cell infection with AIK-C measles strain at an MOI (multiplicity of infection) of 0.005, without glucose regulation and in M199 medium, resulted in a virus titer of 106·²5 TCID50 (median tissue culture infective dose)/ml. To optimize the production process in a 7-l bioreactor, we carried out various perfused cultures using minimum essential medium (MEM) + 5% FCS diluted with phosphate-buffered saline (PBS). We achieved a high cell density level (4.1 × 106 cells/ml) with an efficient use of the medium when MEM + 5% FCS diluted with PBS at 25% was used during the cell amplification step. Optimization of measles production in MRC-5 cells grown on Cytodex 1 beads in a 7-l bioreactor showed that perfusion was the most efficient when compared to repeated-batch culture. Perfusion at a rate of 0.25 V (reactor volume)/day showed the highest specific productivity (1.6 IVP [infectious virus particle] cell⻹ day⻹). Testing of several stabilizers containing pharmaceutically improved components such as sugars, amino acids, and charged ions showed that the formulation composed of sucrose and MgCl2, led to the maintenance of the infectivity of the AIK-C measles virus strain to a significant level, when stored at +28 °C, +4 °C and -60 °C.
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Técnicas de Cultura de Células/métodos , Dextranos , Fibroblastos/virologia , Vacina contra Sarampo , Vírus do Sarampo/crescimento & desenvolvimento , Cultura de Vírus/métodos , Reatores Biológicos , Biotecnologia/métodos , Técnicas de Cultura de Células/instrumentação , Linhagem Celular , Meios de Cultura , Humanos , Microesferas , Replicação ViralRESUMO
Since its isolation, the human granulocyte-macrophage colony-stimulating factor (hGM-CSF) has been proposed as a new class of therapeutic biological products in the treatment of various diseases. However, the toxicity of this cytokine towards its expression host constitutes a major obstacle to bioprocess development for large-scale production. In this work, the optimized gene encoding hGM-CSF was expressed in the yeast Yarrowia lipolytica in one and two copies under the control of the fatty acid-inducible POX2 promoter. Protein secretion was directed by the targeting sequence of the extracellular lipase (LIP2): preXALip2. After 48 h of induction, Western blot analysis revealed the presence of a nonglycosylated form of 14.5 kDa and a trail of hGM-CSF hyperglycosylated varying from 23 kDa to more than 60 kDa. The two-copy transformants produced hGM-CSF level which was sevenfold higher compared to the single-copy ones. Deglycosylation with PNGase F showed two forms: a mature form of 14.5 kDa and an unprocessed form of 18 kDa. The addition of two alanines to the signal sequence resulted in correct hGM-CSF processing. The production level was estimated at 250 mg/l after preliminary optimization studies of the cultivation and induction phases. The purified hGM-CSF was identified by N-terminal sequencing and LC-MS/MS analysis; its biological activity was confirmed by stimulating the proliferation of TF1 cell line. This study demonstrated that Y. lipolytica is a promising host for the efficient production of active toxic proteins like hGM-CSF.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Fator Estimulador de Colônias de Granulócitos e Macrófagos/isolamento & purificação , Yarrowia/genética , Biotecnologia/métodos , Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/química , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Humanos , Peso Molecular , Sinais Direcionadores de Proteínas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Vibrio parahaemolyticus and V. alginolyticus, marine foodborne pathogens, were treated with UVC-radiation (240 J/m(2)) to evaluate alterations in their outer membrane protein profiles. Outer membrane protein patterns of UVC-irradiated bacteria were found altered when analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Altered proteins were identified by mass spectrometry (MS and MS/MS) and analysis revealed that OmpW, OmpA, Long-chain fatty acid transport protein, Outer membrane receptor protein, Putative uncharacterized protein VP0167, Maltoporin (lamB), Polar flagellin B/D, Agglutination protein Peptidoglycan-associated lipoprotein and MltA-interacting protein MipA were appeared, thereby they can be considered as UVC-stress proteins in some vibrios. In addition, expression of OmpK decreased to non-detectable level. Furthermore, we observed a decrease or an increase in the expression level of other outer membrane proteins.
RESUMO
The commonly used host for industrial production of recombinant proteins Pichia pastoris, has been used in this work to produce the rabies virus glycoprotein (RABV-G). To allow a constitutive expression and the secretion of the expressed recombinant RABV-G, the PichiaPink™ commercialized expression vectors were modified to contain the constitutive GAP promoter and the α secretion signal sequences. Recombinant PichiaPink™ strains co-expressing the RABV-G and the protein chaperone PDI, have been then generated and screened for the best producer clone. The influence of seven carbon sources on the expression of the RABV-G, has been studied under different culture conditions in shake flask culture. An incubation temperature of 30°C under an agitation rate of 250 rpm in a filling volume of 10:1 flask/culture volume ratio were the optimal conditions for the RABV-G production in shake flask for all screened carbon sources. A bioreactor Fed batch culture has been then carried using glycerol and glucose as they were good carbon sources for cell growth and RABV-G production in shake flask scale. Cells were grown on glycerol during the batch phase then fed with glycerol or glucose defined solutions, a final RABV-G concentration of 2.7 µg/l was obtained with a specific product yield (YP/X) of 0.032 and 0.06 µg/g(DCW) respectively. The use of semi-defined feeding solution enhanced the production and the YP/X to 12.9 µg/l and 0.135 µg/g(DCW) respectively. However, the high cell density favored by these carbon sources resulted in oxygen limitation which influenced the glycosylation pattern of the secreted RABV-G. Alternatively, the use of sucrose as substrate for RABV-G production in large scale culture, resulted in less biomass production and a YP/X of 0.310 µg/g(DCW) was obtained. A cation exchange chromatography was then used for RABV-G purification as one step method. The purified protein was correctly folded and glycosylated and able to adopt trimeric conformation. The knowledges gained through this work offer a valuable insight into the bioprocess design of RABV-G production in Pichia pastoris to obtain a correctly folded protein which can be used during an immunization proposal for subunit Rabies vaccine development.
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Manganese (Mn) is an essential trace element required for ubiquitous enzymatic reactions. Chronic overexposure to this metal may promote potent neurotoxic effects. The mechanism of Mn toxicity is not well established, but several studies indicate that oxidative stress play major roles in the Mn-induced neurodegenerative processes. Silymarin (SIL) has antioxidant properties and stabilizes intracellular antioxidant defense systems. The aim of this study was to evaluate the toxic effects of MnCl(2) on the mouse neuroblastoma cell lines (Neuro-2A), to characterize the toxic mechanism associated with Mn exposure and to investigate whether SIL could efficiently protect against neurotoxicity induced by Mn. A significant increase in LDH release activity was observed in Neuro-2A cells associated with a significant decrease in cellular viability upon 24 h exposure to MnCl(2) at concentrations of 200 and 800 µM (P < 0.05) when compared with control unexposed cells. In addition, exposure cells to MnCl(2) (200 and 800 µM), increases oxidant biomarkers and alters enzymatic and non enzymatic antioxidant systems. SIL treatment significantly reduced the levels of LDH, nitric oxide, reactive oxygen species and the oxidants/antioxidants balance in Neuro-2A cells as compared to Mn-exposed cells. These results suggested that silymarin is a powerful antioxidant through a mechanism related to its antioxidant activity, able to interfere with radical-mediated cell death. SIL may be useful in diseases known to be aggravated by reactive oxygen species and in the development of novel treatments for neurodegenerative disorders such as Alzheimer or Parkinson diseases.
Assuntos
Adenosina Trifosfatases/metabolismo , Linhagem Celular Tumoral/efeitos dos fármacos , Manganês/toxicidade , Neuroblastoma/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Silimarina/farmacologia , Animais , Antioxidantes/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Humanos , Peroxidação de Lipídeos , Camundongos , Oxirredução , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND: The non conventional yeast Yarrowia lipolytica has aroused a strong industrial interest for heterologous protein production. However most of the studies describing recombinant protein production by this yeast rely on the use of complex media, such media are not convenient for large scale production particularly for products intended for pharmaceutical applications. In addition medium composition can also affect the production yield. Hence it is necessary to design an efficient medium for therapeutic protein expression by this host. RESULTS: Five different media, including four minimal media and a complex medium, were assessed in shake flasks for the production of human interferon alpha 2b (hIFN α2b) by Y. lipolytica under the control of POX2 promoter inducible with oleic acid. The chemically defined medium SM4 formulated by Invitrogen for Pichia pastoris growth was the most suitable. Using statistical experimental design this medium was further optimized. The selected minimal medium consisting in SM4 supplemented with 10 mg/l FeCl3, 1 g/l glutamate, 5 ml/l PTM1 (Pichia Trace Metals) solution and a vitamin solution composed of myo-inositol, thiamin and biotin was called GNY medium. Compared to shake flask, bioreactor culture in GNY medium resulted in 416-fold increase of hIFN α2b production and 2-fold increase of the biological activity. Furthermore, SM4 enrichment with 5 ml/l PTM1 solution contributed to protect hIFN α2b against the degradation by the 28 kDa protease identified by zymography gel in culture supernatant. The screening of the inhibitory effect of the trace elements present in PTM1 solution on the activity of this protease was achieved using a Box-Behnken design. Statistical data analysis showed that FeCl3 and MnSO4 had the most inhibitory effect. CONCLUSION: We have designed an efficient medium for large scale production of heterologous proteins by Y. lipolytica. The optimized medium GNY is suitable for the production of hIFN α2b with the advantage that no complex nitrogen sources with non-defined composition were required.
Assuntos
Meios de Cultura/farmacologia , Interferon-alfa/biossíntese , Proteínas Recombinantes/biossíntese , Yarrowia/metabolismo , Cloretos/farmacologia , Compostos Férricos/farmacologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Ácido Glutâmico/farmacologia , Humanos , Interferon alfa-2 , Interferon-alfa/genética , Ácido Oleico/farmacologia , Regiões Promotoras Genéticas , Projetos de Pesquisa , Yarrowia/genética , Yarrowia/crescimento & desenvolvimentoRESUMO
BACKGROUND: As an oleaginous yeast, Yarrowia lipolytica is able to assimilate hydrophobic substrates. This led to the isolation of several promoters of key enzymes of this catabolic pathway. Less is known about the behavior of Y. lipolytica in large bioreactors using these substrates. There is therefore a lack of established know-how concerning high cell density culture protocols of this yeast. Consequently, the establishment of suitable induction conditions is required, to maximize recombinant protein production under the control of these promoters. RESULTS: Human interferon α2b (huIFN α2b) production in Yarrowia lipolytica was used as a model for the enhancement of recombinant protein production under the control of the oleic acid (OA)-inducible promoter POX2. Cell viability and heterologous protein production were enhanced by exponential glucose feeding, to generate biomass before OA induction. The optimal biomass level before induction was determined (73 g L(-1)), and glucose was added with oleic acid during the induction phase. Several oleic acid feeding strategies were assessed. Continuous feeding with OA at a ratio of 0.02 g OA per g dry cell weight increased huIFNα2b production by a factor of 1.88 (425 mg L(-1)) and decreased the induction time (by a factor of 2.6, 21 h). huIFN α2b degradation by an aspartic protease secreted by Y. lipolytica was prevented by adding pepstatin (10 µM), leading to produce a 19-fold more active huIFN α2b (26.2 × 10(7) IU mg(-1)). CONCLUSION: Y. lipolytica, a generally regarded as safe (GRAS) microorganism is one of the most promising non conventional yeasts for the production of biologically active therapeutic proteins under the control of hydrophobic substrate-inducible promoter.
Assuntos
Microbiologia Industrial/métodos , Interferon-alfa/biossíntese , Yarrowia/metabolismo , Biomassa , Reatores Biológicos/microbiologia , Engenharia Genética , Glucose/metabolismo , Humanos , Interferon alfa-2 , Interferon-alfa/genética , Ácido Oleico/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Yarrowia/genética , Yarrowia/crescimento & desenvolvimentoRESUMO
In this work, we investigated the effect of codon bias and consensus sequence (CACA) at the translation initiation site on the expression level of heterologous proteins in Yarrowia lipolytica; human interferon alpha 2b (hIFN-α2b) was studied as an example. A codon optimized hIFN-α2b gene was synthesized according to the frequency of codon usage in Y. lipolytica. Both wild-type (IFN-wt) and optimized hIFN-α2b (IFN-op) genes were expressed under the control of a strong inducible promoter acyl-co-enzyme A oxidase (POX2). Protein secretion was directed by the targeting sequence of the extracellular lipase (LIP2): pre-proLIP2. Codon optimization increased protein production by 11-fold, whereas the insertion of CACA sequence upstream of the initiation codon of IFN-op construct resulted in 16.5-fold increase of the expression level; this indicates that translational efficiency plays an important part in the increase of hIFN-α2b production level. The replacement of the pre-proLIP2 signal secretion with the LIP2 pre-region sequence followed by the X-Ala/X-Pro stretch but without the pro-region also increased the secretion of the target protein by twofold, suggesting therefore that the LIP2 pro-region is not necessary for extracellular secretion of small heterologous proteins in Yarrowia lipolytica.
Assuntos
Espaço Extracelular/metabolismo , Interferon-alfa/genética , Interferon-alfa/metabolismo , Engenharia de Proteínas/métodos , Yarrowia/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Códon , Espaço Extracelular/genética , Expressão Gênica , Humanos , Interferon alfa-2 , Interferon-alfa/química , Dados de Sequência Molecular , Sinais Direcionadores de Proteínas , Transporte Proteico , Proteínas Recombinantes , Yarrowia/metabolismoRESUMO
Manganese (Mn) is an essential element for biological systems, nevertheless occupational exposure to high levels of Mn can lead to neurodegenerative disorders, characterized by serious oxidative and neurotoxic effects with similarities to Parkinson's disease. The aim of this study was to investigate the potential effects of silymarin (SIL), an antioxidant flavonoid, against manganese chloride induced neurotoxicity both in vivo (cerebral cortex of rats) and in vitro (Neuro2a cells). Twenty-eight male Wistar rats were randomly divided into four groups: the first group (C) received vehicle solution (i.p.) served as controls. The second group (Mn) received orally manganese chloride (20 mg/ml). The third group (Mn + SIL) received both Mn and SIL. The fourth group (SIL) received only SIL (100 mg/kg/day, i.p.). Animals exposed to Manganese chloride showed a significant increase in TBARS, NO, AOPP and PCO levels in cerebral cortex. These changes were accompanied by a decrease of enzymatic (SOD, CAT, GPx) and non-enzymatic (GSH, NpSH, Vit C) antioxidants. Co-administration of silymarin to Mn-treated rats significantly improved antioxidant enzyme activities and attenuated oxidative damages observed in brain tissue. The potential effect of SIL to prevent Mn induced neurotoxicity was also reflected by the microscopic study, indicative of its neuroprotective effects. We concluded that silymarin possesses neuroprotective potential, thus validating its use in alleviating manganese-induced neurodegenerative effects.
Assuntos
Antioxidantes/farmacologia , Cloretos/toxicidade , Síndromes Neurotóxicas/prevenção & controle , Silimarina/farmacologia , Acetilcolinesterase/metabolismo , Animais , Antioxidantes/metabolismo , Células Cultivadas , Córtex Cerebral/metabolismo , Glutationa Peroxidase/metabolismo , Peróxido de Hidrogênio/metabolismo , Peroxidação de Lipídeos , Masculino , Compostos de Manganês , Nitritos/metabolismo , Carbonilação Proteica/efeitos dos fármacos , Ratos , Ratos Wistar , Superóxido Dismutase/metabolismoRESUMO
Yarrowia lipolytica lipase has been assumed to be a good candidate for the treatment of fat malabsorption in patients with pancreatic insufficiency. Nevertheless, no systematic studies on its stability under physiological conditions pertaining to the human GI (gastrointestinal) tract have been published. Stability of various Y. lipolytica lipase powder formulations at various physiological pH values as well as the effect of digestive proteases and bile salts on enzyme activity were investigated. Results were compared with those obtained from another competing fungal lipase sourced from Candida rugosa. Among the studied formulations, Y. lipolytica lipase stabilized with gum arabic and skimmed milk powder was the most promising powder formulation. Under acidic conditions (pH 3-5), this formulation showed higher stability than those observed with the other Y. lipolytica lipase formulations and C. rugosa lipase. In addition, in the presence of gum arabic and skimmed milk powder as additives, Y. lipolytica lipase exhibited markedly higher resistance to pepsin, trypsin and chymotrypsin actions. Resistance to proteolytic degradation by digestive proteases was also by far higher than that observed with C. rugosa lipase. Similar behaviour was, however, observed when these two fungal lipases were incubated with increased concentrations of bile salts. Residual lipase activity of both fungal lipases showed a slight decrease in NaTDC (sodium taurodeoxycholate) concentration above 4 mM. Consequently, Y. lipolytica lipase formulated with gum arabic and milk powder seemed to have great potential for use as a therapeutic tool for patients with pancreatic insufficiency.
Assuntos
Terapia de Reposição de Enzimas/métodos , Insuficiência Pancreática Exócrina/tratamento farmacológico , Lipase/administração & dosagem , Lipase/química , Yarrowia/enzimologia , Animais , Ácidos e Sais Biliares/metabolismo , Candida/enzimologia , Química Farmacêutica , Quimotripsina/metabolismo , Estabilidade Enzimática , Insuficiência Pancreática Exócrina/enzimologia , Goma Arábica/metabolismo , Concentração de Íons de Hidrogênio , Lipase/metabolismo , Leite/metabolismo , Pepsina A/metabolismo , Tripsina/metabolismoRESUMO
Acute pancreatitis (AP) is a real clinical challenge. Acute pancreatitis remains a common cause of emergency department consultations and a major cause for hospitalization. Gallstones and drinking a lot of alcohol are the most frequent causes of AP. Moreover, AP can be induced by diabetic ketoacidosis (DKA) complicated by hypertriglyceridemia. We report 4 cases of DKA with hypertriglyceridemia complicated by AP in previously undiagnosed diabetes patients. All of our patients presented to the emergency ward with abdominal pain. Their physical exam showed epigastric tenderness. An abdominal CT scan was performed for each patient, showing an AP grade E. Laboratory samples showed high serum glucose levels. They had metabolic acidosis with elevated anion gap. They had high lipasemia and amylasemia. Their lipid panel was disturbed with a high level of cholesterol (from 12.8 mmol/l to 33 mmol/l) and triglyceridemia (from 53 to 133 mmol/l). Our patients were admitted into our ICU where they received fluid resuscitation and intravenous insulin, and their triglycerides rates decreased gradually. Two patients recovered to a good health state, and the two others developed septic shock, requiring the use of large-spectrum antibiotics, and acute kidney injury (AKI) with refractory metabolic acidosis, requiring hemodialysis. Despite the intensive treatment, they developed an unrecoverable multiorgan failure. Through our case series, we aim to highlight the importance of making an early diagnosis, which can be difficult in some situations due to overlapping signs; however, it is crucial for a good recovery. A good understanding of the pathway of hypoinsulinemic states causing hypertriglyceridemia then AP is important because it is the key to best management.
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BACKGROUND: Venous thromboembolism (VTE) is a well-established complication of trauma. So far, the factors that are related to early post-traumatic pulmonary embolism (PE) occurrence have been given little attention. AIMS: We have conducted this literature review in order to analyze the incidence and the physiopathology of post-traumatic PE among intensive care unit (ICU) trauma patients, analyze the incidence of early post-traumatic PE, and elucidate risk factors associated with post-traumatic PE. Moreover, we aim to study the impact/outcome of post-traumatic PE in the ICU. METHODS: We used the PubMed and EMBASE databases and entered the following key words in MeSH research: Deep vein thrombosis (DVT), Post-traumatic Pulmonary embolism, Early pulmonary-embolism, risk factors, and Prognosis. RESULTS: The incidence of PE among trauma patients varies considerably, ranging from 0.35% to 24%. The incidence of early post-traumatic PE varies widely from 10 to 42%. After a traumatic injury, many factors have been found to be responsible for the formation of DVT and PE. In addition to the risk factors of hypercoagulability described by Virchow in his original triad, inflammation acting via endothelial damage may be considered as a fourth factor. The literature review showed that lower limb fractures and age are the most frequent factors associated with PE (particularly in early PE). The heterogeneity among studies limits reliable conclusions regarding the true risk factors for the timing of the occurrence of post-traumatic PE. Fatality from pulmonary embolism (PE) is close to 50% in some series. Moreover, high mortality rates, a high rate of nosocomial infections, and a prolonged stay in an ICU and/or in a hospital were found to be associated with the development of PE. CONCLUSION: Post-traumatic PE is frequent in ICUs. Inflammation acting via endothelial damage may be considered as a fourth factor in addition to the Virchow's triad of risk factors for venous thrombosis. Fractures of the lower extremities, obesity, and age happen to be the most frequent factors associated with PE (in particular early PE). PE development was associated with high rates of mortality, nosocomial infections, and a prolonged stay in an ICU and/or in a hospital. Therefore, prevention is warranted.
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Developing vaccine technology platforms to respond to pandemic threats or zoonotic diseases is a worldwide high priority. The risk of infectious diseases transmitted from wildlife and domestic animals to humans makes veterinary vaccination and animal health monitoring highly relevant for the deployment of public health global policies in the context of "one world, one health" principles. Sub-Saharan Africa is frequently impacted by outbreaks of poultry diseases such as avian influenza and Newcastle Disease (ND). Here, an adenovirus-vectored vaccine technology platform is proposed for rapid adaptation to ND or other avian viral threats in the region. Ethiopian isolates of the Newcastle Disease virus (NDV) were subjected to sequence and phylogenetic analyses, enabling the construction of antigenically matched vaccine candidates expressing the fusion (F) and hemagglutinin-neuraminidase (HN) proteins. A cost-effective vaccine production process was developed using HEK293 cells in suspension and serum-free medium. Productive infection in bioreactors (1-3L) at 2 × 106 cells/mL resulted in consistent infectious adenoviral vector titers of approximately 5-6 × 108 TCID50/mL (approximately 1011VP/mL) in the harvest lysates. Groups of chickens were twice immunized with 1 × 1010 TCID50 of the vectors, and full protection against a lethal NDV challenge was provided by the vector expressing the F antigen. These results consolidate the basis for a streamlined and scalable-vectored vaccine manufacturing process for deployment in low- and medium-income countries.
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The marine bacteria Vibrio parahaemolyticus and V. alginolyticus were incubated in seawater for 8 months to evaluate their adaptative responses to starvation. The starved cells showed an altered biochemical and enzymatic profiles, respectively, on Api 20E and Api ZYM systems and an evolution to the filterable minicells state capable to pass membrane pore size 0.45 microm. Outer membrane proteins patterns of stressed bacteria were also altered. Indeed, these modifications were manifested by the appearance and/or disappearance of bands as well as in the level of expression of certain proteins. Plasmids profiles analysis showed that V. alginolyticus ATCC 33787 lost three plasmids, whereas other tested strains conserved their initial profiles.