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1.
J Environ Sci (China) ; 85: 94-106, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31471036

RESUMO

Titanium dioxide nanoparticles (TiO2 NPs) are subjected to various transformation processes (chemical, physical and biological processes) in the environment, potentially affecting their bioavailability and toxic properties. However, the size variation of TiO2 NPs during aging process and subsequent effects in mammalian cells are largely unknown. The aim of this study was to illustrate the adverse effects of TiO2 NPs in different sizes (5, 15 and <100 nm) during aging process on human-hamster hybrid (AL) cells. There was an aging-time dependent enhancement of average hydrodynamic size in TiO2 NPs stock suspensions. The cytotoxicity of fresh TiO2 NPs increased in a size-dependent manner; in contrast, their genotoxicity decreased with the increasing sizes of NPs. No significant toxicity difference was observed in cells exposed to either fresh or 60 day-aged TiO2 NPs. Both Fresh and aged TiO2 NPs efficiently induced mitochondrial dysfunction and activated Caspase-3/7 in a size-dependent manner. Using mitochondrial-DNA deficient (ρ0) AL cells, we further discovered that mitochondrial dysfunction made significant contribution to the size-dependent toxicity induced by TiO2 NPs during the aging process. Taken together, our data indicated that TiO2 NPs could significantly induced the cytotoxicity and genotoxicity in an aging time-independent and size-dependent manner, which were triggered by mitochondrial dysfunction. Our study suggested the necessity to include size as an additional parameter for the cautious monitoring of TiO2 NPs disposal before entering the environment.


Assuntos
Nanopartículas/toxicidade , Titânio/toxicidade , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Dano ao DNA , Humanos , Testes de Toxicidade
2.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(6): 557-562, 2019 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-31292061

RESUMO

Objective To construct a random mutagenesis library of 3E1D7, a chimerical antibody against c-mesenchymal epithelial transition factor (c-Met), using mammalian cell surface display. Methods Antibody genes with randomly mutated complementarity-determining region 3 (CDR3) part were inserted into the mammalian expression plasmid pSZI-CD to construct the random mutagenesis library using double enzyme digestion. Reconstructed plasmids were then cloned into CHO cells by transfection. The expression level of antibodies on the surface of CHO cells was checked by C6 PLUS flow cytometry. Results 3E1D7 random mutagenesis library was successfully constructed with a volume of 5.52×106 in diversity on gene level. Sequence analysis showed that all 20 clones randomly picked from the library coded for 20 different mutated amino acid sequences in open reading frames. After transfection, the expression of full-length antibodies on CHO cell surfaces could be detected by flow cytometry. Conclusion A random mutagenesis library of a certain anti-c-Met antibody has been successfully constructed with an exhibitable diversity of 5.52×106, which would be a useful platform for further screening of therapeutic antibodies.


Assuntos
Biblioteca Gênica , Vetores Genéticos , Mutagênese , Sequência de Aminoácidos , Animais , Anticorpos/química , Cricetinae , Cricetulus , Fases de Leitura Aberta , Plasmídeos/genética , Transfecção
3.
Adv Exp Med Biol ; 1140: 225-236, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31347050

RESUMO

Selection of high-producing lead and backup cell lines with high-fidelity primary structure is a major goal of cell line development of protein therapeutics. Conventional techniques for sequence variant analysis, such as mass spectrometry (MS) and next-generation sequencing (NGS) have limitations on the sample number and turnaround time, thus often are only applied at the final stages of development, where an undesired lead or backup clone could cause a significant delay in project timeline. Here we presented a high-throughput (HT) peptide mapping workflow which can be applied at early stages of cell line selection for testing of a batch of 30-40 clones within 2-week turnaround while reporting valuable information on sequence variants and posttranslational modifications (PTMs). The successful application of this workflow was demonstrated for two mAb programs. Multiple clones were removed from a total of 33 mAb-1 clones using various criteria: nine clones contained at least one >1% upregulated unknown peptide ions, 11 clones contained at least eight >0.1% upregulated unknowns, and six clones contained upregulated critical PTMs. For mAb-2, light chain (LC) sequence extension of approximately 30 amino acids were detected in 6 out of 36 clones at levels up to 11%. Besides, a Q to H mutation at ~30% was detected in the heavy chain (HC) of a single clone. Q to H mutation has mass change of 9.00 Da and failed to be detected by intact mass analysis. Rapid PTM quantitation also facilitated the selection of clones with desirable quality attributes, such as N-glycan profile. Hence, we demonstrated a risk-reducing strategy where abnormal clones could be detected at earlier stages of cell line selection, which should result in reduced and more predictable timeline of cell line development.


Assuntos
Anticorpos Monoclonais/química , Sequenciamento de Nucleotídeos em Larga Escala , Mapeamento de Peptídeos , Processamento de Proteína Pós-Traducional , Animais , Células CHO , Cricetinae , Cricetulus , Espectrometria de Massas
4.
Acta Crystallogr D Struct Biol ; 75(Pt 6): 554-563, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31205018

RESUMO

HER2, a member of the epidermal growth factor receptor (EGFR) family, has been associated with human breast, ovarian and gastric cancers. Anti-HER2 monoclonal antibodies (mAbs) have demonstrated clinical efficacy for HER2-overexpressing breast cancer. A chimeric antibody chA21 that specifically inhibits the growth of HER2-overexpressing cancer cells both in vitro and in vivo has previously been developed. To reduce a potential human anti-mouse immune response, the humanized antibody HuA21 was developed and was further subjected to affinity maturation by phage display on the basis of chA21. Here, the crystal structure of HuA21-scFv in complex with the extracellular domain of HER2 is reported, which demonstrates that HuA21 binds almost the same epitope as chA21 and also provides insight into how substitutions in HuA21 improve the binding affinity compared with chA21, which could facilitate structure-based optimization in the future. Furthermore, the effects of HuA21 variants with constant domains of different lengths were explored and it was noticed that the deletion of constant domain 1 could improve the inhibition efficacy in a cell-proliferation assay, possibly functioning via increased internalization, which might guide the design of other monoclonal antibodies.


Assuntos
Anticorpos Monoclonais Humanizados/química , Complexo Antígeno-Anticorpo/química , Antineoplásicos Imunológicos/química , Neoplasias/terapia , Receptor ErbB-2/antagonistas & inibidores , Animais , Anticorpos Monoclonais Humanizados/genética , Anticorpos Monoclonais Humanizados/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Sítios de Ligação de Anticorpos , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Cristalização , Cristalografia por Raios X/métodos , Feminino , Humanos , Domínios Proteicos , Receptor ErbB-2/imunologia
5.
Exp Parasitol ; 203: 23-29, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31150654

RESUMO

In Brazil, Leishmania amazonensis is one of the etiological agents of tegumentary leishmaniasis and can cause a wide spectrum of diseases in humans, resulting in cutaneous, mucosal, diffuse, and even visceral leishmaniasis. Besides, this species has also been reported to affect dogs, causing typical symptoms of visceral disease. Unfortunately, the diagnostic of the Leishmania species is not routinely performed due to the difficulties of the available methods. In view of this, different molecular methods have been used in an attempt to solve the problem of diagnosis. Loop-mediated isothermal amplification (LAMP) is a relatively new nucleic acid amplification method, which has been successfully applied in the diagnosis of Leishmania spp. infections. However, this is the first work that standardizes a specific LAMP reaction for L. amazonensis. The set of primers selected were designed from the kDNA minicircle sequence of the L. amazonensis (GenBank: U19810.1). The LAMP assay developed in the present study showed 100% specificity and 89% sensitivity when compared with conventional PCR and was more sensitive than qPCR. In addition, the LAMP reaction developed here was able to amplify a qPCR sample with a parasite load of only 28 parasites in 50 ng of DNA. Consequently, considering the LAMP reaction specific to L. amazonensis and several advantages of the method (such as high efficiency, sensitivity and specificity), we believe that this reaction can be used as a promising diagnostic tool in clinical practice, field studies, and research.


Assuntos
Leishmania mexicana/isolamento & purificação , Leishmaniose Cutânea/diagnóstico , Pele/parasitologia , Animais , Sequência de Bases , Colorimetria , Cricetinae , DNA de Cinetoplasto/química , DNA de Cinetoplasto/genética , DNA de Protozoário/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Feminino , Leishmania mexicana/genética , Leishmaniose Cutânea/parasitologia , Masculino , Mesocricetus , RNA Ribossômico 18S/química , RNA Ribossômico 18S/genética , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Coloração pela Prata
6.
Dokl Biochem Biophys ; 485(1): 101-103, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31201624

RESUMO

In this paper, we present an approach to optimize the heterologous expression of the receptor tyrosine kinase IRR, which further simplifies the purification of the IRR from the medium and increases the final yield. The approach proposed by us can find application in the biotechnological production of other large-scale recombinant proteins produced for medical purposes.


Assuntos
Receptor de Insulina/biossíntese , Animais , Células CHO , Cricetinae , Cricetulus , Humanos , Domínios Proteicos , Receptor de Insulina/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética
7.
BMC Infect Dis ; 19(1): 482, 2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-31146699

RESUMO

BACKGROUND: To assess the immune persistence conferred by a Chinese hamster ovary (CHO)-derived hepatitis B vaccine (HepB) 17 to 20 years after primary immunization during early life. METHODS: Participants born between 1997 and 1999 who received a full course of primary vaccination with HepB (CHO) and who had no experience with booster vaccination were enrolled. Blood samples were required from each participant for measurement of hepatitis B surface antibody (anti-HBs), surface antigen and core antibody levels. For those who possessed an anti-HBs antibody < 10 mIU/mL, a single dose of HepB was administered, and 30 days later, serum specimens were collected to assess the booster effects. RESULTS: A total of 1352 participants were included in this study. Of these, 1007 (74.5%) participants could retain an anti-HBs antibody ≥10 mIU/mL, with a geometric mean concentration (GMC) of 57.4 mIU/mL. HBsAg was detected in six participants, resulting in a HBsAg carrier rate of 0.4% (6/1352). Of those participants with anti-HBs antibodies < 10 mIU/mL, after a challenge dose, 231 (93.1%) presented an anti-HBs antibody ≥10 mIU/mL, with a GMC of 368.7 mIU/mL. A significant increase in the anti-HBs positive rate (≥ 10 mIU/mL) after challenge was observed in participants with anti-HBs antibodies between 2.5 and 10 mIU/mL and participants boosted with HepB (CHO), rather than those with anti-HBs antibodies < 2.5 mIU/mL and those boosted with HepB (SC). CONCLUSION: Since satisfactory immune protection against HBV infection conferred by primary vaccination administered 17-20 years ago was demonstrated, there is currently no urgent need for booster immunization.


Assuntos
Anticorpos Anti-Hepatite B/sangue , Vacinas contra Hepatite B/administração & dosagem , Hepatite B/prevenção & controle , Imunização Secundária , Prevenção Primária , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/uso terapêutico , Adolescente , Adulto , Animais , Células CHO , Cricetinae , Cricetulus , Feminino , Seguimentos , Hepatite B/imunologia , Antígenos de Superfície da Hepatite B/imunologia , Vacinas contra Hepatite B/imunologia , Humanos , Recém-Nascido , Masculino , Prevenção Primária/métodos , Estudos Retrospectivos , Fatores de Tempo , Adulto Jovem
8.
Bioelectrochemistry ; 129: 218-227, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31200252

RESUMO

In this study the evolution in the efficiency of electrochemotherapy (reversible electroporation) with pulse number was assessed in vitro. Experiments were performed using 100 µs pulses at different electric field intensities and the chemotherapeutic agent bleomycin. Additionally, electrical impedance spectroscopy measurements were used as a different method to study in real time the changes produced on cells with pulse number during trains of consecutive pulses. Our results show that the relation between pulse number and the observed outcome is complex and difficult to fully characterize. This relation can display a highly linear behaviour up to a certain number of pulses and/or field intensity applied. However, the relation between the number of pulses and the observed outcome always evolves to a saturation or at least a reduction in the electric field effects that is displayed when either electric field intensity or pulse number are increased. An exponential model was found to best describe this relation within the range of experimental conditions considered. Electrical impedance measurements confirmed the results and gave a more precise quantification of this dependence. The study highlights the importance that pulse number has in the electrochemotherapy protocols and establishes some limits in the use of this parameter.


Assuntos
Eletroquimioterapia/métodos , Animais , Antibióticos Antineoplásicos/administração & dosagem , Antibióticos Antineoplásicos/farmacologia , Bleomicina/administração & dosagem , Bleomicina/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Impedância Elétrica , Modelos Biológicos , Neoplasias/tratamento farmacológico
9.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(5): 412-418, 2019 May.
Artigo em Chinês | MEDLINE | ID: mdl-31223110

RESUMO

Objective To measure the effect of duck viperin protein on the proliferation of duck Tembusu virus (DTMUV). Methods We analyzed the duck viperin gene using a bioinformatics. Plasmids pGEX-6P-viperin and pEGFP-N1-viperin were constructed and transformed into Rosseta and transfected into BHK-21 cells, respectively. BHK-21 cells transfected with plasmid pEGFP-N1-viperin and empty vector were infected with DTMUV. The content of DTMUV in cell precipitation and supernatant was analyzed by real-time fluorescent quantitative PCR. Finally, the effect of viperin on DTMUV proliferation and the binding proteins captured by EGFP monoclonal antibody were evaluated using mass spectrometry. Results Significant differences were observed between the expression levels of viperin gene in duck compared to other species. Duck viperin was found to inhibit the budding of DTMUV in BHK-21 cells. Further, we identified six proteins that might be involved in the inhibition of the proliferation and budding of DTMUV, possibly indicative of the viperin anti-Tembusu virus pathway in ducks. Conclusion Expression patterns of duck viperin reveal how the budding of duck Tembusu virus is inhibited.


Assuntos
Patos , Flavivirus/fisiologia , Doenças das Aves Domésticas/virologia , Proteínas/metabolismo , Liberação de Vírus , Animais , Anticorpos Monoclonais , Linhagem Celular , Cricetinae , Proteínas/genética , Transfecção
10.
Vet Microbiol ; 233: 124-132, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31176398

RESUMO

Leptospirosis is a zoonosis, caused by pathogenic spirochetes of the genus Leptospira. Although cattle are usually the maintenance hosts of serovar Hardjo, Pomona is the most frequent serovar circulating in Argentina. The understanding of bovine innate immune response and the virulence of this serovar is important for future control measures. This work compares infection of bovine macrophages with the virulent L. interrogans sv Pomona strain AKRFB (P1) and its attenuated counterpart (P19). First, we confirmed attenuation in the hamster model. Mortality and lung hemorrhages occurred after P1 inoculation, while the survival rate was 100% in P19-infected animals. Cells infected with both strains showed statistically upregulated gene expression of pro-inflammatory cytokines, IL-1ß, IL-6 and TNFα. The level of expression of anti-inflammatory cytokine IL-10 was statistically different between strains. Increased expression of IL-10 was observed only in P1-infected cells. For the first time, we describe macrophages extracellular traps induced by infection of bovine macrophages (bMETs) with both, the virulent and attenuated Leptospira interrogans Pomona strains. P1 was found higher internalized when the phagocytosis was inhibited, suggesting a cell entrance of this strain also by an independent-phagocytosis pathway. Furthermore, P1 was higher colocalized with acidic and late endosomal compartments compared with P19. This data emphasizes the importance to deepen in Leptospira bovine macrophages particular invasion mechanisms and, furthermore, underline the value of studying the main hosts.


Assuntos
Imunidade Inata , Leptospira interrogans serovar pomona/patogenicidade , Macrófagos/imunologia , Macrófagos/microbiologia , Animais , Argentina , Bovinos , Células Cultivadas , Cricetinae , Citocinas/genética , Citocinas/imunologia , Interleucina-10/genética , Interleucina-10/imunologia , Leptospirose/imunologia , Pulmão/microbiologia , Pulmão/patologia , Sorogrupo , Virulência
11.
Sheng Wu Gong Cheng Xue Bao ; 35(6): 1071-1078, 2019 Jun 25.
Artigo em Chinês | MEDLINE | ID: mdl-31232003

RESUMO

The aim of this study is to investigate the effect of the chimeric intron in different directions on the expression of the nerve growth factor (NGF) in recombinant Chinese hamster ovary (CHO) cells. The chimeric intron that contained the splice sequence of the first intron of the human ß-globin and the human immunoglobulin heavy chain variable region intron was used. NGF gene was cloned into the expression vectors containing the chimeric intron in the forward or reverse direction, followed by transfecting into CHO cells, and screened under G418 to produce the stable transfected CHO cells. Fluorescence quantitative PCR, ELISA, and Western blotting were performed to detect the recombinant NGF gene expression in CHO cells. The results showed that the chimeric introns could significantly enhance the expression of NGF in recombinant CHO cells. Moreover, the enhancing effect on NGF expression level by the intron in the forward direction showed stronger than that of the reverse direction both at mRNA and protein level. In conclusion, the chimeric intron could increase NGF expression in stably transfected CHO cells and the effect is associated with the direction of the intron insertion.


Assuntos
Íntrons , Animais , Animais Geneticamente Modificados , Células CHO , Cricetinae , Cricetulus , Expressão Gênica , Humanos , Transfecção
12.
Acta Virol ; 63(2): 162-168, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31230445

RESUMO

Foamy viruses (FVs) or spumaviruses are retroviruses that are explored as vectors for gene therapy. The good feature of foamy viruses is its broad tropism; however, their infections result in non-targeted gene expression. Here, we attempted to design the liver targeted viral gene delivery by employing liver specific gene promoters like albumin (ALB), transthyretin (TTR) and hepatitis B virus (HBV) promoters. We compared the relative gene expression of liver specific promoters versus the U3 promoter in liver cell line (HepG2) and non-liver cell lines: human fibrosarcoma cell line (HT1080), baby hamster kidney cell line (BHK), human embryonic kidney cell line (HEK 293T) and cervical cancer cell line (HeLa). We have found that the promoter exchange didn't affect viral assembly. The ability to drive gene expression was best with TTR promoter which was followed by HBV and ALB promoter. The use of TTR, HBV and ALB promoters are helpful in achieving liver specific gene expression. Keywords: foamy virus; gene therapy; liver; albumin; transthyretin promoter; HBV promoter.


Assuntos
Fígado , Regiões Promotoras Genéticas , Spumavirus , Adulto , Animais , Linhagem Celular , Cricetinae , Terapia Genética , Vetores Genéticos , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Fígado/metabolismo , Regiões Promotoras Genéticas/genética , Spumavirus/genética
13.
Chem Biol Interact ; 309: 108712, 2019 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-31201777

RESUMO

The recent intentional use of nerve agents and pesticides in Europe and Afghanistan highlights the need for an effective countermeasure against organophosphates (OP) toxins. The most developed pretreatment candidate to date is plasma (native) human butyrylcholinesterase (HuBChE), which is limited in availability and because of its 1:1 stoichiometry with OPs, a large dose will present challenges when delivered parenterally both in terms of pharmacokinetics and manageability in the field. A tetrameric recombinant (r) form of human BChE produced in CHO-K1 cells with similar structure, in vivo stability and antidotal efficacy as the native form, has been developed to deliver rHuBChE as an aerosol (aer) to form a pulmonary bioshield capable of neutralizing inhaled OPs in situ and prevent AChE inhibition in the blood and in the brain; the latter associated with the symptoms of OP toxicity. Previous proof-of-concept macaque studies demonstrated that delivery of 9 mg/kg using a microsprayer inserted down the trachea, resulted in protection against an inhaled dose of 15ug/kg of aer-paraoxon (aer-Px) given 72 h later. In the present studies, pulmonary delivery of rHuBChE in macaques was achieved using Aerogen vibrating mesh nebulizers, similar to that used for human self-administration. The promising findings indicate that despite the poor lung deposition observed in macaques using nebulizers (13-20%), protective levels of RBC-AChE were still present in the blood even when exposure aer-Px (55 µg/kg) was delayed for five days. This long term retention of 5 mg/kg rHuBChE deposited in the lung bodes well for the use of an aer-rHuBChE pretreatment in humans where a user-friendly customized nebulizer with increased lung deposition up to 50% will provide even longer protection at a lower dose.


Assuntos
Aerossóis/química , Butirilcolinesterase/química , Paraoxon/química , Animais , Butirilcolinesterase/genética , Butirilcolinesterase/metabolismo , Células CHO , Cricetinae , Cricetulus , Feminino , Humanos , Pulmão/metabolismo , Macaca , Masculino , Nebulizadores e Vaporizadores , Paraoxon/toxicidade , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/sangue , Proteínas Recombinantes/química
14.
J Chromatogr A ; 1602: 284-299, 2019 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-31230875

RESUMO

A great number of protein-binding peptides are known and utilized as drugs, diagnostic reagents, and affinity ligands. Recently, however, peptide mimetics have been proposed as valuable alternative to peptides by virtue of their excellent biorecognition activity and higher biochemical stability. This poses the need to develop a strategy for translating known protein-binding peptides into peptoid analogues with comparable or better affinity. This work proposes a route for translation utilizing the IgG-binding peptide HWRGWV as reference sequence. An ensemble of peptoid analogues of HWRGWV were produced by adjusting the number and sequence arrangement of residues containing functional groups that resemble both natural and non-natural amino acids. The variants were initially screened via IgG binding tests in non-competitive mode to select candidate ligands. A set of selected peptoids were studied in silico by docking onto putative binding sites identified on the crystal structures of human IgG1, IgG2, IgG3, and IgG4 subclasses, returning values of predicted binding energy that aligned well with the binding data. Selected peptoids PL-16 and PL-22 were further characterized by binding isotherm analysis to determine maximum capacity (Qmax ˜ 48-57 mg of IgG per mL of adsorbent) and binding strength on solid phase (KD ˜ 5.4-7.8 10-7 M). Adsorbents PL-16-Workbeads and PL-22-Workbeads were used for purifying human IgG from a cell culture supernatant added with bovine serum, affording high values of IgG recovery (up to 85%) and purity (up to 98%) under optimized binding and elution conditions. Both peptoid ligands also proved to be stable against proteolytic enzymes and strong alkaline agents. Collectively, these studies form a method guiding the design of peptoid variants of cognate peptide ligands, and help addressing the challenges that, despite the structural similarity, the peptide-to-peptoid translation presents.


Assuntos
Anticorpos/metabolismo , Afinidade de Anticorpos , Peptídeos/química , Peptoides/química , Adsorção , Álcalis/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Bovinos , Cricetinae , Cricetulus , Humanos , Imunoglobulina G/isolamento & purificação , Imunoglobulina G/metabolismo , Ligantes , Simulação de Acoplamento Molecular , Ligação Proteica , Proteólise , Temperatura Ambiente
15.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1118-1119: 194-202, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-31059926

RESUMO

A rapid and sensitive liquid chromatography-mass spectrometry assay was developed and used to quantify emetic cereulide peptide exotoxin, which can be related to possible Bacillus cereus contamination in monoclonal antibody (mAb) bioprocess feeds. The assay limit of detection was 0.05 ng/mL (~1 fmol injected) and limit of quantification 0.16 ng/mL (~3 fmol injected) over a standard curve with >3 orders of magnitude linear dynamic range. The assay allowed quantification of toxin removal in an established two-step mAb purification process consisting of Protein A affinity chromatography followed by multi-modal anion exchange chromatography. Toxin content was ascertained in process stream sample fractions as well as on the Protein A affinity column. An optimized analytical method allowed separation of cereulide toxin from other mAb cell culture components within 6 min. Spiking experiments showed that samples should be collected in high (80% v/v) content acetonitrile to reduce nonspecific losses of the cereulide. The majority of mAb purification process-associated cereulide was detected in the Protein A flow through fraction, whereas only residual amounts were found in wash, strip, and elution fractions. Column cleaning-in-place (CIP) procedures were evaluated to prevent carryover between affinity capture cycles. No carryover was detected between cycles, however trace amounts of cereulide were extracted from the Protein A resin. Increasing the CIP NaOH concentration from 0.1 M to 0.5 M, and contact time from 15 min to 1 h, improved removal of residual cereulide from the resin. Applicability of CIP clearance of cereulide during Protein A chromatography was confirmed with three different mAb feeds. Post Protein A polishing, via target flow through on a multi-modal anion exchange chromatography column, resulted in a product pool with no detectable cereulide. Approximately 5 logs of reduction in cereulide concentration was obtained over the two-step chromatography process. Cereulide contamination is well known and of concern in food processing, however this research may be the first LC-MS quantification of cereulide contamination, and its clearance, in biopharmaceutical mAb processing. The analytical method may also be used to rapidly screen for cereulide contamination in upstream cell culture process streams, prior to downstream product purification. This will allow appropriate measures to be taken to reduce toxin exposure to downstream bioprocess raw materials, consumables and equipment.


Assuntos
Anticorpos Monoclonais/química , Toxinas Bacterianas/isolamento & purificação , Cromatografia de Afinidade/métodos , Depsipeptídeos/isolamento & purificação , Proteína Estafilocócica A/metabolismo , Animais , Bacillus cereus , Células CHO , Cricetinae , Cricetulus , Depsipeptídeos/metabolismo , Limite de Detecção , Modelos Lineares , Reprodutibilidade dos Testes
16.
Exp Parasitol ; 201: 78-89, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31047987

RESUMO

Hamsters are a suitable experimental model for visceral leishmaniasis (VL) because they mimic the features of the human disease. However, the infection after inoculation can only be verified after sacrifice of the animal or several months following infection, when obvious signs of the disease appear, compromising animal welfare in both cases. Unlike other studies, the present work used an inoculum of 5 × 108 promastigotes to induce Leishmania infantum infection, which are easier to produce than amastigotes, in in vitro culture. The infection in hamsters was detected using non-invasive methods such as ultrasound imaging (USG) and blood gases, in addition to alterations in hematological parameters and weight loss. USG imaging identified changes in the size and echogenicity of the spleen, liver, and kidney as early as week 9 (W9) after experimental inoculation. However, blood gases, specially lactate, was increased in response to the infection, with statistically significant differences between W9 and W0 (before infection) (p < 0.0001). The conventional hematological parameters showed progressive pancytopenia and weight loss of 15% and 10% in infected males and females respectively, at W9 versus W0 (p < 0.0001). Histological changes in the liver, kidney, and spleen correlated with changes detected by USG imaging and the number of parasites increased proportionately to the progression of infection, being higher at W24. In conclusion, USG imaging, lactate levels, hematocrit and hemoglobin parameters, along with weight loss allowed early detection of infection, which was then confirmed by the identification and quantification of parasites in the blood, liver, and spleen by qRT-PCR. In contrast, blood chemistry was not a useful tool in the early detection of VL infection because it did not correlate with alterations evident in other techniques. The use of non-invasive tools eliminates the need for animal sacrifice to confirm infection, thus reducing the number of animals required for a given study and eliminating the need to wait until the appearance of severe signs of infection, which affect animal welfare. These tools are therefore advantageous for use in preclinical studies, for studying pathogenesis as also for vaccine and drug development.


Assuntos
Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/diagnóstico , Animais , Análise Química do Sangue , Gasometria , Cricetinae , Modelos Animais de Doenças , Feminino , Testes Hematológicos , Rim/diagnóstico por imagem , Rim/patologia , Leishmania infantum/classificação , Leishmaniose Visceral/sangue , Leishmaniose Visceral/diagnóstico por imagem , Fígado/diagnóstico por imagem , Fígado/parasitologia , Fígado/patologia , Masculino , Mesocricetus , Reação em Cadeia da Polimerase em Tempo Real , Fatores Sexuais , Baço/diagnóstico por imagem , Baço/parasitologia , Baço/patologia , Ultrassonografia , Ganho de Peso
17.
Nat Commun ; 10(1): 1785, 2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-31040271

RESUMO

Lysosomal replacement enzymes are essential therapeutic options for rare congenital lysosomal enzyme deficiencies, but enzymes in clinical use are only partially effective due to short circulatory half-life and inefficient biodistribution. Replacement enzymes are primarily taken up by cell surface glycan receptors, and glycan structures influence uptake, biodistribution, and circulation time. It has not been possible to design and systematically study effects of different glycan features. Here we present a comprehensive gene engineering screen in Chinese hamster ovary cells that enables production of lysosomal enzymes with N-glycans custom designed to affect key glycan features guiding cellular uptake and circulation. We demonstrate distinct circulation time and organ distribution of selected glycoforms of α-galactosidase A in a Fabry disease mouse model, and find that an α2-3 sialylated glycoform designed to eliminate uptake by the mannose 6-phosphate and mannose receptors exhibits improved circulation time and targeting to hard-to-reach organs such as heart. The developed design matrix and engineered CHO cell lines enables systematic studies towards improving enzyme replacement therapeutics.


Assuntos
Lisossomos/enzimologia , Animais , Células CHO , Cricetinae , Cricetulus , Modelos Animais de Doenças , Doença de Fabry/tratamento farmacológico , Doença de Fabry/enzimologia , Doença de Fabry/metabolismo , Glicosilação , Masculino , Camundongos , Camundongos Knockout , Proteínas Recombinantes/uso terapêutico , alfa-Galactosidase/uso terapêutico
18.
Einstein (Sao Paulo) ; 17(2): eAO4576, 2019 May 02.
Artigo em Inglês, Português | MEDLINE | ID: mdl-31066794

RESUMO

OBJECTIVE: To evaluate the effect of red propolis and L-lysine on angiogenesis and tumor growth in a new model of hamster cheek pouch inoculated with Walker 256 tumor cells. METHODS: The study consisted of two experiments with four groups each (total: 57 hamsters). In the experiment 1, the animals were inoculated with Walker tumor cells, followed by administration of test substances (red propolis 200mg/5mL/kg or L-lysine 150mg/kg) or control substances (gum arabic 5mL/kg or water 5mL/kg) for 10 days. The animals in the experiment 2 received red propolis, L-lysine, gum arabic or water at the same doses, for 33 days prior to inoculation of Walker tumor cells, followed by 10 days of treatment with the same substances. Based on single-plane images, angiogenesis was quantified (mean vascular area), in percentage, and tumor area (mm2) and perimeter (mm). RESULTS: In the experiment 1, compared to animals receiving water, the mean vascular area expressed in percentage was significantly smaller in animal treated with propolis (p<0.05) and L-lysine (p<0.001). CONCLUSION: Both red propolis and L-lysine inhibited tumor angiogenesis in the new hamster cheek pouch model when administered after tumor inoculation.


Assuntos
Inibidores da Angiogênese/uso terapêutico , Lisina/uso terapêutico , Neovascularização Patológica/tratamento farmacológico , Própole/uso terapêutico , Animais , Antioxidantes , Carcinoma 256 de Walker/irrigação sanguínea , Bochecha , Cricetinae , Feminino , Mesocricetus , Modelos Animais , Neoplasias Bucais/irrigação sanguínea , Neoplasias Bucais/induzido quimicamente , Neoplasias Bucais/tratamento farmacológico , Resultado do Tratamento , Ganho de Peso
19.
J Agric Food Chem ; 67(22): 6150-6159, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31117552

RESUMO

Consumptions of fruit seed oils and meals could potentially improve cardiovascular health by reducing plasma total cholesterol and low-density lipoprotein (LDL). The study objective was to compare the effectiveness of expeller-pressed and solvent-extracted grape, tomato, pomegranate seed oils, and defatted pomegranate meals in lowering plasma and hepatic cholesterol using hamster models. Hamsters were fed with fruit seed oils (FSO), defatted pomegranate seed meals (PDM), or control diets. After a 3-week feeding period, plasma total triglycerides of treatment diets were significantly lower. FSO also reduced total, very-low-density lipoprotein- (VLDL), and LDL-cholesterols, while PDM only lowered VLDL-cholesterols. Decreases in low-density and high-density lipoproteins (LDL/HDL) ratios were also observed in most treatments. In liver, triglycerides, total, and free cholesterol levels did not vary between control and treatments. There were no significant differences in lipid modulating properties between solvent-extracted and expeller-pressed oils. In conclusion, partial replacements of saturated fat in high-fat diets with tomato, pomegranate, and grape seed oils could effectively reduce plasma triglyceride levels and improve HDL/LDL ratios.


Assuntos
Anticolesterolemiantes/metabolismo , Hipercolesterolemia/dietoterapia , Óleos Vegetais/metabolismo , Punicaceae/química , Sementes/química , Animais , Anticolesterolemiantes/química , Colesterol/metabolismo , Cricetinae , Humanos , Hipercolesterolemia/metabolismo , Lipoproteínas HDL/sangue , Fígado/metabolismo , Masculino , Mesocricetus , Óleos Vegetais/química , Punicaceae/metabolismo , Sementes/metabolismo , Triglicerídeos/metabolismo
20.
Environ Pollut ; 251: 13-21, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31071628

RESUMO

Cigarette smoking (CS) and betel quid (BQ) chewing are two known risk factors that have synergistic potential for the enhancing the development of oral squamous cell carcinoma (OSCC) in Taiwan. Most mutagens and carcinogens are metabolically activated by cytochrome P450 (CYP450) to exert their mutagenicity or carcinogenicity. Previous studies have shown that metabolic activation of the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), by CYP2A6 activity determines NNK-induced carcinogenesis. In addition, safrole affects cytochrome P450 activity in rodents. However, the effect of BQ safrole on the metabolism of tobacco-specific NNK and its carcinogenicity remains elusive. This study demonstrates that safrole (1 mg/kg/d) induced CYP2A6 activity, reduced urinary 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) levels, and increased NNK-induced DNA damage, including N7-methylguanine, 8-OH-deoxyguanosine and DNA strand breaks in a Syrian golden hamster model. Furthermore, altered NNK metabolism and increased NNK-induced DNA damage were also observed in healthy subjects with CS and BQ chewing histories compared to healthy subjects with CS histories. In conclusion, BQ containing safrole induced tobacco-specific NNK metabolic activation, resulting in higher NNK-induced genotoxicity. This study provides valuable insight into the synergistic mechanisms of CS- and BQ-induced OSCC.


Assuntos
Nitrosaminas/urina , Safrol/toxicidade , Uso de Tabaco/urina , Tabaco/metabolismo , Ativação Metabólica/efeitos dos fármacos , Animais , Carcinoma de Células Escamosas/induzido quimicamente , Carcinoma de Células Escamosas/metabolismo , Cricetinae , Citocromo P-450 CYP2A6/metabolismo , Feminino , Humanos , Fígado/efeitos dos fármacos , Fígado/enzimologia , Neoplasias Bucais/induzido quimicamente , Neoplasias Bucais/metabolismo , Taiwan , Tabaco/toxicidade
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