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1.
Int J Mol Sci ; 23(18)2022 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-36142480

RESUMO

Type 2 diabetes mellitus (T2D) affects millions of people around the world, and its complications have serious health consequences. In addition to external factors, the causes of morbidity and increased risk were also sought in the variability of the human genome. A phenomenon that can answer these questions is the occurrence of single-nucleotide polymorphisms (SNP). They constitute a field for research into genetic determinants responsible for the increase in the risk of the discussed metabolic disease. This article presents the outline of two enzymes: metalloproteinases 2 and 9 (MMP-2, MMP-9), their biological activity and the effect caused by differences in individual alleles in the population, as well as the reports on the importance of these DNA sequence variations in the occurrence of diabetes mellitus type 2 and associated conditions. The results of the conducted research indicate a relationship between two MMP-2 polymorphisms (rs243865, rs243866) and two MMP-9 polymorphisms (rs3918242, rs17576) and the presence of T2D. This could offer a promising possibility to use them as predictive and diagnostic markers. However, due to the low number of reports, more research is needed to clearly confirm the link between these SNPs and diabetes.


Assuntos
Complicações do Diabetes , Diabetes Mellitus Tipo 2 , Metaloproteinase 2 da Matriz , Metaloproteinase 9 da Matriz , Humanos , Estudos de Casos e Controles , Complicações do Diabetes/complicações , Complicações do Diabetes/genética , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/genética , Predisposição Genética para Doença , Genótipo , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/genética , Polimorfismo de Nucleotídeo Único
2.
Biologicals ; 54: 1-7, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29861269

RESUMO

BACKGROUND: Recombinant proteins expressed in host cell systems may contain host cell proteins (HCP) as impurities. While there is no clear evidence of clinical adverse events attributable to HCP, HCP levels and profiles must be documented to meet regulatory requirements and to understand the consistency of the biological product and manufacturing process. We present a general strategy for HCP characterization applied to a recombinant protein antigen, Hepatitis B surface antigen (HBsAg) used in a multivalent vaccine. METHODS: Polyclonal antisera raised against HCPs in process fractions from a mock preparation of the HBsAg yeast expression host, Hansenula polymorpha, were used to develop a quantitative sandwich ELISA to measure HCP content in batches of purified recombinant HBsAg. Batches were also subjected to SDS-PAGE and LC-MS/MS to identify detectable proteins. Batch consistency was further assessed by SDS-PAGE/densitometry purity analysis and by the ratio of specific HBsAg content (by ELISA) to total protein. RESULTS: Using the quantitative HCP ELISA, the HCP content showed no discernable trend in multiple HBsAg batches manufactured over a 5-year period. All batches were ≥95% pure by SDS-PAGE/densitometry, with consistent HBsAg/total protein ratios. In addition to the expected HBsAg antigen protein, LC-MS/MS analysis of three HBsAg batches identified several yeast proteins, none of which are known to cause adverse reactions in humans. CONCLUSIONS: Analysis of multiple HBsAg batches showed consistent HCP content and identification profiles, as well as product purity and specific antigen content, demonstrating consistent manufacturing process. Recombinant vaccines, unlike therapeutic products, are administered infrequently with only small amounts of protein injected at a time. With limited potential for adverse reactions to small quantities of HCPs in purified recombinant vaccine antigens, and considering the relevant regulatory guidelines, we conclude that once consistent manufacturing process has been demonstrated, routine HCP testing in recombinant vaccine antigens is no longer required.


Assuntos
Expressão Gênica , Antígenos de Superfície da Hepatite B/biossíntese , Vacinas contra Hepatite B/biossíntese , Vírus da Hepatite B/genética , Pichia/metabolismo , Antígenos de Superfície da Hepatite B/genética , Antígenos de Superfície da Hepatite B/imunologia , Vacinas contra Hepatite B/genética , Vírus da Hepatite B/imunologia , Pichia/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Vacinas Sintéticas/biossíntese , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia
3.
Neurodegener Dis ; 16(3-4): 235-44, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26954557

RESUMO

BACKGROUND: Sporadic amyotrophic lateral sclerosis (SALS) is a fatal motor neuron degenerative disease of unclear pathogenesis. Disturbances of intracellular transport are possible causes of the disease. OBJECTIVE: We evaluated the expression of motor proteins involved in the anterograde (kinesins KIF1B, KIF5C) and retrograde (KIFC3, dynactin subunits DCTN1 and DCTN3) intracellular transport in peripheral blood mononuclear cells (PBMCs). MATERIALS AND METHODS: PBMCs were obtained from 74 SALS patients with different clinical phenotypes, 65 blood donors (healthy control I), and 29 cases with other neurological diseases (disease control II) divided into subgroups IIA (atypical parkinsonism) and IIB (ALS-mimicking disorders). mRNA expression was studied by real-time qPCR, and protein level by Western blotting. RESULTS: In SALS, KIF5C and KIFC3 expression was significantly lower and DCTN1 higher than in control I, and dependent of age. KIF1B expression was significantly higher in SALS than in subgroup IIB, whereas DCTN1 and DCTN3 were higher in SALS than in subgroup IIA. All changes in the studied proteins were statistically significant in classic ALS but not in progressive muscular atrophy. CONCLUSION: In SALS, and especially in classic ALS, the changes in motor protein expression may alter bidirectional intracellular transport in PBMCs. More studies are needed to find out whether the levels of KIF5C and DCTN1 may be useful in ALS diagnosis, and whether KIF1B expression may discriminate ALS from ALS-mimicking disorders.


Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Complexo Dinactina/metabolismo , Cinesinas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/metabolismo , Esclerose Lateral Amiotrófica/diagnóstico , Biomarcadores/sangue , Western Blotting , Feminino , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Adulto Jovem
4.
Immunopharmacol Immunotoxicol ; 37(1): 90-102, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25519469

RESUMO

Thiram (TMTD) is a widely used dithiocarbamate pesticide and fungicide and is one of potent contact allergens. In the light of known properties, thiram is also considered to be used as an inhibitor of inflammation. To investigate whether known pro-oxidative properties of thiram might be involved in immunogenic mechanisms, we carried out an in vitro study aimed at analysis of reactive oxygen species (ROS) generation, activation of NF-κB, expression of iNOS and COX-2, production of NO, PGE2 and IL-1ß in murine macrophage cells (RAW 264.7). The cells were treated by thiram alone (0.5 µg/mL; 2 µM and 2 µg/mL; 8 µM) or concomitantly with bacterial endotoxin (LPS; 1 µg/mL). LPS was used as an endotoxin that triggers changes characteristic for inflammatory state of the cell. TMTD increased ROS production, level of oxidized glutathione (GSSG) and activated NF-κB. The consequence of NF-κB activation was the increase of IL-1ß and NO production characteristic for inflammation. However, we did not observe changes in PGE2 concentration. We observed expression of iNOS, COX-2 proteins and NO and PGE2 production in macrophages treated with thiram concomitantly with LPS lower than those in cells stimulated with LPS alone. Thiram (2 µg/mL) decreased NF-κB activation and production of LPS-induced IL-1ß. In conclusion, we demonstrated changes induced by TMTD characteristic for inflammation. Hence, it can be supposed that they may participate in the elicitation phase of allergic contact dermatitis induced by thiram. However, when TMTD acts concomitantly with LPS, it decreases the intensity of inflammation state in RAW 264.7.


Assuntos
Fungicidas Industriais/toxicidade , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Tiram/toxicidade , Animais , Western Blotting , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Dermatite Alérgica de Contato/imunologia , Dermatite Alérgica de Contato/metabolismo , Relação Dose-Resposta a Droga , Endotoxinas/toxicidade , Glutationa/metabolismo , Macrófagos/metabolismo , Camundongos , Microscopia Confocal , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Espécies Reativas de Oxigênio/metabolismo
5.
Neurodegener Dis ; 12(2): 71-80, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23006449

RESUMO

BACKGROUND: Amyotrophic lateral sclerosis is a fatal motor neuron degenerative disease. Most cases are sporadic (SALS), and approximately 10% are familial (FALS) among which over 20% are linked to the SOD1 mutation. Both SALS and FALS have been associated with retrograde axonal transport defects. Kinesins (KIFs) are motor proteins involved mainly in anterograde transport; however, some also participate in retrograde transport. OBJECTIVE: The purpose of the study was to investigate and compare the expression of kinesins involved in anterograde (KIF5A, 5C) and retrograde (KIFC3/C2) axonal transport in SALS in humans and FALS in mice with the hSOD1G93A mutation. METHODS: The studies were conducted on various parts of the CNS from autopsy specimens of SALS patients, and transgenic mice at presymptomatic and symptomatic stages using real-time quantitative PCR and reverse-transcription PCR. RESULTS: All KIF expression in the motor cortex of individual SALS subjects was higher than in the adjacent sensory cortex, in contrast to the expression in control brains. It was also significantly higher in the frontal cortex of symptomatic but not presymptomatic mice compared to wild-type controls. However, the mean KIF expression in the SALS motor and sensory cortexes was lower than in control cortexes. To a lesser extent the decrease in KIF mean expression also occurred in human but not in mouse ALS spinal cords and in both human and mouse cerebella. CONCLUSION: Disturbances in kinesin expression in the CNS may dysregulate both anterograde and retrograde axonal transports leading to motor neuron degeneration.


Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Encéfalo/metabolismo , Cerebelo/metabolismo , Cinesinas/metabolismo , Medula Espinal/metabolismo , Idoso , Idoso de 80 Anos ou mais , Esclerose Lateral Amiotrófica/genética , Animais , Feminino , Humanos , Cinesinas/genética , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Superóxido Dismutase/genética , Superóxido Dismutase-1
6.
Vaccines (Basel) ; 11(2)2023 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-36851153

RESUMO

There is increasing interest to replace animal-based potency assays used routinely to test vaccines, since they are highly variable, are costly, and present ethical concerns. The development of relevant in vitro assays is part of the solution. Using pertactin (PRN) antigen as an example in DTaP-IPV (diphtheria, tetanus, acellular pertussis, and inactivated poliovirus) vaccines, a PRN antigenicity ELISA was developed using two monoclonal antibodies with a high affinity to unique PRN epitopes, relevance to human immune responses, and evidence of functionality. The ELISA measured consistent PRN antigenicity between the vaccine lots and was validated to demonstrate its accuracy, precision, linearity, and specificity. Notably, the PRN antigenicity ELISA was more sensitive than the mouse-based potency test and could more effectively differentiate between degraded and intact vaccine lots compared to the in vivo test. From these studies, the PRN antigenicity ELISA is proposed as an in vitro replacement for the in vivo potency test for PRN in DTaP-IPV-based formulations. Important considerations in this study included comprehensive antibody characterization, testing of multiple vaccine lots, method validation, and comparison to animal-based potency. Together, these factors form part of an overall strategy that ensures reliable and relevant in vitro assays are developed to replace animal tests.

7.
Materials (Basel) ; 15(4)2022 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-35208102

RESUMO

Road layers should be properly compacted to obtain an adequate bearing capacity and durability. Both the unbound and hydraulically bound mixtures used in the layers require compaction. After compaction and hardening, soil mixed with a binder acquires mechanical features that unbound soil lacks, including tensile strength (Rit) and unconfined compressive strength (Rc). The effect of the compaction ratio (DPr) of the low-strength cement-stabilised soils on these features has rarely been investigated. This study investigates the influence of the compaction ratio on the mechanical properties of hardened, stabilised mixtures of medium-grained sand with 5%, 6.5%, and 8% Portland cement. Cement-soil stabilisation tests showed that compressive strength depends exponentially on the compaction ratio, whereas tensile strength and the stiffness modulus depend linearly on the compaction ratio. For tensile strength and the dynamic stiffness modulus, the effect is not statistically significant, and the usual practice of ignoring compaction dependence is justified. For compressive strength, however, the effect is significant, especially when DPr = 98-100%. When the values of Rc and Rit strengths at various DPr were normalised by those at 100%, it was found that mixtures with higher strengths are the least resistant to changes in the compaction ratio. Knowing the percentage by which the value of a given parameter changes with compaction can be extremely valuable in engineering practice.

8.
Vaccines (Basel) ; 10(5)2022 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-35632531

RESUMO

In the vaccine industry, multiple physicochemical, immunological, in vitro and in vivo analytical methods are applied throughout the manufacturing process to characterize and monitor the quality of vaccines. Presented here is the Single Epitope Antigenicity Test (SEAT), an innovative, quantitative epitope profiling method which provides an extended immunochemical analysis for diphtheria toxoid (DTxd) to be used for consistency testing during manufacturing process changes. The method uses BioLayer Interferometry (BLI) and a panel of monoclonal antibodies (mAbs) to independently assess nine individual antigenic sites of DTxd. The panel includes mAbs which are functional, bind distinct sites on DTxd and are able to distinguish intact DTxd from that which has been exposed to heat treatment. The SEAT method was qualified for precision, accuracy, and linearity, and was used to define a preliminary comparability range for DTxd made using the current manufacturing process. DTxd lots manufactured using alternate processes were assessed in the context of this range to determine the impact on DTxd antigenicity. Epitope profiling by SEAT provides quantitative information on the integrity of multiple important antigenic regions of DTxd, and therefore represents a valuable tool in a comprehensive analytical test package which can be used to support manufacturing process changes for vaccines.

9.
J Pharm Sci ; 111(3): 672-679, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34742727

RESUMO

Various vaccine quality attributes should be monitored to ensure consistency, potency, purity, and safety of vaccine products prior to lot release. Vaccine particle size and protein antigen aggregation are two important considerations for particle-adsorbed vaccines. In this study, we evaluated the use of imaging flow cytometry as a potential all-in-one platform to measure adjuvant particle size and to detect protein aggregates through a combination of brightfield microscopy, side scatter detection, and fluorescence microscopy. An aluminum phosphate adjuvant was analyzed for size using the brightfield function, and the size measurement was compared against laser diffraction. Heat-induced protein aggregates of either unadsorbed antigens or aluminum phosphate adjuvant-adsorbed antigens were stained with the fluorescent ProteoStat aggregation dye, followed by detection and analysis using a combination of the brightfield and fluorescence microscopy functions. The change in aggregation of unadsorbed antigens was confirmed using dynamic light scattering. These results demonstrate the versatility of the imaging flow cytometry platform for the evaluation of multiple vaccine quality characteristics.


Assuntos
Agregados Proteicos , Vacinas , Adjuvantes Imunológicos/farmacologia , Adjuvantes Farmacêuticos , Antígenos , Citometria de Fluxo , Corantes Fluorescentes
10.
Postepy Hig Med Dosw (Online) ; 65: 588-96, 2011 Sep 14.
Artigo em Polonês | MEDLINE | ID: mdl-21918263

RESUMO

Correct cell functioning, division and morphogenesis rely on efficient intracellular transport. Apart from dyneins and myosins, kinesins are the main proteins responsible for intracellular movement. Kinesins are a large, diverse group of motor proteins, which based on phylogenetic similarity were classified into fourteen families. Among these families, due to the location of their motor domains, three groups have been characterized: N-, C- and M-kinesin. As molecular motors, kinesins transport various molecules and vesicles mainly towards the microtubule plus end (from the cell body) participating in anterograde transport, although there are also kinesins involved in retrograde transport (C-kinesins). Kinesins are also involved in spindle formation, chromosome segregation, and spermatogenesis. Because of their great importance for the correct functioning of cells, mutations in kinesin coding genes may lead to such neurodegenerative diseases as dominant hereditary spastic paraplegia or Charcot-Marie-Tooth disease.


Assuntos
Proteínas de Transporte/metabolismo , Cinesinas/classificação , Humanos , Cinesinas/metabolismo , Modelos Biológicos , Transporte Proteico/fisiologia
11.
Pol Merkur Lekarski ; 31(181): 24-30, 2011 Jul.
Artigo em Polonês | MEDLINE | ID: mdl-21870705

RESUMO

UNLABELLED: Quantitative and semi-quantitative determination of gene expression by PCR plays an important role in studying of tumors initiation and progression mechanisms. Selection of appropriate reference gene is a critical factor influencing the results of gene expression analysis. One of the most commonly used reference genes in PCR is beta2-microglobuline (beta2-M). Recent studies showed however that expression of some common reference genes might be unstable, therefore it is necessary to verify again their usefulness. The aim of the study was to determine the level of beta2-M mRNA in normal and tumor tissues of gastrointestinal tract due to adequate selection of reference gene in gene expression studies. MATERIAL AND METHODS: Samples were taken from 253 patients operated on for gastrointestinal tract tumors: 22 with oral cavity cancer, 12 with benign and 50 with malignant liver tumors, 86 with colorectal cancer, and 83 with metachronous metastases to liver. Also 56 patients with liver cirrhosis were studied, which was treated as pre-tumor state. Together 309 patients were studied. RNA was isolated from tissues by Chomczynski method. The expression level of 12-M was determined by reverse transcriptase PCR (RT-PCR) and given in terms of optical density values. RESULTS: Expression of beta2-M was observed in all studied tissues. There were no differences between normal and tumor tissue. The level of expression of beta2-M was different due to type of studied tissue (oral cavity, liver, colon). CONCLUSIONS: The lack of significant differences in beta2-M expression level in normal and tumor tissues indicated that beta2-M can be used as reference gene in studies of gene expression in gastrointestinal tract tumors. On the other hand differences of beta2-M expression level in different types of tissues point to its tissue specificity and suggest application in PCR of more than one reference gene.


Assuntos
Neoplasias Gastrointestinais/genética , Microglobulina beta-2/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Perfilação da Expressão Gênica/métodos , Marcadores Genéticos/genética , Humanos , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase/métodos , Valores de Referência
12.
Materials (Basel) ; 14(14)2021 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-34300935

RESUMO

When designing embankments on a soft ground improved with columns (rigid inclusions) and with a geosynthetically reinforced load transfer platform (LTP), the methods of determining strains in reinforcement reduce the spatial problem to a two-dimensional one, and analytical calculations are carried out for reinforcement strips in the directions along and across the embankment. In addition, the two-dimensional FEM models do not allow for a complete analysis of the behavior of the reinforcement material. The aim of this research was to analyze the work of the membrane in the 3D space modeling of the LTP reinforcement, depending on the interaction with the column, the shape of the column's cap, the value of the Poisson's ratio, the value of the stiffness of the elastic foundation (subgrade reaction k) modeling of the soft soil resistance between the columns and the load distribution over membranes that model the reinforcement. The membranes were modeled in the framework of the theory of large deformations using the finite element method and slender shell elements as three-dimensional objects. This modeling method allowed for the analysis of the behavior of the LTP reinforcement in various directions. The conducted analyses showed, among others, that in the absence of soil resistance between the columns, regardless of the shape of the cap (square, circle), the greatest strains are located near the edge of the cap in the diagonal direction between the columns.

13.
PLoS One ; 16(3): e0248491, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33720984

RESUMO

The microplate assay with Chinese Hamster Ovary (CHO) cells is currently used as a safety test to monitor the residual pertussis toxin (PT) amount in acellular pertussis antigens prior to vaccine formulation. The assay is based on the findings that the exposure of CHO cells to PT results in a concentration-dependent clustering response which can be used to estimate the amount of PT in a sample preparation. A major challenge with the current CHO cell assay methodology is that scoring of PT-induced clustering is dependent on subjective operator visual assessment using light microscopy. In this work, we have explored the feasibility of replacing the microscopy readout for the CHO cell assay with the xCELLigence Real-Time Cell Analysis system (ACEA BioSciences, a part of Agilent). The xCELLigence equipment is designed to monitor cell adhesion and growth. The electrical impedance generated from cell attachment and proliferation is quantified via gold electrodes at the bottom of the cell culture plate wells, which is then translated into a unitless readout called cell index. Results showed significant decrease in the cell index readouts of CHO cells exposed to PT compared to the cell index of unexposed CHO cells. Similar endpoint concentrations were obtained when the PT reference standard was titrated with either xCELLigence or microscopy. Testing genetically detoxified pertussis samples unspiked or spiked with PT further supported the sensitivity and reproducibility of the xCELLigence assay in comparison with the conventional microscopy assay. In conclusion, the xCELLigence RTCA system offers an alternative automated and higher throughput method for evaluating PT-induced clustering in CHO cells.


Assuntos
Bioensaio , Processamento de Imagem Assistida por Computador , Toxina Pertussis/análise , Animais , Células CHO , Cricetulus , Microscopia
14.
J Pharm Sci ; 109(2): 1002-1007, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31589874

RESUMO

Vaccine manufacturers have recently focused on the development of in vitro potency assays to promote 3R's strategy to replace animal testing. To be able to develop an in vitro potency assay, the immunological characteristics of the monoclonal antibodies used in the assay should be well understood as these antibodies likely reflect the biological activity of a vaccine product. The PRN antigen is one of the immunogenic antigens included in many commercialized acellular pertussis vaccines. Development of an in vitro potency assay for PRN is challenging as the biological properties of PRN are not well understood. In addition, binding of Bordetella pertussis to human cells occurs through multiple bacterial molecules, which makes it very challenging to assess if antibodies contribute to prevention of bacterial adhesion. To overcome these challenges, the functionality of several in-house anti-PRN mAbs has been investigated through a novel approach using PRN-coated beads. We were able to consistently quantify the inhibition of PRN-mediated adhesion for each anti-PRN mAb. Application of the protein-coated beads model has not only enabled screening of functional anti-PRN mAbs but can also be expanded for screening of antibodies against other bacterial or viral antigens.


Assuntos
Anticorpos Monoclonais , Fatores de Virulência de Bordetella , Animais , Anticorpos Antibacterianos , Proteínas da Membrana Bacteriana Externa , Bordetella pertussis , Humanos , Vacina contra Coqueluche
15.
Front Immunol ; 11: 605273, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33384692

RESUMO

The pertussis vaccination is highly recommended for infants, children, and pregnant women. Despite a high coverage of vaccination, pertussis continues to be of public health concern as a re-emerging infectious disease. The mechanism by which vaccine-elicited anti-pertussis antibodies mediate direct bactericidal effects is poorly understood. In this study, we showed that the interaction of B. pertussis with A549 epithelial cells induce release of biological factors which enhance bacteria growth. Complement-depleted antisera from vaccine-immunized guinea pigs or monoclonal antibodies targeting FHA and FIM mediate bacteria aggregation and elicit bactericidal effects. Our in vitro results indicated that aggregation of bacteria through anti-FIM and anti-FHA specific antibodies is one of the major biological mechanisms to clear bacterial infections and restore epithelial cell survival in vitro. Our data also indicates that the anti-pertussis antibodies reduce secretion of proinflammatory chemokines and cytokines by preventing interaction of B. pertussis with host cells. The results of this study not only demonstrate mechanism of action of anti-FIM and anti-FHA antibodies, but also opens translational applications for potential therapeutic approaches or development of analytical assays such as in vitro potency assays.


Assuntos
Anticorpos Monoclonais/farmacologia , Antígenos de Bactérias/imunologia , Bordetella pertussis/efeitos dos fármacos , Proteínas de Fímbrias/antagonistas & inibidores , Fatores de Virulência de Bordetella/antagonistas & inibidores , Coqueluche/prevenção & controle , Células A549 , Adesinas Bacterianas/imunologia , Animais , Aderência Bacteriana/efeitos dos fármacos , Bordetella pertussis/crescimento & desenvolvimento , Bordetella pertussis/imunologia , Citocinas/metabolismo , Proteínas de Fímbrias/imunologia , Cobaias , Interações Hospedeiro-Patógeno , Humanos , Imunidade Humoral/efeitos dos fármacos , Imunogenicidade da Vacina , Mediadores da Inflamação/metabolismo , Viabilidade Microbiana , Vacina contra Coqueluche/administração & dosagem , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/metabolismo , Mucosa Respiratória/microbiologia , Vacinação , Fatores de Virulência de Bordetella/imunologia , Coqueluche/imunologia , Coqueluche/metabolismo , Coqueluche/microbiologia
16.
Mol Cell Biol ; 26(17): 6403-11, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16914726

RESUMO

Members of the B7 family of cosignaling molecules regulate T-cell proliferation and effector functions by engaging cognate receptors on T cells. In vitro and in vivo blockade experiments indicated that B7-H4 (also known as B7S1 or B7x) inhibits proliferation, cytokine production, and cytotoxicity of T cells. B7-H4 binds to an unknown receptor(s) that is expressed on activated T cells. However, whether B7-H4 plays nonredundant immune regulatory roles in vivo has not been tested. We generated B7-H4-deficient mice to investigate the roles of B7-H4 during various immune reactions. Consistent with its inhibitory function in vitro, B7-H4-deficient mice mounted mildly augmented T-helper 1 (Th1) responses and displayed slightly lowered parasite burdens upon Leishmania major infection compared to the wild-type mice. However, the lack of B7-H4 did not affect hypersensitive inflammatory responses in the airway or skin that are induced by either Th1 or Th2 cells. Likewise, B7-H4-deficient mice developed normal cytotoxic T-lymphocyte reactions against viral infection. Thus, B7-H4 plays a negative regulatory role in vivo but the impact of B7-H4 deficiency is minimal. These results suggest that B7-H4 is one of multiple negative cosignaling molecules that collectively provide a fine-tuning mechanism for T-cell-mediated immune responses.


Assuntos
Antígeno B7-1/metabolismo , Animais , Doenças Autoimunes/imunologia , Proliferação de Células , Marcação de Genes , Inflamação/imunologia , Vírus da Influenza A/fisiologia , Leishmania major/fisiologia , Vírus da Coriomeningite Linfocítica/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/virologia , Células Th1/citologia , Células Th1/imunologia , Células Th1/parasitologia , Inibidor 1 da Ativação de Células T com Domínio V-Set
17.
Vaccine ; 37(22): 2960-2966, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31010716

RESUMO

Potency assays for vaccine products are an important regulatory requirement, and are used to assess product quality and consistency prior to lot release for clinical testing. Ideally they measure an established correlate of efficacy or protection. In cases where there is no known correlate of protection, however, a functional assay that measures a biological response to a vaccine can be applied as a potency assay. Here we describe an in vitro assay which quantitatively measures human T cell activation as a biological response to the TB vaccine candidate H4-IC31. The Cytokine Secretion Assay (CSA) is based on the ability of peripheral blood mononuclear cells (PBMCs) from a Bacillus Calmette-Guérin (BCG)-vaccinated human donor to process and respond to H4-IC31. The ability of H4-IC31 to stimulate a cellular immune response is measured through the quantification of secreted IFNγ and is reported as relative stimulatory activity (RSA) compared to an in-house reference standard. The CSA is specific to the H4-IC31 vaccine, determines the RSA of H4-IC31 in the range of 50% to 150% of the reference standard, and is stability indicating as it detects differences in RSA between intact and heat treated H4-IC31. Although the CSA does not provide a link to clinical efficacy, it fulfills the critical requirements for a biological potency test to assess TB vaccine candidates and can be used along with biochemical and immunochemical assays to define a product profile during clinical development, while eliminating the use of animals for product testing.


Assuntos
Imunidade Celular/imunologia , Vacinas contra a Tuberculose/imunologia , Vacina BCG/imunologia , Células Cultivadas , Humanos , Imunização , Interferon gama/metabolismo , Mycobacterium tuberculosis/imunologia
18.
J Immunol Methods ; 452: 39-45, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29056527

RESUMO

We have developed an accurate, precise and stability-indicating flow cytometry (FC) based assay to directly measure antigenicity of H4 protein (also known as HyVac4) in a vaccine formulation of H4-IC31, without desorbing the H4 protein from the IC31 adjuvant. This method involves immuno-staining of H4-IC31 complex with anti-H4 monoclonal antibodies (mAbs) followed by FC analysis. The assay is not only able to consistently measure H4 antigenicity levels in H4-IC31 stored under normal condition at 2-8°C, but also able to detect changes in H4 antigenicity after H4-IC31 undergoes heat stress or freeze-thawing. In addition, the FC method is able to characterize particle morphology while measuring antigenicity. The biological relevance of the changes in H4 antigenicity detected by the FC assay was supported by an in vitro cell based functional assay using human PBMCs to measure IFN-gamma (IFN-γ) secretion upon re-stimulation with H4-IC31. Our results show that the FC based antigenicity assay can efficiently monitor the biological and physicochemical properties of H4-IC31 and is an indicator for adjuvanted vaccine product stability.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Citometria de Fluxo/métodos , Leucócitos Mononucleares/imunologia , Vacinas contra a Tuberculose/imunologia , Tuberculose/imunologia , Anticorpos Monoclonais/sangue , Células Cultivadas , Criopreservação , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/imunologia , Combinação de Medicamentos , Temperatura Alta , Humanos , Interferon gama/metabolismo , Ativação Linfocitária , Oligodesoxirribonucleotídeos , Oligopeptídeos , Tamanho da Partícula , Material Particulado/química
19.
Vaccine ; 36(37): 5636-5644, 2018 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-30104115

RESUMO

The first evidence in humans that a safe and effective preventive vaccine for HIV is possible came from the phase III HIV clinical trial RV144 in Thailand. This trial was based on a prime/boost combination of a recombinant canarypox vaccine and two glycoprotein 120 proteins (ALVAC-HIV and AIDSVAX B/E). A pivotal phase IIb/III trial has recently commenced in the Republic of South Africa, for which the infectious titer assay was applied as the quantitative release test for the ALVAC-HIV vaccine. The infectious titer assay measures the ability of the vaccine vector to infect target permissive cells, but does not indicate if the vaccine transgenes are expressed. We have developed a high-throughput biological activity assay that provides results in agreement with the infectious titer assay. This assay uses flow cytometry to quantify expression of ALVAC-HIV encoded proteins gp120 and p24 in human cells. This transgene expression is detected by two cross-clade-reactive, biologically functional human anti-gp120 monoclonal antibodies isolated from clinical trial participants and a commercial mouse anti-p24 monoclonal antibody. The relative biological activity of the vaccine test sample is calculated by comparison of the test sample dose-response curve against that of a reference standard. We show that the novel biological activity assay is specific, accurate, precise, stability-indicating, and robust. The assay is being used for characterization of ALVAC-HIV (vCP2438) product, the efficacy of which is being evaluated in the pivotal phase IIb/III clinical trial HVTN702. The biological activity assay has the potential to indicate vaccine consistency and quality as a complement to the infectious titer assay.


Assuntos
Vacinas contra a AIDS/imunologia , Citometria de Fluxo , Anticorpos Anti-HIV/imunologia , Ensaios de Triagem em Larga Escala , Vacinas contra a AIDS/química , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes , Ensaios Clínicos Fase II como Assunto , Ensaios Clínicos Fase III como Assunto , Proteína do Núcleo p24 do HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Células HeLa , Humanos , Células Jurkat , Sensibilidade e Especificidade
20.
Free Radic Biol Med ; 124: 79-96, 2018 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-29860127

RESUMO

Lipid peroxidation (LPO) products are relatively stable and abundant metabolites, which accumulate in tissues of mammals with aging, being able to modify all cellular nucleophiles, creating protein and DNA adducts including crosslinks. Here, we used cells and mice deficient in the ERCC1-XPF endonuclease required for nucleotide excision repair and the repair of DNA interstrand crosslinks to ask if specifically LPO-induced DNA damage contributes to loss of cell and tissue homeostasis. Ercc1-/- mouse embryonic fibroblasts were more sensitive than wild-type (WT) cells to the LPO products: 4-hydroxy-2-nonenal (HNE), crotonaldehyde and malondialdehyde. ERCC1-XPF hypomorphic mice were hypersensitive to CCl4 and a diet rich in polyunsaturated fatty acids, two potent inducers of endogenous LPO. To gain insight into the mechanism of how LPO influences DNA repair-deficient cells, we measured the impact of the major endogenous LPO product, HNE, on WT and Ercc1-/- cells. HNE inhibited proliferation, stimulated ROS and LPO formation, induced DNA base damage, strand breaks, error-prone translesion DNA synthesis and cellular senescence much more potently in Ercc1-/- cells than in DNA repair-competent control cells. HNE also deregulated base excision repair and energy production pathways. Our observations that ERCC1-deficient cells and mice are hypersensitive to LPO implicates LPO-induced DNA damage in contributing to cellular demise and tissue degeneration, notably even when the source of LPO is dietary polyunsaturated fats.


Assuntos
Senescência Celular , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/fisiologia , Endonucleases/fisiologia , Peroxidação de Lipídeos , Estresse Oxidativo , Animais , Proliferação de Células , Camundongos , Camundongos Knockout , Espécies Reativas de Oxigênio/metabolismo
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