RESUMO
Dengue is a rapidly emerging, mosquito-borne viral infection, with an estimated 400 million infections occurring annually. To gain insight into dengue immunity, we characterized 145 human monoclonal antibodies (mAbs) and identified a previously unknown epitope, the envelope dimer epitope (EDE), that bridges two envelope protein subunits that make up the 90 repeating dimers on the mature virion. The mAbs to EDE were broadly reactive across the dengue serocomplex and fully neutralized virus produced in either insect cells or primary human cells, with 50% neutralization in the low picomolar range. Our results provide a path to a subunit vaccine against dengue virus and have implications for the design and monitoring of future vaccine trials in which the induction of antibody to the EDE should be prioritized.
Assuntos
Anticorpos Neutralizantes/isolamento & purificação , Vírus da Dengue/imunologia , Dengue/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/sangue , Bioensaio , Linhagem Celular , Dengue/sangue , Ensaio de Imunoadsorção Enzimática , Humanos , Immunoblotting , Proteínas do Envelope Viral/metabolismoRESUMO
BACKGROUND: Antibodies against blood group antigens play a key role in the pathophysiology of haemolytic transfusion reactions (HTRs) and haemolytic disease of the fetus and newborn (HDFN). This study aimed to determine the frequencies of alleles, genotypes, and risk of alloimmunisation of clinically significant blood group systems in ethnic northeastern Thais. METHODS: In total, 345 unrelated, healthy, ethnic northeastern Thais were tested using the in-house PCR-sequence specific primers (PCR-SSP) method for simultaneously genotyping of RHCE, Kell, Duffy, Kidd, Diego and MNS glycophorin hybrids and results confirmed by Sanger sequencing. RESULTS: In this cohort, the alleles RHCE*C (81.0%) and RHCE*e (84.8%) were more prevalent than RHCE*c (19.0%) and RHCE*E (15.2%). The most common predicted haplotype combinations of the RHCE alleles were C+c-E-e+(R1R1) (59.4%) followed by the C+c+E+e+ (R1R2) (20.6%) and C+c+E-e+ (R1r) (11.3%). The KEL*01 allele was not found in this study. The frequencies of FY*01 and FY*02 were 88.3% and 11.7%, respectively. The genotype FY*02/02 was found in four samples (1.2%). The frequencies of JK*01 and JK*02 were 52.5% and 47.5%, respectively. Homozygous JK*02/02 was found in 81 samples (23.5%). The frequencies of DI*01 and DI*02 were 0.6% and 99.4%, respectively. In total, 64 samples (18.6%) were found to carry the MNS glycophorin hybrids. CONCLUSIONS: Our results indicated a possible high risk of c, E, Fyb, Jka, Jkb and Mia alloimmunisation in these populations. Moreover, methods established for genotyping clinically significant blood groups in this study can now be utilised in routine clinical application.
Assuntos
Alelos , Sistema do Grupo Sanguíneo Duffy , Glicoforinas , Sistema do Grupo Sanguíneo Rh-Hr , Feminino , Humanos , Masculino , Antígenos de Grupos Sanguíneos/genética , Sistema do Grupo Sanguíneo Duffy/genética , Etnicidade/genética , Frequência do Gene , Perfil Genético , Genótipo , Glicoforinas/genética , Isoanticorpos/sangue , Sistema do Grupo Sanguíneo de Kell/genética , Sistema do Grupo Sanguíneo de Kell/imunologia , Sistema do Grupo Sanguíneo Kidd/genética , Glicoproteínas de Membrana , Metaloendopeptidases , Sistema do Grupo Sanguíneo MNSs/genética , Sistema do Grupo Sanguíneo Rh-Hr/genética , População do Sudeste AsiáticoRESUMO
Opisthorchis viverrini infection is endemic in the lower Mekong subregion. The liver is an organ that worms are drawn to and cause damage. However, the immune-related susceptibility in the liver is poorly understood. In this study, we investigated T helper (Th) cell responses in the liver of BALB/c mice and golden Syrian hamsters during 2-28 days post-infection (DPI). We found that Th cell responses were distinct between mice and hamsters in terms of dynamics and polarization. Mice exhibited the early induction of Th1, Th2, Th17, and regulatory T (Treg) cells responses after the presence of O. viverrini worms at 2 DPI. In hamsters, the late induction of Th1/Th17, downregulation of Th2/Treg responses and early elevation of suppressive cytokine interleukin (IL)-10 were found together with swift reduction of Th cell numbers. Interestingly, expressions of IL-4 (Th2 functional cytokine) and Foxp3 (Treg lineage) were completely different between mice and hamsters which elevated in mice but suppressed in hamsters. These results suggest that early induction and well-regulation are related to host resistance. In contrast, late induction of Th cell response might allow immature worms to develop in the host. Our findings provide a greater understanding in Th cell response-related susceptibility in O. viverrini infection which would be targeting immunity for the development of immune-based intervention such as vaccine.
Assuntos
Opistorquíase , Opisthorchis , Cricetinae , Animais , Camundongos , Opistorquíase/prevenção & controle , Mesocricetus , Citocinas , Linfócitos T Auxiliares-Indutores/metabolismoRESUMO
Killer cell immunoglobulin-like receptors (KIRs) are cell surface receptors on natural killer (NK) cells and subsets of T cells. The interaction between KIRs and their cognate ligands (Human leukocyte antigen class I molecules, HLA class I) modulates the immune response of NK cells, in particular through clearance of virus-infected cells. Here, we investigated the effect of KIRs and HLA ligands on dengue infections and disease severity. The KIRs and HLA ligands were identified in 235 healthy controls (HC) and 253 dengue patients (DEN) using polymerase chain reaction with sequence specific primer (PCR-SSP); moreover, DEN was classified to 100 dengue fever (DF) and 153 dengue haemorrhagic fever (DHF). Risks were expressed as odds ratios (ORs) and 95% confidence intervals (CIs) with significance set at a two-tailed P value of < 0.05. The Bonferroni correction was applied for multiple comparisons. Twelve significant associations were observed in dengue infections and disease severity; however, two outcomes survived after the Bonferroni correction. Of these, HLA-A11 was associated with an increased risk to develop dengue disease (OR 2.41, 95% CI 1.62-3.60, Pc = 0.004), while KIR3DS1+ Bw4 was a protective genotype to developing DHF (OR 0.28, 95% CI 0.16-0.48, Pc < 0.001). This study revealed an important role of KIR and HLA ligands in innate immune responses to dengue viral infections and, in particular, their effect on clinical outcomes and disease severity.
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Dengue/epidemiologia , Antígenos HLA/genética , Células Matadoras Naturais/imunologia , Receptores KIR/genética , Subpopulações de Linfócitos T/imunologia , Adolescente , Adulto , Estudos de Casos e Controles , Suscetibilidade a Doenças , Humanos , Células Matadoras Naturais/citologia , Polimorfismo Genético , Índice de Gravidade de Doença , Subpopulações de Linfócitos T/citologia , Tailândia/epidemiologia , Adulto JovemRESUMO
The four serotypes of dengue virus (DENV) cause the most important mosquito-borne viral disease in humans. The envelope (E) protein is the major target of neutralizing antibodies and contains 3 domains (domain I [DI], DII, and DIII). Recent studies reported that human monoclonal antibodies (MAbs) recognizing DIII, the D1/DII hinge, the E-dimer epitope, or a quaternary epitope involving DI/DII/DIII are more potently neutralizing than those recognizing the fusion loop (FL) of DII. Due to inefficient cleavage of the premembrane protein, DENV suspensions consist of a mixture of mature, immature, and partially immature particles. We investigated the neutralization and binding of 22 human MAbs to DENV serotype 1 (DENV1) virions with differential maturation status. Compared with FL MAbs, DIII, DI/DII hinge, and E-dimer epitope MAbs showed higher maximum binding and avidity to mature particles relative to immature particles; this feature may contribute to the strong neutralizing potency of such MAbs. FL-specific MAbs required 57 to 87% occupancy on mature particles to achieve half-maximal neutralization (NT50), whereas the potently neutralizing MAbs achieved NT50 states at 20 to 38% occupancy. Analysis of the MAb repertoire and polyclonal sera from patients with primary DENV1 infection supports the immunodominance of cross-reactive anti-E antibodies over type-specific antibodies. After depletion with viral particles from a heterologous DENV serotype, the type-specific neutralizing antibodies remained and showed binding features shared by potent neutralizing MAbs. Taken together, these findings suggest that the use of homogeneous mature DENV particles as an immunogen may induce more potent neutralizing antibodies against DENV than the use of immature or mixed particles.IMPORTANCE With an estimated 390 million infections per year, the four serotypes of dengue virus (DENV) cause the most important mosquito-borne viral disease in humans. The dengue vaccine Dengvaxia was licensed; however, its low efficacy among dengue-naive individuals and increased risk of causing severe dengue in children highlight the need for a better understanding of the role of human antibodies in immunity against DENV. DENV suspensions contain mature, immature, and partially immature particles. We investigated the binding of 22 human monoclonal antibodies (MAbs) to the DENV envelope protein on particles with different maturation states. Potently neutralizing MAbs had higher relative maximum binding and avidity to mature particles than weakly neutralizing MAbs. This was supported by analysis of MAb repertoires and polyclonal sera from patients with primary DENV infection. Together, these findings suggest that mature particles may be the optimal form of presentation of the envelope protein to induce more potent neutralizing antibodies against DENV.
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Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vacinas contra Dengue/imunologia , Vírus da Dengue/imunologia , Dengue/prevenção & controle , Proteínas do Envelope Viral/imunologia , Adulto , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , Dengue/imunologia , Dengue/virologia , Vírus da Dengue/metabolismo , Humanos , Vírion/imunologiaRESUMO
The envelope (E) protein of dengue virus (DENV) is the major target of neutralizing antibodies (Abs) and vaccine development. Previous studies of human dengue-immune sera reported that a significant proportion of anti-E Abs, known as group-reactive (GR) Abs, were cross-reactive to all four DENV serotypes and to one or more other flaviviruses. Based on studies of mouse anti-E monoclonal antibodies (MAbs), GR MAbs were nonneutralizing or weakly neutralizing compared with type-specific MAbs; a GR response was thus not regarded as important for vaccine strategy. We investigated the epitopes, binding avidities, and neutralization potencies of 32 human GR anti-E MAbs. In addition to fusion loop (FL) residues in E protein domain II, human GR MAbs recognized an epitope involving both FL and bc loop residues in domain II. The neutralization potencies and binding avidities of GR MAbs derived from secondary DENV infection were stronger than those derived from primary infection. GR MAbs derived from primary DENV infection primarily blocked attachment, whereas those derived from secondary infection blocked DENV postattachment. Analysis of the repertoire of anti-E MAbs derived from patients with primary DENV infection revealed that the majority were GR, low-avidity, and weakly neutralizing MAbs, whereas those from secondary infection were primarily GR, high-avidity, and potently neutralizing MAbs. Our findings suggest that the weakly neutralizing GR anti-E Abs generated from primary DENV infection become potently neutralizing MAbs against the four serotypes after secondary infection. The observation that the dengue immune status of the host affects the quality of the cross-reactive Abs generated has implications for new strategies for DENV vaccination.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vírus da Dengue/imunologia , Dengue/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Reações Cruzadas , Dengue/virologia , Vírus da Dengue/classificação , Epitopos/imunologia , Feminino , Humanos , Masculino , Camundongos , Proteínas do Envelope Viral/químicaRESUMO
Dengue virus infections are still increasing at an alarming rate in tropical and subtropical countries, underlying the need for a dengue vaccine. Although it is relatively easy to generate Ab responses to dengue virus, low avidity or low concentrations of Ab may enhance infection of FcR-bearing cells with clinical impact, posing a challenge to vaccine production. In this article, we report the characterization of a mAb, 2H12, which is cross-reactive to all four serotypes in the dengue virus group. Crystal structures of 2H12-Fab in complex with domain III of the envelope protein from three dengue serotypes have been determined. 2H12 binds to the highly conserved AB loop of domain III of the envelope protein that is poorly accessible in the mature virion. 2H12 neutralization varied between dengue serotypes and strains; in particular, dengue serotype 2 was not neutralized. Because the 2H12-binding epitope was conserved, this variation in neutralization highlights differences between dengue serotypes and suggests that significant conformational changes in the virus must take place for Ab binding. Surprisingly, 2H12 facilitated little or no enhancement of infection. These data provide a structural basis for understanding Ab neutralization and enhancement of infection, which is crucial for the development of future dengue vaccines.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Antivirais/química , Vírus da Dengue/imunologia , Proteínas do Envelope Viral/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Antivirais/metabolismo , Sítios de Ligação de Anticorpos , Reações Cruzadas/imunologia , Cristalografia por Raios X , Vírus da Dengue/química , Vírus da Dengue/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Testes de Neutralização , Estrutura Terciária de Proteína , Sorotipagem , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismo , Vírion/química , Vírion/imunologia , Vírion/metabolismoRESUMO
The major histocompatibility complex (MHC) class I chain-related A (MICA) plays an important role in stress cell recognition. High polymorphisms of MICA are relevant to NKG2D binding capacity, responses of NK cells and tumor progression. In this study, MICA genotyping of 97 cholangiocarcinoma patients was performed using PCR-SSP. MICA*010 was positively associated with a corrected p-value of < 0.001 (RR=2.16 (95 % CI, 1.48-3.14)). MICA*010 was previously reported as a non-expressed allele. Thus, the expression of MICA*010 on the cell surface was studied on both MICA*010 transfected cells (HEK 293 T and L929 cells) and stimulated primary monocytes obtained from homozygous MICA*010 individuals using different clones of antibodies (1H10, 1D10, 1C3.1, 1C3.2, 6D4 and 3H5) for detection. Surprisingly, the expression of MICA*010 could be observed on both transfected cells and stimulated monocytes and effectively bound to the NKG2D-Fc fusion protein. The functional study of various MICA alleles revealed the high relative killing activity of NK cells induced by the MICA*010 transfected C1R cells, not following the previously reported rule of the M129V substitution. The structural analysis highlighted the amino acid at position 36 as another important amino acid relevant to preserving the structural integrity of the MICA protein and NKG2D binding. Our data propose a new aspect of functional MICA contributing motifs and that MICA*010 has a potential effect on NK cell functions and might be applicable to other fields of immune responses.
RESUMO
Major histocompatibility complex class I chain-related gene A (MICA) is an NKG2D ligand that is over-expressed under cellular stress including cancer transformation and viral infection. High expression of MICA in cancer tissues or patients' sera is useful for prognostic or follow-up markers in cancer patients. In this study, phage display technology was employed to improve antigen-binding activities of anti-MICA monoclonal antibodies (WW2G8, WW6B7, and WW9B8). The 12 amino acid residues in the complementarity determining regions (CDRs) on the V domain of the heavy chain CDR3 (HCDR3) of these anti-MICA antibodies were modified by PCR-random mutagenesis, and phages displaying mutated anti-MICA Fab were constructed. After seven rounds of panning, five clones of phages displaying mutant anti-MICA Fab which exhibited 3-7-folds higher antigen-binding activities were isolated. Two clones of the mutants (phage-displayed mutant Fab WW9B8.1 and phage-displayed mutant Fab WW9B8.21) were confirmed to have antigen-binding specificity for cell surface MICA proteins by flow cytometry. These phage clones are able to recognize MICA in a native form according to positive results obtained by indirect ELISA and flow cytometry. Thus, these phage particles could be potentially used for further development of nanomedicine specifically targeting cancer cells expressing MICA proteins.
Assuntos
Anticorpos Antineoplásicos/imunologia , Técnicas de Visualização da Superfície Celular/métodos , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Terapia de Alvo Molecular/métodos , Neoplasias/imunologia , Neoplasias/terapia , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos/imunologia , Sequência de Bases , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Citometria de Fluxo , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/imunologia , Dados de Sequência Molecular , Proteínas Mutantes/isolamento & purificação , Proteínas Mutantes/metabolismo , Reação em Cadeia da Polimerase , Ligação Proteica/imunologiaRESUMO
Dengue virus receptors are relatively poorly characterized, but there has been recent interest in 2 C-type lectin molecules, dendritic cell-specific intercellular adhesion molecule 3 (ICAM-3)-grabbing nonintegrin (DC-SIGN) and its close homologue liver/lymph node-specific ICAM-3-grabbing integrin (L-SIGN), which can both bind dengue and promote infection. In this report we have studied the interaction of dengue viruses produced in insect cells, tumor cell lines, and primary human dendritic cells (DCs) with DC-SIGN and L-SIGN. Virus produced in primary DCs is unable to interact with DC-SIGN but remains infectious for L-SIGN-expressing cells. Skin-resident DCs may thus be a site of initial infection by insect-produced virus, but DCs will likely not participate in large-scale virus replication during dengue infection. These results reveal that differential glycosylation of dengue virus envelope protein is highly dependent on cell state and suggest that studies of virus tropism using virus prepared in insect cells or tumor cell lines should be interpreted with caution.
Assuntos
Células Dendríticas/virologia , Vírus da Dengue/metabolismo , Vírus da Dengue/patogenicidade , Lectinas/metabolismo , Animais , Antígenos CD/metabolismo , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Chlorocebus aethiops , Dengue/virologia , Ensaio de Imunoadsorção Enzimática , Humanos , Insetos/citologia , Lectinas Tipo C/metabolismo , Receptores de Superfície Celular/metabolismo , Células VeroRESUMO
In the aging process, the presence of interleukin (IL)-17-producing CD4+CD28-NKG2D+T cells (called pathogenic CD4+ T cells) is strongly associated with inflammation and the development of various diseases. Thus, their presence needs to be monitored. The emergence of attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy empowered with machine learning is a breakthrough in the field of medical diagnostics. This study aimed to discriminate between the elderly with a low percentage (LP; ≤3%) and a high percentage (HP; ≥6%) of pathogenic CD4+CD28-NKG2D+IL17+ T cells by utilizing ATR-FTIR coupled with machine learning algorithms. ATR spectra of serum, exosome, and HDL from both groups were explored in this study. Only exosome spectra in the 1700-1500 cm-1 region exhibited possible discrimination for the LP and HP groups based on principal component analysis (PCA). Furthermore, partial least square-discriminant analysis (PLS-DA) could differentiate both groups using the 1700-1500 cm-1 region of exosome ATR spectra with 64% accuracy, 69% sensitivity, and 61% specificity. To obtain better classification performance, several spectral models were then established using advanced machine learning algorithms, including J48 decision tree, support vector machine (SVM), random forest (RF), and neural network (NN). Herein, NN was considered to be the best model with an accuracy of 100%, sensitivity of 100%, and specificity of 100% using serum spectra in the region of 1800-900 cm-1. Exosome spectra in the 1700-1500 and combined 3000-2800 and 1800-900 cm-1 regions using the NN algorithm gave the same accuracy performance of 95% with a variation in sensitivity and specificity. HDL spectra with the NN algorithm also showed excellent test performance in the 1800-900 cm-1 region with 97% accuracy, 100% sensitivity, and 95% specificity. This study demonstrates that ATR-FTIR coupled with machine learning algorithms can be used to study immunosenescence. Furthermore, this approach can possibly be applied to monitor the presence of pathogenic CD4+ T cells in the elderly. Due to the limited number of samples used in this study, it is necessary to conduct a large-scale study to obtain more robust classification models and to assess the true clinical diagnostic performance.
Assuntos
Antígenos CD28 , Linfócitos T CD4-Positivos , Idoso , Proteínas Mutadas de Ataxia Telangiectasia , Análise de Fourier , Humanos , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Espectroscopia de Infravermelho com Transformada de Fourier/métodosRESUMO
BACKGROUND: Peripheral eosinophilia (eosinophilia) is observed among systemic sclerosis (SSc) patients. The association between eosinophilia and SSc in terms of pathogenesis remains uncertain. We aimed to determine the prevalence of the clinical, serological, and cytokine associations with eosinophilia in SSc patients. METHODS: A cross-sectional study was conducted among adult SSc patients. We excluded patients having overlap syndrome and other conditions that cause eosinophilia. Investigations into the etiology of eosinophilia were performed on the same study date, including clinical parameters, blood tests for tissue parasites, IgE, interleukin-5, and transforming growth factor-beta. Eosinophilia is defined when the total eosinophil count is > 500 cells/mm3. RESULTS: According to the sample size calculation, 185 patients were enrolled, of whom 57 (30.8%) had eosinophilia. The causes of eosinophilia were based on laboratory indicators without clinical symptoms in 21 cases (10 had a parasitic infection, 9 adrenal insufficiency, and 2 tuberculosis). After excluding suspected causes of eosinophilia, the total prevalence of eosinophilia was 21.9% (95%CI 15.9-29.1). Most of patients (164 cases; 70.6%) had diffuse cutaneous SSc. According to the logistic regression analysis, the factors associated with eosinophilia were being male (OR 3.46), duration of disease increasing every year (OR 1.16), and Raynaud's phenomenon (OR 0.27), while SSc subset, serology (i.e., anti-topoisomerase I, anti-neutrophilic cytoplasmic antibody), inflammatory markers, and cytokine levels were not. CONCLUSIONS: Eosinophilia of unknown causes was detected in 1 in 5 SSc patients, particularly in males with no vasculopathy. Eosinophilia has a nonspecific role vis-à-vis clinical relevance in SSc.
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Eosinofilia , Escleroderma Sistêmico , Adulto , Estudos Transversais , Citocinas , Eosinofilia/epidemiologia , Feminino , Humanos , Masculino , Prevalência , Escleroderma Sistêmico/complicações , Escleroderma Sistêmico/diagnóstico , Escleroderma Sistêmico/epidemiologiaRESUMO
The beneficial physiological effects of traditional Thai massage (TTM) have been previously documented. However, its effect on immune status, particularly in the elderly, has not been explored. This study aimed to investigate the effects of multiple rounds of TTM on senescent CD4+ T cell subsets in the elderly. The study recruited 12 volunteers (61-75 years), with senescent CD4+ T cell subsets, who received six weekly 1-h TTM sessions or rest, using a randomized controlled crossover study with a 30-day washout period. Flow cytometry analysis of surface markers and intracellular cytokine staining was performed. TTM could attenuate the senescent CD4+ T cell subsets, especially in CD4+28null NKG2D+ T cells (n = 12; p < 0.001). The participants were allocated into two groups (low < 2.75% or high ≥ 2.75%) depending on the number of CD4+28null NKG2D+ T cells. After receiving TTM over 6 sessions, the cell population of the high group had significantly decreased (p < 0.001), but the low group had no significant changes. In conclusion, multiple rounds of TTM may promote immunity through the attenuation of aberrant CD4+ T subsets. TTM may be provided as a complementary therapy to improve the immune system in elderly populations.
Assuntos
Linfócitos T CD4-Positivos , Massagem , Idoso , Estudos Cross-Over , Humanos , Subpopulações de Linfócitos T , TailândiaRESUMO
In this study, a key issue to be addressed is the safe disposal of hybridoma instability. Hybridoma technology was used to produce anti-O. viverrini monoclonal antibody. Previous studies have shown that antibody production via antibody phage display can sustain the hybridoma technique. This paper presents the utility of antibody phage display technology for producing the phage displayed KKU505 Fab fragment and using experiments in concomitant with molecular simulation for characterization. The phage displayed KKU505 Fab fragment and characterization were successfully carried out. The KKU505 hybridoma cell line producing anti-O. viverrini antibody predicted to bind to myosin was used to synthesize cDNA so as to amplify the heavy chain and the light chain sequences. The KKU505 displayed phage was constructed and characterized by a molecular modeling in which the KKU505 Fab fragment and -O. viverrini myosin head were docked computationally and it is assumed that the Fab fragment was specific to -O. viverrini on the basis of mass spectrometry and Western blot. This complex interaction was confirmed by molecular simulation. Furthermore, the KKU505 displayed phage was validated using indirect enzyme-linked immunosorbent assays (ELISA) and immunohistochemistry. It is worthy to note that ELISA and immunohistochemistry results confirmed that the Fab fragment was specific to the -O. viverrini antigen. Results indicated that the approach presented herein can generate anti-O. viverrini antibody via the phage display technology. This study integrates the use of phage display technology together with molecular simulation for further development of monoclonal antibody production. Furthermore, the presented work has profound implications for antibody production, particularly by solving the problem of hybridoma stability issues.
Assuntos
Anticorpos Anti-Helmínticos/biossíntese , Anticorpos Anti-Helmínticos/imunologia , Simulação de Dinâmica Molecular , Opisthorchis/imunologia , Biblioteca de Peptídeos , Sequência de Aminoácidos , Animais , Anticorpos Anti-Helmínticos/química , Antígenos de Helmintos/química , Antígenos de Helmintos/imunologia , Sistema Biliar/imunologia , Sistema Biliar/parasitologia , Bovinos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Camundongos , Simulação de Acoplamento Molecular , Miosinas/química , Reprodutibilidade dos Testes , Soroalbumina Bovina/análiseRESUMO
BACKGROUND: Vitamin C is an essential element required for normal metabolic function. We investigated the effect of vitamin C supplementation on circulating miRNA (miR) expression in subjects with poorly controlled type 2 diabetes mellitus (T2DM). Changes in miR expression were also correlated with clinical measures of disease. METHODS: Pre- and post-vitamin C supplementation samples from five participants who had increased vitamin C levels, improved oxidative status and polymorphonuclear (PMN) function after receiving 1,000 mg of vitamin C daily for six weeks were screened for miRNA expression using the NanoString miRNA assay. Differences in miRNA expression identified from the miRNA screen were validated by qRT-PCR. RESULTS: Four miRNAs showed significantly different expression post-vitamin C supplementation relative to baseline, including the down-regulation of miR-451a (-1.72 fold change (FC), p = 0.036) and up-regulation of miR-1253 (0.62 FC, p = 0.027), miR-1290 (0.53 FC, p = 0.036) and miR-644a (0.5 FC, p = 0.042). The validation study showed only miR-451a expression was significantly different from baseline with vitamin C supplementation. MiR-451a expression was negatively correlated with vitamin C levels (r = - 0.497, p = 0.049) but positively correlated with levels of malondialdehyde (MDA) (r = 0.584, p = 0.017), cholesterol (r = 0.564, p = 0.022) and low-density lipoproteins (LDL) (r = 0.522, p = 0.037). Bioinformatics analysis of the putative target genes of miR-451a indicated gene functions related to signaling pathways involved in cellular processes, such as the mammalian target of rapamycin (mTOR) signaling pathway. CONCLUSIONS: Vitamin C supplementation altered circulating miR-451a expression. The results from this pilot study suggest that miRNAs could be used as biomarkers to indicate oxidative status in subjects with T2DM and with poor glycemic control and could lead to a novel molecular strategy to reduce oxidative stress in T2DM.
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(1) Background: KIR2DL4/KIR3DL3 are the framework genes present in all KIR haplotypes, with unique expression patterns being present only in women and CD56bright NK cells. KIR genes have a high degree of DNA sequence identity. Consequently, they are one of the most challenging genes for molecular detection-especially regarding expressions; (2) Methods: We developed an effective method to determine KIR3DL3/KIR2DL4 expressions based on a multiplex quantitative real-time Reverse transcription polymerase chain reaction (qRT-PCR )with fluorescent probes using NK92; (3) Results: Standardizations of the singleplex KIR2DL4 and KIR3DL3 were performed to evaluate the sensitivity and specificity for further development of the multiplex assay. The limit of detection was at 500 copies each. There was cross-amplification with the presence of related KIR genes at a level of 5 × 107 copies. This is not biologically significant because this high level of KIR expression has not been found in clinical samples. The multiplex assay was reproducible equivalent to its singleplex (KIR2DL4; R2 = 0.995, KIR3DL3; R2 = 0.996, but lower sensitivity of 103 copies). Furthermore, the validation of the developed method on samples of blood donors showed high sensitivity (100%) and specificity (99.9%); (4) Conclusions: The developed method is reliable and highly specific suitable for evaluation of the KIR2DL4/3DL3 mRNA expressions in further applications.
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Cardiovascular diseases (CVD), which are major causes of morbidity and mortality worldwide, are characterized by complicated chronic inflammatory manifestation inducing from multi-risk factors. Previously, we have identified a pathological T cell subpopulation producing interleukin (IL)-17 in diabetes. We hypothesized that this T cell subpopulation could exist in the elderly with persistence low grade inflammation related to the risk factors for cardiovascular diseases. Thus, we investigated whether high levels of the natural group 2, member D (NKG2D) expression, IL-17 and interferon (IFN)-γ production by CD4 + T cells and T cell subsets were more prevalent in individuals who had age ≥ 60 years with > 2 risk factors for CVD (dyslipidemia, hypertension and/or diabetes mellitus) compared to subjects who had < 2 risk factors. Using flow cytometric analysis, we found that CD4 + T cells of subjects who had ≥ 2 risk factors had significantly higher NKG2D expression than those of subjects with < 2 risk factors (P = 0.023). Apparently, CD4+CD28null T subset of both two groups preferentially expressed NKG2D, and prominently produced IL-17 and IFN-γ compared to the CD4+CD28+ T subset. Expectedly, there was a statistical significance of IL-17 and IFN-γ production of CD4 + 28nullNKG2D + T cells (P = 0.037 and P = 0.042, respectively). We concluded that cumulative number of CVD risk factors associated with progressive alteration of CD4+ T cell phenotypes and their functions. Handling of metabolic risk factors may be an approach for healthcare of the elderly to prevent cardiovascular morbidity resulting from alteration of immunity.
RESUMO
BACKGROUND: Various cell types undergo activation and stress during atherosclerosis resulting in the development of acute myocardial infarction (AMI) in coronary artery disease (CAD). Major histocompatibility complex class I related chain A and B (MICA/B) can be expressed on the surface of activated and stressed cells and released into blood circulation in several forms including microparticles (MICA/B+ MPs) from various cell types. We aimed to investigate the association of these MICA/B+ MPs with the presence of AMI. Fifty-one AMI and 46 age-matched control subjects were recruited. METHODS: Levels of MICA/B+ MPs derived from various parent cells including endothelial cells, platelets, monocytes, neutrophils, and T lymphocytes were determined by flow cytometry. RESULTS: The levels and proportion of MICA/B+ MPs from all types of cell origin were significantly increased in AMI patients compared to those of the controls. A multivariate regression model showed an independent association between MICA/B+ MPs and AMI (OR = 11.6; 95% CI = 2.8, 47.3). Interestingly, based on the disease severity, we found that the levels of MICA/B+ MPs were significantly elevated in the ST-segment elevation myocardial infarction (STEMI) compared to the non-STEMI (NSTEMI) patients. Moreover, an independent association of MICA/B+ MPs with the occurrence of STEMI was also demonstrated (OR = 4.1; 95% CI = 1.5, 16.7). CONCLUSIONS: These results suggest that MICA/B+ MPs are associated with AMI and disease severity. They may act as mediators contributing to the pathological process of AMI. Alternatively, they are the results of various cell activations contributing to AMI.