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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.
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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.
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Alelos , Sistema del Grupo Sanguíneo Duffy , Glicoforinas , Sistema del Grupo Sanguíneo Rh-Hr , Femenino , Humanos , Masculino , Antígenos de Grupos Sanguíneos/genética , Sistema del Grupo Sanguíneo Duffy/genética , Etnicidad/genética , Frecuencia de los Genes , Perfil Genético , Genotipo , Glicoforinas/genética , Isoanticuerpos/sangre , Sistema del Grupo Sanguíneo de Kell/genética , Sistema del Grupo Sanguíneo de Kell/inmunología , Sistema del Grupo Sanguíneo de Kidd/genética , Glicoproteínas de Membrana , Metaloendopeptidasas , Sistema del Grupo Sanguíneo MNSs/genética , Sistema del Grupo Sanguíneo Rh-Hr/genética , Pueblos del Sudeste AsiáticoRESUMEN
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.
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Opistorquiasis , Opisthorchis , Cricetinae , Animales , Ratones , Opistorquiasis/prevención & control , Mesocricetus , Citocinas , Linfocitos T Colaboradores-Inductores/metabolismoRESUMEN
OBJECTIVE: Chronic Opisthorchis viverrini (OV) infection is the cause of advanced periductal fibrosis (APF), subsequently leading to cholangiocarcinoma (CCA). Natural killer (NK) cells can kill hepatic stellate cells (HSCs), the initiating cells for fibrosis formation, by using the interaction between the natural killer group 2 member D (NKG2D) receptor and its ligand on the HSCs. This can inhibit the fibrosis formation. Major histocompatibility complex class I chain-related A (MICA) is the ligand of the NKG2D receptor and has highly polymorphic characteristics that are involved in NKG2D binding and NK cell activation. This study aimed to investigate the polymorphism of MICA in OV-induced fibrosis. METHOD: MICA typing was performed by polymerase chain reaction- sequence specific primer (PCR-SSP) and sequencing in two groups: OV infection without fibrosis (N = 99) and with fibrosis (N = 290). RESULT: Six alleles were identified and the MICA*010 allele had the highest frequency in both groups. The MICA*00201-02 allele was a protective factor for fibrosis (OR= 0.508, 95%CI= 0.34-0.76, Pc <0.05), while the MICA*019 allele was suggested to be a risk allele for fibrosis (OR=1.95, 95%CI=1.25-3.03, Pc<0.005). In addition, two motifs, glycine (G) at position 14 and glutamine (Q) at position 251, were negatively associated with fibrosis (G14: OR=0.508, 95%CI=0.34-0.76, Pc <0.05 and Q251: OR=0.586, 95%CI=0.41-0.84, Pc <0.05). Moreover, the distribution of the MICA-129 genotype also showed the protective genotype (Pc<0.05, OR=0.319, 95%CI= 0.12-0.54) for fibrosis. The MICA*00201-02 allele encoded all these motifs, and this suggested that it might lead to strong NK cell activation to kill HSCs, subsequently preventing fibrosis formation. CONCLUSION: This study described initial evidence suggesting that the polymorphism of the MICA gene might be a marker for OV-derived periductal fibrosis.
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Neoplasias de los Conductos Biliares , Opistorquiasis , Opisthorchis , Humanos , Animales , Opisthorchis/genética , Tailandia , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Ligandos , Polimorfismo Genético/genética , Antígenos de Histocompatibilidad Clase I/genética , Fibrosis , Opistorquiasis/complicaciones , Opistorquiasis/genética , Conductos Biliares Intrahepáticos , Neoplasias de los Conductos Biliares/genéticaRESUMEN
This study investigated the associations between relative telomere length (RTL) and resting metabolic rate (RMR), resting fat oxidation (RFO), and aerobic capacity and whether oxidative stress and inflammation are the underlying mechanisms in sedentary women. We also aimed to determine whether the correlations depend on age and obesity. Sixty-eight normal weight and 66 obese women participated in this study. After adjustment for age, energy expenditure, energy intake, and education level, the RTL of all participants was negatively correlated with absolute RMR (RMRAB) and serum high-sensitivity C-reactive protein (hsCRP) concentration, and positively correlated with maximum oxygen consumption (VËO2max) (all p < 0.05). After additional adjustment for adiposity indices and fat-free mass (FFM), RTL was positively correlated with plasma vitamin C concentration (p < 0.05). Furthermore, after adjustment for fasting blood glucose concentration, RTL was negatively correlated with age and positively correlated with VËO2max (mL/kg FFM/min). We found that normal weight women had longer RTL than obese women (p < 0.001). We suggest that RTL is negatively correlated with RMRAB and positively correlated with aerobic capacity, possibly via antioxidant and anti-inflammatory mechanisms. Furthermore, age and obesity influenced the associations. We provide useful information for the management of promotion strategies for health-related physical fitness in women.
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Metabolismo Basal , Obesidad , Humanos , Femenino , Estudios Transversales , Ingestión de Energía , Telómero , Composición CorporalRESUMEN
This work demonstrates the ability of the Ion-Sensitive Field-Effect Transistor (ISFET)-based immunosensor to detect antibodies against the human leukocyte antigen (HLA) and the major histocompatibility complex class-I-related chain A (MICA). The sensing membrane of the ISFET devices was modified and functionalized using an APTES-GA strategy. Surface properties, including wettability, surface thickness, and surface topology, were assessed in each module of the modification process. The optimal concentrations of HLA and MICA proteins for the immobilization were 10 and 50 µg/mL. The dose-response curve showed a detection range of 1.98-40 µg/mL for anti-HLA and 5.17-40 µg/mL for anti-MICA. The analytical precision (%CV) was found to be 10.69% and 8.92% for anti-HLA and -MICA, respectively. Moreover, the electrical signal obtained from the irrelevant antibody was considerably different from that of the specific antibodies, indicating the specific binding of the relevant antibodies without noise interference. The sensitivity and specificity in the experimental setting were established for both antibodies (anti-HLA: sensitivity = 80.00%, specificity = 86.36%; anti-MICA: sensitivity = 86.67%, specificity = 88.89%). Our data reveal the potential of applying the ISFET-based immunosensor to the detection of relevant anti-HLA and -MICA antibodies, especially in the field of kidney transplantation.
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Técnicas Biosensibles , Trasplante de Riñón , Anticuerpos , Antígenos HLA , Antígenos de Histocompatibilidad Clase I , Antígenos de Histocompatibilidad Clase II , Humanos , Inmunoensayo , IonesRESUMEN
BACKGROUND AND OBJECTIVES: DEL (D-elute) red blood cells (RBCs) are typed as D- by routine serological methods, as they carry a very weak form of D variant. Asia type DEL (c.1227 G>A) is the most prevalent DEL allele in East Asian populations that can lead to alloimmunization in D negative transfusion recipients. This study aimed to screen D+ and D- Thai blood donors for the Asia type DEL and its zygosity. MATERIALS AND METHODS: Blood samples of 723 D+ , and 191 D- Thai blood donors were collected. D antigen was typed by the routine serological method and adsorption-elution test to screen DEL phenotype. The hybrid Rhesus box, RHD 1227 G, RHD 1227 A and RHD exon 4 were analyzed with polymerase chain reaction using sequence specific primers, PCR-SSP. RESULTS: Among 191 D- blood donors, 52 (27.2%) RHD 1227 A allele carriers were detected, and 39 out of these 52 (75.0%) were positive for the hybrid Rhesus box (i.e. hemizygous RHD 1227 A). The remaining carriers were RHD 1227 A homozygous. RHD zygosity analysis in Thai D+ blood donors showed that only 4% (29/723) had a Dd genotype (hemizygous RHD 1227 G). Five (0.69%) blood donors were detected with RHD 1227 A alleles among 723 D+ blood donors. They were all G/A heterozygous at the RHD 1227 site. CONCLUSION: This study indicates that the high prevalence of RHD 1227 A allele among serologically apparent D- blood donors in Thais. Furthermore, the screening of hybrid Rhesus box, RHD 1227 A combined with RHD exon 4 is useful for analyses of Asia type DEL and its zygosity.
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Donantes de Sangre , Sistema del Grupo Sanguíneo Rh-Hr , Humanos , Sistema del Grupo Sanguíneo Rh-Hr/genética , Tailandia , Prevalencia , Alelos , Genotipo , FenotipoRESUMEN
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.
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Antígenos CD28 , Linfocitos T CD4-Positivos , Anciano , Proteínas de la Ataxia Telangiectasia Mutada , Análisis de Fourier , Humanos , Subfamilia K de Receptores Similares a Lectina de Células NK , Espectroscopía Infrarroja por Transformada de Fourier/métodosRESUMEN
Killer immunoglobulin-like receptors (KIRs) are a family of receptors expressed on Natural killer (NK) cells. The extensive polymorphism of KIR is involved in the immune responses of NK cells and influences dengue infections. We investigated the diversity of KIR copy numbers in dengue-infected patients from northeastern Thailand. Copy numbers of KIRs were determined by quantitative polymerase chain reaction in 137 dengue-infected patients, comprising 63 dengue fever (DF) and 74 dengue hemorrhagic fever (DHF). The distribution of KIRs was observed to be between 0 and 4 copies. The KIR AA genotype with heterozygous KIR2DS4D/WT was the most common in dengue patients, 25.4% DF and 23% DHF. Forty KIR profiles were determined in dengue patients, including 31 usual, 6 expanded, and 3 contracted profiles. Investigation of KIR copy number and dengue severity indicated that two copies of KIR2DL3 combined with HLA-C1C1 associated with an increased risk of DHF (OR 2.32, 95% CI 1.159-4.624, P = 0.016), whereas one copy of KIR2DL2 and KIR2DL3 together with HLA-C1C1 associated with a reduced risk of DHF (OR 0.17, 95% CI 0.058-0.482, P < 0.001). The outcomes of this study will contribute to the understanding of KIR complexity and innate immune responses in dengue infections.
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Variaciones en el Número de Copia de ADN , Dengue , Dengue/genética , Genotipo , Antígenos HLA/genética , Humanos , Receptores KIR/genética , TailandiaRESUMEN
AIM: Donor-recipient antigen mismatching for anti-human leucocyte antigen (HLA) and MICA is one of the risk factors for antibody induction leading to graft rejection. Our aim was to analyze the incidence and specificity of the different DSAs developing and to investigate the impact of HLA and MICA allele mismatches on antibody production in kidney transplant patients experiencing antibody-mediated rejection (AMR). METHODS: We retrospectively reviewed 253 consecutive recipients of kidney transplant who were diagnosed as experiencing AMR. RESULTS: Our results showed that around 27% of our patients were positive for DSAs over a median follow-up period of 24 months. Antibody to HLA-DQ7 was the most prevalent DSA detected. The allele mismatch number was significantly lower for DQ loci than -A and -B loci (DQ vs. A, p < .001; DQ vs. B, p = .002). Considering each HLA antigen, the incidence rate of DQ-DSA [41.9 (32.92-51.46; 95%CI)] was much higher than the rate observed for DSA directed to -A, -DR and -B loci. Half of the recipients in the DQ-DSA-only group, and the DQ-DSA together with non-DQ group, had MFI > 5000. Only one case developed de novo MICA-DSA (MICA002). CONCLUSION: Our study indicates that mismatching for HLA and MICA alleles leads to the development of HLA and MICA antibodies in some kidney transplant recipients. We have also demonstrated that DSA to the DQ locus is the most prevalent in kidney transplant patients with AMR. Thus, matching the DQ locus in kidney allocation algorithms may reduce post-transplant development of DSA.
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Rechazo de Injerto/inmunología , Antígenos HLA/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Histocompatibilidad , Isoanticuerpos/sangre , Trasplante de Riñón/efectos adversos , Adulto , Biomarcadores/sangre , Femenino , Frecuencia de los Genes , Rechazo de Injerto/sangre , Rechazo de Injerto/diagnóstico , Antígenos HLA/genética , Antígenos de Histocompatibilidad Clase I/genética , Prueba de Histocompatibilidad , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Tailandia , Factores de Tiempo , Resultado del Tratamiento , Adulto JovenRESUMEN
Mucositis is one of the most adverse effects of 5-fluorouracil (5-FU) and had no standard drug for treatment. Melatonin is a neurohormone, and can ameliorate radiotherapy-induced small intestinal mucositis. Melatonin encapsulated in niosomes improved its poor bioavailability. Succinyl melatonin, a melatonin derivative, showed prolonged release compared with melatonin. This study investigated the efficacy of melatonin niosome gel (MNG) and succinyl melatonin niosome gel (SNG) in 5-FU-induced small intestinal mucositis treatment in mice. MNG and SNG with particle sizes of 293 and 270 nm were shown to have mucoadhesive potentials. The effect of a daily oral application of MNG, SNG, or fluocinolone acetonide gel (FAG, positive control) was compared to that of the normal group. The body weight, food consumption, histology, Fourier transform infrared (FTIR) spectroscopy, inflammatory cytokines (tumor necrosis factor (TNF)-α and interleukin (IL)-1ß), and malondialdehyde (MDA) in the small intestine were monitored. The results showed decreased %body weight and food consumption in all 5-FU-injected groups compared with the normal group. The MNG and SNG treatments maintained the food consumption and the normal integrity of the small intestines, as evidenced by villus length and crypt depth, similar to the observations in the normal groups. The FTIR spectra showed no change in lipids of the MNG and SNG groups compared with the normal group. Moreover, SNG could reduce IL-1ß content to a level that was not different from the level in the normal groups. Therefore, the oral application of MNG and SNG could protect against 5-FU-induced small intestinal mucositis in mice.
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Liposomas/química , Melatonina/administración & dosificación , Mucositis/tratamiento farmacológico , Administración Oral , Animales , Fluorouracilo/toxicidad , Interleucina-1beta/metabolismo , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Intestino Delgado/patología , Peroxidación de Lípido/efectos de los fármacos , Masculino , Melatonina/química , Melatonina/farmacología , Ratones , Ratones Endogámicos ICR , Mucositis/inducido químicamente , Mucositis/patología , Tamaño de la Partícula , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
OBJECTIVES: In our study group of Thai PLT apheresis donors, we assessed the prevalence of anti-leucocyte antibodies. BACKGROUND: Antibodies against human leucocyte antigens (anti-HLA), neutrophil antigens (anti-HNA), and major histocompatibility complex class I related chain A (anti-MICA) in blood products can lead to transfusion-related acute lung injury (TRALI). To reduce the risk of TRALI, some blood centres are implementing strategies based on screening platelet (PLT) apheresis donors for the presence of anti-leucocyte antibodies. METHODS/MATERIALS: Blood samples were collected from non-transfused individuals, 340 males and 63 females (50 nulliparous and 13 parous). Anti-HLA class I and II and anti-MICA were analysed using the Luminex assay, and anti-HNA-3 was detected using the granulocyte agglutination test. RESULTS: Anti-HLA was found in 14 of 403 subjects (3.5%). Ten subjects (2.5%) tested positive for HLA class I, 2 (0.5%) for HLA class II, and 2 (0.5%) for both HLA class I and HLA class II. Anti-HLA class I or II were detected in 2 of 13 (15.4%) parous females and only anti-HLA class I was found in 4 (8.0%) nulliparous females. Six of 327 subjects tested (1.8%), all males, were positive for anti-MICA. Anti-HNA-3 was not found in any of the 403 individuals. CONCLUSIONS: Screening for anti-HLA class I and II should be implemented for Thai PLT apheresis donors. Although immunisation against HNA and MICA seems to be a rare event in Thais, further work is necessary to decide whether our PLT apheresis donors should be screened for HNA and MICA antibodies.
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Plaquetoferesis , Anticuerpos , Donantes de Sangre , Femenino , Antígenos HLA , Humanos , Masculino , Prevalencia , Lesión Pulmonar Aguda PostransfusionalRESUMEN
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.
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Linfocitos T CD4-Positivos , Masaje , Anciano , Estudios Cruzados , Humanos , Subgrupos de Linfocitos T , TailandiaRESUMEN
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.
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Anticuerpos Antihelmínticos/biosíntesis , Anticuerpos Antihelmínticos/inmunología , Simulación de Dinámica Molecular , Opisthorchis/inmunología , Biblioteca de Péptidos , Secuencia de Aminoácidos , Animales , Anticuerpos Antihelmínticos/química , Antígenos Helmínticos/química , Antígenos Helmínticos/inmunología , Sistema Biliar/inmunología , Sistema Biliar/parasitología , Bovinos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/inmunología , Ratones , Simulación del Acoplamiento Molecular , Miosinas/química , Reproducibilidad de los Resultados , Albúmina Sérica Bovina/análisisRESUMEN
This study aimed to assess the effect of Vitamin C on blood pressure (BP), and subsequently on oxidative stress and nitric oxide (NO) release, following the low-intensity exercise in the patients. This study included 24 patients with type 2 diabetes mellitus (T2D) (age, 53 ± 7 years; hemoglobin A1c, 10.1% ± 0.9%) randomized into two 6-week daily arms based on the consumption of either placebo or 1000 mg Vitamin C. The crossover trial occurred after a 6-week washout. Before and after both supplementation arms, all patients performed cycling exercise at 33% of peak oxygen consumption for 20 min. BP was measured before, immediately, and 60 min after the exercise. Blood samples were drawn immediately before and after the exercise to determine plasma ascorbate, malondialdehyde (MDA), F2-isoprostanes (F2-IsoPs), and NO concentrations. Data showed significant lower BP in the Vitamin C arm when compared with the placebo arm (systolic BP [SBP] P < 0.001 at every time point, diastolic BP [DBP] P < 0.001 except at immediately after exercise, P < 0.05). Plasma ascorbate concentration (P < 0.05 at every time point) and plasma NO (at resting P < 0.001, immediately after exercise P < 0.05) were significantly increased in the Vitamin C arm than in the placebo arm. Plasma MDA (P < 0.05 at every time point) and F2-IsoPs (P < 0.05 at every time point) concentrations were significantly lower in the Vitamin C arm than in the placebo arm. In addition, data showed significantly lower SBP (P < 0.001 at every time point), DBP (P < 0.001 except at immediately after exercise P < 0.05), plasma MDA (P < 0.001 at every time point), and F2-IsoPs (P < 0.05 at every time point) at post-supplementation than at pre-supplementation. Besides, there were significantly higher plasma ascorbate (P < 0.05 at every time point) and NO (at rest P < 0.01, immediately after exercise P < 0.05) concentrations at post-supplementation than at pre-supplementation. This is in contrast to the placebo treatment arm which demonstrated no statistical difference in all outcomes throughout the experiment. This study suggests that 6-week Vitamin C supplementation decreased preexercise and postexercise BPs, possibly due to improved oxidative stress and NO release. However, exercise had no effect on any outcome measures.
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Diabetes Mellitus Tipo 2 , F2-Isoprostanos , Antioxidantes , Ácido Ascórbico , Presión Sanguínea , Estudios Cruzados , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Suplementos Dietéticos , Ejercicio Físico , Humanos , Persona de Mediana Edad , Estrés OxidativoRESUMEN
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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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.
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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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This study investigated the effect of vitamin C on polymorphonuclear (PMN) cell functions in type 2 diabetes mellitus patients with poor glycemic control. We hypothesized that oral vitamin C treatment improves PMN cell functions. Patients (14) received either a vitamin C (1000 mg/d) or placebo (anhydrous calcium hydrogen phosphate) tablet for 6 weeks and were subjected to a 6-week washout period followed by a 6-week treatment crossover period. Blood samples were collected at pretreatment and posttreatment for PMN cell functions (by flow cytometry) and plasma vitamin C concentration. Phagocytosis was examined by incubating whole blood samples with fluorescein isothiocyanate-labeled Staphylococcus aureus, and oxidative burst was simultaneously evaluated by adding hydroethidine. In comparison with placebo, vitamin C increased both PMN cell phagocytosis (pretreatment: placebo, 17.8% ± 1.6% and vitamin C, 19.0% ± 3.4%, P = .70; posttreatment: placebo, 16.6% ± 1.7% and vitamin C, 27.1% ± 2.9%, P = .005) and oxidative burst (pretreatment: placebo, 6.4% ± 0.8% and vitamin C, 7.1% ± 1.2%, P = .60; posttreatment: placebo, 6.9% ± 1.3% and vitamin C, 12.1% ± 1.6%, P = .02). The plasma vitamin C concentration was elevated after vitamin C treatment as compared with that before treatment (P < .001) and was higher than that observed in the placebo treatment group (P < .01). Plasma vitamin C concentration and PMN cell functions were not significantly different before both treatments. We conclude that the 6-week 1000-mg/d vitamin C increased PMN phagocytosis and oxidative burst in type 2 diabetes mellitus patients with poor glycemic control.