RESUMO
Adaptive immune responses protect against infection with dengue virus (DENV), yet cross-reactivity with distinct serotypes can precipitate life-threatening clinical disease. We found that clonotypes expressing the T cell antigen receptor (TCR) ß-chain variable region 11 (TRBV11-2) were 'preferentially' activated and mobilized within immunodominant human-leukocyte-antigen-(HLA)-A*11:01-restricted CD8+ T cell populations specific for variants of the nonstructural protein epitope NS3133 that characterize the serotypes DENV1, DENV3 and DENV4. In contrast, the NS3133-DENV2-specific repertoire was largely devoid of such TCRs. Structural analysis of a representative TRBV11-2+ TCR demonstrated that cross-serotype reactivity was governed by unique interplay between the variable antigenic determinant and germline-encoded residues in the second ß-chain complementarity-determining region (CDR2ß). Extensive mutagenesis studies of three distinct TRBV11-2+ TCRs further confirmed that antigen recognition was dependent on key contacts between the serotype-defined peptide and discrete residues in the CDR2ß loop. Collectively, these data reveal an innate-like mode of epitope recognition with potential implications for the outcome of sequential exposure to heterologous DENVs.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Reações Cruzadas/imunologia , Vírus da Dengue/imunologia , Mutação em Linhagem Germinativa/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Imunidade Adaptativa/genética , Imunidade Adaptativa/imunologia , Sequência de Aminoácidos , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Dengue/genética , Dengue/imunologia , Dengue/virologia , Vírus da Dengue/classificação , Vírus da Dengue/genética , Epitopos de Linfócito T/química , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Antígenos HLA-A/química , Antígenos HLA-A/genética , Antígenos HLA-A/imunologia , Humanos , Modelos Moleculares , Estrutura Terciária de Proteína , Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Serina Endopeptidases/genética , Serina Endopeptidases/imunologia , Sorotipagem , Ressonância de Plasmônio de SuperfícieRESUMO
This study aimed to assess the prevalence and associated risk factors for extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae (ESBL-EK) acquisition among patients staying in medical and surgical Intensive Care Units (ICU) in Northern Thailand. Rectal swabs were collected from 206 ICU patients upon admission and discharge. Overall, the ESBL-EK acquisition rate among patients during ICU stay was 29.6%. Acquisition rate was significantly higher for K. pneumoniae (20.9%) than that of E. coli (12.1%) (p = 0.024). Multivariate logistic regression analysis identified the use of third generation cephalosporin (p = 0.008) as a risk factor for ESBL-EK acquisition. Sixty-eight ESBL-EK isolates (25 E. coli and 43 K. pneumoniae) were recovered. The majority of ESBL-EK isolates (≥88%) were resistant to ceftazidime, cefepime and aztreonam. Fifty-two acquired ESBL-EK isolates (76.5%) were positive for blaCTX-M and 4 K. pneumoniae isolates simultaneously carried blaNDM-1. Our results reveal that ICU patients could acquire ESBL-EK during hospitalization and the use of third generation cephalosporin should be strictly controlled to prevent the acquisition of ESBL-EK among ICU patients.
Assuntos
Infecções por Escherichia coli , Infecções por Klebsiella , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Escherichia coli , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/epidemiologia , Humanos , Unidades de Terapia Intensiva , Infecções por Klebsiella/tratamento farmacológico , Infecções por Klebsiella/epidemiologia , Klebsiella pneumoniae , Testes de Sensibilidade Microbiana , Tailândia , beta-LactamasesRESUMO
This study was conducted to investigate the prevalence of and risk factors for colonization and acquisition of carbapenem-resistant (CR) Gram-negative bacteria (GNB) among patients admitted to intensive care units (ICUs) in two tertiary care hospitals in northern Thailand. Screening of rectal swab specimens for CR-GNB was performed on patients at ICU admission and discharge. The phenotypes and genotypes of all isolates were determined. Risk factors were analyzed by logistic regression analysis. The overall carriage rate of CR-GNB at admission was 11.6% (32/275), with the most predominant species carried being Acinetobacter baumannii (n = 15), followed by Klebsiella pneumoniae (n = 9). The risk factor for CR-GNB colonization was hospitalization within the previous 6 months (P = 0.002). During the ICU stay, the rate of CR-GNB acquisition was 25.2% (52/206), with the most predominant species carried being A. baumannii (n = 28) and K. pneumoniae (n = 13). Risk factors associated with CR-GNB acquisition were the use of an enteral feeding tube (P = 0.008) and administration of third-generation cephalosporins (P = 0.032) and carbapenems (P = 0.045). The most common carbapenemase genes in A. baumannii and K. pneumoniae were blaOXA-23/51 and blaNDM, respectively. Patient-to-patient transmission was demonstrated in three cases, resulting in the acquisition of CR A. baumannii (2 cases) and K. pneumoniae (1 case) isolates from other patients who were admitted during the same period of time. This is the first Indochinese study screening patients, examining patients for the carriage of CR-GNB, and further demonstrating the transfer of CR-GNB isolates in ICUs. Our study suggests that effective infection control measures are required to limit the spread of CR-GNB within hospitals.
Assuntos
Infecções por Acinetobacter/tratamento farmacológico , Antibacterianos/uso terapêutico , Carbapenêmicos/uso terapêutico , Infecção Hospitalar/tratamento farmacológico , Gastroenteropatias/tratamento farmacológico , Infecções por Klebsiella/tratamento farmacológico , Resistência beta-Lactâmica , Infecções por Acinetobacter/epidemiologia , Infecções por Acinetobacter/microbiologia , Infecções por Acinetobacter/transmissão , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/crescimento & desenvolvimento , Acinetobacter baumannii/patogenicidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/microbiologia , Infecção Hospitalar/transmissão , Feminino , Gastroenteropatias/epidemiologia , Gastroenteropatias/microbiologia , Humanos , Unidades de Terapia Intensiva , Infecções por Klebsiella/epidemiologia , Infecções por Klebsiella/microbiologia , Infecções por Klebsiella/transmissão , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/crescimento & desenvolvimento , Klebsiella pneumoniae/patogenicidade , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Fatores de Risco , Centros de Atenção Terciária , Tailândia/epidemiologiaRESUMO
The cytotoxicity of the ethyl acetate fraction of the Calotropis gigantea (L.) Dryand. (C. gigantea) stem bark extract (CGEtOAc) has been demonstrated in many types of cancers. This study examined the improved cancer therapeutic activity of sorafenib when combined with CGEtOAc in HepG2 cells. The cell viability and cell migration assays were applied in HepG2 cells treated with varying concentrations of CGEtOAc, sorafenib, and their combination. Flow cytometry was used to determine apoptosis, which corresponded with a decline in mitochondrial membrane potential and activation of DNA fragmentation. Reactive oxygen species (ROS) levels were assessed in combination with the expression of the phosphatidylinositol-3-kinase (PI3K)/ protein kinase B (Akt)/ mammalian target of rapamycin (mTOR) pathway, which was suggested for association with ROS-induced apoptosis. Combining CGEtOAc at 400 µg/mL with sorafenib at 4 µM, which were their respective half-IC50 concentrations, significantly inhibited HepG2 viability upon 24 h of exposure in comparison with the vehicle and each single treatment. Consequently, CGEtOAc when combined with sorafenib significantly diminished HepG2 migration and induced apoptosis through a mitochondrial-correlation mechanism. ROS production was speculated to be the primary mechanism of stimulating apoptosis in HepG2 cells after exposure to a combination of CGEtOAc and sorafenib, in association with PI3K/Akt/mTOR pathway suppression. Our results present valuable knowledge to support the development of anticancer regimens derived from the CGEtOAc with the chemotherapeutic agent sorafenib, both of which were administered at half-IC50, which may minimize the toxic implications of cancer treatments while improving the therapeutic effectiveness toward future medical applications.
Assuntos
Acetatos , Calotropis , Neoplasias Hepáticas , Humanos , Sorafenibe/farmacologia , Sorafenibe/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células Hep G2 , Calotropis/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Casca de Planta/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Apoptose , Linhagem Celular Tumoral , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Calotropis gigantea (L.) Dryand. (C. gigantea) is a traditional medicinal plant, recognized for its effectiveness in managing diabetes, along with its notable antioxidant, anti-inflammatory, and anticancer properties. Type II diabetes mellitus (T2DM) is characterized by chronic metabolic disorders associated with an elevated risk of hepatocellular carcinoma (HCC) due to hyperglycemia and impaired insulin response. The scientific validation of C. gigantea's ethnopharmacological efficacy offers advantages in alleviating cancer progression in T2DM complications, enriching existing knowledge and potentially aiding future clinical cancer treatments. AIM: This study aimed to investigate the preventive potential of the dichloromethane fraction of C. gigantea stem bark extract (CGDCM) against diethylnitrosamine (DEN)-induced HCC in T2DM rats, aiming to reduce cancer incidence associated with diabetes while validating C. gigantea's ethnopharmacological efficacy. MATERIALS AND METHODS: Spontaneously Diabetic Torii (SDT) rats were administered DEN to induce HCC (SDT-DEN-VEH), followed by treatment with CGDCM. Metformin was used as a positive control (SDT-DEN-MET). All the treatments were administered for 10 weeks after the initial DEN injection. Diabetes-related parameters, including serum levels of glucose, insulin, and glycosylated hemoglobin (HbA1c), as well as liver function enzymes (aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and gamma-glutamyl transferase), were quantified. Serum inflammation biomarkers interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were evaluated. Liver tissue samples were analyzed for inflammation protein expression (IL-6, TNF-α, transforming growth factor-ß1 (TGF-ß1), and α-smooth muscle actin (α-SMA)). Histopathological evaluation was performed to assess hepatic necrosis, inflammation, and fibrosis. Liver cell proliferation was determined using immunohistochemistry for Ki-67 expression. RESULTS: Rats with SDT-DEN-induced HCC treated with CGDCM exhibited reduced serum glucose levels, elevated insulin levels, and decreased HbA1c levels. CGDCM treatment also reduced elevated hepatic IL-6, TNF-α, TGF-ß1, and α-SMA levels in SDT-DEN-VEH rats. Additionally, CGDCM treatment prevented hepatocyte damage, fibrosis, and cell proliferation. No adverse effects on normal organs were observed with CGDCM treatment, suggesting its safety for the treatment of HCC complications associated with diabetes. Additionally, the absence of adverse effects in SD rats treated with CGDCM at 2.5 mg/kg further supports the notion of its safe usage. CONCLUSIONS: These findings suggest that C. gigantea stem bark extract exerts preventive effects against the development of HCC complications in patients with T2DM, expanding the potential benefits of its ethnopharmacological advantages.
Assuntos
Calotropis , Diabetes Mellitus Experimental , Dietilnitrosamina , Insulina , Cloreto de Metileno , Casca de Planta , Extratos Vegetais , Animais , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Casca de Planta/química , Masculino , Ratos , Dietilnitrosamina/toxicidade , Cloreto de Metileno/química , Insulina/sangue , Calotropis/química , Diabetes Mellitus Experimental/tratamento farmacológico , Neoplasias Hepáticas/prevenção & controle , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/patologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Hipoglicemiantes/farmacologia , Hipoglicemiantes/isolamento & purificação , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/prevenção & controle , Fígado/efeitos dos fármacos , Fígado/patologia , Fígado/metabolismo , Glicemia/efeitos dos fármacos , Caules de Planta/química , Neoplasias Hepáticas Experimentais/prevenção & controle , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Neoplasias Hepáticas Experimentais/patologiaRESUMO
BACKGROUND: The first COVID-19 vaccination campaign in Thailand began in April 2020, with healthcare workers receiving two doses of inactivated COVID-19 vaccine (CoronaVac). However, the emergence of the delta and omicron variants raised concerns about vaccine effectiveness. The Thai Ministry of Public Health provided the first booster dose (third dose) and second booster dose (fourth dose) of the mRNA vaccine (BNT162b2) for healthcare workers. This study investigated the immunity and adverse reactions elicited by a heterologous second booster dose of BNT162b2 after a two-dose CoronaVac vaccination for COVID-19 in healthcare workers of the Faculty of Medicine, Naresuan University. METHODS: IgG titres against the SARS-CoV-2-spike protein were measured four and 24 weeks after the second booster dose of BNT162b2 in the study participants. Adverse reactions were recorded during the first three days, four weeks and 24 weeks after the second booster dose of BNT162b2. RESULTS: IgG against the SARS-CoV-2-spike protein was positive (>10 U/ml) in 246 of 247 participants (99.6 %) at both four and 24 weeks after the second booster dose of BNT162b2. The median specific IgG titres at four and 24 weeks after the second booster dose of BNT162b2 were 299 U/ml (min: 2, max: 29,161) and 104 U/ml (min: 1, max: 17,920), respectively. The median IgG level declined significantly 24 weeks after the second booster dose of the BNT162b2 vaccine. Of the 247 participants, 179 (72.5 %) experienced adverse reactions in the first three days after the second booster dose of BNT162b2. Myalgia, fever, headache, injection site pain and fatigue were the most common adverse reactions. CONCLUSION: This study demonstrated that a heterologous second booster dose of BNT162b2 after two doses of CoronaVac induced elevated IgG against the SARS-CoV-2-spike protein and caused minor adverse reactions in healthcare workers of the Faculty of Medicine, Naresuan University. This study was registered as Thailand Clinical Trials No. TCTR20221112001.
Assuntos
Vacina BNT162 , Vacinas contra COVID-19 , COVID-19 , Humanos , Anticorpos Antivirais , COVID-19/prevenção & controle , Vacinas contra COVID-19/efeitos adversos , Docentes , Pessoal de Saúde , Imunoglobulina G , Mialgia , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Universidades , Vacinação/efeitos adversosRESUMO
Calotropis gigantea stem bark extract, particularly the dichloromethane fraction (CGDCM), demonstrated the most potent antiproliferative effects on hepatocellular carcinoma HepG2 and colorectal HCT116 cells. The current study focused on enhancing the effectiveness of cancer treatment with CGDCM at concentrations close to the IC50 in HCT116 cells by reducing their nutrient supply. CGDCM (2, 4, and 8 µg/mL) treatment for 24 h under glucose conditions of 4.5 g/L without fetal bovine serum (FBS) supplementation or serum starvation (G+/F-), glucose 0 g/L with 10% FBS or glucose starvation (G-/F+), and glucose 0 g/L with 0% FBS or complete starvation (G-/F-) induced a greater antiproliferative effect in HCT116 cells than therapy in complete medium with glucose 4.5 g/L and 10% FBS (G+/F+). Nonetheless, the anticancer effect of CGDCM at 4 µg/mL under (G-/F-) showed the highest activity compared to other starvation conditions. The three starvation conditions showed a significant reduction in cell viability compared to the control (G+/F+) medium group, while the inhibitory effect on cell viability did not differ significantly among the three starvation conditions. CGDCM at 4 µg/mL in (G-/F-) medium triggered apoptosis by dissipating the mitochondrial membrane potential and arresting cells in the G2/M phase. This investigation demonstrated that a decrease in intracellular ATP and fatty acid levels was associated with enhanced apoptosis by treatment with CGDCM at 4 µg/mL under (G-/F-) conditions. In addition, under (G-/F-), CGDCM at 4 µg/mL increased levels of reactive oxygen species (ROS) and was suggested to primarily trigger apoptosis in HCT116 cells. Thus, C. gigantea extracts may be useful for the future development of alternative, effective cancer treatment regimens.
RESUMO
The 95% ethanolic extract of the dry powder of Calotropis gigantea (C. gigantea) stem bark was separated by fractionation with different solutions to yield 4 fractions: dichloromethane (CGDCM), ethyl acetate (CGEtOAc), and water (CGW). This research focused on CGDCM-induced apoptosis in HepG2 cells with IC50 and above-IC50 values, which provide useful information for future anticancer applications. CGDCM had lower cytotoxicity on normal lung fibroblast IMR-90 cells than on HepG2 cells. Apoptotic induction of CGDCM was mediated by decreased fatty acid and ATP synthesis while increasing reactive oxygen species production. The effects of the four extracts on the activity of the four major CYP450 isoforms (CYP1A2, CYP2C9, CYP2E1 and CYP3A4) were determined using the CYP-specific model activity of each isoform. All four fractions of the extract were shown to be poor inhibitors of CYP1A2 and CYP2E1 (IC50 > 1000 µg/mL) and moderate inhibitors of CYP3A4 (IC50 = 56.54-296.9 µg/mL). CGDCM and CGW exerted moderate inhibition activities on CYP2C9 (IC50 = 59.56 and 46.38 µg/mL, respectively), but CGEtOH and CGEtOAc exhibited strong inhibition activities (IC50 = 12.11 and 20.43 µg/mL, respectively). It is proposed that C. gigantea extracts at high doses have potential for further studies to develop alternative anticancer applications. Inhibiting CYP2C9 activity may also lead to drug-herb interactions.
RESUMO
Binding of platelet-derived growth factor-BB (PDGF-BB) to its cognate receptor (PDGFR) promotes lens epithelial cell (LEC) proliferation and migration. After cataract surgery, these LEC behaviors have been proposed as an influential cause of posterior capsule opacification (PCO). Stimulated PDFGR undergoes dimerization and tyrosine phosphorylation providing docking sites for a SH2-domain-containing noncatalytic region of tyrosine kinase (Nck). Nck is an adaptor protein acting as a linker of the proximal and downstream signaling events. However, the functions of Nck1 protein in LEC have not been investigated so far. We reported here a crucial role of Nck1 protein in regulating PDGFR-mediated LEC activation using LEC with a silenced expression of Nck1 protein. The knockdown of Nck1 suppressed PDGF-BB-stimulated LEC proliferation and migration and disrupted the cell cycle progression especially G1/S transition. LEC lacking Nck1 protein failed to exhibit actin polymerization and membrane protrusions. The downregulation of Nck1 protein in LEC impaired PDGFR-induced phosphorylation of intracellular signaling proteins, including Erk1/2, Akt, CREB and ATF1, which resulted in inhibition of LEC responses. Therefore, these data suggest that the loss of Nck1 expression may disturb LEC activation and Nck1 may potentially be a drug target to prevent PCO and lens-related disease.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Becaplermina , Células Epiteliais/metabolismo , Proteínas Oncogênicas/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Opacificação da Cápsula/etiologia , Linhagem Celular , Proliferação de Células , Células Epiteliais/citologia , Inativação Gênica , Humanos , Cristalino/citologia , Proteínas Oncogênicas/genética , Fosforilação , Transdução de SinaisRESUMO
BACKGROUND: The non-catalytic region of tyrosine kinase (Nck) is an adaptor protein, which is ubiquitously expressed in many types of cells. In T cells, the Nck1 isoform promotes T cell receptor signalling as well as actin polymerisation. However, the role of Nck1 in the lipid metabolism in T cells is unknown. In the present study, we investigated the effect of the Nck1 protein and Nck-CD3 interaction on lipid metabolism and on the physical and biological properties of Jurkat T cells, using a newly developed holotomographic microscope. RESULTS: Holotomographic microscopy showed that Nck1-knocked-out cells had membrane blebs and were irregular in shape compared to the rounded control cells. The cell size and volume of Nck1-deficient cells were comparable to those of the control cells. Nck1-knocked-out Jurkat T cells had a greater lipid content, lipid mass/cell mass ratio, and lipid metabolite levels than the control cells. Interestingly, treatment with a small molecule, AX-024, which inhibited Nck-CD3 interaction, also caused an increase in the lipid content in wild-type Jurkat T cells, as found in Nck1-deficient cells. CONCLUSIONS: Knockout of Nck1 protein and hindrance of the Nck-CD3 interaction cause the elevation of lipid content in Jurkat T cells.