RESUMEN
Follicular helper T (Tfh) cells are crucial for humoral immunity. Dysregulation of Tfh cell differentiation can cause infectious, allergic, and autoimmune diseases. To elucidate the molecular mechanisms underlying Tfh cell differentiation, we attempted to establish an in vitro mouse model of Tfh cell differentiation in the absence of other cell types. Various cytokines and cell surface molecules are suggested to contribute to the differentiation. We found that stimulating naïve CD4+ T cells with immobilized antibodies to CD3, ICOS, and LFA-1 in the presence of soluble anti-CD28 antibody, IL-6, and antibodies that block IL-2 signaling for 3 days induced the expression of Bcl6 and Rorc(γt), master regulator genes of Tfh and Th17 cells, respectively. TGF-ß significantly enhanced cell proliferation and Bcl6 and Rorc(γt) expression. An additional 2 days of culture without immobilized antibodies selectively downregulated Rorc(γt) expression. These cells produced IL-21 and promoted B cells to produce IgG antibodies. Adding the aryl hydrocarbon receptor (AhR) antagonist CH-223191 to the T cell culture further downregulated Rorc(γt) expression without significantly affecting Bcl6 expression, and upregulated expression of a key Tfh marker, CXCR5. Although their CXCR5 expression levels were still not high, the CH-223191-treated cells showed chemotactic activity towards the CXCR5 ligand CXCL13. On the other hand, AhR agonists upregulated Rorc(γt) expression and downregulated CXCR5 expression. These findings suggest that AhR activity and the duration of T cell receptor stimulation contribute to regulating the balance between Tfh and Th17 cell differentiation. Although this in vitro system needs to be further improved, it may be useful for elucidating the mechanisms of Tfh cell differentiation as well as for screening physiological or pharmacological factors that affect Tfh cell differentiation including CXCR5 expression.
Asunto(s)
Interleucina-6 , Linfocitos T Colaboradores-Inductores , Animales , Ratones , Interleucina-6/metabolismo , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismo , Anticuerpos Inmovilizados , Diferenciación Celular , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores CXCR5/genética , Receptores CXCR5/metabolismoRESUMEN
The PCR technique is indispensable in biology and medicine, but some difficulties are associated with its use, including false positive or false negative amplifications. To avoid these issues, a non-amplification nucleic acid detection protocol is needed. In the present study, we propose a method in which nucleic-acid probe hybridization is combined with thio-NAD cycling to detect nucleic acids without amplification. We report our application of this method for the detection of the gene of MPT64 in Mycobacterium tuberculosis. Two different cDNA probes targeted the mpt64 gene: the first probe was used to immobilize the mpt64 gene, and the second probe, linked with alkaline phosphatase (ALP), was hybridized to a target sequence in the mpt64 gene. A substrate was then hydrolyzed by ALP, and a cycling reaction was conducted by a dehydrogenase with its co-factors (thio-NAD and NADH). The single-stranded DNA, double-stranded DNA, plasmid DNA for the mpt64 gene, and whole genome of M. tuberculosis var. BCG were detected at the level of 105-106 copies/assay, whereas the non-tuberculosis mycobacteria (e.g., M. avium, M. intracellulare, M. kansasii, and M. abscessus) were below the limits of detection. The present method enables us to avoid the errors inherent in nucleic acid amplification methods.
Asunto(s)
Mycobacterium tuberculosis , NAD , Mycobacterium tuberculosis/genética , Micobacterias no Tuberculosas/genética , Técnicas de Amplificación de Ácido Nucleico , Hibridación de Ácido Nucleico , ADN Bacteriano/genética , ADN Bacteriano/análisisRESUMEN
Objective: The chronic kidney disease (CKD) is widely diagnosed on the basis of albuminuria and the glomerular filtration rate. A more precise diagnosis of CKD, however, requires the assessment of other factors. Urinary adiponectin recently attracted attention for CKD assessment, but evaluation is difficult due to the very low concentration of urinary adiponectin in normal subjects. Research design and methods: We developed an ultrasensitive ELISA coupled with thionicotinamide-adenine dinucleotide cycling to detect trace amounts of proteins, which allows us to measure urinary adiponectin at the subattomole level. We measured urinary adiponectin levels in 59 patients with diabetes mellitus (DM) and 24 subjects without DM (normal) to test our hypothesis that urinary adiponectin levels increase with progression of CKD due to DM. Results: The urinary adiponectin levels were 14.88±3.16 (ng/mg creatinine, mean±SEM) for patients with DM, and 3.06±0.33 (ng/mg creatinine) for normal subjects. The threshold between them was 4.0 ng/mg creatinine. The urinary adiponectin levels increased with an increase in the CKD risk. Furthermore, urinary adiponectin mainly formed a medium-molecular weight multimer (a hexamer) in patients with DM, whereas it formed only a low-molecular weight multimer (a trimer) in normal subjects. That is, the increase in urinary adiponectin in patients with DM led to the emergence of a medium-molecular weight form in urine. Conclusions: Our new assay showed that urinary adiponectin could be a new diagnostic index for CKD. This assay is a non-invasive test using only urine, thus reducing the patient burden.