Molecular Control of Follicular Helper T cell Development and Differentiation.

Follicular helper T cells (Tfh) are specialized helper T cells that are predominantly located in germinal centers and provide help to B cells. The development and differentiation of Tfh cells has been shown to be regulated by transcription factors, such as B-cell lymphoma 6 protein (Bcl-6), signal transducer and activator of transcription 3 (STAT3) and B lymphocyte-induced maturation protein-1 (Blimp-1). In addition, cytokines, including IL-21, have been found to be important for Tfh cell development. Moreover, several epigenetic modifications have also been reported to be involved in the determination of Tfh cell fate. The regulatory network is complicated, and the number of novel molecules demonstrated to control the fate of Tfh cells is increasing. Therefore, this review aims to summarize the current knowledge regarding the molecular regulation of Tfh cell development and differentiation at the protein level and at the epigenetic level to elucidate Tfh cell biology and provide potential targets for clinical interventions in the future.

Overexpression of Wilms' tumor 1 in skin lesions of psoriasis is associated with abnormal proliferation and apoptosis of keratinocytes.

Psoriasis vulgaris (PV) is a chronic inflammatory skin disease, which is characterized by the abnormal proliferation and apoptosis of keratinocytes. Previous studies have demonstrated that transcription factor Wilms' tumor 1 (WT1) is involved in a number of pathophysiological processes, including organ development, tumorigenesis and cell proliferation. However, the role of WT1 in PV remains unclear. In the present study, WT1 expression was analyzed by reverse transcription­quantitative polymerase chain reaction and western blot analyses. WT1 was stably overexpressed or inhibited in HaCaT cells using Lipofectamine® 2000, and cell proliferation and apoptosis were determined using a Cell Counting Kit­8 or Fluorescein Isothiocyanate Annexin V Apoptosis Detection kit II, respectively. We demonstrated that compared with normal controls, the mRNA and protein expression levels of WT1 were significantly increased in non­lesional skins (human, P<0.0001 and P=0.0291, respectively; mouse, P=0.0020 and P=0.0150, respectively) and lesional skins (human, P<0.0001 and P=0.0060, respectively; mouse, P=0.0010 and P=0.0172, respectively) of patients with PV, in addition to the imiquimod (IMQ)­induced psoriasis­like mouse model. WT1 mRNA and protein expression levels in lesional skins were slightly increased compared with those in non­lesional skins from patients with psoriasis (P=0.2510 and P=0.1690, respectively) and IMQ­treated mice (P=0.9590 and P=0.2552, respectively), although there were no statistical differences. Knockdown of WT1 inhibited the proliferation of HaCaT cells [day (D)4, P=0.0454; D5, P=0.0021] and promoted their apoptosis (P=0.0007), while overexpressing WT1 exhibited the opposite effects (proliferation D3, P=0.0216; D4, P=0.0356; D5, P=0.0188; apoptosis, P=0.0003). Furthermore, it was identified that the inflammatory cytokines interleukin­17A (IL­17A), interferon­Î³ and IL­22 induced the overexpression of WT1 in HaCaT cells. The results of the present study suggested that inflammatory cytokine­induced WT1 overexpression may promote the formation of psoriatic skin lesions via regulation of the proliferation and apoptosis of keratinocytes.

Oral JS-38, a metabolite from Xenorhabdus sp., has both anti-tumor activity and the ability to elevate peripheral neutrophils.

AIM: JS-38 (mitothiolore), a synthetic version of a metabolite isolated from Xenorhabdus sp., was evaluated for its anti-tumor and white blood cell (WBC) elevating activities. METHOD: These anti-proliferative activities were assessed in vitro using a panel of ten cell lines. The anti-tumor activities were tested in vivo using B16 allograft mouse models and xenograft models of A549 human lung carcinoma and QGY human hepatoma in nude mice. The anti-tumor interactions of JS-38 and cyclophosphamide (CTX) or 5-fluorouracil (5-Fu) were studied in a S180 sarcoma model in ICR mice. Specific stimulatory effects were determined on peripheral neutrophils in normal and CTX- and 5-Fu-induced neutropenic mice. RESULTS: The IC50 values ranged from 0.1 to 2.0 µmol·L(-1). JS-38 (1 µmol·L(-1)) caused an increase in A549 tumor cell apoptosis. Multi-daily gavage of JS-38 (15, 30, and 60 mg·kg(-1)·d(-1)) inhibited in vivo tumor progression without a significant effect on body weight. JS-38 additively enhanced the in vivo anti-tumor effects of CTX or 5-Fu. JS-38 increased peripheral neutrophil counts and neutrophil rates in normal BALB/c mice almost as effectively as granulocyte colony-stimulating factor (G-CSF). In mice with neutropenia induced by CTX or 5-Fu, JS-38 rapidly restored neutrophil counts. CONCLUSION: These results suggest that JS-38 has anti-tumor activity, and also has the ability to increase peripheral blood neutrophils.

Development and validation of a rapid and sensitive liquid chromatography-tandem mass spectrometry method for benvitimod quantification in human plasma.

Benvitimod is a newly synthesized non-steroid small molecule being developed as a candidate drug for the treatment of inflammatory skin diseases. Here a rapid, sensitive and specific high performance liquid chromatography-tandem mass spectrometry (LC/ESI/MS/MS) method was developed for the determination of benvitimod in human plasma. The samples were alkalified with disodium tetraborate firstly, and then extracted by methyl tert-butyl ether. Fluorophenyl-benvitimod was used as internal standard (I.S.). Chromatographic separation was performed on an Ultra C(18) column (150mm×2.1mm, 5.0µm). The mixed mobile phase delivered at 300µl/min was CH3CN/H2O, 76.65:23.35 (v/v), containing 0.2mmol/L NH(4)COOH. Detection and quantitation was performed by electrospray ionization (ESI) and multiple reaction monitoring (MRM) in the negative ion mode. The most intense [M-H](-) MRM transition of benvitimod at m/z 253.1→211.0 was used for benvitimod quantitation and the transition at m/z 270.9→229.2 was used to monitor I.S. The calibration curve was linear within the concentration range of 0.1-10.0ng/mL (r>0.99). The lower limit of quantification (LLOQ) was 0.1ng/mL. The extraction recovery was above 80%. The accuracy expressed as relative error (RE) was less than 1.03%. The intra- and inter-day precisions were less than 11.81%. The freeze-thaw stability was also investigated and it was found that both benvitimod and the I.S. were quite stable. This method is especially useful for the pharmacokinetic study of benvitimod.