Renbing Jia

Accumulation of FoxP3‐expressing CD4+CD25+ T cells with distinct chemokine receptors in synovial fluid of patients with active rheumatoid arthritis

Objective: To explore the presence and characteristics of FoxP3‐expressing CD4+CD25+ regulatory T cells in synovial fluid (SF) of patients with active rheumatoid arthritis (RA). Methods: The frequency and chemokine receptors expression profile of FoxP3‐expressing CD4+CD25+ regulatory T cells in SF and peripheral blood (PB) from RA patients and PB from healthy controls were investigated by flow cytometry using three‐ or four‐colour intracellular staining. Results: The frequency of CD4+CD25+ FoxP3+ T cells was increased significantly in SF compared with paired PB from RA patients and PB from healthy controls (p<0.05). However, the frequency in PB from RA patients was significantly lower than in PB from healthy controls (p<0.05). Notably, CD4+CD25+FoxP3+ T cells in SF expressed increased levels of inflammation‐related trafficking chemokine receptors, such as CCR4, CCR5, and CXCR4. Conclusion: There is an accumulation of FoxP3‐expressing regulatory T cells in RA SF, and such recruitment may be dependent on the distinct chemokine receptors expressed on regulatory T cells.

Microarray-based analysis: identification of hypoxia-regulated microRNAs in retinoblastoma cells.

Hypoxia is an essential feature of retinoblastoma and contributes to poor prognosis and resistance to conventional therapy. MicroRNAs (miRNAs) are small non-coding RNAs involved in a wide variety of biological processes, including cell differentiation, proliferation, death and metabolism. However, the relationship between hypoxia and the expression of miRNAs in retinoblastoma is not well understood. In this study, we aimed to analyze the pattern of miRNA expression in a retinoblastoma cell line under hypoxic conditions and to identify the miRNAs regulated by hypoxia, as well as their possible functions. miRNA expression profiling in retinoblastoma cells (HXO-RB44) under normal and hypoxic conditions was assessed by microarray techniques. The differentially expressed miRNAs were subjected to bioinformatic analyses to predict and categorise the key miRNAs and their target genes. A quantitative real-time RT-PCR approach was used to validate their expression. A Cell Counting kit was used to evaluate the functional significance of miR-181b in RB cell proliferation. There were 46 miRNAs that changed expression more than 2-fold in response to hypoxia (34 up-regulated and 12 down-regulated). We identified a cluster of miRNAs that includes miR-181b, miR-125a-3p, miR-30c-2, miR-497 and miR-491-3p as hypoxia-regulated miRNAs (HRMs) in retinoblastoma cells, of which miR-181b was the most typically differentially expressed miRNA under hypoxic conditions. Functionally, these HRMs are involved in apoptosis, cell adhesion, cell proliferation and mRNA processing, all processes that associate closely with the hypoxia response of cancer cells. Additionally, we found that administration of miR-181b inhibitor can suppress proliferation of retinoblastoma cells. These findings provide the first evidence that miRNAs play an important role in the hypoxia response of retinoblastoma cells. MiR-181b, the most typically up-regulated miRNA may aid in future clinical intervention of retinoblastoma.

WWOX-mediated apoptosis in A549 cells mainly involves the mitochondrial pathway

The human WWOX gene, known as WW domain-containing oxidoreductase, is located on 16q23.3-24.1, a chromosome region that spans the common fragile site, FRA16D. Abnormal transcripts or even loss of expression are frequently found in a number of cancer cell types, including breast, ovarian, prostate and lung cancer cells. It has therefore been proposed that the WWOX gene encodes a candidate tumor suppressor, possibly a pro-apoptotic protein. However, the mechanism behind this is not entirely clear. In the present study, we examined the pro-apoptotic action of WWOX using transient expression in A549 cells. We observed that the ectopic expression of WWOX caused apoptosis in A549 cells. We further observed procaspase-3 and procaspase-9 activation and the release of cytochrome C from the mitochondria in A549 cells transfected with pcDNA3.0-WWOX. These data indicate that WWOX induces apoptosis in A549 cells via the mitochondrial pathway.

Pingyangmycin as First-Line Treatment for Low-Flow Orbital or Periorbital Venous Malformations: Evaluation of 33 Consecutive Patients

IMPORTANCE Low-flow orbital or periorbital venous malformation (OVM) is the most common periorbital vascular lesion that may produce an appearance defect, visual dysfunction, internal hemorrhage, and thrombosis. Intralesional injection of pingyangmycin as a minimally invasive, gentle intervention may have better outcomes in treating low-flow OVMs compared with other currently used methods. OBJECTIVE To investigate the efficacy and safety of intralesional injection of pingyangmycin for treatment of low-flow OVM. DESIGN, SETTING, AND PARTICIPANTS A retrospective, noncomparative, interventional case series was conducted in a single medical center. Thirty-three consecutive patients with low-flow OVMs undergoing intralesional injection of pingyangmycin were included in the study. INTERVENTIONS Injections of 1 to 5 mL of a pingyangmycin 1.5-mg/mL mixture with lidocaine hydrochloride, 2%, were given. Each patient received 1 to 4 injections at an interval of 6 to 8 weeks between February 2002 and January 2013. Mixture volume was determined on a basis of 0.5 mL of solution per cubic centimeter of the lesion. The maximum dose for 1 injection was 8 mg. Clinical observations were well documented before and after treatment. MAIN OUTCOMES AND MEASURES Reduction of lesion volume based on ultrasound-measured volume; overall appearance, including blue color and thickness of lesions before and after treatment; and adverse events were evaluated. RESULTS Patients received a median of 2 (range, 1-4) intralesional injections of pingyangmycin. The mean pretreatment volume was 4.4 cm3 and posttreatment volume was 1.0 cm3 (t = 4.63; P < .001), with a mean decrease of 84% (range, 28%-100%). Marked to moderate improvement in the volume of the lesions was noticed in 31 eyes (94%; 25 of 33 [76%] with marked improvement and 6 of 33 [18%] with moderate improvement). Improvement occurred in 95% (18 of 19) of superficial lesions, 100% (3 of 3) of deep lesions, and 91% (10 of 11) of combined lesions. We noticed significant improvements in blue color and thickness on the basis of investigator scores from clinical photographs taken before and after treatment. None of the patients had recurrence noted at their final follow-up. Adverse events were limited to swelling of the conjunctiva and localized subcutaneous atrophy. CONCLUSIONS AND RELEVANCE The results of intralesional pingyangmycin injection for treatment of low-flow OVM are encouraging and associated with a low risk of adverse events.

Inhibition of retinoblastoma in vitro and in vivo with conditionally replicating oncolytic adenovirus H101.

PURPOSE To determine the therapeutic effect of oncolytic adenovirus H101 on retinoblastoma in vitro and in vivo. METHODS The expression of coxsackievirus-adenovirus receptor (CAR) in human retinoblastoma cell line HXO-RB(44) was determined by RT-PCR, Western blot, immunofluorescence, and immunocytochemistry staining. Appropriate multiplicity of infection was determined using flow cytometry in retinoblastoma cells with green fluorescent protein-expressing adenovirus (AdGFP). The viability of HXO-RB(44) cells treated with H101 or AdGFP was measured using a cell counting kit-8-based procedure. Viral proliferation in vitro was measured by end point dilution titration and real-time PCR. Cell cycle and apoptotic activity of HXO-RB(44) were analyzed by flow cytometry. NOD-SCID mice bearing retinoblastoma xenografts were treated with intratumoral injection of H101, AdGFP, or PBS. Tumor volume and survival time were recorded. Immunohistochemistry for adenoviral fiber protein and Western blot for adenoviral Hexon protein of retinoblastoma xenografts were performed to evaluate H101 virus replication in vivo. RESULTS HXO-RB(44) cells expressed CAR and were sensitive to adenoviral infection. HXO-RB(44) cells treated with H101 had reduced cell viability compared with AdGFP-treated cells (P < 0.01). Abundant replication of H101 in HXO-RB(44) cells resulted in G(2)/M-phase arrest and finally tumor cell lysis, but the apoptosis pathway was not activated. Tumor-bearing mice treated with H101 had reduced tumor burdens and prolonged survival times compared with PBS and AdGFP controls (both P < 0.01). Immunohistochemical and Western blot examination revealed widespread replication of H101 within the tumor. CONCLUSIONS These results suggest that H101 effectively inhibits the growth of retinoblastoma cells in vitro and in mice and may serve as a novel therapy for retinoblastoma.

Combination of transorbital and endoscopic transnasal approaches to repair orbital medial wall and floor fractures.

Purpose This study aimed to illustrate the effectiveness of the combination of the transorbital and the endoscopic transnasal approach in the repair of medial wall and floor orbital fractures in Chinese patients. Methods A retrospective study was carried out on 25 Chinese patients (18 men and 7 women) with orbital medial wall and floor fractures. All patients had enophthalmos more than 2 mm, 23 had diplopia, and 11 had eye movement restriction. Bone defect involving both medial and inferior walls was found with computed tomographic scans in all patients. In all 25 patients, surgery was done by 1 surgeon group using the transorbital and the endoscope-assisted transnasal approach. The endoscope was used to give a clear view of the posterior edge of the fracture. Titanium meshes were used to repair fractures of the orbital floor and the medial wall. Porous polyethylene sheet implants were used to recover the orbital volume. All patients were followed up 12 months after surgery. Results Enophthalmos was corrected in all 25 patients immediately, diplopia disappeared or improved in 21 of 23 cases, and eye movement restriction was released or improved in all 11 patients. No significant complications occurred. The titanium mesh was completely covered by nasal mucosa at 1 month after surgery by an endoscopic check. Conclusions The endoscope-assisted transnasal approach allows for excellent visualization of the extent of the fracture, particularly in areas that are difficult to visualize by conventional methods. The combination of the transorbital and the endoscope-assisted transnasal approach is a good way to reconstruct a large orbital wall fracture involving the floor and the medial wall.

Combination of oncolytic adenovirus and dacarbazine enhances antitumor ability against uveal melanoma cells via cell cycle block

Uveal melanoma is the most common primary intraocular malignancy in adults; however, current therapeutic modalities, including chemotherapy, have not been successful. Oncolytic viruses serve as an emerging gene therapy tool for cancer treatment because they specifically kill tumor cells while sparing normal cells. The oncolytic virus H101 has been approved by the Chinese State Food and Drug Administration for the treatment of certain malignancies. Unfortunately, the monotherapy of adenovirus has demonstrated limited efficacy in a clinical setting. Thus, novel treatment strategies in which an oncolytic virus is combined with existing chemicals are advancing toward potential clinical use. In this study, we chose the combination of oncolytic virus H101 and the alkylating agent dacarbazine (DTIC) to treat uveal melanoma cells in vitro. Our results demonstrated that the combination exerted a synergistic antitumor effect without enhanced toxicity to normal cells via a type of cell cycle block other than the induction of apoptosis. Further investigation is warranted to elucidate the specific underlying mechanisms of this co-treatment therapy. Our study suggests the viro-chemo combination therapy is feasible and is a potentially promising approach for the treatment of uveal melanoma.

Recombinant oncolytic adenovirus H101 combined with siBCL2: cytotoxic effect on uveal melanoma cell lines

Background Deregulation of Bcl2 pathway is implicated in the pathogenesis of uveal melanoma (UM). Oncolytic adenovirus H101 is the worlds first oncolytic viral therapy for cancer approved for clinical use. We aimed to explore a potential synergy of downregulating Bcl2 pathway using a small interfering RNA (siBCL2) combined with H101 therapy on UM cell lines. Methods The sensitivity to adenovirus infection was analysed by flow cytometry. PCR, real-time-PCR and western blot were used to detect Bcl2, p53, Bax and fibre expression. Appropriate multiplicity of H101 infection and cell survival rate were measured by a cell counting kit-8 assay. UM cells were stained with Annexin-V and propidium iodide for apoptosis assay and cell cycle distribution. Results VUP cells (without elevation of Bcl2) exhibited greater sensitivity to adenovirus infection than OM431 cells (Bcl2 elevated cell line). Bcl2 expression was markedly reduced by siBCL2 or siBCL2 plus H101. Combined treatment with siBCL2 and H101 produced substantial growth inhibition of OM431 cells by enhancing apoptosis and cell cycle arrest through Bax-p53-induced apoptotic pathway. Conclusions SiBCL2 and H101 exhibited synergistic cytotoxic effect in Bcl2 elevated UM cell lines and could potentially serve as a novel targeted molecular therapy for UM.

BAP1 regulates cell cycle progression through E2F1 target genes and mediates transcriptional silencing via H2A monoubiquitination in uveal melanoma cells

Uveal melanoma (UM) is the most common form of primary intraocular malignancy in adult and has the tendency to metastasize. BAP1 mutations are frequently found in UM and are associated with a poor prognosis. The role of BAP1 in cell cycle regulation is currently a research highlight, but its underlying mechanism is not well understood. Here, we report that BAP1 knockdown can lead to G1 arrest and is accompanied by a decrease in the expression of S phase genes in OCM1 cells. Furthermore, in chromatin immunoprecipitation experiments, BAP1 could bind to E2F1 responsive promoters and the localization of BAP1 to E2F1-responsive promoters is host cell factor-1 dependent. Moreover, BAP1 knockdown leads to increased H2AK119ub1 levels on E2F responsive promoters. Together, these results provide new insight into the mechanisms of BAP1 in cell cycle regulation.

Functional significance of B7-H1 expressed by human uveal melanoma cells

B7-H1, a recently described B7 family member, has been reported to negatively regulate T-cell function in most cancer cells. In this study, we sought to investigate B7-H1 expression in four uveal melanoma (UM) cells (OCM1, SP6.5, OM431 and VUP) to determine the functional significance of B7-H1 expression in T-cell immune response. Using flow cytometry (FCM), we demonstrated that SP6.5 cells had high B7-H1 protein expression, while the other three UM cell lines had none. However, all four UM cell lines expressed B7-H1 mRNA, as confirmed by reverse transcription-polymerase chain reaction. In co-culture experiments using B7-H1-expressing UM cells with T-cells, FCM to determine CD69 expression in T-cells revealed that SP6.5 cell-related B7-H1 inhibited T-cell activation. This effect was eliminated by B7-H1-targeted RNA interference. An Annexin V/PI double staining assay further showed that B7-H1 expressed by SP6.5 cells did not increase the apoptosis of T-cells, though it was found in a variety of other solid tumors. In conclusion, all the UM cell lines constitutively expressed B7-H1 mRNA, while B7-H1 protein was expressed at different levels. UM-related B7-H1 expression negatively regulated T-cell immune response through the inhibition of T-cell activation, and not through the promotion of T-cell apoptosis. This provides new insight into anti-tumor immunity against B7-H1-expressing UM cells.