Clinical Observation of Double Percutaneous Nephrostomy Combined with Ureter Occlusion Stent for Treating Cervical Cancer Complicated with Vesicovaginal Fistula.

Background: To evaluate the efficacy of double percutaneous nephrostomy (PCN) combined with ureter occlusion stent for treating cervical cancer complicated with vesicovaginal fistula (VVF). Materials and Methods: A retrospective analysis was performed for 12 patients with cervical cancer complicated with VVF. Regardless of surgical resection, radiotherapy alone or combined chemoradiotherapy were carried out in all patients. After VVF was diagnosed by gynecological examination, imaging, and cystoscopy, concurrent double PCN and ureter occlusion stent implantation were performed for all patients. Results: All patients successfully received ureter occlusion stent implantation after nephrostomy. The success rate of nephrostomy and stent placement was 100% (12/12). After intervention, urinary fistula immediately disappeared in all patients. One week post-surgery, bilateral hydronephrosis disappeared in 4 patients, and their renal insufficiency and renal function returned to normal. One month after operation, 6 patients with genital eczema or ulcer and 5 patients with urinary tract infection were cured. During follow-up, there were no recurrence in urinary fistula, renal dysfunction, and other complications. Conclusion: Double PCN combined with ureter occlusion stent could effectively treat cervical cancer complicated with VVF hydronephrosis, urinary tract infection, and renal insufficiency and contribute to alleviate all kinds of clinical discomfort.

An Optimal Ablative Margin of Small Single Hepatocellular Carcinoma Treated with Image-Guided Percutaneous Thermal Ablation and Local Recurrence Prediction Base on the Ablative Margin: A Multicenter Study.

OBJECTIVE: To explore the best ablative margin (AM) for single hepatocellular carcinoma (HCC) patients with image-guided percutaneous thermal ablation (IPTA) based on MRI-MRI fusion imaging, and to develop and validate a local tumor progression (LTP) predictive model based on the recommended AM. METHODS: Between March 2014 and August 2019, 444 treatment-naïve patients with single HCC (diameter ≤3 cm) who underwent IPTA as first-line treatment from three hospitals were included, which were randomly divided into training (n= 296) and validation (n = 148) cohorts. We measured the ablative margin (AM) by MRI-MRI fusion imaging based on pre-ablation and post-ablation images. Then, we followed up their LPT and verified the optimal AM. Risk factors related to LTP were explored through Cox regression models, the nomogram was developed to predict the LTP risk base on the risk factors, and subsequently validated. The predictive performance and discrimination were assessed and compared with conventional indices. RESULTS: The median follow-up was 19.9 months (95% CI 18.0-21.8) for the entire cohort. The results revealed that the tumor size (HR: 2.16; 95% CI 1.25-3.72; P = 0.003) and AM (HR: 0.72; 95% CI, 0.61-0.85; P < 0.001) were independent prognostic factors for LTP. The AM had a pronounced nonlinear impact on LTP, and a cut-off value of 5-mm was optimal. We developed and validated an LTP predictive model based on the linear tumor size and nonlinear AM. The model showed good predictive accuracy and discrimination (training set, concordance index [C-index] of 0.751; validation set, C-index of 0.756) and outperformed other conventional indices. CONCLUSION: The 5-mm AM is recommended for the best IPTA candidates with single HCC (diameter ≤3 cm). We provided an LTP predictive model that exhibited adequate performance for individualized prediction and risk stratification.

Hepatic Arterial Chemoembolization With Arsenic Trioxide Eluting CalliSpheres Microspheres Versus Lipiodol Emulsion: Pharmacokinetics And Intratumoral Concentration In A Rabbit Liver Tumor Model.

BACKGROUND: The objective of this study was to investigate the plasma pharmacokinetic profiles, intratumoral concentration and tissue distribution of arsenic trioxide (ATO) by drug-eluting beads (DEB)-transcatheter arterial chemoembolization (TACE) compared with conventional TACE (cTACE) in a rabbit liver tumor model. METHODS: Sixty-four rabbits with VX2 liver tumor were established and randomly assigned to four groups equally. The calliSpheres microspheres (CSM)-ATO group received DEB-TACE treatment using ATO-loaded CSM; the cTACE-ATO group received cTACE treatment using ATO mixed with lipiodol; the CSM-normal control (NC) group received DEB-TACE treatment using blank CSM; the TAE-lipiodol group received cTACE treatment using saline mixed with lipiodol. ATO concentration in plasma, tumor and normal tissues, and liver and kidney function indexes were evaluated. RESULTS: The CSM-ATO group exhibited lower plasma ATO concentrations at 10 minutes and 20 minutes post treatment compared with the cTACE-ATO group. Meanwhile, intratumoral ATO concentrations were higher in the CSM-ATO group compared with the cTACE-ATO group at 3-, 7- and 14-days post treatment. In normal liver tissue, heart and muscle tissues, ATO concentrations between the CSM-ATO and cTACE groups were similar at each time point; in kidney and lung tissues, ATO concentrations were lower in the CSM-ATO group at 1-day post treatment while they were similar at 3, 7 and 14 days post treatment. Also, liver or kidney function indexes were of no difference at each time point between CSM-ATO and cTACE-ATO groups. CONCLUSION: Administration of ATO via DEB-TACE decreases systemic concentration while increasing intratumoral concentration of ATO without increasing liver or kidney toxicity compared with cTACE.

Upregulation of IL-6 is involved in moderate hyperthermia induced proliferation and invasion of hepatocellular carcinoma cells.

Increasing evidences suggested that insufficient radiofrequency ablation (RFA) can paradoxically promote tumor invasion and metastatic processes, while the effects of moderate hyperthermia on cancer progression are not well illustrated. Our present study confirmed moderate hyperthermia treatment can promote the proliferation, migration and invasion of hepatocellular carcinoma (HCC) cells, which was evidenced by the results that moderate hyperthermia induced up regulation of proliferating cell nuclear antigen (PCNA) and matrix metalloproteinase-2 (MMP-2). Cellular studies indicated that moderate hyperthermia treatment can increase the mRNA and protein expression of IL-6 and IL-10, while not IL-2, IL-4, IL-8, IL-22, VEGF, TGF-ß, or TNF-α, in HCC cells. Silencing of IL-6, while not IL-10, attenuated moderate hyperthermia treatment induced proliferation and cell invasion. Furthermore, our data revealed the inhibition of NF-κB, while not ERK1/2 or PI3K/Akt, abolished moderate hyperthermia treatment induced production of IL-6. Collectively, our data showed that activation of NF-κB/IL-6 is involved in moderate hyperthermia treatment induced progression of HCC cells.

CD8(+) T cells specific for both persistent and non-persistent viruses display distinct differentiation phenotypes but have similar level of PD-1 expression in healthy Chinese individuals.

Anti-viral CD8(+) T cell responses involve an initial expansion and effector phase, followed by contraction phase and formation of CD8(+) memory T cells. During this contraction phase, increased surface expression of the negative regulator PD-1 is associated with functional exhaustion of CD8(+) T cells. Although its role in T cell suppression has been established, the importance of PD-1 in the differentiation of CD8(+) T cells remains unclear. In this study, we examine PD-1 expression in relation to viral specificity of CD8(+) T cells against persistent or non-persistent viruses, and further define differentiation phenotypes of CD8(+) T cells by CD27 and CD28 expression. Surprisingly, the inhibitory receptor PD-1 was expressed by Flu-specific CD8(+) T cells in a level comparable to HCMV-and EBV-specific cells. Moreover, in virus-specific CD8(+) T cells, CD127(+)/CD127(-) and CD62L(+)/CD62L(-) cells expressed similar levels of PD-1 molecules. These results suggest that the PD-1/PD-L1 pathway may play a regulatory role in memory T cell subsets in addition to its association with T-cell exhaustion.

[Improved expression of HLA-A* 2402-BSP in Escherichia coli and its tetramer preparation].

HLA-A* 2402 is one of the most frequently encountered HLA-A alleles in East Asian populations. In order to study the CD8+ T cell responses in Chinese populations, we have described the generation and functional test of HLA-A* 2402 tetramer loaded with HCMV pp65(341-349) peptide (QYDPVAALF, QYD). The cDNA of HLA-A* 2402 heavy chain was cloned by RT-PCR from one of the donors. DNA fragment encoding the ectodomain of HLA-A* 2402 heavy chain fused at its carboxyl-terminal a BirA substrate peptide (BSP) was amplified by PCR with the cloned heavy chain cDNA as a template. The wild-type gene of HLA-A* 2402-BSP was not expressed in Escherichia coli (E. coli), while mutant HLA-A* 2402-BSP gene with optimized codons was overexpressed as inclusion bodies in E. coli. Furthermore, the soluble HLA-A* 2402-QYD monomers were generated by in vitro refolding of washed inclusion bodies in the presence of beta2-microglobulin and QYD peptide. The tetramer was subsequently formed by mixing HLA-A* 2402-QYD monomers with streptavidin-PE at a molar ratio of 4:1. Flow cytometry analysis indicated that this tetramer possessed binding activity with specific CTL from HLA-A24+ donors and the frequencies of tetramer-binding CTL were 0.09% - 0.37% within total CD8+ T cells. This tetrameric agent provides a powerful tool to explore the secrets of CTL responses against HCMV antigens in HLA-A* 2402 individuals.

[Preparation and identification of human soluble sPD-L1 and its antibodies].

This study reports the preparation and identification of soluble programmed death-1 (PD-1) ligand-1 (sPD-L1) and its antibodies of mouse origin. Immobilized metal ion affinity chromatography was used to perform on-column refolding with simultaneous purification of denatured sPD-L1, and soluble sPD-L1 with purity of 95% was obtained. The purified sPD-L1 was verified by immunoblotting using a commercial goat-anti-human PD-L1 antibody. An ELISA-based assay showed that it also had high binding activity for its cognate receptor PD-1. Furthermore, mouse anti-sPD-L1 antiserum of high titer was raised using the purified sPD-L1 as an immunogen, and the specific IgG antibodies were purified using sPD-L1-HiTrap affinity chromatography. In addition, a sensitive sandwich ELISA was established using the purified IgG antibodies together with the commercial goat antibodies. In conclusion, the preparation of soluble sPD-Ll and its antibodies provide the basis for detection of the potential anti-PD-L1 antibodies and soluble PD-L1 in humans as well as for further investigation of its in vivo bioactivities and characterization of its potential receptors.

[Expression and refolding of a HLA-A*0203-BSP fusion protein and identification of its tetramers].

AIM: To optimize expression condition of HLA-A*0203 heavy chain ectodomain fused with a BirA substrate peptide (BSP) (HLA-A*0203-BSP) for E.coli BL21(DE3) transformant and to prepare a functional HLA-A*0203 tetramer loaded with an antigenic peptide derived from EBNA3(596-604) of Epstein-Barr virus (EBV). METHODS: The temperature, IPTG concentration and inductive duration of HLA-A*0203-BSP fusion protein expressed for E.coli BL21(DE3) transformant were optimized. SDS-PAGE and Western blot analyses were employed to detect the expressed fusion protein. The monomer of soluble HLA-A*0203-peptide was generated from the fusion protein by in vitro refolding of washed inclusion bodies in the presence of beta2-microglobulin (beta2m) and HLA-A*0203 restricted EBV EBNA3(596-604) peptide (SVRDRLARL, SVR). Refolded and purified monomer was then biotinylated with BirA. Following the purification of the obtained biotinylated monomer, the tetramer was formed by incubation with streptavidin-PE at a ratio of 4:1. Flow cytometry (FCM) analysis was performed to determine its binding activity with specific cytotoxic T lymphocytes (CTL). RESULTS: SDS-PAGE and Western blot showed that the optimized expression condition was overnight induction at 37 degrees C with 0.4 mmol/L IPTG. The expressed protein of about 34 kDa in the form of inclusion bodies accumulated up to about 30% of total bacterial protein under the optimized expression condition. The monomer of soluble HLA-A*0203/SVR was successfully generated and purified. Non-reducing SDS-PAGE analysis showed that the biotinylation was above 85%. HLA-A*0203/SVR tetramer was constructed by mixing the monomer with streptavidin-PE at a ratio of 4:1. FCM analysis indicated that this tetramer could bind specific CTL from HLA-A2+ donors. CONCLUSION: HLA-A*0203-BSP fusion protein was overexpressed in E.coli under the optimized condition. The tetramers of HLA-A*0203/SVR were prepared from this fusion protein and it possessed binding activity with specific CTL, which provided a powerful tool for direct visualization and quantification of specific CTL from HLA-A*0203 donors.