Genome-wide characterization of heavy metal-associated isoprenylated plant protein gene family from Citrus sinensis in response to huanglongbing.

Introduction: Heavy metal-associated isoprenylated plant proteins (HIPPs) play vital roles in maintaining heavy metal balance and responding to both biotic and abiotic stresses in vascular plants. However, the role of HIPPs in the response to Huanglongbing (HLB), a harmful disease of citrus caused by the phloem-colonizing bacterium Candidatus Liberibacter asiaticus (CLas), has not been examined. Methods and results: In this study, a total of 26 HIPP genes were identified in Citrus sinensis, and they were grouped into 5 clades. The CsHIPP genes are distributed on 8 chromosomes and exhibited considerable synteny with HIPPs found in Arabidopsis thaliana. Additionally, we analyzed the gene structure, conserved motifs and domains of the CsHIPPs. Various cis-acting elements related to plant hormones and stress responses were identified in the promoters of CsHIPPs. Public transcriptome data and RT-qPCR analysis showed that the expression level of CsHIPP03 was significantly reduced in samples infected by CLas and Xanthomonas citri ssp. citri (Xcc). Furthermore, silencing the homologous gene of CsHIPP03 in Nicotiana benthamiana increased the disease resistance of plants to bacteria. Discussion: Our results provide a basis for functional studies of HIPP gene family in C. sinensis, highlighting their functions in bacterial resistance, and improve our understanding to the susceptibility mechanism of HLB.

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.

PSMC2/ITGA6 axis plays critical role in the development and progression of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a type of malignant tumor with sixth highest incidence and causes the third most cancer-related deaths in the world, whose treatment is limited by the unclear molecular mechanism. Currently, the correlation between PSMC2 and HCC is still unclear. Herein, we found that the expression of PSMC2 in HCC tissues was significantly higher than normal tissues. We also discovered the significant association between PSMC2 expression and tumor infiltrate as well as tumor stage. Further investigations indicated that PSMC2 knockdown contributed to impaired proliferation, colony formation, migration, and enhanced cell apoptosis in HCC cells. Moreover, PSMC2 could also suppress tumorigenicity of HCC cells in vivo. Gene microarray analysis followed by ingenuity pathway analysis was performed for exploring downstream of PSMC2 and identified ITGA6 as a potential target. Furthermore, our study revealed that ITGA6 knockdown exhibited similar inhibitory effects with PSMC2 on HCC cells in vitro. More importantly, our results proved the direct interaction and showed the mutual regulation between PSMC2 and ITGA6, and that PSMC2 knockdown could significantly aggravate the inhibition of HCC by ITGA6 depletion. Based on these intriguing results, this is the first time ever that PSMC2 is pinpointed as a tumor promotor to interfere HCC development and progression via interacting with ITGA6 directly.

Comparison of Treatment Response, Survival Profiles, as Well as Safety Profiles Between CalliSpheres® Microsphere Transarterial Chemoembolization and Conventional Transarterial Chemoembolization in Huge Hepatocellular Carcinoma.

PURPOSE: CalliSpheres® microspheres (CSM) are the first drug-eluting beads (DEB) developed in China. This study aimed to compare treatment response, survival, and safety profiles between DEB transarterial chemoembolization (DEB-TACE) with CSM and conventional TACE (cTACE) in huge hepatocellular carcinoma (HCC) patients. METHODS: A total of 71 patients with huge HCC who underwent DEB-TACE or cTACE were consecutively enrolled in this retrospective cohort study. Treatment response was assessed at first month (M1), third month (M3), and sixth month (M6) after TACE therapy; progression-free survival (PFS) and overall survival (OS) were evaluated; liver function indexes were recorded before TACE operation (M0), at first week (W1), M1 and M6 after TACE therapy; adverse events which occurred after TACE operation were recorded. RESULTS: DEB-TACE presented with higher objective response rate (60.0% vs. 29.7%, p < 0.05) and disease control rate (86.7% vs. 59.4%, p < 0.05) compared with cTACE at M3. Regarding survival profiles, PFS [median: 3.3 months (95% CI: 2.8-3.7) vs. 2.1 months (95% CI: 1.7-2.5)] as well as OS [median: 7.8 months (95% CI: 4.6-11.0) vs. 5.7 months (95% CI: 5.0-6.3)] were longer in DEB-TACE group compared with cTACE group (both p < 0.01). Multivariate Cox's regression further illustrated that DEB-TACE vs. cTACE was an independent protective factor for PFS and OS (both p < 0.01). As for safety profiles, patients' liver function injury was reduced in the DEB-TACE group compared with the cTACE group. The incidence of fever was lower, and CINV was less severe in the DEB-TACE group compared with the cTACE group (both p < 0.05), while no difference in occurrence of liver abscess, increase of ascites, or moderate pain between two groups was observed. CONCLUSION: DEB-TACE with CSM presents with better treatment response, survival profiles, as well as safety profiles compared with cTACE in treatment for huge HCC patients.

Antitumor properties of arsenic trioxide-loaded CalliSpheres® microspheres by transarterial chemoembolization in VX2 liver tumor rabbits: suppression of tumor growth, angiogenesis, and metastasis and elongation of survival.

This study aimed to investigate the antitumor effect of arsenic trioxide (ATO)-loaded CalliSpheres® microspheres (CSM) by transarterial chemoembolization (TACE) in rabbits with VX2 liver tumors. A total of 120 VX2 liver tumor rabbits were randomized into four groups (N = 30 for each group), which received ATO-loaded CSM by TACE (CSM-ATO group), ATO by conventional TACE (cTACE-ATO group), transcatheter arterial embolization using CSM (TAE-CSM group), and saline arterial injection (control group). Five rabbits in each group were sacrificed at 12 h, 3 d, 7 d and 14 d, and then tumor proliferation, apoptosis, and angiogenesis/epithelial-mesenchymal transition (EMT) markers were detected. Tumor volume, metastasis status and ascites were assessed at 14 d. Ten rabbits in each group were observed until death for accumulating survival calculation. Tumor volume and ascites were decreased in the CSM-ATO group compared to the cTACE-ATO and TAE-CSM groups. Pulmonary, abdominal wall and omentum metastases were reduced while accumulating survival was increased in the CSM-ATO group compared to the TAE-CSM group. However, no difference in metastasis foci or survival between the CSM-ATO and cTACE-ATO groups was discovered. Meanwhile, tumor apoptosis was promoted while proliferation was suppressed in the CSM-ATO group compared to the cTACE-ATO and TAE-CSM groups. Additionally, HIF-1α, VEGF and microvessel density were decreased in the CSM-ATO group compared to the cTACE-ATO and TAE-CSM groups. Additionally, twist, N-cadherin, vimentin and MMP-9 were reduced while E-cadherin was enhanced in the CSM-ATO group compared to the cTACE-ATO and TAE-CSM groups. In conclusion, ATO-loaded CSM by TACE suppressed tumor growth, angiogenesis, and metastasis and elongated survival in VX2 liver tumor rabbits.

Acute Blood Pressure and Cardiovascular Effects of Near-Roadway Exposures With and Without N95 Respirators.

BACKGROUND: The risk for cardiovascular events increases within hours of near-roadway exposures. We aimed to determine the traffic-related air pollution (TRAP) and biological mechanisms involved and if reducing particulate matter <2.5 µm (PM2.5) inhalation is protective. METHODS: Fifty healthy-adults underwent multiple 2-hour near-roadway exposures (Tuesdays to Fridays) in Ann Arbor during 2 separate weeks (randomized to wear an N95 respirator during 1 week). Monday both weeks, participants rested 2 hours in an exam room (once wearing an N95 respirator). Brachial blood pressure, aortic hemodynamics, and heart rate variability were repeatedly measured during exposures. Endothelial function (reactive hyperemia index [RHI]) was measured post-exposures (Thursdays). Black carbon (BC), total particle count (PC), PM2.5, noise and temperature were measured throughout exposures. RESULTS: PM2.5 (9.3 ± 7.7 µg/m3), BC (1.3 ± 0.6 µg/m3), PC (8,375 ± 4,930 particles/cm3) and noise (69.2 ± 4.2 dB) were higher (P values <0.01) and aortic hemodynamic parameters trended worse while near-roadway (P values<0.15 vs. exam room). Other outcomes were unchanged. Aortic hemodynamics trended towards improvements with N95 respirator usage while near-roadway (P values<0.15 vs. no-use), whereas other outcomes remained unaffected. Higher near-roadway PC and BC exposures were associated with increases in aortic augmentation pressures (P values<0.05) and trends toward lower RHI (P values <0.2). N95 respirator usage did not mitigate these adverse responses (nonsignificant pollutant-respirator interactions). Near-roadway outdoor-temperature and noise were also associated with cardiovascular changes. CONCLUSIONS: Exposure to real-world combustion-derived particulates in TRAP, even at relatively low concentrations, acutely worsened aortic hemodynamics. Our mixed findings regarding the health benefits of wearing N95 respirators support that further studies are needed to validate if they adequately protect against TRAP given their growing worldwide usage.

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.

Reduction of personal PM2.5 exposure via indoor air filtration systems in Detroit: an intervention study.

The adverse health effects of fine particulate matter (PM < 2.5 µm in diameter [PM2.5]) air pollution are well-documented. There is a growing body of evidence that high-efficiency particulate arrestance (HEPA) filtration can reduce indoor PM2.5 concentrations and deliver some health benefits via the reduction of exposure to PM. However, few studies have tested the ability of portable air filtration systems to lower overall personal-level PM2.5 exposures. The Reducing Air Pollution in Detroit Intervention Study (RAPIDS) was designed to evaluate cardiovascular health benefits and personal PM2.5 exposure reductions via indoor portable air filtration systems among senior citizens in Detroit, Michigan. We evaluated the utility of two commercially available high-efficiency (HE: true-HEPA) and low-efficiency (LE: HEPA-type) indoor air filtration to reduce indoor PM2.5 concentrations and personal PM2.5 exposures for 40 participants in a double-blinded randomized crossover intervention. Each participant was subjected to three intervention scenarios: HE, LE, or no filter (control) of three consecutive days each, during which personal, indoor, and outdoor PM2.5 concentrations were measured daily. For mean indoor PM2.5 concentrations, we observed 60 and 52% reductions using HE and LE filters, respectively, relative to no filtration. Personal PM2.5 exposures were reduced by 53 and 31% using HE and LE filters, respectively, when compared with the control scenario. To our knowledge, this is the first indoor air filtration intervention study to examine the effectiveness of both HE and LE filters in reducing personal PM2.5 exposures.

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.

Direct Evidence for the Origin of Bis-Gold Intermediates: Probing Gold Catalysis with Mass Spectrometry.

Gold-catalyzed alkyne hydration was studied by using in situ reacting mass spectrometry (MS) technology. By monitoring the reaction process in solution under different conditions (regular and very diluted catalyst concentrations, different pH values) and examining the reaction occurrence in the early reaction stage (1-2 ms after mixing) with MS, we collected a series of experimental evidence to support that the bis-gold complex is a potential key reaction intermediate. Furthermore, both experimental and computational studies confirmed that the σ,π-bis-gold complexes are not active intermediates toward nucleophilic addition. Instead, formation of geminally diaurated complex C is crucial for this catalytic process.

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.

High level expression of HLA-A*0203-BSP fusion protein in Escherichia coli and construction of soluble HLA-A*0203 monomer and tetramer loaded with Epstein-Barr virus peptide.

Major histocompatibility complex (MHC) tetramer technology is critical for characterization of antigen-specific T cells. In the present study we reported the successful generation of HLA-A*0203 tetramer loaded with Epstein-Barr virus EBNA3(596-604) peptide (SVRDRLARL, SVR). Prokaryotic expression vector for the ectodomain of the heavy chain of HLA-A*0203 fused with a BirA substrate peptide (HLA-A*0203-BSP) was constructed and the expression conditions of the fusion protein in Escherichia coli (E. coli) were optimized. The fusion protein was highly expressed in inclusion bodies within E. coli. It was then refolded in the presence of beta2-microglobulin and SVR peptide to form a soluble HLA-A*0203-SVR monomer. After biotinylation with BirA, the monomer was purified by anion-exchange chromatography and its purity was up to 95%. The tetramer was then formulated by mixing the biotinylated monomer with streptavidin-PE at a ratio of 4:1. Flow cytometry showed that this tetramer could specifically react with antigen-specific CD8+ T cells, indicating that it was biologically functional. These results provide a foundation for further characterization of antigen-specific CD8+ T cells from HLA-A*0203 subjects.

Preparation and identification of HLA-A*1101 tetramer loading with human cytomegalovirus pp65 antigen peptide.

MHC/peptide tetramer technology has been widely used to study antigen-specific T cells, especially for identifying virus-specific CD8+ T cells in humans. The tetramer molecule is composed of HLA heavy chain, beta2-microglobulin (beta2m), an antigenic peptide, and fluorescent-labeled streptavidin. To further investigate the HLA-A*1101-restricted CD8+ T cell responses against human cytomegalovirus (HCMV), we established an approach to prepare HLA-A*1101 tetramer complexed with a peptide from HCMV. The cDNA encoding HLA-A*1101 heavy chain was cloned and the prokaryotic expression vector for the ectodomain of HLA-A*1101 fused with a BirA substrate peptide (HLA-A*1101-BSP) at its carboxyl terminus was constructed. The fusion protein was highly expressed as inclusion bodies under optimized conditions in Escherichia coli. Moreover, HLA-A*1101-BSP protein was refolded in the presence of beta2m and an HCMV peptide pp65(16-24) (GPISGHVLK, GPI). Soluble HLA-A*1101-GPI monomer was biotinylated and purified to a purity of 95%, which was subsequently combined with streptavidin to form tetramers at a yield of > 80%. The HLA-A*1101-GPI tetramers could bind to virus-specific CD8+ T cells, suggesting soluble HLA-A*1101-GPI tetramers were biologically functional. This study provides the basis for further evaluation of HLA-A*1101-restricted CD8+ T cell responses against HCMV infection.

[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.

Preparation and characterization of HLA-A*0201 tetramer loaded with IE-1316-324 antigenic peptide of human cytomegalovirus.

Major histocompatibility complex (MHC) class I tetramer technology has become the central technique for analyzing antigen-specific CD8(+) T cell responses and it has been widely used to explore the differentiation and formation of memory CD8(+) T cells. Previously, a simplified and efficient procedure for preparing high quality HLA-A*0201 tetramers has been established in our lab and the tetramers loaded with HCMV peptide pp65(495-503) has been successfully applied to investigate HCMV-specific CD8(+) T cells in Chinese populations. Using similar procedure we reported here the construction of HLA-A*0201 tetramer loaded with another dominant epitope derived from immediate early (IE)-1(316-324) (VLEETSVML, VLE) of HCMV (A2-VLE) and characterization of this tetramer. After A2-VLE monomer was prepared and purified, its tetramer was then formed at a yield of 83%. The optimized amount of A2-VLE tetramer for staining 100 microl whole blood was 0.5 microg with incubation at 4 degrees C for 1 h. Furthermore, the dissociation constant of the tetramer binding to the specific CD8(+) T cells of one HLA-A2(+) donor was estimated to be 32.7 nmol/L, which is markedly higher than that of MHC monomer. The construction of A2-VLE tetramer provides an alternative choice for investigating HCMV-specific CD8(+) T cell responses and will deepen our understanding of the differentiation and formation of HCMV-specific memory CD8(+) T cells.