Andrographolide sensitizes KRAS-mutant colorectal cancer cells to cetuximab by inhibiting the EGFR/AKT and PDGFRß/AKT signaling pathways.

BACKGROUND: Cetuximab, an inhibitor targeting EGFR, is widely applied in clinical management of colorectal cancer (CRC). Nevertheless, drug resistance induced by KRAS-mutations limits cetuximab's anti-cancer effectiveness. Furthermore, the persistent activation of EGFR-independent AKT is another significant factor in cetuximab resistance. Nevertheless, the mechanism that EGFR-independent AKT drives cetuximab resistance remains unclear. Thus, highlighting the need to optimize therapies to overcome cetuximab resistance and also to explore the underlying mechanism. PURPOSE: This work aimed to investigate whether and how andrographolide enhance the therapeutic efficacy of cetuximab in KRAS-mutant CRC cells by modulating AKT. METHODS: The viabilities of CRC cell lines were analyzed by CCK-8. The intracellular proteins phosphorylation levels were investigated by Human Phospho-kinase Antibody Array analysis. Knockdown and transfection of PDGFRß were used to evaluate the role of andrographolide on PDGFRß. The western blotting was used to investigate Wnt/ß-catenin pathways, PI3K/AKT, and EMT in KRAS-mutant CRC cells. The animal models including subcutaneous tumor and lung metastasis were performed to assess tumor response to therapy in vivo. RESULTS: Andrographolide was demonstrated to decrease the expression of PI3K and AKT through targeting PDGFRß and EGFR, and it enhanced cetuximab effect on KRAS-mutant CRC cells by this mechanism. Meanwhile, andrographolide helped cetuximab to inhibit Wnt/ß-catenin, CRC cell migration and reduced Vimentin expression, while increasing that of E-cadherin. Lastly, co-treatment with cetuximab and andrographolide reduced the growth of KRAS-mutant tumors and pulmonary metastases in vivo. CONCLUSIONS: Our findings suggest that andrographolide can overcome the KRAS-mutant CRC cells' resistance to cetuximab through inhibiting the EGFR/PI3K/AKT and PDGFRß /AKT signaling pathways. This research provided a possible theory that andrographolide sensitizes KRAS-mutant tumor to EGFR TKI.

Impact of pericoronary adipose tissue attenuation on recurrence after radiofrequency catheter ablation for atrial fibrillation.

BACKGROUND: Inflammation plays a vital role in the occurrence and progression of atrial fibrillation (AF). The association between pericoronary adipose tissue attenuation (PCATA) and AF recurrence following ablation has not been fully clarified. HYPOTHESIS: We aimed to evaluate the association between PCATA and AF recurrence after radiofrequency catheter ablation (RFCA). METHODS: Patients who underwent the first RFCA for AF and performed coronary computed tomography angiography before ablation between 2018 and 2021 were enrolled. The predictive values of PCATA for AF recurrence after ablation were investigated. The area under curve (AUC), relative integrated discrimination improvement (IDI), and categorical free net reclassification improvement (NRI) were used to assess the discrimination ability of different models for AF recurrence. RESULTS: During 1-year follow-up, 34.1% patients experienced AF recurrence. The multivariable analysis model revealed that PCATA of the right coronary artery (RCA) was an independent risk factor for AF recurrence. Patients with a high level of RCA-PCATA had a high risk of recurrence, after adjusting for other risk factors by restricted cubic splines. The performance in predicting AF recurrence was significantly improved by adding the marker of RCA-PCATA to the clinical model (AUC: 0.724 vs. 0.686, p = .024), with a relative IDI of 0.043 (p = .006) and continuous NRI of 0.521 (p < .001). CONCLUSIONS: PCATA of RCA was independently associated with AF recurrence after ablation. PCATA may be helpful for risk classification for AF ablation patients.

Effect of Insulin Resistance on Recurrence after Radiofrequency Catheter Ablation in Patients with Atrial Fibrillation.

BACKGROUND: Whether there are many risk factors for recurrence of atrial fibrillation (AF) after ablation is unclear. The aim of this study was to investigate the relationship between insulin resistance (IR) and AF recurrence in patients without diabetes who underwent catheter ablation. METHODS: This retrospective study included patients who underwent AF ablation between 2018 and 2019 at the First Affiliated Hospital of Zhengzhou University. Homeostasis model assessment of insulin resistance (HOMA-IR) was calculated, and a value of ≥2.69 was defined as IR. The patients were divided into two groups (group 1 HOMA-IR < 2.69, n = 163; group 2 HOMA-IR ≥ 2.69, n = 69). AF recurrence was defined as the occurrence of atrial arrhythmias of more than 30 s after the first 3 months. Univariate and multivariable Cox regression models were used to analyse the risk of AF recurrence. RESULTS: Overall, 232 patients were enrolled (mean age, 59.9 ± 10.2 years old; female, 37.5%; paroxysmal AF, 71.6%). We found that dyslipidaemia, antiarrhythmic drug use, fasting blood glucose and fasting insulin were significantly higher in the IR group (P < 0.05). During the follow-up 1 year after ablation, 62 (26.7%) patients experienced AF recurrence. After adjusting for traditional risk factors, multivariable analysis showed that the HOMA-IR value (HR 1.259, 95% CI 1.086-1.460, P = 0.002) and left atrial diameter (LAD; HR 1.043, 95% CI 1.005-1.083, P = 0.026) were independently associated with AF recurrence. CONCLUSIONS: The present results provide evidence that IR patients are more likely to experience AF recurrence. Improving IR status may be a potential target for reducing the postoperative recurrence rate.

Gut mucosal microbiota profiles linked to colorectal cancer recurrence.

BACKGROUND: Emerging evidence links gut microbiota to various human diseases including colorectal cancer (CRC) initiation and development. However, gut microbiota profiles associated with CRC recurrence and patient prognosis are not completely understood yet, especially in a Chinese cohort. AIM: To investigate the relationship between gut mucosal microbiota profiles and CRC recurrence and patient prognosis. METHODS: We obtained the composition and structure of gut microbiota collected from 75 patients diagnosed with CRC and 26 healthy controls. The patients were followed up by regular examination to determine whether tumors recurred. Triplet-paired samples from on-tumor, adjacent-tumor and off-tumor sites of patients diagnosed with/without CRC recurrence were analyzed to assess spatial-specific patterns of gut mucosal microbiota by 16S ribosomal RNA sequencing. Next, we carried out bioinformatic analyses, Kaplan-Meier survival analyses and Cox regression analyses to determine the relationship between gut mucosal microbiota profiles and CRC recurrence and patient prognosis. RESULTS: We observed spatial-specific patterns of gut mucosal microbiota profiles linked to CRC recurrence and patient prognosis. A total of 17 bacterial genera/families were identified as potential biomarkers for CRC recurrence and patient prognosis, including Anaerotruncus, Bacteroidales, Coriobacteriaceae, Dialister, Eubacterium, Fusobacterium, Filifactor, Gemella, Haemophilus, Mogibacteriazeae, Pyramidobacter, Parvimonas, Porphyromonadaceae, Slackia, Schwartzia, TG5 and Treponema. CONCLUSION: Our work suggests that intestinal microbiota can serve as biomarkers to predict the risk of CRC recurrence and patient death.

Concurrent chemoradiotherapy using gemcitabine and nedaplatin in recurrent or locally advanced head and neck squamous cell carcinoma.

BACKGROUND: Patients with recurrent or locally advanced head and neck squamous cell carcinoma (HNSCC) typically have limited treatment options and poor prognosis. AIM: To evaluate the efficacy and safety of two drugs with potent radio-sensitization properties including gemcitabine and nedaplatin as concurrent chemoradiotherapy regimens in treating HNSCC. METHODS: This single-arm prospective study enrolled patients with HNSCC to receive gemcitabine on days 1 and 8 and nedaplatin on days 1 to 3 for 21 days. Intensity-modulated radiation therapy with a conventional fraction was delivered 5 days per week. Objective response rate (ORR), disease control rate, and toxicity were observed as primary endpoints. Overall survival (OS) and progression free survival were recorded and analyzed as secondary endpoints. RESULTS: A total of 24 patients with HNSCC were enrolled. During the median 22.4-mo follow-up, both ORR and disease control rate were 100%. The one-year OS was 75%, and one-year progression-free survival (PFS) was 66.7% (median PFS was 15.1 mo). Recurrent HNSCC patients had a poorer prognosis than the treatment-naïve patients, and patients who achieved complete response had better survival than those in the PR group (all P < 0.05). The most common grade 1-4 (100%) or grade 3-4 toxicities (75%) were hematological, and the most common grade 3-4 non-hematological toxicity was mucositis in 17 (71%) patients. CONCLUSION: Gemcitabine plus nedaplatin with concurrent chemoradiotherapy is a therapeutic option for HNSCC with predictable tolerability. Considering the high adverse event rate, the optimized dose and schedule must be further explored.

The value of the left atrial appendage orifice perimeter of 3D model based on 3D TEE data in the choice of device size of LAmbre™ occluder.

Preoperative optimal selection of the occluder size is crucial in percutaneous left atrial appendage (LAA) occlusion, and the maximal width of the LAA orifice is the main reference index, however it can not fully meet the practical operation requirements. We retrospectively analyzed three-dimensional (3D) transesophageal echocardiography (TEE) and computed tomography (CT) imaging dataset of the 41 patients who underwent LAA occlusion with LAmbre™ system. The LAA orifice parameters were overall evaluated to determine their role in device size selection. Eight LAA 3D models of the four cases who had been replaced their device during the procedure based on TEE and CT were printed out to verify the optimal parameter decision strategy. There was a significant concordance of the results between 3D TEE and CT in the LAA orifice evaluation. The correlations between the perimeter and maximal width measurements by 3D TEE and the closure disk of the device were stronger than that between the area measurements and the closure disk (r = 0.93, 0.95, 0.86, respectively and p < 0.001 all), and the result was similar to that by CT (r = 0.92, 0.93, 0.84, respectively and p < 0.001 all). The ratios of the maximal width to the minimal width of the four cases were all > 1.4, however the rest 37 cases were all ≤ 1.4. Based on the comprehensive assessment of the LAA orifice perimeter and maximal width of the 3D printed models, the experiments were all succeed just for one try. The LAA orifice perimeter of 3D printed model based on 3D TEE may help in choosing the optimal device size of LAmbre™, especially for the LAA with flater ostial shape.

Comparison of the inhibition potential of parthenolide and micheliolide on various UDP-glucuronosyltransferase isoforms.

Parthenolide (PTL) and micheliolide (MCL) are sesquiterpene lactones with similar structures, and both of them have been reported to exhibit multiple biochemical and pharmacological activities. This study aims to investigate the inhibition of these two compounds on the activity of UDP-glucuronosyltransferases (UGTs). In vitro incubation mixture for recombinant UGTs-catalyzed glucuronidation metabolism of 4-methylumbelliferone (4-MU) was utilized to investigate the inhibition potential. Inhibition kinetics (including inhibition type and parameters) were determined, and in silico docking was employed to elucidate the inhibition difference between PTL and MCL on UGT1A1. MCL showed no inhibition toward all the UGT isoforms, and PTL showed strong inhibition toward UGT1A1. The half-maximal inhibitory concentration (IC50) of PTL on the activity of UGT1A1 was determined to be 64.4 µM. Inhibition kinetics determination showed that PTL exerted noncompetitive inhibition toward UGT1A1, and the inhibition kinetic constant (Ki) was determined to be 12.1 µM. In silico docking method has been employed to show that hydrogen bonds between PTL and the activity cavity of UGT1A1 contributed to the stronger inhibition of PTL on the activity of UGT1A1 than MCL. In conclusion, PTL can more easily induce drug-drug interaction (DDI) with clinical drugs mainly undergoing UGT1A1-catalyzed glucuronidation.

A high-sensitivity electrochemical aptasensor of carcinoembryonic antigen based on graphene quantum dots-ionic liquid-nafion nanomatrix and DNAzyme-assisted signal amplification strategy.

In this work, we have developed an electrochemical aptasensor for high-sensitivity determination of carcinoembryonic antigen (CEA) based on lead ion (Pb2+)-dependent DNAzyme-assisted signal amplification and graphene quantum dot-ionic liquid-nafion (GQDs-IL-NF) composite film. We designed hairpin DNA containing CEA-specific aptamers and DNAzyme chains. In the presence of CEA, hairpin DNA recognized the target and performed a DNAzyme-assisted signal amplification reaction to yield a large number of single-stranded DNA. The GQDs-IL-NF composite film was immobilized on the glassy carbon electrode for the interaction with single-stranded DNA through noncovalent π-π stacking interaction. Therefore, the methylene blue-labeled substrate DNA (MB-substrate) was fixed on the electrode and exhibited an initial electrochemical signal. Under optimal conditions, the response current change was proportional to the concentration of CEA, demonstrating a wide linear range from 0.5fgmL-1 to 0.5ngmL-1, with a low detection limit of 0.34fgmL-1. Furthermore, the proposed aptasensor was successfully applied in determining CEA in serum samples, showing its superior prospects in clinical diagnosis.

Ultrasound­targeted microbubbles combined with a peptide nucleic acid binding nuclear localization signal mediate transfection of exogenous genes by improving cytoplasmic and nuclear import.

The development of an efficient delivery system is critical for the successful treatment of cardiovascular diseases using non­viral gene therapies. Cytoplasmic and nuclear membrane barriers reduce delivery efficiency by impeding the transfection of foreign genes. Thus, a gene delivery system capable of transporting exogenous genes may improve gene therapy. The present study used a novel strategy involving ultrasound­targeted microbubbles and peptide nucleic acid (PNA)­binding nuclear localization signals (NLS). Ultrasound­targeted microbubble destruction (UTMD) and PNA­binding NLS were used to improve the cytoplasmic and nuclear importation of the plasmid, respectively. Experiments were performed using antibody­targeted microbubbles (AT­MCB) that specifically recognize the SV40T antigen receptor expressed on the membranes of 293T cells, resulting in the localization of ultrasound microbubbles to 293T cell membranes. Furthermore, PNA containing NLS was inserted into the enhanced green fluorescent protein (EGFP)­N3 plasmid DNA (NLS­PNA­DNA), which increased nuclear localization. The nuclear import and gene expression efficiency of the AT­MCB with PNA­binding NLS were compared with AT­MCB alone or a PNA­binding NLS. The effect of the AT­MCB containing PNA­binding NLS on transfection was investigated. The ultrasound and AT­MCB delivery significantly enhanced the cytoplasmic intake of exogenous genes and maintained high cell viability. The nuclear import and gene expression of combined microbubble­ and PNA­transfected cells were significantly greater compared with cells that were transfected with AT­MCB or DNA with only PNA­binding NLS. The quantity of EGFP­N3 plasmids in the nuclei was increased by >5.0­fold compared with control microbubbles (CMCB) and NLS­free plasmids. The gene expression was ~1.7­fold greater compared with NLS­free plasmids and 1.3­fold greater compared with control microbubbles. In conclusion, UTMD combined with AT­MCB and a PNA­binding NLS plasmid significantly improved transfection efficiency by increasing cytoplasmic and nuclear DNA import. This method is a promising strategy for the noninvasive and effective delivery of target genes or drugs for the treatment of cardiovascular diseases.

Efficient gene therapy with a combination of ultrasound­targeted microbubble destruction and PEI/DNA/NLS complexes.

Current strategies of gene transfection are not efficient at achieving a notable therapeutic effect. The aim of the present study was to combine ultrasound­targeted microbubble destruction (UTMD) with a polyethylenimine/pEGFP­N3 plasmid/nuclear localization sequence (PEI/DNA/NLS) complex gene delivery system, and evaluate the transfection efficiency of enhanced green fluorescent protein (EGFP) gene delivery to 293T cells using this system. The formation of PEI/DNA/NLS complexes and the protective effects of PEI/NLS were verified by gel electrophoresis. Solutions consisting of the plasmid alone, PEI/DNA complexes, PEI/DNA/NLS complexes, UTMD+DNA, UTMD+PEI/DNA complexes, and UTMD+PEI/DNA/NLS complexes were transduced into 293T cells via ultrasound irradiation. The expression of GFP was observed using an inverted microscope and transfection efficiency was detected by flow cytometry following 24 h incubation in vitro. Cell activity was detected using a Cell Counting kit (CCK)­8 assay. Gel electrophoresis confirmed the formation of PEI/DNA/NLS complexes and demonstrated that PEI/NLS exhibited protective effects on plasmid integrity for a limited time. Inverted microscope observations revealed that a greater GFP signal was observed with the combined action of PEI/DNA/NLS complexes with UTMD, and flow cytometry analysis demonstrated the highest level of transfection efficiency in this group. In addition, the viability of the cells detected by CCK­8 and treated with PEI/DNA/NLS complexes with UTMD was >80%. In conclusion, the combination of UTMD and PEI/DNA/NLS complexes was highly effective for the efficient transfection of 293T cells without causing excessive cell damage. This method may provide a novel and effective gene transduction system to be applied in clinical treatments.

Ultrasound combined with targeted cationic microbubble-mediated angiogenesis gene transfection improves ischemic heart function.

The present study aimed to construct targeted cationic microbubbles (TCMBs) by synthesizing cationic microbubbles conjugated to an intercellular adhesion molecule-1 (ICAM-1) antibody, and then to use the TCMBs to deliver the angiopoietin-1 (Ang-1) gene into infarcted heart tissue using ultrasound-mediated microbubble destruction. It was hypothesized that the TCMBs would accumulate in higher numbers than non-targeted cationic microbubbles (CMBs) in the infarcted heart, and would therefore increase the efficiency of targeted Ang-1 gene transfection and promote angiogenesis. The results of the study demonstrated that the ability of TCMBs to target inflammatory endothelial cells was 18.4-fold higher than that of the CMBs in vitro. The accumulation of TCMBs was greater than that of CMBs in TNF-α-stimulated human umbilical cord veins, indicated by a 212% higher acoustic intensity. In vivo, the TCMBs specifically accumulated in the myocardial infarct area in a rabbit model. Three days after ultrasound microbubble-mediated gene transfection, Ang-1 protein expression in the TCMB group was 2.7-fold higher than that of the CMB group. Angiogenesis, the thickness of the infarct region and the heart function of the TCMB group were all significantly improved compared with those in the CMB and control groups at 4 weeks following gene transfection (all P<0.01). Therefore, the results of the current study demonstrate that ultrasound-mediated TCMB destruction effectively delivered the Ang-1 gene to the infarcted myocardium, resulting in improved cardiac morphology and function in the animal model. Ultrasound-mediated TCMB destruction is a promising strategy for improving gene therapy in the future.

Risk factors for incomplete healing of the uterine incision after cesarean section.

OBJECTIVE: To analyze related risk factors of post-cesarean scar defects (PCSDs). METHODS: A retrospective study of full-term women delivered by cesarean with singleton infants at our hospital from April 2014 to December 2015 was performed. 69 cases of diagnosed PCSDs and 107 cases with no PCSD who accepted cesarean were recruited for analysis. Individual medical case and operative report review were retrieved for maternal clinical characteristics analysis. RESULTS: There was no difference in age, gestational age, BMI and baby's weight between the two groups. PCSD group has more cases of anemia, higher neutrophil percentage and more cases of elected cesarean and emergency cesarean than controls (all p < 0.05). For woman who received emergency cesarean, there were more cases with cervix dilated larger than 3 cm in operation and more cases received cesarean at least one time before. In addition, women with cesarean interval of at least 5 years, women with ultrasonic measured echo longer than 3 cm, women with poor healing in uterine incision, women with retroposition of uterus and women who had intrauterine separation are more prone to develop PCSDs. CONCLUSIONS: The occurrence of a defective uterine scar after cesarean section is primarily a by-product of the combination of multiple factors: age ≥30 years, BMI ≥27.30, premature rupture of membranes, elective cesarean section, post-operative anemia, WBC count ≥12.5 × 109 g/L and retroposition of uterus. These are high risk factors of PCSDs.

Enhanced effect of nuclear localization signal peptide during ultrasound­targeted microbubble destruction­mediated gene transfection.

Ultrasound­targeted microbubble destruction (UTMD) can promote the entry of plasmid DNA (pDNA) into the cell cytoplasm, by increasing the permeability of the cell membrane. But the transfection efficiency remains low due to inability of the pDNA to enter the nucleus. Various methods have been explored to improve the UTMD transfection efficiency, but with little success. In cells, the classic nuclear localization signal (cNLS) peptide is an amino acid sequence that signals proteins that are due for nuclear transport. The present study aimed to investigate whether binding of a cNLS peptide to the pDNA may improve the transfection efficiency of UTMD. Four experimental groups were analyzed: Control group (UTMD + pDNA), group with cNLS (UTMD + pDNA + cNLS), group with mutated NLS (mNLS; UTMD + pDNA + mNLS), and group with cNLS and the nuclear import blocker, wheat germ agglutinin (WGA; UTMD + pDNA + cNLS + WGA). The NLS was labeled by fluorescein isothiocyanate, whereas pDNA was labeled with Cy3. Different molar ratios were tested for the NLS and pDNA combination in order to achieve optimal binding of the two molecules. Human umbilical vein endothelial cells were then transfected using the optimum ultrasonic irradiation parameters and NLS/pDNA molar ratio. At 6 h post­transfection, the rates of Cy3­labeled pDNA inside the cells and their nuclei were detected by flow cytometry and laser confocal microscopy, and the cellular vs. nuclear uptake of pDNA was calculated. In order to further evaluate the effect of NLS on UTMD­mediated gene transfection, the transfection efficiency and relative expression levels of mRNA and protein were detected at 48 h post­transfection. The results demonstrated that the optimal molar ratio of NLS with pDNA was 104:1. The rates of pDNA successful entry into the cell and nucleus were significantly higher in the cNLS group compared with the control group. The transfection efficiency, and relative expression levels of mRNA and protein from the plasmid were significantly increased in the cNLS group compared with the control group. The mNLS group displayed no significant difference compared with the control group, while the WGA group exhibited significant inhibition in most indicators of transfection efficiency compared to the cNLS group. These results suggest that combining a cNLS peptide with pDNA during UTMD­mediated transfection significantly improved transfection efficiency. Thus, a cNLS peptide may be an important mediator and a new strategy in enhancing the efficiency of UTMD­mediated gene transfection.

Selective, switchable fluorescent probe for heparin based on aggregation-induced emission.

A positively charged tetraphenylethene (TPE) derivative, TPE-4MN, was synthesized as a probe for heparin based on aggregation induced emission. On the addition of 5.0 µg/mL of heparin, TPE-4MN showed an enhanced emission of about 10-fold. The change in fluorescence at 475 nm was linear over a range of heparin concentrations of 0-1.0 µg/mL with an R = 0.99988 and the limit of detection (LOD) was calculated to be 0.75 µg/mL. The mechanism of the detection was proven to be through an ion pairing interaction. TPE-4MN showed good selectivity for heparin over other types of polysaccharides and could easily distinguish heparin from heparan sulfate, a glycosaminoglycan having a similar structure to that of heparin.

A macrocyclic 1,4-bis(4-pyridylethynyl)benzene showing unique aggregation-induced emission properties.

A box-like macrocycle based on 1,4-bis(4-pyridylethynyl)benzene was derived in high yield. The macrocyclic fluorogen shows unique aggregation-induced emission properties.

A Red to Near-IR Fluorogen: Aggregation-Induced Emission, Large Stokes Shift, High Solid Efficiency and Application in Cell-Imaging.

A tetraphenylethene (TPE) derivative modified with the strong electron acceptor 2-dicyano-methylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran (TCF) was obtained in high yield by a simple two-step reaction. The resultant TPE-TCF showed evident aggregation-induced emission (AIE) features and pronounced solvatochromic behavior. Changing the solvent from apolar cyclohexane to highly polar acetonitrile, the emission peak shifted from 560 to 680 nm (120 nm redshift). In an acetonitrile solution and in the solid powder, the Stokes shifts are as large as 230 and 190 nm, respectively. The solid film emits red to near-IR (red-NIR) fluorescence with an emission peak at 670 nm and a quantum efficiency of 24.8 %. Taking the advantages of red-NIR emission and high efficiency, nanoparticles (NPs) of TPE-TCF were fabricated by using tat-modified 1,2-distearoylsn-glycero-3-phosphor-ethanol-amine-N-[methoxy-(polyethyl-eneglycol)-2000] as the encapsulation matrix. The obtained NPs showed perfect membrane penetrability and high fluorescent imaging quality of cell cytoplasm. Upon co-incubation with 4,6-diamidino-2-phenylindole (DAPI) in the presence of tritons, the capsulated TPE-TCF nanoparticles could enter into the nucleus and displayed similar staining properties to those of DAPI.

Conjugates of tetraphenylethene and diketopyrrolopyrrole: tuning the emission properties with phenyl bridges.

Diketopyrrolopyrrole (ACQ-gen) and tetraphenylethenes (AIE-gen) are linked together with phenyl bridges. The derivatives show substantially enhanced and red-shifted emission in the solid state.

[Development of IgHV family specific nucleic acid vaccine against lymphoma by construction of fusion gene of immunoglobulin heavy chain variable region and cytokine].

OBJECTIVE: To construct the gene co-expression vector of immunoglobulin heavy chain variable region (IgHV) gene and GM-CSF or IL-2 as IgHV family specific nucleic acid vaccine to lymphoma, and study the immune response of the immunized mice against lymphoma. METHODS: Six clones with significantly different length in gene fragments were selected from a gene bank constructed from normal fetal umbilical cord blood. These gene fragments in the clones were sequenced. The sequences were translated into IgHV peptides. T cell epitopes in the IgHV were predicted by bioinformatics. Meanwhile 6 clones of IgHV1 constructed before were analyzed. The most typical clone of IgHV1 and IgHV3 containing most of the T cell epitopes were selected. The IgHV gene fragments whose complementary determining region 3 (CDR3) was cut out and composed mainly of framework region were cloned into eukaryotic expression vector. The gene fragments of framework region of IgHV linking with gene of GM-CSF or IL-2 were cloned into pcDNA3.0 to form fusion genes of IgHV (FR)-GM-CSF/IL-2. Then they were transected into COS cells, African green monkey renal cells, by Lipofectin and their transient expression product was detected by ELISA. Twenty male Balb/c mice were randomly divided into 5 groups of 4 mice to be injected with different vectors at the weeks 0, 2, and 4: with IgHV3 (FR)/pcDNA3.0, with IgHV3 (FR)-IL-2/pcDNA 3.0, with blank vector pcDNA3.0 as control, with IgHV1 (FR)/pcDNA3.0, and with IgHV1 (FR)-IL-2/pcDNA3.0. At the weeks 0, 2, 4, 6, and 8 serum was collected from the mice to undergo indirect immunofluorescence examination to detect the antibodies against Namalwa cells, lymphoma cells from Africa green monkey, and normal lymphocytes. ELISA was used to examine the serum interferon (IFN)-gamma. RESULTS: About 30 of T cell epitopes existed in each IgHV peptides predicted by bioinformatics. Among the bioinformatics prediction, about 90% of the T cell epitopes were in the framework region (FR) of IgHV, and the first 10% with higher prediction score were in FR. The CDR3 of two typical IgHV sequences were cut down and the remaining sequences, mainly composed of FR, were used to construct the IgHV (FR)/pcDNA3.0 expression vectors. The 3' end of IgHV1 (FR) and IgHV3 (FR) were linked to GM-CSF or IL-2 gene successfully. The expression of GM-CSF in the group transfected with IgHV (FR)-GM-CSF/pcDNA3.0 were 200 times higher than in the group transfected with control pcDNA3.0. The expression of IL-2 in the group transfected with IgHV (FR)-IL-2/pcDNA3.0 were 60 times higher than in the group transfected with control pcDNA3.0. The antibody against IgHV1 family lymphoma cell line Namalwa could be detected since the second week in the mice immunized with IgHV1 (FR)-IL-2/pcDNA3.0. The antibody could be detected since the fourth week in the mice immunized with IgHV1 (FR). The antibody against lymphocyte line of IgHV3 family could be detected since the second week in the mice immunized with IgHV3 (FR)-IL-2/pcDNA3.0. The antibody could be detected since the fourth week in the mice immunized with IgHV3 (FR)/pcDNA3.0. The contents of serum IFN-gamma the mice immunized with IgHV1 (FR)-IL-2/pcDNA3.0 and with IgHV3 (FR)-IL-2/pcDNA3.0 was significantly higher than those of the mice immunized with IgHV (FR)/pcDNA3.0 or pcDNA3.0 (all P < 0.01). CONCLUSION: The gene fragments of IgHV (FR) can be used to construct IgHV family specific nucleic acid vaccine that induces anti-lymphoma immune response in mice by muscle injection. The expressing vectors of IgHV (FR)-GM-CSF/IL-2 induces stronger immunoreaction.

[The variation of T-cell clonal repertoire in patients with systemic lupus erythematosus (SLE) following autologous peripheral blood hematopoietic stem cell transplantation].

OBJECTIVE: Analyze the variation of T cell receptor(beta) (TCRbeta) CDR3 gene expressing repertoire in systemic lupus erythematosus (SLE) patients before and after autologous peripheral blood hematopoietic stem cell transplantation (APBSCT), to search the pathogenesis of SLE and characteristics of T cell reconstitute immune after APBSCT. METHODS: Reverse transcriptase - polymerase chain reaction (RT-PCR) amplified 25 subfamily genes of TCRbeta prom peripheral blood lymphocytes (PBLs) of SLE patient with APBSCT and run denaturation polyacrylamide sequencing gel electrophoresis, set up a map of TCRbetaCDR3 gene expressing repertoire. The bands of TCRbeta gene repertoire in the electrophoresis gel can be used to analyze the variety of T cell clones which expanded and disappeared in cases of SLE. Cut the bands and sequencing. Compare sequences of TCRbetagenes in normal and abnormal T cell clones to understand the relationship between TCRbeta gene and autoimmune diseases. RESULTS: In the normal control group, TCRbeta gene repertoires show more than 10 bands in each of 25 TCRbetaV families, characterized as Gaussian distribution. TCRbeta gene repertoire in the 8 patients with SLE were abnormally. Four patients expressed TCRbetaV13.1 gene preferentially. Three patients had TCRbetaV8, TCRbetaV9 and TCRbetaV18 genes over expressed. These over expressive genes represented the oligoclonal expansion of T cell populations. After APBSCT the TCRbeta gene repertoires regained CDR3 polymorphism in 5 patients, some of them recovered to normal completely. The sequencing analyse show dense and strong band in map of TCRbeta gene repertoires were T cell monoclonal or oligoclonal expansion frequently. In a case of SLE an amino acid sequence of TCRbetaV8 CDR3 was same to a sequence of disease correlative T cell clone with ankylosing spondylitis that had been reported in a literature before. CONCLUSION: SLE patients have oligoclonal expansion of T cell that related with the pathogenesis of autoimmune disease. After APBSCT, T cell clones recover to normal distribution. T cell clones that related with disease have been suppressed during immunological reconstitution. It may be a main reason the SLE get remission after transplantation.