Understanding the dynamics of TKI-induced changes in the tumor immune microenvironment for improved therapeutic effect.

BACKGROUND: The dynamic interplay between tyrosine kinase inhibitors (TKIs) and the tumor immune microenvironment (TME) plays a crucial role in the therapeutic trajectory of non-small cell lung cancer (NSCLC). Understanding the functional dynamics and resistance mechanisms of TKIs is essential for advancing the treatment of NSCLC. METHODS: This study assessed the effects of short-term and long-term TKI treatments on the TME in NSCLC, particularly targeting epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) mutations. We analyzed changes in immune cell composition, cytokine profiles, and key proteins involved in immune evasion, such as laminin subunit γ-2 (LAMC2). We also explored the use of aspirin as an adjunct therapy to modulate the TME and counteract TKI resistance. RESULTS: Short-term TKI treatment enhanced T cell-mediated tumor clearance, reduced immunosuppressive M2 macrophage infiltration, and downregulated LAMC2 expression. Conversely, long-term TKI treatment fostered an immunosuppressive TME, contributing to drug resistance and promoting immune escape. Differential responses were observed among various oncogenic mutations, with ALK-targeted therapies eliciting a stronger antitumor immune response compared with EGFR-targeted therapies. Notably, we found that aspirin has potential in overcoming TKI resistance by modulating the TME and enhancing T cell-mediated tumor clearance. CONCLUSIONS: These findings offer new insights into the dynamics of TKI-induced changes in the TME, improving our understanding of NSCLC challenges. The study underscores the critical role of the TME in TKI resistance and suggests that adjunct therapies, like aspirin, may provide new strategies to enhance TKI efficacy and overcome resistance.

ß-Glucan alleviates goal-directed behavioral deficits in mice infected with Toxoplasma gondii.

BACKGROUND: Toxoplasma gondii (T. gondii) is a neuroinvasive parasite causing neuroinflammation, which in turn is associated with a higher risk for several psycho-behavioral disorders. There is an urgent need to identify drugs capable of improving cognitive deficits induced by T. gondii infection. ß-Glucan, an active ingredient in mushrooms, could significantly enhance immunity. However, the effects of ß-glucan against neuroinflammation and cognitive decline induced by T. gondii infection remain unknown. The present study aimed to investigate the neuroprotective effect of ß-glucan on goal-directed behavior of mice chronically infected by T. gondii Wh6 strain. METHODS: A mice model of chronic T. gondii Wh6 infection was established by infecting mice by oral gavage with 10 cysts of T. gondii Wh6. Intraperitoneal injection of ß-glucan was manipulated 2 weeks before T. gondii infection. Performance of the infected mice on the Y-maze test and temporal order memory (TOM) test was used to assess the goal-directed behavior. Golgi-Cox staining, transmission electron microscopy, immunofluorescence, real-time PCR and western blot assays were used to detect prefrontal cortex-associated pathological change and neuroinflammation. RESULTS: The administration of ß-glucan significantly prevented T. gondii Wh6-induced goal-directed behavioral impairment as assessed behaviorally by the Y-maze test and TOM test. In the prefrontal cortex, ß-glucan was able to counter T. gondii Wh6-induced degeneration of neurites, impairment of synaptic ultrastructure and decrease of pre- and postsynaptic protein levels. Also, ß-glucan significantly prevented the hyperactivation of pro-inflammatory microglia and astrocytes, as well as the upregulation of proinflammatory cytokines caused by chronic T. gondii Wh6 infection. CONCLUSIONS: This study revealed that ß-glucan prevents goal-directed behavioral impairment induced by chronic T. gondii infection in mice. These findings suggest that ß-glucan may be an effective drug candidate to prevent T. gondii-associated psycho-behavioral disorders including goal-directed behavioral injury.

Polyene phosphatidylcholine ameliorates synovial inflammation: involvement of PTEN elevation and glycolysis suppression.

BACKGROUND: Synovial inflammation, characterized by the activation of synovial fibroblasts (SFs), is a crucial factor to drive the progression of rheumatoid arthritis (RA). Polyene phosphatidylcholine (PPC), the classic hepatoprotective drug, has been reported to ameliorate arthritis in animals. However, the molecular mechanism remains poorly understood. METHODS AND RESULTS: Using in vitro primary synovial fibroblast (SFs) culture system, we revealed that phosphatase and tension homolog deleted on chromosome 10 (PTEN), a tumor suppressor, mediates the anti-inflammatory effect of PPC in lipopolysaccharide (LPS)-stimulated primary SFs. PPC decreased the production of TNF-α and IL-6 production while elevating the level of IL-10 and TGF-ß. Furthermore, PPC up-regulated the expression of PTEN, but inhibited the expression of p-AKT (ser473) and PI3K-p85α. Moreover, pre-treatment of SF1670 (the inhibitor of PTEN) or 740Y-P (the agonist of AKT/PI3K pathways) partially abrogated the anti-inflammatory effect of PPC. In addition, PPC could inhibit the expression of GLUT4, a key transporter of glucose that fuels the glycolysis, which is accompanied by the expression downregualtion of glycolytic enzymes PFKFB3 and PKM2. Furthermore, PPC could reduce ROS production and mitochondrial membrane potential in LPS-stimulated SFs and MH7A cell line. CONCLUSION: The present study supported that PPC can alleviate synovial inflammation, which involves in the elevation of PTEN and blockage of glycolysis.

The non-oral infection of larval Echinococcus granulosus induces immune and metabolic reprogramming in the colon of mice.

Background: The intestinal tract serves as a critical regulator for nutrient absorption and overall health. However, its involvement in anti-parasitic infection and immunity has been largely neglected, especially when a parasite is not transmitted orally. The present study investigated the colonic histopathology and functional reprogramming in mice with intraperitoneal infection of the larval Echinococcus granulosus (E. granulosus). Results: Compared with the control group, the E. granulosus-infected mice exhibited deteriorated secreted mucus, shortened length, decreased expression of tight junction proteins zonula occludens-1 (ZO-1), and occludin in the colon. Moreover, RNA sequencing was employed to characterize colonic gene expression after infection. In total, 3,019 differentially expressed genes (1,346 upregulated and 1,673 downregulated genes) were identified in the colon of infected mice. KEGG pathway and GO enrichment analysis revealed that differentially expressed genes involved in intestinal immune responses, infectious disease-associated pathways, metabolism, or focal adhesion were significantly enriched. Among these, 18 tight junction-relative genes, 44 immune response-associated genes, and 23 metabolic genes were annotated. Furthermore, mebendazole treatment could reverse the colonic histopathology induced by E. granulosus infection. Conclusions: Intraperitoneal infection with E. granulosus induced the pathological changes and functional reprogramming in the colon of mice, and mebendazole administration alleviated above alternations, highlighting the significance of the colon as a protective barrier against parasitic infection. The findings provide a novel perspective on host-parasite interplay and propose intestine as a possible target for treating parasitic diseases that are not transmitted orally.

Schistosoma japonicum Infection Leads to the Reprogramming of Glucose and Lipid Metabolism in the Colon of Mice.

The deposition of Schistosoma japonicum (S. japonicum) eggs commonly induces inflammation, fibrosis, hyperplasia, ulceration, and polyposis in the colon, which poses a serious threat to human health. However, the underlying mechanism is largely neglected. Recently, the disorder of glucose and lipid metabolism was reported to participate in the liver fibrosis induced by the parasite, which provides a novel clue for studying the underlying mechanism of the intestinal pathology of the disease. This study focused on the metabolic reprogramming profiles of glucose and lipid in the colon of mice infected by S. japonicum. We found that S. japonicum infection shortened the colonic length, impaired intestinal integrity, induced egg-granuloma formation, and increased colonic inflammation. The expression of key enzymes involved in the pathways regulating glucose and lipid metabolism was upregulated in the colon of infected mice. Conversely, phosphatase and tensin homolog deleted on chromosome ten (PTEN) and its downstream signaling targets were significantly inhibited after infection. In line with these results, in vitro stimulation with soluble egg antigens (SEA) downregulated the expression of PTEN in CT-26 cells and induced metabolic alterations similar to that observed under in vivo results. Moreover, PTEN over-expression prevented the reprogramming of glucose and lipid metabolism induced by SEA in CT-26 cells. Overall, the present study showed that S. japonicum infection induces the reprogramming of glucose and lipid metabolism in the colon of mice, and PTEN may play a vital role in mediating this metabolic reprogramming. These findings provide a novel insight into the pathogenicity of S. japonicum in hosts.

TLR-2-mediated metabolic reprogramming participates in polyene phosphatidylcholine-mediated inhibition of M1 macrophage polarization.

This study aimed to investigate whether the classic hepatoprotective drug polyene phosphatidylcholine (PPC) regulates macrophage polarization and explores the potential role of TLR-2 in this process. In RAW264.7 macrophages and murine bone marrow-derived macrophages (BMDMs) stimulated by lipopolysaccharide (LPS), PPC significantly inhibited the production of IL-6, TNF-α, and the mRNA expression of M1-type macrophage markers. Consistently, PPC reduced the mRNA expression of several key enzymes in the pathways of glycolysis and lipid synthesis while increasing the expression of key enzymes associated with lipid oxidation. Moreover, blocking the glycolytic pathway using 2-deoxy-D-glucose (2-DG) significantly enhanced the anti-inflammatory effect of PPC. However, inhibition of lipid oxidation using GW9662 (an inhibitor of PPAR-γ) and GW6471 (an inhibitor of PPAR-α) abolished the anti-inflammatory effect of PPC. Interestingly, TLR-2 expression in macrophages was significantly downregulated after exposure to PPC. Moreover, pre-activation of TLR-2 hampered the anti-inflammatory effect of PPC. In addition, PPC did not inhibit the secretion of IL-6 and TNF-α in TLR-2-/- BMDMs that were activated by LPS. This was consistent with the increased expression of M1 markers and glycolytic and lipid synthesis enzymes but decreased lipid oxidation-related enzymes. These results showed that PPC inhibits the differentiation of M1-type macrophages, which was most likely related to TLR-2-mediated metabolic reprogramming.

Metformin reduces HGF-induced resistance to alectinib via the inhibition of Gab1.

Alectinib is a second-generation anaplastic lymphoma kinase (ALK) inhibitor that has sufficient clinical efficacy and satisfactory safety in ALK-positive non-small cell lung cancer (NSCLC) patients with or without brain metastasis. Alectinib has now become an important drug in the first-line treatment of advanced ALK-positive NSCLC; however, resistance is almost inevitable. The increased expression of hepatocyte growth factor (HGF) and its physiological receptor tyrosine kinase MET have been shown to be linked to acquired resistance to various tyrosine kinase inhibitors (TKIs), and this phenomenon has been observed in some ALK-positive NSCLC tumour tissues. In this study, we found that HGF levels in the culture supernatant of an ALK-positive cell line tended to increase with time and could be further increased by alectinib in a time-dependent manner. Exogenous or endogenous HGF did not cause resistance to the ALK/MET double-targeted small molecule inhibitor crizotinib, but it was an important cause of alectinib resistance. Furthermore, Gab1 was a key effector in the HGF/MET signal transduction pathway that mediated alectinib resistance. The antidiabetic drug metformin combined with alectinib overcame alectinib resistance triggered by HGF/MET through disrupting the complex between MET and Gab1, thereby inhibiting Gab1 phosphorylation and the activation of downstream signal transduction pathways. These results suggest that metformin combined with alectinib may be useful for overcoming alectinib resistance induced by the activation of the HGF/MET signalling pathway and improving the efficacy of alectinib.

The Metabolic Reprogramming Profiles in the Liver Fibrosis of Mice Infected with Schistosoma japonicum.

Disordered glucose and lipid metabolism contributes to the progression of several liver diseases, while the upregulation of phosphatase and tensin homology deleted on chromosome ten (PTEN), a well-known tumour suppressor gene, can improve the condition through metabolic programming. This study first characterized the metabolic profiles and the involvement of PTEN in the hepatic fibrosis induced by Schistosoma japonicum (S. japonicum) to provide a novel clue for metabolism-targeted treatment. Compared with control mice, infected mice showed infiltrated immune cells in their livers, increased levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and decreased glucose levels in their sera. The expression of key enzymes in the glycolytic pathway was significantly increased, and the expression of gluconeogenic genes was distinctly decreased. Moreover, the infection upregulated the hepatic expression of enzymes involved in fatty acid oxidation, which was consistent with the decreased number of lipid droplets in livers and the lowered levels of triglyceride in sera. Consistently, PTEN and its downstream signalling were significantly inhibited. In vitro, soluble egg antigen (SEA) downregulated the expression of PTEN in both the macrophage RAW264.7 cell line and the murine hepatocellular carcinoma HEP1-6 cell line, and induced a metabolic phenotype similar to the in vivo results. Overall, this study showed that S. japonicum infection induced the reprogramming of glucose and lipid metabolism in mice during the period of liver fibrosis and that SEA could act as a modulator to trigger such a metabolic switch in macrophages and hepatocytes. PTEN might play an essential role in mediating these metabolic reprogramming events.

The diagnostic effect of sequential detection of ADA screening and T-SPOT assay in pleural effusion patients.

Purpose: To evaluate the diagnostic effect of sequential detection of Adenosine deaminase (ADA) screening and T-SPOT assay on tuberculosis (TB) pleurisy in pleural effusion patients. Materials and methods: 248 pleural effusion patients (172 TB and 76 non-TB) were retrospectively analyzed in the study. The concentrations of ADA and lactate dehydrogenase (LDH) were measured in pleural fluids and serum samples of the patients. T-SPOTT assays were performed in pleural fluids. The relationship between ADA, T-SPOT and the occurrence of TB pleurisy was evaluated using logistic regression analysis. Results: The level of pleural ADA and positive rate of T-SPOT were all higher in TB pleurisy group than non-TB pleurisy group (p < .001). The positive rate of T-SPOT detection reached 98.83% in the TB pleurisy group while only 40.7% in non-TB pleurisy group (p < .001). Additionally, 8 patients (4.65%) in the TB pleurisy group showed the level of pleurisy ADA exceeded 40 IU/L while only one patient (1.31%) in the non-TB pleurisy group. Conclusion: The sequential detection of ADA screening and T-SPOT assay was found to be an accurate and rapid method for identifying TB pleurisy from pleural effusion, which would promote effective treatment.

The excretory-secretory products of Echinococcus granulosus protoscoleces stimulated IL-10 production in B cells via TLR-2 signaling.

BACKGROUND: Excretory-secretory products released by Echinococcus granulosus protoscoleces (EgPSC-ESPs) are well-known to regulate T cell responses. However, their direct influence on the differentiation of B cell subsets remains largely elusive. This study investigated the effects of EgPSC-ESPs on the differentiation of IL-10-producing B cells (B10), and explored the possible role of Toll-like receptor 2 (TLR-2) signaling in this process. RESULTS: In comparison to phosphate buffered saline (PBS), B cells exposed to the excretory-secretory products (ESPs) generated higher percentages of B10 cells, with higher expression of IL-10 mRNA, and larger amount of IL-10 production, which were in a dose dependent way. The mRNA and protein expression of TLR-2 in the ESPs-stimulated B cells were significantly higher than those in PBS, which was consistent to the results in B cells isolated from EgPSC infected mice. Moreover, TLR-2-/- B cells in response to ESPs stimulation expressed lower levels of IL-10 mRNA and produced undetectable IL-10 in comparison to those in normal B cells. In addition, Phosphatase and tensin homolog deleted on chromosome ten/AKT/Phosphatidylinositol-3 kinase (PTEN/AKT/PI3K) pathway was activated in ESPs-treated B cells, which was also dependent on TLR-2 signaling. Pam3CSK4, the agonist of TLR-2, could mock the effects of ESPs on the expression of PTEN, AKT and PI3K. CONCLUSION: Overall, this study revealed that TLR-2 signaling was required for B10 induction mediated by EgPSC-ESPs, which might be an immunomodulatory target against the parasite infection.

The excretory-secretory products of Echinococcus granulosus protoscoleces directly regulate the differentiation of B10, B17 and Th17 cells.

BACKGROUND: Excretory-secretory products (ESPs) released by helminths are well-known to regulate T cell responses in the host. However, their direct influence in the differentiation of naïve T cells, and especially B cells, remains largely unknown. This study investigated the effects of Echinococcus granulosus protoscoleces ESPs (EgPSC-ESPs) on the differentiation of IL-10-producing B cells (B10), IL-17A-producing B cells (B17) and Th17 cells. METHODS: BALB/c mice injected with EgPSC were used to evaluate the in vivo profiles of B10, B17 and Th17 cells. In vitro purified CD19+ B and naïve CD4+ T cells were cultured in the presence of native, heat-inactivated or periodate-treated EgPSC-ESPs, and the differentiation of these cell subsets were compared. RESULTS: In contrast to the control group, infected mice showed higher frequencies of B10, B17 and Th17 cells, and higher levels of IL-10 and IL-17A in the sera. Interestingly, B17 cells were first identified to express CD19+CD1dhigh. In vitro, B cells cultured with native ESPs exhibited a higher percentage of B10 cells but lower percentage of B17 and Th17 cells compared to the PBS group. Moreover, the relative expression of IL-10 and IL-17A mRNA were consistent with the altered frequencies. However, ESPs subjected to heat-inactivation or periodate treatment exhibited an inverse effect on the induction of these cell subsets. CONCLUSIONS: Our findings indicate that ESPs released by EgPSC can directly regulate the differentiation of B10, B17 and Th17 cells, which appear to be heat-labile and carbohydrate-dependent.

Bioinformatic prediction of the epitopes of Echinococcus granulosus antigen 5.

The aim of the present study was to predict and analyze the secondary structure, and B and T cell epitopes of Echinococcus granulosus antigen 5 (Ag5) using online software in order to investigate its immunogenicity and preliminarily evaluate its potential as an effective antigen peptide vaccine for cystic echinococcosis. The PortParam program was used to analyze molecular weight, the theoretical isoelectric point, instability index and other physicochemical properties. The secondary structure of the Ag5 protein was predicted using Self-Optimized Prediction method With Alignment and the tertiary structure of the Ag5 protein was predicted using 3DLigandSite together with Center for Biological Sequence Analysis Prediction Servers. Furthermore, the Immune Epitope Database software was used to predict B cell epitopes, and T cell epitopes were predicted with the BioInformatics and Molecular Analysis Section and SYFPEITHI programs. The results demonstrated that α-helixes, ß-turns, random coils and extended strands account for 23.35, 10.95, 41.32, and 24.38% of the secondary structure of the Ag5 protein, respectively. Ten potential B cell epitopes of Ag5 were identified as the amino acids sequences 27-39, 70-80, 117-130, 146-168, 250-262, 284-293, 339-349, 359-371, 403-412 and 454-462, and seven potential T cell epitopes were identified as the amino acid sequences 52-60, 57-65, 182-190, 231-239, 273-281, 318-326 and 467-475. Thus, ten B cell epitopes and seven T cell epitopes were identified on Ag5, suggesting the strong immunogenicity of this protein, which could be applied to design antigen peptide vaccines for echinococcosis.

[Changes of Phenotype and Phagocytosis of Peritoneal Macrophages in Mice Infected with the Larval-stage of Echinococcus granulosus].

Objective: To investigate the phenotype and phagocytosis changes of the peritoneal macrophages (Mφ) in mice infected with the larval-stage Echinococcus granulosus, and explore the role of Mφ in the responses to parasite infection. Methods: Twenty-four female BALB/c mice (age of 6-8 weeks) were randomly assigned into control group and infection group (n=12 in each group). The mice in the infection group were intraperitoneally injected with 2 000 protoscoleces, while the control mice were injected with equal volume of PBS. Five months after infection, the peritoneal mononuclear cells were collected, and the percentage of Mφ and the expression of surface markers CD40, CD80, CD86, and major histocompatibility complex Ⅱ (MHCⅡ) were determined by flow cytometry. The absorbance（A490 value） of Mφ at different concentrations（1×106, 5×105, 1×105） was determined by the neutral red assay to evaluate the phagocytic ability of Mφ. Results: The Mφ constituted（30.40±3.15）% and（20.75±5.91）% in mononuclear cells in the infection and the control groups, respectively. The percentages of Mφ expressing CD40, CD80, CD86, and MHC Ⅱ were（45.33±5.51）%, （61.00±10.61）%, （56.88±10.66）% and （27.00±3.82）% in the infection group, which were all significantly higher than those in the control ［（41.43±6.19）%, （59.23±8.65）%, （10.91±1.82）% and （13.67±3.01%）］ （P<0.05）. The A490 values of Mφ at 1×106, 5×105, 1×105 were 0.41±0.03， 0.24±0.05 and 0.16±0.01 in the infection group, which were significantly lower than those in the control （0.61±0.15, 0.47±0.07 and 0.18±0.01）（P<0.01）. Conclusion: The phagocytic ability of peritoneal Mφ is dramatically weakened after infection, but the expression of activation-associated surface markers is significantly up-regulated after infection.

Metformin attenuates gefitinib-induced exacerbation of pulmonary fibrosis by inhibition of TGF-ß signaling pathway.

Interstitial lung disease (ILD) is a serious side-effect of epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) treatment. Therefore, it is necessary to study underlying mechanisms for the development of pulmonary fibrosis induced by EGFR-TKI and potential approaches to attenuate it. Metformin is a well-established and widely prescribed oral hypoglycemic drug, and has gained attention for its potential anticancer effects. Recent reports have also demonstrated its role in inhibiting epithelial-mesenchymal transition and fibrosis. However, it is unknown whether metformin attenuates EGFR-TKI-induced pulmonary fibrosis. The effect of metformin on EGFR-TKI-induced exacerbation of pulmonary fibrosis was examined in vitro and in vivo using MTT, Ki67 incorporation assay, flow cytometry, immunostaining, Western blot analysis, and a bleomycin-induced pulmonary fibrosis rat model. We found that in lung HFL-1 fibroblast cells, TGF-ß or conditioned medium from TKI-treated lung cancer PC-9 cells or conditioned medium from TKI-resistant PC-9GR cells, induced significant fibrosis, as shown by increased expression of Collegen1a1 and α-actin, while metformin inhibited expression of fibrosis markers. Moreover, metformin decreased activation of TGF-ß signaling as shown by decreased expression of pSMAD2 and pSMAD3. In vivo, oral administration of gefitinib exacerbated bleomycin-induced pulmonary fibrosis in rats, as demonstrated by HE staining and Masson staining. Significantly, oral co-administration of metformin suppressed exacerbation of bleomycin-induced pulmonary fibrosis by gefitinib. We have shown that metformin attenuates gefitinib-induced exacerbation of TGF-ß or bleomycin-induced pulmonary fibrosis. These observations indicate metformin may be combined with EGFR-TKI to treat NSCLC patients.

Metformin sensitizes EGFR-TKI-resistant human lung cancer cells in vitro and in vivo through inhibition of IL-6 signaling and EMT reversal.

PURPOSE: The EGF receptor tyrosine kinase inhibitors (EGFR-TKI) have become a standard therapy in patients with EGFR-activating mutations. Unfortunately, acquired resistance eventually limits the clinical effects and application of EGFR-TKIs. Studies have shown that suppression of epithelial-mesenchymal transition (EMT) and the interleukin (IL)-6/STAT3 pathway may abrogate this acquired mechanism of drug resistance of TKIs. This study aims to investigate the effect of metformin on sensitizing EGFR-TKI-resistant human lung cancer cells in vitro and in vivo through inhibition of IL-6 signaling and EMT reversal. EXPERIMENTAL DESIGN: The effect of metformin on reversing TKI resistance was examined in vitro and in vivo using MTT, BrdUrd incorporation assay, invasion assay, flow cytometry analysis, immunostaining, Western blot analysis, and xenograft implantation. RESULTS: In this study, metformin, a widely used antidiabetic agent, effectively increased the sensitivity of TKI-resistant lung cancer cells to erlotinib or gefitinib. Metformin reversed EMT and decreased IL-6 signaling activation in TKI-resistant cells, while adding IL-6 to those cells bypassed the anti-TKI-resistance effect of metformin. Furthermore, overexpression or addition of IL-6 to TKI-sensitive cells induced TKI resistance, which could be overcome by metformin. Finally, metformin-based combinatorial therapy effectively blocked tumor growth in xenografts with TKI-resistant cancer cells, which was associated with decreased IL-6 secretion and expression, EMT reversal, and decreased IL-6-signaling activation in vivo. CONCLUSION: Metformin, generally considered nontoxic and remarkably inexpensive, might be used in combination with TKIs in patients with non-small cell lung cancer, harboring EGFR mutations to overcome TKI resistance and prolong survival.

Novel sequences of subgroup J avian leukosis viruses associated with hemangioma in Chinese layer hens.

BACKGROUND: Avian leukosis virus subgroup J (ALV-J) preferentially induces myeloid leukosis (ML) in meat-type birds. Since 2008, many clinical cases of hemangioma rather than ML have frequently been reported in association with ALV-J infection in Chinese layer flocks. RESULTS: Three ALV-J strains associated with hemangioma were isolated and their proviral genomic sequences were determined. The three isolates, JL093-1, SD09DP03 and HLJ09MDJ-1, were 7,670, 7,670, and 7,633 nt in length. Their gag and pol genes were well conserved, with identities of 94.5-98.6% and 97.1-99.5%, respectively, with other ALV-J strains at the amino acid level (aa), while the env genes of the three isolates shared a higher aa identity with the env genes of other hemangioma strains than with those of ML strains. Interestingly, two novel 19-bp insertions in the U3 region in the LTR and 5' UTR, most likely derived from other retroviruses, were found in all the three isolates, thereby separately introducing one E2BP binding site in the U3 region in the LTR and RNA polymerase II transcription factor IIB and core promoter motif ten elements in the 5' UTR. Meanwhile, two binding sites in the U3 LTRs of the three isolates for NFAP-1 and AIB REP1 were lost, and a 1-base deletion in the E element of the 3' UTR of JL093-1 and SD09DP03 introduced a binding site for c-Ets-1. In addition to the changes listed above, the rTM of the 3' UTR was deleted in each of the three isolates. CONCLUSION: Our study is the first to discovery the coexistence of two novel insertions in the U3 region in the LTR and the 5' UTR of ALV-J associated with hemangioma symptoms, and the transcriptional regulatory elements introduced should be taken into consideration in the occurrence of hemangioma.

[Screening proteins interacting with infectious bursa disease virus Gt VP2 from cDNA library of B lymphoid cells of the bursa of Fabricius].

To screen the interactive proteins with IBDV Gt VP2 protein from cDNA library of B Lymphoid cells of the bursa of Fabricius. The expression cDNA library plasmids was transformed to the yeast competent cells, which have the bait plasmid-Gt VP2. After testing for growth in synthetic complete medium lacking histidine and uracil and for production of beta-galactosidase (X-gal), we obtained 16 positive clones. We searched the gene sequences of positive clones in the NCBI website. The blast results showed that five positive clones were the gallus sequences. They were Gallus gallus breed mitochondrial DNA, O_G1cNAc transferase, Tumor protein p53 binding protein, Stathmin and Chondroitin sulfate Ga1NAcT-2, respectively. This study is helpful for the further identifying the receptors of IBDV in B Lymphoid cells of the bursa of Fabricius.

Mitochondrial and energy metabolism-related properties as novel indicators of lung cancer stem cells.

Energy metabolism is the foundation of survival for all organisms, and mitochondria are the most important energy-supplying organelles in eukaryotic cells. However, the mitochondrial and energy/metabolism-related properties of cancer stem cells (CSCs), the stem cell-like subpopulation in tumor masses, remain unknown. In our study, we compared the masses of mitochondria and mitochondrial DNA (mtDNA), the mitochondrial membrane potential (Δψm), oxygen/glucose consumption, and the concentration of reactive oxygen species (ROS) and ATP between lung CSCs (LCSCs) and non-LCSCs. In addition, the change in features during differentiation was examined. Some mitochondrial and energy metabolism-related properties, such as perinuclear mitochondrial distribution, a lower quantity of mtDNA, higher Δψm, lower oxygen/glucose consumption, and lower intracellular concentrations of ROS and ATP, can be used as indicators of LCSCs.

[Prokaryotic expression, purification and identification of the recombinant gp90 protein of Reticuloendotheliosis virus].

OBJECTIVE: To obtain the recombinant gp90 protein of Reticuloendotheliosis virus (REV) and the anti-gp90 serum with high titer. METHODS: Using the plasmid pMD18T-env as template, we amplified the gp90 gene and then cloned it into pET-28a(+). The recombinant plasmid pET28a-gp90 was transformed into Escherichia coli BL21 (DE3), which was induced with isopropylthio-beta-D-galactoside(IPTG). After identification by SDS-PAGE and Western blotting, the purified gp90 protein was injected into Balb/c mice to prepare anti-gp90 serum. The specificity and titer of the antiserum were evaluated by IFA and the enzyme-linked immunosorbant assay (ELISA). RESULTS: SDS-PAGE and Western blotting showed that the gp90 protein was expressed successfully in the form of inclusion body in the recombinant E coli. ELISA showed the mouse anti-gp90 serum had a titer of 1:12800. Successful expression of recombinant gp90 protein and preparation of its antiserum laid the foundation for the development of diagnostic reagent of REV.