Early stage of antibody-mediated rejection after lung transplantation： A case report and literature review / 中南大学学报(医学版)

Antibody-mediated rejection (AMR) is a rare and serious complication after lung transplantation, with no characteristic of pathological manifestation, no systematic standard treatment, and the poor efficacy and prognosis. We reported a case of early AMR after lung transplantation and the relevant literature has been reviewed. A male patient presented with symptoms of cold 99 days after transplantation and resolved after symptomatic treatment. He admitted to the hospital 14 days later because of a sudden dyspnea and fever. Anti-bacteria, anti-fungi, anti-virus, and anti-pneumocystis carinii treatment were ineffective, and a dose of 1 000 mg methylprednisolone did not work too. The patient's condition deteriorated rapidly and tracheal intubation was done to maintain breathing. Serum panel reactive antibody and donor specific antibody showed postive in humen leukocyte antigen (HLA) II antibody. Pathological examination after transbronchial transplantation lung biopsy showed acute rejection. Clinical AMR was diagnosed combined the donor-specific antibody with the pathological result. The patient was functionally recovered after combined treatment with thymoglobuline, rituximab, plasmapheresis, and immunoglobulin. No chronic lung allograft dysfunction was found after 3 years follow up. We should alert the occurrence of AMR in lung transplantation recipient who admitted to hospital with a sudden dyspnea and fever while showed no effect after common anti-infection and anti-rejection treatment. Transbronchial transplantation lung biopsy and the presence of serum donor-specific antibody are helpful to the diagnosis. The treatment should be preemptive and a comprehensive approach should be adopted.

PTP1B up-regulates EGFR expression by dephosphorylating MYH9 at Y1408 to promote cell migration and invasion in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers worldwide. Protein tyrosine phosphatase 1B (PTP1B) is a member of protein tyrosine phosphatases (PTPs) family. In our previous work, PTP1B was found to be overexpressed in ESCC tissues and made contributions to the the cell migration and invasion as well as lung metastasis of ESCC. In this study, we explored the underlying molecular mechanisms. PTP1B enhanced cell migration and invasion by promoting epidermal growth factor receptor (EGFR) expression in ESCC, which was relied on phosphatase activity of PTP1B. Using GST-pulldown combined with LC/MS/MS, we found that nonmuscle myosin IIA (MYH9) was a novel substrate of PTP1B in ESCC cells. PTP1B dephosphorylated MYH9 at Y1408, by which PTP1B up-regulated EGFR expression and enhanced cell migration and invasion in ESCC. In conclusion, our study first reported that PTP1B was the positive regulator of EGFR by dephosphorylating MYH9 at Y1408 to promote cell migration and invasion, which revealed the regulatory mechanism of PTP1B-MYH9-EGFR axis in ESCC.

Tumor infiltrating T lymphocyte components in malignant pleural effusion of lung adenocarcinoma and their killing activities to autologous tumor cells / 中南大学学报(医学版)

To analyze the components of tumor infiltrating T lymphocyte (TIL) cells in malignant pleural effusion of lung adenocarcinoma, and evaluate their killing activities to autologous tumor cells. 
 Methods: Malignant pleural effusions were collected from 17 patients with lung adenocarcinoma. Mononuclear cells were isolated by Ficoll density gradient centrifugation and flow cytometer was used to analyze TIL cell components. TIL and tumor cells were separated through adherent culture. The tumor cells were identified via intramuscular injection of adherent cells into nude mice and the killing effect of cultured lymphocytes on autologous tumor cells was studied.
 Results: Of the TIL in malignant pleural effusions, T cells accounted for 60.6%-79.3%, while T helper cells were significantly higher than T killer cells (36.63%±1.90% vs 24.64%±2.32%, P<0.001). There were also natural killer (NK) cells and NK T cells in the effusions. Tumor cells were successfully isolated and cultured. The killing activity of cultured TIL to autologous tumor cells was 39.14%±12.04%, and the killing activity of TIL with high proliferation rate to autologous tumor cells was higher than that of low proliferation group (50.51%±3.80% vs 29.04%±5.77%, P<0.001).
 Conclusion: T lymphocytes are the major components of TIL in malignant pleural effusions derived from lung adenocarcinoma, and T helper cells are more than T killer cells. The killing activity of TIL with strong proliferation ability to autologous tumor cells is higher than that of TIL with weak proliferation ability. Therefore, cells from malignant pleural effusions could be used for cellular immunotherapy against tumor.

Co-targeting PLK1 and mTOR induces synergistic inhibitory effects against esophageal squamous cell carcinoma.

Both polo-like kinase 1 (PLK1) and mammalian/mechanistic target of rapamycin (mTOR) are attractive therapeutic targets for cancer therapy. However, the efficacy of the combined inhibition of both pathways for treating esophageal squamous cell carcinoma (ESCC), an aggressive malignancy with poor prognosis, remains unknown. In this study, we found that suppression of PLK1 by specific siRNA or inhibitor attenuated mTOR activity in ESCC cells. Phosphorylated S6, a downstream effector of mTOR signaling, was significantly correlated with overexpression of PLK1 in a subset of ESCC. These data suggest that PLK1 activates mTOR signaling in vitro and in vivo. More importantly, the mTOR inhibitor rapamycin synergized with PLK1 inhibitor BI 2536 to inhibit ESCC cell proliferation in culture and in mice. Notably, combined treatment with BI 2536 and rapamycin produced more potent inhibitory effects on the activation of S6 and AKT than either alone. Further analysis reveals that PLK1 modulates both mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) cascades. Therefore, dual inhibition of PLK1 and mTOR yields stronger antitumor effects, at least partially due to synergistic abrogated the activation of S6, eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), and AKT by cooperatively blocking mTORC1 and mTORC2 cascades. These results provide evidence that the mTOR inhibitor rapamycin synergistically enhances the antitumor effect of PLK1 inhibitor BI 2536 in ESCC cells. Simultaneous targeting of PLK1 and mTOR may thus be a novel and promising therapeutic strategy for ESCC. KEY MESSAGES: PLK1 potentiates both mTORC1 and mTORC2 activities in ESCC cells. PLK1 expression positively correlated with mTOR activity in a subset of ESCC. Co-targeting of PLK1 and mTOR produced stronger antitumor effects partially due to synergistic inhibition of AKT, 4E-BP1 and S6 through cooperatively blocking mTORC2 and mTORC1 cascades. Combination targeting of PLK1 and mTOR may be a novel and promising therapeutic strategy for ESCC treatment.

Sequestosome 1 protects esophageal squamous carcinoma cells from apoptosis via stabilizing SKP2 under serum starvation condition.

Esophageal squamous cell carcinoma (ESCC) is one of the malignancies in digestive system, with a low 5-year survival rate. We previously revealed that Sequestosome 1 (SQSTM1/p62) protein levels were upregulated in ESCC tissues. However, it is unclear about the function of p62 and the underlying mechanism. Here, we used immunofluorescence and immunohistochemistry to investigate the expression of p62 in ESCC. Western blotting, quantitative RT-PCR, colony formation assay, flow cytometry, immunoprecipitation and xenograft tumor assay were used to analyze the role of p62 in vitro and vivo. Here, we showed that p62 serves as a regulator of cell apoptosis under serum starvation condition in ESCC cells. Through activating the protein kinase C iota (PKCiota)-S-phase kinase-associated protein 2 (SKP2) signaling pathway, p62 enhances cell apoptosis resistance and colony formation in vitro and tumor growth in mouse models. Through interaction with the domains PB1, p62 upregulated the expression of PKCiota and then depressed the ubiquitin-mediated proteasomal degradation of SKP2. p62-silencing combined with a PKCiota inhibitor ATM significantly enhanced cell apoptosis and inhibited cell survival. Immunohistochemical analysis revealed a positive association between the expression of p62 and SKP2 in primary ESCC tissues. And importantly, p62 presented a markedly cytoplasmic translocation in cancerous cells, including in 16 (30.76%) tumors at stage T1, as compared with its nuclear location in normal esophageal epithelial cells. In summary, p62 plays an anti-apoptotic role in ESCC cells via stabilizing SKP2 under serum starvation condition. These data suggest that p62 might be an early biomarker and a candidate therapeutic target of ESCC.

mTOR: An attractive therapeutic target for osteosarcoma?

Osteosarcoma (OS) is a common primary malignant bone tumor with high morbidity and mortality in children and young adults. How to improve poor prognosis of OS due to resistance to chemotherapy remains a challenge. Recently, growing findings show activation of mammalian target of rapamycin (mTOR), is associated with OS cell growth, proliferation, metastasis. Targeting mTOR may be a promising therapeutic approach for treating OS. This review summarizes the roles of mTOR pathway in OS and present research status of mTOR inhibitors in the context of OS. In addition, we have attempted to discuss how to design a better treatment project for OS by combining mTOR inhibitor with other drugs.

[IGHMBP2 overexpression promotes cell migration and invasion in esophageal squamous carcinoma].

Immunoglobulin mu binding protein 2 (IGHMBP2) is located in 11q13.2, which is frequently amplified in esophageal squamous cell carcinoma (ESCC). IGHMBP2 encodes a helicase involved in DNA replication and repair. IGHMBP2 protein also regulates gene transcription. The present study aims to explore the amplification of IGHMBP2 and its potential role in ESCC. A further analysis of our previously reported array-CGH data showed that IGHMBP2 was amplified in 28.9% of primary ESCC tumors. Fluorescence in situ hybridization (FISH) and Western blot showed that IGHMBP2 was amplified and overexpressed in KYSE30, KYSE180, KYSE510 and KYSE150 esophageal cancer cell lines. Transwell assays demonstrated that knockdown of IGHMBP2 in KYSE30 and KYSE150 inhibited cell invasion and migration, and increased the expression levels of E-cadherin. When rescue plasmids expressing IGHMBP2 were introduced, the abilities of cell invasion and migration were restored. These data suggest that IGHMBP2 overexpression may promote invasion and migration of ESCC cells through down-regulation of E-cadherin.

Calreticulin promotes migration and invasion of esophageal cancer cells by upregulating neuropilin-1 expression via STAT5A.

PURPOSE: We previously revealed that the calreticulin (CRT) gene is a candidate oncogene promoting cell migration and invasion and that neuropilin-1 (NRP1) is a possible effector downstream of CRT in esophageal squamous carcinoma cells. This study aims to explore the mechanisms underlying the migration and invasion of esophageal cancer cells regulated by CRT through NRP1. EXPERIMENTAL DESIGN: Quantitative reverse-transcription polymerase chain reaction, Western blot analysis, chromatin immunoprecipitation, and reporter gene assays were used to investigate the relationship between CRT and NRP1. In vitro and in vivo assays were carried out to evaluate the effects of NRP1 on malignant phenotypes of ESCC cells and tumor metastasis in NOD/SCID mice. Immunohistochemistry was performed to analyze the expression of CRT and NRP1 in esophageal squamous cell carcinomas (ESCC). RESULTS: Knockdown of CRT decreased the expression of NRP1. Inhibition of NRP1 reduced ESCC cell motility in vitro and experimental metastasis in vivo. Ectopic expression of NRP1 rescued the defects of cell migration and invasion in CRT-shRNA cells. CRT depletion inhibited STAT5A phosphorylation at the Y694 site via a CaMKII-independent pathway. Moreover, STAT5A directly regulated NRP1 transcription. Knockdown of CRT or NRP1 led to a downregulation of MMP2, MMP9, and FAK. Notably, positive correlation was found between CRT and NRP1 expression in ESCC tissues (P = 5.87 × 10(-5)). CRT and NRP1 coexpression was significantly associated with lymph node metastasis (P = 0.025). CONCLUSIONS: Our findings suggest that NRP1 is a critical downstream effector of CRT in promoting cell migration and invasion, which might contribute to the metastasis of ESCC.

[Primary culture and morphological characteristics of human lacrimal adenoid cystic carcinoma cells].

OBJECTIVE: To explore a method of primary culturing human adenoid cystic carcinoma cells of lacrimal gland. METHODS: Experiment research. Tumor tissue was obtained from the surgical material of a patient diagnosed as lacrimal adenoid cystic carcinoma in Tianjin Medical University Eye Hospital during May 16th to June 1st.We gained primary cells via tissue culture techniques. Mixed cells were removed through several ways.Observed cell morphological characteristics by phase contrast microscope, scanning electron microscope and transmission electron microscope. Cyto-immunochemical staining was applied to detect the expressions of vimentin, desmin, S-100, cytokeratin, pan and CD34. Their expressions were also detected in the tumor tissue except CD34. Made cell growth curve and calculated cell doubling time. RESULTS: The outgrowth of cells was observed by day 5 after seeding tissues, and then cells generated slowly. The first passage proceeded by day 32, and the classical epithelioid cell colonies was observed by day 69 after inoculation. Purified cells of human lacrimal adenoid cystic carcinoma were obtained after the removal of mixed cells through several ways, which have been successfully subcultured for more than 100 passages. The 25th passage LACC cells appeared to be typically epithelioid cells, they showed contact inhibition as the density high enough.SEM and TEM showed the 25th passage LACC cells were malignant tumor cells poorly differentiated. They showed positive reaction with vimentin, cytokeratin (pan) as well as S-100, but negative reaction with desmin and CD34, which were consistent with the tumor tissue. The cell growth curve turned like a sigmoid one, and the cell doubling time was 37.1 h. CONCLUSIONS: We gained purified LACC cells, and understood the morphological characteristics, laying the foundation for the establishment of a human lacrimal adenoid cystic carcinoma cell line.

[The application of serum free medium in the research of isolating cancer stem cells].

The isolation and identification of cancer stem cells are the key points in exploring characteristics of cancer stem cells at present. Several species of cancer stem cells, for instance, retinoblastoma tumor stem cells, cancer stem cells of melanoma of choroid, breast cancer stem cells, lung cancer stem cells, colon cancer stem cells, etc., have been isolated and cultured successfully by serum free medium while their biological functions and characteristics are acquired. This review focus on the application of serum free medium in the research of isolating cancer stem cells, both ocular and general, in terms of providing foundation for further research on ocular cancer stem cells.

A population-based study of acquired bilateral nevus-of-Ota-like macules in Shanghai, China.

Acquired bilateral nevus-of-Ota-like macule (ABNOM) is a common skin dyspigmentation in Asian females. Although its clinical characteristics are well defined, its epidemiology and pathogenesis remain unclear. A large population-based cross-sectional study was conducted to determine the prevalence and risk factors of ABNOM. A total of 8,680 subjects (ages ranging from newborn to 99 years old; 54% female) were selected from urban areas in Shanghai, China, using a multistage sampling method. All participants (response rate=97%) were interviewed and examined for the presence of various pigmentary disorders by board-certified dermatologists. ABNOM was found in 2.5% of the study population (0.5% of males and 4.2% of females), and 90% of cases observed were female. In females, prevalence rose after the age of 15 years and sharply declined after the age of 50 years, with nearly half of the cases observed within ages 45-55 years (prevalence=8.5%). Age, contraceptive use, and sun exposure were independently associated with ABNOM. Women with ABNOM were less likely to have facial nevomelanocytic nevi and seborrheic keratoses. These findings indicate that sex hormone alteration and UV exposure may independently have an important role on the pathogenesis of ABNOM.

[Vascular endothelial growth factor D promoting the growth of lymphatic vessel in gastric carcinoma].

OBJECTIVE: To investigate the relationship between vascular endothelial growth factor D (VEGF-D) and metastasis of lymphatic vessel in gastric carcinoma. METHODS: The human VEGF-D cDNA was amplified from total RNA isolated from human normal gastric tissue, then it was inserted into T-A clone plasmid and subcloned into pEGFP eukaryotic expression vector. After the full-length sequence expected was confirmed by enzymatic digestion and sequencing,the human gastric carcinoma cell line SGC7901, which expressed a low level of VEGF-D, was transfected with the pEGFP/VEGF-D expression vector. Stable SGC7901 clones with high expression of VEGF-D were selected in vitro with G418, which were then combined and subcutaneously injected into nude mice to observe the density and morphology of lymphatic vessel. The outcomes were later compared with those of SGC7901 cells transfected with null vector(pEGFP) by immunostaining with a specific antibody LYVE-1. RESULTS: The average weight of tumors in the pEGFP group (1.13+/-0.40) g at day 35, was significantly lower than that in the pEGFP/VEGF-D group (2.24+/-0.82)g (P<0.05). The lymphatic vessel density (LVD) in the pEGFP group (2.89+/-1.32) was significantly lower than that in the pEGFP/VEGF-D group (5.74+/-1.30)(P<0.01). There were dilated functional lymphatic vessels around the tumor margin. CONCLUSION: VEGF-D may promote the growth and metastasis of tumor in gastric carcinoma by increasing the growth of lymphatic vessels.

Vascular endothelial growth factor D promoting the growth of lymphatic vessel in gastric carcinoma / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To investigate the relationship between vascular endothelial growth factor D (VEGF-D) and metastasis of lymphatic vessel in gastric carcinoma.</p><p><b>METHODS</b>The human VEGF-D cDNA was amplified from total RNA isolated from human normal gastric tissue, then it was inserted into T-A clone plasmid and subcloned into pEGFP eukaryotic expression vector. After the full-length sequence expected was confirmed by enzymatic digestion and sequencing,the human gastric carcinoma cell line SGC7901, which expressed a low level of VEGF-D, was transfected with the pEGFP/VEGF-D expression vector. Stable SGC7901 clones with high expression of VEGF-D were selected in vitro with G418, which were then combined and subcutaneously injected into nude mice to observe the density and morphology of lymphatic vessel. The outcomes were later compared with those of SGC7901 cells transfected with null vector(pEGFP) by immunostaining with a specific antibody LYVE-1.</p><p><b>RESULTS</b>The average weight of tumors in the pEGFP group (1.13+/-0.40) g at day 35, was significantly lower than that in the pEGFP/VEGF-D group (2.24+/-0.82)g (P<0.05). The lymphatic vessel density (LVD) in the pEGFP group (2.89+/-1.32) was significantly lower than that in the pEGFP/VEGF-D group (5.74+/-1.30)(P<0.01). There were dilated functional lymphatic vessels around the tumor margin.</p><p><b>CONCLUSION</b>VEGF-D may promote the growth and metastasis of tumor in gastric carcinoma by increasing the growth of lymphatic vessels.</p>