[Clinical features and risk factors of anastomotic leakage after radical esophagectomy].

OBJECTIVE: To analyze the clinical features and risk factors of anastomotic leakage after radical esophagectomy of esophageal carcinoma. METHODS: The clinical data of 547 esophageal cancer patients underwent radical esophagectomy in Tianjin Medical University Cancer Hospital from January 2012 to December 2013 was analyzed retrospectively. There were 421 male and 126 female patients, with a median age of 65 years (ranging from 29 to 82 years). There were 155 cases of upper esophageal carcinoma, 340 cases of middle esophageal carcinoma and 52 cases of lower esophageal carcinoma. The surgical procedures included 41 cases completed through Sweet, 145 cases completed through McKeown, 279 cases completed through Ivor Lewis, 82 cases completed through minimally invasive esophagectomy. Moreover, 24 of 547 cases underwent preoperative neoadjuvant radiochemotherapy. χ² test and Cox's proportional hazards regression model were used for univariate analysis and multivariate analysis of the risk factors of postoperative anastomotic leakage. RESULTS: Twenty-seven of 547 cases with esophagectomy occurred anastomotic leakage and the incidence rate was 4.94% (27/547). One of 27 cases died and the mortality rate was 3.70% (1/27). The time of anastomotic leakage found was 4 to 45 days, with a median time of 10 days. There were 0 case of early leakage, 20 cases of mid-term leakage, 7 cases of late leakage. Three of 27 cases with anastomotic leakage had tracheoesophageal fistula, while 3 cases had contralateral pleural fistula. As to the incidence rate of anastomotic leakage, there was statistically significant difference between cervical anastomotic leakage (8.14%, 18/221) and intrathoracic anastomotic leakage (2.76%, 9/326) (χ² =7.41, P=0.000), among Sweet (4.88%, 2/41), McKeown (9.66%, 14/145), Ivor Lewis (2.51%, 7/279) and MIE (4.88%, 4/82) (χ² =21.48, P=0.000), and between with (16.67%, 4/24) and without (4.40%, 23/523) neoadjuvant radiochemotherapy (χ² =9.20, P=0.000). The multivariate analysis showed that anastomotic site (HR=2.594, P=0.048), surgical approach (HR=5.689, P=0.003) and preoperative neoadjuvant radiochemotherapy (HR=3.604, P=0.027) are independent risk factors for anastomotic leakage after esophagectomy. CONCLUSIONS: The mid-term anastomotic leakage after esophagectomy occurs higher. McKeown is a main surgical procedure and neoadjuvant radiochemotherapy is an important factor for the anastomotic leakage.

The Effects of the Recombinant CCR5 T4 Lysozyme Fusion Protein on HIV-1 Infection.

BACKGROUND: Insertion of T4 lysozyme (T4L) into the GPCR successfully enhanced GPCR protein stability and solubilization. However, the biological functions of the recombinant GPCR protein have not been analyzed. METHODS: We engineered the CCR5-T4L mutant and expressed and purified the soluble recombinant protein using an E.coli expression system. The antiviral effects of this recombinant protein in THP-1 cell lines, primary human macrophages, and PBMCs from different donors were investigated. We also explored the possible mechanisms underlying the observed antiviral effects. RESULTS: Our data showed the biphasic inhibitory and promotion effects of different concentrations of soluble recombinant CCR5-T4L protein on R5 tropic human immunodeficiency virus-1 (HIV-1) infection in THP-1 cell lines, human macrophages, and PBMCs from clinical isolates. We demonstrated that soluble recombinant CCR5-T4L acts as a HIV-1 co-receptor, interacts with wild type CCR5, down-regulates the surface CCR5 expression in human macrophages, and interacts with CCL5 to inhibit macrophage migration. Using binding assays, we further determined that recombinant CCR5-T4L and [125I]-CCL5 compete for the same binding site on wild type CCR5. CONCLUSIONS: Our results suggest that recombinant CCR5-T4L protein marginally promotes HIV-1 infection at low concentrations and markedly inhibits infection at higher concentrations. This recombinant protein may be helpful in the future development of anti-HIV-1 therapeutic agents.

Is tumor length a prognostic indicator for esophageal squamous cell carcinoma? A single larger study among Chinese patients.

OBJECTIVE: In esophageal cancer, depth of wall penetration, reflected by T classification, represents the most important prognostic variable. Our study aimed to investigate the impact of tumor length, measured as the longitudinal length, on the outcome of esophageal squamous cell carcinoma (ESCC) patients. METHODS: The survival data of 362 ESCC patients who underwent surgical resection as the primary treatment between 1999 and 2007 were collected retrospectively. Receiver-operator characteristic analysis was applied to identify the optimal cut-off values. RESULTS: 4.0 cm was identified as the optimal cut-off value within the whole group. Tumor length greater than 4.0 cm was associated with increasing T stage (P=0.001), N stage (P=0.046), and tumor differentiation (P=0.033). Univariate analysis and multivariate analysis both found that tumor length greater than 4.0 cm was associated with worse overall survival compared with shorter tumors (P<0.001). It appeared to have a greater impact on N0-N1 (P<0.001, P=0.026, respectively) than N2-N3 and appeared to have a higher impact on the lower-stage patients than the higher-stage patients. CONCLUSIONS: Tumor length proved to be an independent prognostic parameter for ESCC patients, especially for node-negative and lower-stage patients. More attention should be paid to its role in the management of ESCC.

Role for the conserved N-terminal cysteines in the anti-chemokine activities by the chemokine-like protein MC148R1 encoded by Molluscum contagiosum virus.

Molluscum contagiosum poxvirus (MCV) type 1 and type 2 encode two chemokine-like proteins MC148R1 and MC148R2. It is believed that MC148R proteins function by blocking the inflammatory response. However, the mechanism of the proposed biological activities of MC148R proteins and the role of the additional C-terminal cysteines that do not exist in other chemokines are not understood. Here, we demonstrated in two different assay systems that His-tagged MC148R1 displaces the interaction between CXCL12α and CXCR4. The N-terminal cysteines but not the additional C-terminal cysteines modulate this displacement. His-tagged MC148R1 blocked both CXCL12α-mediated and MIP-1α-mediated chemotaxis. In contrast, MC148R2 blocked MIP-1α-mediated but not CXCL12α-mediated chemotaxis. Immunoprecipitation by antibodies to MC148R1 or CXCL12α followed by immunoblotting and detection by antibodies to the other protein demonstrated physical interaction of His-tagged CXCL12α and His-tagged MC148R1. Interaction with chemokines might mask the receptor interaction site resulting in decreased binding and impairment of the biological activities.

Alternate receptor usage of neuropilin-1 and glucose transporter protein 1 by the human T cell leukemia virus type 1.

Recent studies have demonstrated that neuropilin 1 (NP-1) is involved in HTLV-1 entry; however, the role NP-1 plays in this process is not understood. We demonstrated that ectopic expression of human NP-1 but not NP-2 cDNA increased susceptibility to HTLV-1. SiRNA-mediated inhibition of NP-1 expression correlated with significant reduction of HTLV-1 Env-mediated fusion. The vascular endothelial growth factor (VEGF(165)) caused downmodulation of surface NP-1 and inhibited HTLV-1 infection of U87 cells. In contrast, VEGF(165) partially inhibited infection of primary astrocytes and had no significant effect on infection of HeLa cells. VEGF(165) and antibodies to the glucose transporter protein 1 (anti-GLUT-1) were both needed to block infection of primary astrocytes, however, only anti-GLUT-1 antibodies were sufficient to block infection of HeLa cells. HTLV-1 Env forms complexes with both NP-1 and GLUT-1 in primary human astrocytes. The alternate usage of these two cellular receptors may have important implications regarding HTLV-1 neuro-tropism.

Resistance to human immunodeficiency virus type 1 (HIV-1) generated by lentivirus vector-mediated delivery of the CCR5{Delta}32 gene despite detectable expression of the HIV-1 co-receptors.

It has previously been demonstrated that there are two distinct mechanisms for genetic resistance to human immunodeficiency virus type 1 (HIV-1) conferred by the CCR5Delta32 gene: the loss of wild-type CCR5 surface expression and the generation of CCR5Delta32 protein, which interacts with CXCR4. To analyse the protective effects of long-term expression of the CCR5Delta32 protein, recombinant lentiviral vectors were used to deliver the CCR5Delta32 gene into human cell lines and primary peripheral blood mononuclear cells that had been immortalized by human T-cell leukemia virus type 1. Blasticidin S-resistant cell lines expressing the lentivirus-encoded CCR5Delta32 showed a significant reduction in HIV-1 Env-mediated fusion assays. It was shown that CD4(+) T lymphocytes expressing the lentivirus-encoded CCR5Delta32 gene were highly resistant to infection by a primary but not by a laboratory-adapted X4 strain, suggesting different infectivity requirements. In contrast to previous studies that analysed the CCR5Delta32 protective effects in a transient expression system, this study showed that long-term expression of CCR5Delta32 conferred resistance to HIV-1 despite cell-surface expression of the HIV co-receptors. The results suggest an additional unknown mechanism for generating the CCR5Delta32 resistance phenotype and support the hypothesis that the CCR5Delta32 protein acts as an HIV-suppressive factor by altering the stoichiometry of the molecules involved in HIV-1 entry. The lentiviral-CCR5Delta32 vectors offer a method of generating HIV-resistant cells by delivery of the CCR5Delta32 gene that may be useful for stem cell- or T-cell-based gene therapy for HIV-1 infection.

A naturally occurring splice variant of CXCL12/stromal cell-derived factor 1 is a potent human immunodeficiency virus type 1 inhibitor with weak chemotaxis and cell survival activities.

CXCL12/stromal cell-derived factor 1 is a member of the CXC family of chemokines that plays an important role in hematopoiesis and signals through CXCR4 and CXCR7. Two splice variants of human CXCL12 (CXCL12alpha and CXCL12beta) induce chemotaxis of CXCR4(+) cells and inhibit X4 infection. Recent studies described four other novel splice variants of human CXCL12; however, their antiviral activities were not investigated. We constructed and expressed all of the CXCL12 splice variants in Escherichia coli. Recombinant proteins were purified through a His affinity column, and their biological properties were analyzed. All six CXCL12 variants induced chemotaxis of CXCR4(+) and CXCR7(+) cell lines. Enhancement of survival and replating capacity of human hematopoietic progenitor cells were observed with CXCL12alpha, CXCL12beta, and CXCL12epsilon but not with the other variants. CXCL12gamma showed the greatest antiviral activity in X4 inhibition assays and the weakest chemotaxis activity through CXCR4. The order of potency in X4 inhibition assays was as follows: CXCL12gamma > CXCL12beta > CXCL12alpha > CXCL12theta > CXCL12epsilon > CXCL12delta. The order of anti-human immunodeficiency virus (HIV) activity was associated with the number of BBXB motifs present in each variant; the most potent inhibitor was CXCL12gamma, with five BBXB domains. The results suggest that the different C termini of CXCL12 variants may contain important molecular determinants for the observed differences in antiviral effects and other biological functions. These studies implicate CXCL12gamma as a potent HIV-1 entry inhibitor with significantly reduced chemotaxis activity and small or absent effects on progenitor cell survival or replating capacity, providing important insight into the structure-function relationships of CXCL12.

CCR5Delta32 protein expression and stability are critical for resistance to human immunodeficiency virus type 1 in vivo.

Human immunodeficiency virus type 1 (HIV-1) infection of individuals carrying the two alleles of the CCR5Delta32 mutation (CCR5(-/-)) has rarely been reported, but how the virus overcomes the CCR5Delta32 protective effect in these cases has not been delineated. We have investigated this in 6 infected (HIV(+)) and 25 HIV(-) CCR5(-/-) individuals. CD4(+) T lymphocytes isolated from HIV(-) CCR5(-/-) peripheral blood mononuclear cells (PBMCs) showed lower levels of CXCR4 expression that correlated with lower X4 Env-mediated fusion. Endogenous CCR5Delta32 protein was detected in all HIV(-) CCR5(-/-) PBMC samples (n = 25) but not in four of six unrelated HIV(+) CCR5(-/-) PBMC samples. Low levels were detected in another two HIV(+) CCR5(-/-) PBMC samples. The expression of adenovirus 5 (Ad5)-encoded CCR5Delta32 protein restored the protective effect in PBMCs from three HIV(+) CCR5(-/-) individuals but failed to restore the protective effect in PBMCs isolated from another three HIV(+) CCR5(-/-) individuals. In the latter samples, pulse-chase analyses demonstrated the disappearance of endogenous Ad5-encoded CCR5Delta32 protein and the accumulation of Ad5-encoded CCR5 during the chase periods. PBMCs isolated from CCR5(-/-) individuals showed resistance to primary X4 but were readily infected by a lab-adapted X4 strain. Low levels of Ad5-encoded CCR5Delta32 protein conferred resistance to primary X4 but not to lab-adapted X4 virus. These data provide strong support for the hypothesis that the CCR5Delta32 protein actively confers resistance to HIV-1 in vivo and suggest that the loss or reduction of CCR5Delta32 protein expression may account for HIV-1 infection of CCR5(-/-) individuals. The results also suggest that other cellular or virally induced factors may be involved in the stability of CCR5Delta32 protein.

GLUT-1-independent infection of the glioblastoma/astroglioma U87 cells by the human T cell leukemia virus type 1.

The human glucose transporter protein 1 (GLUT-1) functions as a receptor for human T cell leukemia virus (HTLV). GLUT-1 is a twelve-transmembrane cell surface receptor with six extracellular (ECL) and seven intracellular domains. To analyze HTLV-1 cytotropism, we utilized polyclonal antibodies to a synthetic peptide corresponding to the large extracellular domain of GLUT-1. The antibodies caused significant blocking of envelope (Env)-mediated fusion and pseudotyped virus infection of HeLa cells but had no significant effect on infection of U87 cells. This differential effect correlated with the detection of high-level surface expression of GLUT-1 on HeLa cells and very weak staining of U87 cells. To investigate this in terms of viral cytotropism, we cloned GLUT-1 cDNA from U87 cells and isolated two different versions of cDNA clones: the wild-type sequence (encoding 492 residues) and a mutant cDNA with a 5-base pair deletion (GLUT-1Delta5) between nucleotides 1329 and 1333. The deletion, also detected in genomic DNA, resulted in a frame-shift and premature termination producing a truncated protein of 463 residues. Transfection of the wild-type GLUT-1 but not GLUT-1Delta5 cDNA into CHO cells resulted in efficient surface expression of the human GLUT-1. Co-expression of GLUT-1 with GLUT-1Delta5 produces a trans-inhibition by GLUT-1Delta5 of GLUT-1-mediated HTLV-1 envelope (Env)-mediated fusion. Co-immunoprecipitation experiments demonstrated physical interaction of the wild-type and mutant proteins. Northern blot and RT-PCR analyses demonstrated lower GLUT-1 RNA expression in U87 cells. We propose two mechanisms to account for the impaired cell surface expression of GLUT-1 on U87 cells: low GLUT-1 RNA expression and the formation of GLUT-1/GLUT-1Delta5 heterodimers that are retained intracellularly. Significant RNAi-mediated reduction of endogenous GLUT-1 expression impaired HTLV-1 Env-mediated fusion with HeLa cells but not with U87 cells. We propose a GLUT-1-independent mechanism of HTLV-1 infection of U87 cells. The results may have important implications for HTLV-1 neurotropism and pathogenesis.

Infection of CD4+ T lymphocytes by the human T cell leukemia virus type 1 is mediated by the glucose transporter GLUT-1: evidence using antibodies specific to the receptor's large extracellular domain.

To analyze HTLV-1 cytotropism, we developed a highly sensitive vaccinia virus-based assay measuring activation of a reporter gene upon fusion of two distinct cell populations. We used this system in a functional cDNA screening to isolate and confirm that the glucose transporter protein 1 (GLUT-1) is a receptor for HTLV-1. GLUT-1 is a ubiquitously expressed plasma membrane glycoprotein with 12 transmembrane domains and 6 extracellular loops (ECL). We demonstrate for the first time that peptide antibodies (GLUT-IgY) raised in chicken to the large extracellular loop (ECL1) detect GLUT-1 at the cell surface and inhibit envelope (Env)-mediated fusion and infection. Efficient GLUT-IgY staining was detected with peripheral blood CD4(+) lymphocytes purified by positive selection. Further, GLUT-IgY caused efficient inhibition of Env-mediated fusion and infection of CD4(+) T and significantly lower inhibition of CD8(+) T lymphocytes. The specificity of GLUT-IgY antibodies to GLUT-1 was demonstrated by ECL1 peptide competition studies. Grafting ECL1 of GLUT-1 onto the receptor-negative GLUT-3 conferred significant receptor activity. In contrast, grafting ECL1 of GLUT-3 onto GLUT-1 resulted in a significant loss of the receptor activity. The ECL1-mediated receptor activity was efficiently blocked with four different human monoclonal antibody (HMab) to HTLV-1 Env. The ECL1-derived peptide blocked HTLV-1 Env-mediated fusion with several nonhuman mammalian cell lines. The results demonstrate the utilization of cell surface GLUT-1 in HTLV-1 infection of CD4(+) T lymphocytes and implicate a critical role for the ECL1 region in viral tropism.