[Surgical management strategy for metastatic cervical lymph nodes surrounding the carotid artery in head and neck squamous cell carcinoma].

Objective: To explore the surgical intervention strategy for metastatic cervical lymph nodes surrounding the carotid artery in head and neck squamous cell carcinoma. Methods: A total of 62 patients with advanced head and neck tumors and carotid wrap by disease treated in Department of Otorhinolaryngology and Head and Neck Surgery, the Affiliated BenQ Hospital of Nanjing Medical University between June 2019 and December 2023 were reviewed, of whom 9 patients presented with metastatic squamous cell carcinoma in cervical lymph nodes of unknown primary or with no recurrence of primary lesion and all the 9 patients were males, aged from 48 to 79 years old, with≤level 2 of Eastern Cooperative Oncology Group-Performance Status (ECOG-PS). Radiographically common carotid artery (CCA) and/or internal carotid artery (ICA) were surrounded by≥270° with tumor. All the 9 patients received implantation of covered stent in carotid artery and radical resection of metastatic cervical lymph nodes. The success rate, complications, surgery-related complications, local recurrence rate, quality of life (QOL) and overall survival (OS) were analyzed. The QOL of patients was compared by paired rank sum test, and P<0.05 indicated statistically significant difference. The OS was analyzed by Kaplan-Meier. Results: The success rate of stent implantation was 100%, with no implantation-related complications. R0 resection was performed in 8 cases and R1 resection in 1 case. The QOL of patients after surgery was improved, and the improvements in "pain", "mood" and "anxiety" were statistically significant(Z values were -2.236, -2.460 and -2.200, respectively, and all P values were<0.05). Follow-up was 1-18 months, with a median of 7 months, and 1 case was lost to follow-up. Local recurrence occurred in 3 patients with an incidence of 37.5% (3/8). OS was 59.9% at 12 months after surgery. Conclusion: Implantation of covered stent in carotid artery combined with radical resection is an effective method for the treatment of cervical lymph node metastasis.

[The significance of circulating tumor cells in head and neck squamous cell carcinoma: a preliminary study].

Objective: To investigate the significance of circulating tumor cells (CTC) in squamous cell carcinoma of the head and neck (HNSCC). Methods: Twenty-four patients with HNSCC treated between October 2016 and July 2017 in our department were selected (experimental group), including 23 males and 1 females, aged 47-81 years. There were 14 cases of squamous cell carcinoma of larynx and 10 cases of hypopharynx, including I-Ⅱ stage (5 cases) and Ⅲ- Ⅳ stage (19 cases). All patients were primary and/or relapsed after treatment. Nine healthy volunteers were selected as control group. A novel in vivo capture technique (CellCellector system) was used to detect CTC. SPSS23.0 was used for statistical analysis. Results: The total capture rate of CTC in patients with HNSCC before treatment was 70.8% (17/24), with 40% (2/5) for patients at I-Ⅱ stage, and 78.9% (15/19) for patients at Ⅲ- Ⅳ stage, and was 0 in patients of control group. The total capture rate of CTC in patients with HNSCC after treatment was 50% (8/16). There was no significant correlation between CTC and age, sex, location of tumor or lymph node metastasis (P>0.05). CTC was related to tumor staging and tumor differentiation (P<0.05). The positive rate of EGFRVⅢ in CTC was 26.3% (5/19). Conclusions: The CellCollector system is a very efficient way of detecting CTC, and CTC plays an important role in the occurrence, progression and metastasis of HNSCC.

[Preliminary study on the expression of EGFRvâ ¢ and CXCR4 in head and neck squamous cell carcinoma].

Objective:The aim of this study is to detect the expression of EGFRvⅢ and CXCR4 in HNSCC and to explore its possible mechanism. Method:We selected 60 cases of HNSCC from May 2013 to November 2016 in our hospital for surgical treatment, including 44 cases of laryngeal squamous cell carcinoma, 14 cases of hypopharyngeal squamous cell carcinoma, and 2 cases of nasal squamous cell carcinoma. pTNM staging according to AJCC 2010, Ⅲ stage 17 cases, Ⅳa stage 40 cases, and Ⅳb stage 3 cases; according to tumor differentiation degree: 13 cases of high differentiation, moderately differentiation in 31 cases, and 16 cases of low differentiated; primary tumor, positive lymph node metastasis as the experimental group, and 10 cases of adjacent tissues are taken as control (control group). The expression of EGFRvⅢ and CXCR4 in primary tumor, metastasis and control group was observed by immunohistochemistry. The immunohistochemical results are semi quantitatively analyzed by Image-Pro Plus. All analyses were conducted using the SPSS 23.0 and Graphpad-Prism 5.0. Result:The expression of EGFRvⅢ and CXCR4 in HNSCC is higher than control, which was not correlated with age, gender, location, degree of differentiation, T grading, and TNM stage (P>0.05). But there was a significant correlation with N grading (P<0.05). The pathology result shows lymph node metastasis in cancer tissues were high expression, but the difference in expression between primary and metastatic tumors are not statistically significant. There is no significant positive correlation between EGFRvⅢ and CXCR4 (r=0.144, P>0.05). Conclusion: Both EGFRvⅢ and CXCR4 promote the invasion and metastasis of advanced HNSCC, however, there may be some synergistic effect between them.

[The role of CD4 ⁺ CD25 ⁺ T regs and CCL17, CCL22 in the pathogenesis of head and neck squamous cell carcinoma].

Objective:To investigate the role of CD4 ⁺ CD25 ⁺ T regs and CCL17 and CCL22 in the pathogenesis of HNSCC.Method:Twenty cases of HNSCC were enrolled. All patients were primary or recurrent after treatment (chemotherapy, surgery). The primary tumor was taken as the experimental group, and the adjacent normal tissues from the primary tumor 1-3 cm were taken as control group. CD4 ⁺ /Foxp3 and CD25⁺/Foxp3 were detected by immunofluorescence, while CCL17 and CCL22 were detected by ELISA. The difference and correlation between the amount of CD4⁺,CD25⁺ and the expression of CCL17, CCL22 were observed and analyzed.Result:The difference of mean optical density between CD4⁺/Foxp3 and CD25⁺/Foxp3 was statistically significant between the experimental group and the control group (P<0.05). The concentration of CCL17 and CCL22 was statistically different between the two groups (P<0.01). There was a positive correlation between CD25⁺and CCL17,CCL22(r=0.595, 0.720,P<0.01).Conclusion:CD4⁺CD25⁺T regs and CCL17,CCL22 played an important role in the pathogenesis of head and neck squamous cell carcinoma,both of which interacted with each other,and promoted the recurrence and metastasis of HNSCC.

[Preliminary study on the expression of CD4⁺CD25⁺Tregs and Foxp3 in peripheral blood of patients with head and neck squamous cell carcinoma].

Objective:This paper discusses the expression and significance of CD4⁺CD25⁺ Tregs and Foxp3 in peripheral blood of patients with head and neck squamous cell carcinoma. Method:We have collected 40 cases of head and neck squamous cell carcinoma patients with newly diagnosed or relapse after treatment, all of them underwent surgery, 39 males and 1 females, aged 41-79 years, in our department from January 2014 to December 2015. At the same time, 10 healthy volunteers are enrolled as control group. 2 ml peripheral blood has been detected by flow cytometry, and the ratio of CD4⁺CD25⁺/CD4⁺ and CD4⁺CD25⁺Fxop3⁺/CD4⁺ are calculated, respectively. SPSS 23.0 is used for statistical analysis. Result:CD4⁺CD25⁺ Tregs is highly expressed in head and neck tumors, compared with that in the healthy control, and the difference is statistically significant (P<0.01). There is significant difference between the early and late stage (P<0.05). The positive rate of Foxp3+ is higher in CD4⁺CD25⁺ Tregs positive cells than in control group (P<0.01). The difference of positive rate between late stage and early stage head and neck tumors is statistically significant (P<0.05). There is a significant positive correlation between CD4⁺CD25⁺ Tregs and Foxp3 (r=0.95). Conclusion:CD4⁺CD25⁺ Tregs and Foxp3 are highly expressed in the peripheral blood of patients with head and neck squamous cell carcinoma. Through the inhibition of the immune system in patients with head and neck squamous cell carcinoma, the development of carcinoma were promoted.

The purified 14-kilodalton envelope protein of vaccinia virus produced in Escherichia coli induces virus immunity in animals.

Vaccinia virus (VV) was successfully used as a live vaccine to eradicate smallpox, but the nature of viral proteins involved in eliciting viral immunity has not yet been identified. A potential candidate is a 14-kDa VV envelope protein that is involved in virus penetration at the level of virus-cell fusion, in cell-cell fusion late in infection, and in virus dissemination. The 14-kDa envelope protein has been produced in Escherichia coli, with properties similar to those of the native protein found in the virus particle and in infected cells (C. Lai, S. Gong, and M. Esteban, J. Biol. Chem. 256:22174-22180, 1990). In this investigation, we showed that mice immunized with purified VV 14-kDa protein synthesized in E. coli in the form of a monomer or a trimer develop high-titer neutralizing antibodies and are protected when challenged with lethal doses of wild-type VV. Our findings demonstrate that it is possible to confer protection against VV through immunization with the 14-kDa envelope protein.

The 32-kilodalton envelope protein of vaccinia virus synthesized in Escherichia coli binds with specificity to cell surfaces.

The nature of interaction between vaccinia virus and the surface of host cells as the first step in virus infection is undefined. A 32-kDa virus envelope protein has been identified as a cell surface binding protein (J.-S. Maa, J. F. Rodriguez, and M. Esteban, J. Biol. Chem. 265:1569-1577, 1990). To carry out studies on the structure-function relationship of this protein, the 32-kDa protein was obtained from Escherichia coli cells harboring the expression plasmid pT7Ek32. The recombinant polypeptide was found to have structural properties similar to those of the native virus envelope protein. Binding studies of 125I-labeled 32-kDa protein to cultured cells of various origins revealed that the E. coli-produced 32-kDa protein exhibited selectivity, specificity, and saturability. Scatchard analysis indicated about 4.5 x 10(4) sites per cell with a high affinity (Kd = 1.8 x 10(-9) M), suggesting interaction of the 32-kDa protein with a specific receptor. The availability of large quantities of the 32-kDa virus protein in bacteria will permit further structural and functional studies of this virus envelope protein and facilitate identification of the specific cell surface receptor.

Structural and functional properties of the 14-kDa envelope protein of vaccinia virus synthesized in Escherichia coli.

Vaccinia virus is a highly cytocidal virus, but the steps that lead to virus penetration into cells, the first event in virus pathogenesis, have not been elucidated. We have shown that a 14-kDa envelope protein of vaccinia virus might play a major role in virus-penetration acting at the level of cell fusion (Rodriguez, J. F., Paez, E., and Esteban, M. (1987) J. Virol. 61, 395-404; Gong, S., Lai, C., and Esteban, M. (1990) Virology 178, 81-91). To carry out structural and functional studies on the vaccinia 14-kDa protein, it would be desirable to have a high level expression system, since the amount of protein that can be obtained from purified virus or from infected cells is very limited. In this investigation we demonstrate that the 14-kDa envelope protein of vaccinia virus is expressed in Escherichia coli in soluble form and at high levels. We establish, by several criteria, that the 14-kDa vaccinia virus protein expressed in E. coli is similar to the protein found in the virus particle based on apparent molecular mass, occurrence of disulfide-linked oligomers, reactivity against specific monoclonal antibody, and identity in amino-terminal sequence with the predicted DNA sequence of the gene. We define several structural and functional properties concerning the 14-kDa envelope protein of vaccinia virus. 1) 14 kDa is a trimer of identical subunits. 2) A monomer binds to itself more strongly than to a dimer or a trimer. 3) Oligomerization does not require cellular factors. 4) Trimers induce high titer neutralizing antibodies in animals which correlate with overall immunogenicity. 5) 14-kDa binds with specificity to the cell surface of cultured cells.

Vaccinia virus induces cell fusion at acid pH and this activity is mediated by the N-terminus of the 14-kDa virus envelope protein.

The mechanism by which the large-size poxviruses enter animal cells is not known. In this investigation we show that acid pH treatment of wild-type vaccinia virus-infected cells triggers strong fusion of cells in culture, with an optimum at pH 4.8. We have identified the virus-induced fusion protein as a 14-kDa envelope protein, based on the ability of a 14-kDa specific monoclonal antibody (mAbC3) to block vaccinia virus-induced fusion-from-within and fusion-from-without. We provide genetic evidence for a role of the 14-kDa protein in cell fusion, since insertion of the 14-kDa encoding gene into the genome of nonfusogenic mutant viruses generates heterozygous viruses that now acquire acid pH-dependent fusion activity. DNA sequence analyses of the 14-kDa encoding gene of the mutant viruses, 65-16 and 101-14, reveal N-terminal deletions of 46 and 10 amino acids, respectively. These deletions remove a small hydrophobic region at the N-terminus of the 14-kDa protein and prevent fusion. Our findings demonstrate that vaccinia virus can induce strong fusion of cells in culture at acid pH implying some entry of the virus by endocytosis, that the 14-kDa virus envelope protein is the fusogenic protein, and that the N-terminal proximal region is involved in fusion.

A single point mutation of Ala-25 to Asp in the 14,000-Mr envelope protein of vaccinia virus induces a size change that leads to the small plaque size phenotype of the virus.

The molecular defect responsible for a structural and functional abnormality of the 14,000-molecular-weight (14K) envelope protein of vaccinia virus has been identified. Through DNA sequence analysis of the entire 14K gene from wild-type vaccinia virus and three vaccinia virus mutants, a single base change of C to A was found that resulted in the substitution of Asp for Ala-25. This mutation is responsible for protein size abnormality, as documented by cell-free translation in a rabbit reticulocyte lysate of in vitro mRNA transcripts. In addition, through marker rescue experiments we show that this mutation is responsible for the small plaque size phenotype of vaccinia virus mutants. The structural consequence of the point mutation is a possible turn in an alpha-helix domain with destabilization of a hydrophobic interaction at the N terminus, resulting in monomers and trimers of vaccinia virus 14K protein with decreased electrophoretic mobilities. The functional consequence of the point mutation is a reduction in virulence of the virus.