HSP70、eIF4E、DNMT1在宫颈癌中的表达及意义

摘要 目的：探讨热休克蛋白70（HSP70）、真核细胞翻译起始因子4E（eIF4E）、DNA甲基转移酶1（DNMT1）在宫颈癌中的表达及意义。方法：选择2015年2月至2017年2月我院接诊的40例女性宫颈癌患者为本研究对象,收集所有患者手术切除病理组织制作石蜡切片,并选择我院同期接受其他手术的35例标本作为对照组,使用SP免疫组织化学法观察乳腺癌组织中HSP70、eIF4E、DNMT1染色结果,并分析其和临床病理因素之间的关系。结果：在免疫组化学法结果中显示,40例宫颈癌组织中,HSP70阳性表达率为65.00%（26/40）,eIF4E阳性表达率为67.50%（27/40）,DNMT1阳性表达率为72.50%（29/40）,均显著高于对照组（P＜0.05）;在宫颈癌组织中,HSP70、eIF4E、DNMT1和分化程度、临床分期及淋巴转移均有密切关系,（P＜0.05）;将分化程度、临床分期、淋巴转移、HSP70、eIF4E、DNMT1进行相关分析,结果显示,HSP70、eIF4E、DNMT1和分化程度、临床分期及淋巴转移之间均呈正相关（P＜0.05）,且HSP70和eIF4E、DNMT1均呈正相关（P＜0.05）,eIF4E和DNMT1呈正相关（P＜0.05）。结论：在宫颈癌组织中HSP70、eIF4E、DNMT1的高表达和临床病理之间存在着密切关系。     

The point mutation analysis of Cyp2C9*2 (Arg144Cys C>T), Cyp2C9*3 (Ile359Leu A>C) and VKORC1 (1639G>A) in women with cervical cancer related to HPV: A case-control study

Human papillomavirus (HPV) is the most common sexually transmitted viral infection worldwide. HPV tumorigenesis genotypes are the causative agents of cervical cancer and genital malignancies. The scientific literature has demonstrated that life style, environmental, epigenetic accompanied with HR-HPV genotypes are potential risk factors for cervical cancer progression. The frequencies of the Cyp2C9*², Cyp2C9*³, and vitamin K epoxide reductase complex subunit 1 (VKORC1) genotypes as potential molecular biomarkers have been investigated on Iranian women with cervical malignancy related to HPV genotypes. As a case-control study, the mutations were appraised using a polymerase chain reaction-restriction fragment length polymorphism procedure on women suffering from HPV infection (60 cases), CC (46 cases), and 40 subjects of as healthy control. The outcomes demonstrated that Cyp2C9*³ showed a meaningful relationship between women diagnosed with cervical cancer and the healthy population (AA vs. AC; OR, 7.15; 95% CI, 1.94-26.3; p = .003). It was also observed that the Cyp2C9*³ mutation in women with cervical cancer and VKORC1 in healthy population with HPV (+), did not follow the Hardy–Weinberg equilibrium. Our findings aid understanding the genetic polymorphism distribution of Cyp2C9*², Cyp2C9*³, and VKORC1 in women with genital malignancies. This can also be useful in predicting the susceptibility risk factors for developing cervical cancer. However, allelic discrimination as a molecular biomarker requires further research.     

Caractérisation des adénopathies médiastinales en TEP/TDM au 18F-FDG

The mediastinum is a complex anatomical region located at the junction of the cervical, thoracic and abdominal regions. It is an important lymphatic gateway that has an impact in many pathologies with nodal involvement and for which ¹⁸F-FDG PET/CT is a central examination. In this review article, we propose to recall the normal and pathological lymphatic drainage, mediastinum anatomy, the International Association for the Study of Lung Cancer (IASLC) lymph node classification and its implications in lung cancer. Then we will detail the morphological and metabolic characteristics, principal etiologies and the procedures to access mediastinal lymph nodes.     

Immunization with a poly (lactide co-glycolide) encapsulated plasmid DNA expressing antigenic regions of HPV 16 and 18 results in an increase in the precursor frequency of T cells that respond to epitopes from HPV 16, 18, 6 and 11

A phase II trial was conducted in subjects with human papillomavirus (HPV) associated high-grade cervical dysplasia testing the safety and efficacy of a microparticle encapsulated pDNA vaccine. Amolimogene expresses T cell epitopes from E6 and E7 proteins of HPV types 16 and 18. An analysis was performed on a subset of HLA-A2+ subjects to test whether CD8+ T cells specific to HPV 16, 18, 6 and 11 were increased in response to amolimogene immunization. Of the 21 subjects receiving amolimogene, 11 had elevated CD8+ T cell responses to HPV 16 and/or 18 peptides and seven of these also had increases to corresponding HPV 6 and/or 11 peptides. In addition, T cells primed and expanded in vitro with an HPV 18 peptide demonstrated cross-reactivity to the corresponding HPV 11 peptide. These data demonstrate that treatment with amolimogene elicits T cell responses to HPV 16, 18, 6 and 11.     

Human papillomavirus type 16 and 18 E7-pulsed dendritic cell vaccination of stage IB or IIA cervical cancer patients: a phase I escalating-dose trial

The safety and immunogenicity of the human papillomavirus type 16 (HPV16) or HPV18 (HPV16/18) E7 antigen-pulsed mature dendritic cell (DC) vaccination were evaluated for patients with stage IB or IIA cervical cancer. Escalating doses of autologous DC (5, 10, and 15 × 10⁶ cells for injection) were pulsed with recombinant HPV16/18 E7 antigens and keyhole limpet hemocyanin (KLH; an immunological tracer molecule) and delivered in five subcutaneous injections at 21-day intervals to 10 cervical cancer patients with no evidence of disease after they underwent radical surgery. Safety, toxicity, delayed-type hypersensitivity (DTH) reaction, and induction of serological and cellular immunity against HPV16/18 E7 and KLH were monitored. DC vaccination was well tolerated, and no significant toxicities were recorded. All patients developed CD4⁺ T-cell and antibody responses to DC vaccination, as detected by enzyme-linked immunosorbent spot (ELISpot) and enzyme-linked immunosorbent assays (ELISA), respectively, and 8 out of 10 patients demonstrated levels of E7-specific CD8⁺ T-cell counts, detected by ELISpot during or immediately after immunization, that were increased compared to prevaccination baseline levels. The vaccine dose did not predict the magnitude of the antibody or T-cell response or the time to detection of HPV16/18 E7-specific immunity. DTH responses to intradermal injections of HPV E7 antigen and KLH were detected for all patients after vaccination. We conclude that HPV E7-loaded DC vaccination is safe and immunogenic for stage IB or IIA cervical cancer patients. Phase II E7-pulsed DC-based vaccination trials with cervical cancer patients harboring a limited tumor burden, or who are at significant risk of tumor recurrence, are warranted.     

HLA DOA1 and DOB1 loci in Honduran women with cervical dysplasia and invasive cervical carcinoma and their relationship to human papillomavirus infection.

Molecular and epidemiological studies have demonstrated that certain types of human papillomavirus (HPV), mainly HPV-16 and HPV-18, are the primary causes of cervical cancer and its precursor lesions; there is now evidence for a clear association with specific HLA class I and class II loci contributing independently to the expression of cervical cancer. Among Honduran women carcinoma of the cervix is the most common type of cancer, and infections with high-risk HPV types are highly prevalent. To study the interactive role of viral-host genetics, we performed PCR amplification of DNA and sequence-specific oligonucleotide probe typing on cervical scrapes from 49 women [24 with cervical intraepithelial neoplasia stage III or cervical cancer (severe cases) and 25 with stage I or II cervical intraepithelial neoplasia (mild cases)] and 75 control subjects to look for possible associations between HPV and HLA class II DQA1 and DQB1 alleles in the development of dysplasias and invasive cancer. This analysis revealed a predominance of HLA-DQA1*0301 among severe-case patients [relative risk (RR) = 3.45, p = 0.008), whereas DQA1*0501 was negatively associated (RR = 0.30, p = 0.03), suggesting a protective effect of this allele. HPV typing showed a decreased relative risk among the HPV-16 or HPV-18 carrying patients and other HPV-related positive patients in the presence of DQB1*0602 compared with positive control subjects (p = 0.04). No statistically significant allele frequency difference was observed between mild dysplasia cases and control subjects. The results suggest that DQA1*03011, which is in linkage desequilibrium with all HLA-DR4 alleles, confers an increased risk for severe cervical dysplasia and invasive cancer, whereas DQA1*0501, which is in several DR52 haplotypes, has a protective effect. Furthermore, specific HLA-DQB1 sequences may be important in determining the immune response to HPV peptides and may affect the risk for cervical cancer after HPV infection in mestizo Honduran women.     

A novel CD4 T-cell epitope described from one of the cervical cancer patients vaccinated with HPV 16 or 18 E7-pulsed dendritic cells

Previously, safety and immunogenicity of human papillomavirus type 16 (HPV16) or 18 E7-pulsed dendritic cells (DC) vaccinations were demonstrated in a dose-escalation Phase I clinical trial which enrolled ten patients diagnosed with stage IB or IIA cervical cancer (nine HPV 16-positive, one HPV 18-positive). The goal of the study was to define the T-cell epitopes of HPV 16 or 18 E7 protein in these patients in order to develop new strategies for treating HPV-associated malignancies. This was accomplished through establishing T-cell lines by stimulating peripheral blood mononuclear cells with autologous mature DC pulsed with the HPV 16 or 18 E7 protein, examining the T-cell responses using ELISPOT assays, and isolating E7-specific T-cell clones based on IFN-γ secretion. Then, the epitope was characterized in terms of its core sequence and the restriction element. Twelve T-cell lines from eight subjects (seven HPV 16-positive, one HPV 18-positive) were evaluated. Positive T-cell responses were demonstrated in four subjects (all HPV 16-positive). All four were positive for the HPV 16 E7 46-70 (EPDRAHYNIVTFCCKCDSTLRLCVQ) region. T-cell clones specific for the E7 47–70 region were isolated from one of the subjects. Further analyses revealed a novel, naturally processed, CD4 T-cell epitope, E7 58–68 (CCKCDSTLRLC), restricted by the HLA-DR17 molecule. This work was supported by the National Institutes of Health (R21CA094507). An erratum to this article can be found at     

Beryllium Increases the CD14dimCD16+ Subset in the Lung of Chronic Beryllium Disease

CD14^dimCD16+ and CD14^brightCD16+ cells, which compose a minor population of monocytes in human peripheral blood mononuclear cells (PBMC), have been implicated in several inflammatory diseases. The aim of this study was to investigate whether this phenotype was present as a subset of lung infiltrative alveolar macrophages (AMs) in the granulomatous lung disease, chronic beryllium disease (CBD). The monocytes subsets was determined from PBMC cells and bronchoalveolar lavage (BAL) cells from CBD, beryllium sensitized Non-smoker (BeS-NS) and healthy subjects (HS) using flow cytometry. The impact of smoking on the AMs cell phenotype was determined by using BAL cells from BeS smokers (BeS-S). In comparison with the other monocyte subpopulations, CD14^dimCD16+ cells were at decreased frequency in PBMCs of both BeS-NS and CBD and showed higher HLA-DR expression, compared to HS. The AMs from CBD and BeS-NS demonstrated a CD14^dimCD16+phenotype, while CD14^brightCD16+ cells were found at increased frequency in AMs of BeS, compared to HS. Fresh AMs from BeS-NS and CBD demonstrated significantly greater CD16, CD40, CD86 and HLA-DR than HS and BeS-S. The expression of CD16 on AMs from both CBD and BeS-NS was downregulated significantly after 10μM BeSO₄ stimulation. The phagocytic activity of AMs decreased after 10μM BeSO₄ treatment in both BeS-NS and CBD, although was altered or reduced in HS and BeS-S. These results suggest that Be increases the CD14^dimCD16+ subsets in the lung of CBD subjects. We speculate that Be-stimulates the compartmentalization of a more mature CD16+ macrophage phenotype and that in turn these macrophages are a source of Th1 cytokines and chemokines that perpetuate the Be immune response in CBD. The protective effect of cigarette smoking in BeS-S may be due to the low expression of co-stimulatory markers on AMs from smokers as well as the decreased phagocytic function.     

HLA-A2-restricted peripheral blood cytolytic T lymphocyte response to HPV type 16 proteins E6 and E7 from patients with neoplastic cervical lesions

 The DNA from human papillomavirus (HPV) can be detected in 90% of cervical carcinomas. To address whether patients infected with HPV can mount efficient T cell responses to this pathogen we examined the cytotoxic T lymphocyte (CTL) response of peripheral blood mononuclear cells (PBMC) from patients with abnormal genital epithelial cells. PBMC from 11 HLA-A2⁺ patients were stimulated with CaSki, a cervical carcinoma cell line that is HPV 16⁺ and HLA-A2⁺. The CTL were screened for reactivity to the cervical carcinoma cell line C33A (HPV^–, HLA-A2⁺) transfected with the HPV 16 E6 or E7 genes or the plasmid without insert. The CTL of 1 patient showed particularly strong CaSki and HPV E6 or E7 protein-specific cytotoxicity in a HLA-A2-restricted fashion. In contrast, these CTL lysed neither a vector-only transfectant, the natural killer cell (NK) target, K562 nor the lymphokine-activated killer cell (LAK) target, Daudi. HLA-A2 restriction was demonstrated by the lack of recognition of a HLA-A2^– CaSki cell line developed in our laboratory. The CTL line was cloned and 99 clones were harvested and screened; 51 clones lysed CaSki, of which 17 did not lyse the A2^– CaSki. Of these HLA-A2^– restricted clones, 8 did not lyse C33A transfectants, 6 lysed all C33A transfectants, 3 lysed C33A-E7 only and none lysed C33A-E6 only. These data imply that, within the bulk CTL line, HLA-A2-restricted recognition of antigens was restricted to CaSki antigens, antigens common to cervical carcinoma (CaSki plus C33A), or HPV-16-E7-derived antigen on the clonal level. The E7-restricted clones were negative for recognition of known HLA-A2-binding peptides from E7. Received: 16 November 1995 / Accepted: 15 January 1996     

Inhibition of Lymphangiogenesis of Endothelial Progenitor Cells with VEGFR-3 siRNA Delivered with PEI-alginate Nanoparticles

Lymphangiogenesis is implicated in lymphatic metastasis of tumor cells. Recently, growing evidences show that endothelial progenitor cells (EPCs) are involved in lymphangiogenesis. This study has investigated effects of VEGF-C/VEGFR-3 (vascular endothelial growth factor receptor-3) signaling pathway on EPC differentiation and effectiveness of inhibiting lymphatic formation of EPCs with VEGFR-3 siRNA delivered in PEI (polyethylenimine)-alginate nanoparticles. CD34⁺VEGFR-3⁺ EPCs were sorted from mononuclear cells of human cord blood. Under induction with VEGF-C, the cells differentiated toward lymphatic endothelial cells. The nanoparticles were formulated with 25 kDa branched PEI and alginate. The size and surface charge of PEI-alginate nanoparticles loading VEGFR-3 siRNA (N/P = 16) are 139.1 nm and 7.56 mV respectively. VEGFR-3 siRNA specifically inhibited expression of VEGFR-3 mRNA in the cells. After treatment with PEI-alginate/siRNA nanocomplexes, EPCs could not differentiate into lymphatic endothelial cells, and proliferation, migration and lymphatic formation of EPC-derived cells were suppressed significantly. These results demonstrate that VEGFR-3 signaling plays an important role in differentiation of CD34⁺VEGFR-3⁺ EPCs. VEGFR-3 siRNA delivered with PEI-alginate nanoparticles can effectively inhibit differentiation and lymphangiogenesis of EPCs. Inhibiting VEGFR-3 signaling with siRNA/nanocomplexes would be a potential therapy for suppression of tumor lymphangiogenesis and lymphatic metastasis.     

Local lymphogenic migration pathway in normal mouse spleen

Although the immunological and hemodynamical significance of the spleen is of great importance, few reports detail the lymphatic vessels in this organ. We have used an immunohistochemical three-dimensional imaging technique to characterize lymphatic vessels in the normal mouse spleen and have successfully demonstrated their spatial relationship to the blood vascular system for the first time. Lymphatic markers, such as LYVE-1, VEGFR-3, and podoplanin, show different staining patterns depending on their location in the spleen. LYVE-1-positive lymphatic vessels run reverse to the arterial blood flow along the central arteries in the white pulp and trabecular arteries and exit the spleen from the hilum. These lymphatic vessels are surrounded by type IV collagen, indicating that they are collecting lymphatic vessels rather than lymphatic capillaries. Podoplanin is expressed not only in lymphatic vessels, but also in stromal cells in the white pulp. These podoplanin-positive cells form fine meshworks surrounding the lymphatic vessels and central arteries. Following intravenous transplantation of lymphocytes positive for green fluorescent protein (GFP⁺) into normal recipient mice, donor cells appear in the meshworks within 1 h and accumulate in the lymphatic vessels within 6 h after injection. The GFP⁺ cells further accumulate in a draining celiac lymph node through the efferent lymphatic vessels from the hilum. These meshworks might therefore act as an extravascular lymphatic pathway and, together with ordinary lymphatic vessels, play a primary role in the cell traffic of the spleen, additional to the blood circulatory system.     

Enhancement of immunotherapeutic effects of HPV16E7 on cervical cancer by fusion with CTLA4 extracellular region

Cervical cancer is caused by infection by high-risk human papillomavirus (HPV), especially HPV16. Limitations in current treatments of cervical cancers call for the development of new and improved immunotherapies. This study aims at investigating the efficacy of a novel vaccine consisting of modified HPV 16E7 fused with human cytotoxic T-lymphocyte antigen 4 (CTLA4). The regions in HPV16 E7 gene associated with its transformation and CTL-enhanced response were modified; the resultant HPV16mE7 was fused with extracellular region of CTLA4 to generate HPVml6E7-eCTLA4 fusion protein. Binding of this fusion protein to B7 molecules expressed on antigen presenting-cells (APCs) was demonstrated. C57BL/6 (H-2^b) mice immunized with low dose of the fusion protein (10 μg) produced higher titer antibody and stronger specific CTL response, and expressed higher levels of IFN-γ and IL-12, compared with those immunized with HPVml6E7 only or admixture of HPVml6E7 and CTLA4, or PBS; and were protected from lethal dose tumor challenge. Tumor growth was retarded and survival prolonged in mouse models with the fusion protein treatment. Our results demonstrate that fusion of HPV16 E7 with eCTLA4 targeting APCs resulted in enhanced immunity, and that this fusion protein may be useful for improving the efficacy of immunotherapeutic treatments of cervical cancer and other HPV16 infection-associated tumors.     

Up-regulation of Foxp3 participates in progression of cervical cancer

Foxp3 was identified as a key protein in mediating inhibitory functions of regulatory T cell (Treg). Foxp3 was thought to express only in the T cell lineage until recently when some researches reported that Foxp3 was also expressed by cancer cells. In this study, we describe for the first time the expression of Foxp3 in cervical cancer. Progression from cervical intraepithelial neoplasia (CIN) to cervical cancer is a multistep process initiated by persistent infection with high-risk human papillomavirus (HPV). P16^INK4a is a crucial marker of HPV integration into host cells. In the present study, expressions of Foxp3 and P16^INK4a in CIN and cervical cancer were detected by immunohistochemistry. Our results found expression level of Foxp3 was increased during the progression of cervical neoplasia. Moreover, up-regulation of Foxp3 appeared to be correlated with the expression of P16^INK4a. Examination of the role of Foxp3 in differentiation by double immunostaining for cytokeratin 10 (CK10) showed significant association between Foxp3 expression and differentiation (Foxp3 vs CK10). Furthermore, positive expression of Foxp3 was correlated with tumor size. These data suggest that Foxp3 may play an important role in differentiation and growth of cervical cancer cells. Our findings provide new insights regarding the role of Foxp3 in differentiation and its association with HPV infection during the development of cervical cancer.     

Growth of the jugular lymph sacs and establishment of the topography of the cervical portion of the thoracic duct during early human ontogenesis

In 196 human embryos, prefetuses, fetuses and newborns, by means of a complex of morphological methods, development of the jugular lymphatic sacs and the process of settling of the thoracic duct cervical part topography have been studied. The jugular lymphatic sac anlages take place on the 6th week of the development. From the lymphatic cleft, situating in the mesenchyme near the anterior cardinal veins, multichambered cavities laid with endotheliocytes are forming,--the jugular lymphatic sacs. Connection of the initially close lymphatic sacs with the venous system takes place secondarily by the end of the embryonic period of development. In the area of the sac ostia a valve is formed, that makes morphological premises for unidirected lymph flow into the venous system. The lymph nodes developing at the place of the reducing jugular lymphatic sacs, ensure formation: from the left jugular lymphatic sac--the cervical part of the thoracic duct, from the right jugular lymphatic sac--the right lymphatic duct and the jugular and the subclavicular lymphatic trunks. Variability in the form and topography of these structures are determined both by the form and construction of the jugular lymphatic sacs and by developmental peculiarities of the lymph nodes at their place. The process of settling of the thoracic duct cervical part topography depends on age changes of its size and form, as well as on development of structures situating nearby, and by the time of birth it is not completed.     

Clinical significance of peritumoral lymphatic vessel density and lymphatic vessel invasion detected by D2-40 immunostaining in FIGO Ib1-IIa squamous cell cervical cancer

The clinical significance of lymphangiogenesis in cervical cancer remains controversial. Our aim was to investigate the correlation between lymphangiogenesis, lymphatic vessel invasion (LVI) and tumor metastasis, invasion and prognosis in squamous cell cervical cancer. Paraffin sections of 90 patients with FIGO (Fédération Internationale de Gynécologie et d'Obstétrique) Ib1-IIa squamous cell cervical cancer were stained for immunohistochemistry with a D2-40 monoclonal antibody against the carcinoembryonic antigen M2A. The lymphatic vessel density (LVD) and LVI were measured, and their relationship with the clinicopathological data was analyzed. D2-40-positive lymphatic vessels were found in 75 of the 90 patients (83.3 %). All D2-40-positive vessels were located in peritumoral areas. The mean±SD of the peritumoral LVD was 10.08±4.16. The positive rate of LVI was 32.0 % (24/75). The recurrence rate of patients with LVD >10 (62.1 %, 18/29) was significantly higher than that of patients with LVD ≤10 (34.8 %, 16/46, P = 0.021). The 5-year recurrence-free survival rate of patients with LVD >10 (41.0 %) was significantly lower than that of patients with LVD ≤10 (67.0 %, P = 0.045). Univariate analysis showed that the peritumoral LVD (≤10 vs >10) was correlated with LVI (absent vs present, P = 0.016). The peritumoral LVD and LVI showed no correlation with age, FIGO stage, tumor size, tumor grade, depth of invasion, or pelvic lymph node metastasis (all: P > 0.05). Peritumoral lymphangiogenesis was correlated with the recurrence and recurrence-free survival in patients with squamous cell cervical cancer. Examination of peritumoral LVD in these patients might therefore help to estimate the risk of recurrence.     

Sonoporation Delivery of Monoclonal Antibodies against Human Papillomavirus 16 E6 Restores p53 Expression in Transformed Cervical Keratinocytes

High-risk types of human papillomavirus (HPV), such as HPV16, have been found in nearly all cases of cervical cancer. Therapies targeted at blocking the HPV16 E6 protein and its deleterious effects on the tumour suppressor pathways of the cell can reverse the malignant phenotype of affected keratinocytes while sparing uninfected cells. Through a strong interdisciplinary collaboration between engineering and biology, a novel, non-invasive intracellular delivery method for the HPV16 E6 antibody, F127-6G6, was developed. The method employs high intensity focused ultrasound (HIFU) in combination with microbubbles, in a process known as sonoporation. In this proof of principle study, it was first demonstrated that sonoporation antibody delivery into the HPV16 positive cervical carcinoma derived cell lines CaSki and SiHa was possible, using chemical transfection as a baseline for comparison. Delivery of the E6 antibody using sonoporation significantly restored p53 expression in these cells, indicating the antibody is able to enter the cells and remains active. This delivery method is targeted, non-cytotoxic, and non-invasive, making it more easily translatable for in vivo experiments than other transfection methods.     

外周血Treg细胞、T淋巴细胞及其亚群与早期宫颈癌的关系及对淋巴结转移的预测价值研究

摘要 目的：分析外周血Treg细胞、T淋巴细胞及其亚群与早期宫颈癌的关系及对淋巴结转移的预测价值。方法：选择我院自2017年1月至2020年12月接诊的60例接受子宫颈癌根治术及盆腔淋巴清扫术的早期宫颈癌患者作为观察组,另选同期的60例健康体检者作为对照组。比较两组外周血Treg细胞、T淋巴细胞及其亚群水平,使用受试者工作特征曲线（ROC）下面积（AUC）评价外周血Treg细胞、T淋巴细胞及其亚群对淋巴结转移的预测效能。结果：观察组外周血Treg细胞、CD8⁺T细胞水平高于对照组,CD3⁺T细胞、CD4⁺T细胞、CD4⁺/CD8⁺比值均低于对照组（P＜0.05）;观察组术后外周血Treg细胞、CD8⁺T细胞水平较术前降低,CD3⁺T细胞、CD4⁺T细胞、CD4⁺/CD8⁺比值均较术前升高（P＜0.05）;在60例早期宫颈癌患者中,发生淋巴结转移12例;淋巴结转移组术前外周血Treg细胞水平、CD8⁺T细胞高于非淋巴结转移组,CD3⁺T细胞、CD4⁺T细胞、CD4⁺/CD8⁺比值均低于非淋巴结转移组（P＜0.05）;经多因素Logistic回归分析,外周血Treg细胞、CD3⁺T细胞、CD4⁺/CD8⁺比值均是早期宫颈癌患者发生淋巴结转移的独立预测因素（P＜0.05）;经ROC曲线分析,外周血Treg细胞、CD3⁺T细胞联合CD4⁺/CD8⁺比值预测早期宫颈癌患者发生淋巴结转移的AUC为0.910。结论：外周血Treg细胞、T淋巴细胞及其亚群水平与早期宫颈癌的病情演变有关,其中外周血Treg细胞、CD3⁺T细胞联合CD4⁺/CD8⁺比值预测淋巴结转移的效能较好,值得进一步研究应用。     

Assessment of the association between the frequency of micronucleus and p16INK4a/Ki-67 co-expression in patients with cervical intraepithelial lesions

Human papilloma virus (HPV) infection is the main etiological factor for cervical intraepithelial lesions (CIN). An important characteristic of this process is the loss of genome stability. Therefore, it is imperative to use biomarkers of DNA damage caused by genomic instability to identify high risk individuals. We investigated the frequency of micronuclei (MN) in peripheral blood lymphocytes (PBL) of 20 patients, diagnosed as histologically CIN 1 and 10 healthy controls. We also examined the frequency of other nuclear anomalies including nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) in PBL of patients with CIN 1 and healthy controls, and evaluated the benefits of p16^INK4a and Ki-67 (p16^INK4a/Ki-67) immunohistochemical double staining for identifying cervical squamous cells that express HPV E6/E7 oncogenes. We analyzed the association between the frequency of MN in PBL and the amount of p16^INK4a/Ki-67 co-expression in CIN 1 patients to establish genomic instability. Among CIN 1 subjects, 15% exhibited diffuse p16^INK4a/Ki-67 co-expression and were considered high positive, 25% of the CIN 1 cases exhibited p16^INK4a/Ki-67 co-expression restricted to the lower part of the epithelium and were considered low positive and the remaining 60% of cases were negative. The frequency of MN, NPBs and NBUDs differed significantly among groups. We found a statistically significant positive correlation between p16^INK4a/Ki-67 co-expression and the frequency of MN, NPBs and NBUDs in PBL. Our findings demonstrate the efficacy of p16^INK4a/Ki-67 double immunostaining for histological samples with CIN 1. MN frequency in PBL might be useful for detecting genomic instability in cases of HPV infection and CIN.     

Analysis of nerve supply pattern in thoracic duct in young and elderly men

BACKGROUND: Analysis of the innervation pattern of the thoracic duct in young and elderly human subjects has been performed. The subdivision of the vessels in cervical and lumbar region were taken in consideration. MeTHODS AND RESULTS: Immunostaining for general nerve fibers with a PGP 9.5 marker disclosed a diffuse innervation of the thoracic duct in young subjects, which was strongly reduced in elderly subjects. In young subjects, tyrosine hydroxylase (TH) and neuropeptide Y (NPY) immunoreactive fibers, markers of noradrenergic postganglionic sympathetic fibers, were frequent; choline acetyltransferase (ChAT) immunoreactive fibers, marker of cholinergic parasympathetic nerve fibers, were also well represented. Therefore, the influence of sympathetic and parasympathetic nerve systems on the thoracic duct can be confirmed. The immunoreactivity of vasoactive intestinal peptide (VIP), a neuropeptide frequently present in cholinergic parasympathetic nerve fibers, was scarcely present. Dopamine-positive fibers were observed in few short nerve fibers. Substance P (SP)-positive fibers were widely distributed in the medial and intimal smooth muscle layers, suggesting their involvement as contractile modulating fibers and sensitive fibers. In elderly subjects, an evident reduction of all specific nerve fibers analyzed was detected, the ChAT-positive fibers being the most affected. CONCLUSIONS: The lymphatic vessel thoracic duct is able to regulate hydrodynamic lymph flow by intrinsic contraction of its smooth muscle layer. Therefore, analysis of the thoracic duct innervation pattern may be important in assessing the regulation of vessel contraction. These findings called attention to the reduction of lymphatic drainage functionality affecting fluid balance in the elderly.