Bromodeoxyuridine, an analogue of thymidine, can be detected by means of monoclonal antibodies and utilized as a marker of the S-phase of the cell cycle. In vitro immunohistochemical application of the BrdU/anti-BrdU-MAb method permits a quantitative assessment of the proliferative activity of a tissue as well as the direct location of the actively replicating cells in histological sections. In this paper, a method for the detection of the labeling index of S-phase cells in normal and neoplastic tissues with in vitro BrdU labeling and standard immunohistochemical techniques using anti-BrdU-MAb and avidin-biotin peroxidase complex is described. We have employed this method in 47 human solid tumor samples, including squamous cell carcinomas of head and neck and cervix uteri, adenocarcinomas and malignant lymphomas, and also evaluated the possible application of the BrdU labeling index to estimate the cycling S-phase cells in neoplastic cell populations. In our data, the in vitro labeling index varied greatly in an individual case (3.56-29.2%) and from an area to an area within the same case. Squamous cell carcinomas of the head and neck showed higher LI than those of the cervix uteri. A case of metastatic carcinoma to the lung from ductal carcinoma of the breast had the highest LI (29.2%), in contrast to the low LI (3.6%) in the primary ductal carcinoma of breast.
Adenocarcinoma/immunology/pathology ; Antibodies, Monoclonal/*immunology ; Breast Neoplasms/immunology/pathology ; Bromodeoxyuridine/*immunology ; Carcinoma, Squamous Cell/immunology/pathology ; Cell Nucleus/immunology/*physiology ; Head and Neck Neoplasms/immunology/pathology ; Humans ; Immunohistochemistry ; *Interphase ; Lymphoma/immunology/pathology ; Neoplasms/immunology/*pathology

PURPOSE: Forkhead box p3 (Foxp3) positive T regulatory cells (Tregs) have a functionally immunosuppressive property that prevents effector cells from acting against self in autoimmune diseases or a tumor. It is known that Tregs may be highly relevant in cancer progression. Dendritic cells (DCs) induce cutaneous immune response, however several studies have suggested that DCs are involved in immunosuppression. The aim of this study is to evaluate the prevalence of Tregs and DCs infiltration in cutaneous premalignant and malignant squamous lesions. MATERIALS AND METHODS: We evaluated Tregs and DCs in skin tissue samples obtained from 83 patients with actinic keratosis, Bowen's disease or squamous cell carcinoma by immunohistochemistry. RESULTS: The prevalence of Tregs and DCs was significantly higher in squamous cell carcinoma and Bowen's disease than in actinic keratosis. In addition, the number of DCs was closely correlated with the prevalence of Tregs, and DCs were also located in direct proximity to Tregs. CONCLUSION: Tregs is related to cutaneous squamous tumor progression.
Bowen's Disease/immunology/metabolism/pathology ; Carcinoma, Squamous Cell/immunology/metabolism/pathology ; Dendritic Cells/immunology/metabolism/pathology ; Forkhead Transcription Factors/immunology/*metabolism ; Humans ; Immune Tolerance ; Keratosis, Actinic/immunology/metabolism/pathology ; Skin Neoplasms/*immunology/metabolism/pathology ; T-Lymphocytes, Regulatory/*immunology/metabolism/pathology

OBJECTIVE: This research aims to investaigate the effect of cetuximab and dendritic cells (DCs) to kill the head and neck squamous cell (HNSCC), in order to provide a new way for the patients of HNSCC. METHOD: DCs were induced from peripheral blood monocytes by rhIL-4, rhGM-CSF and TNF-alpha in vitro, 7days later, detecting the surface marks of DCs for example CD83, CD86, and then using MTT and flow cytometry detecting the effect T lymphocytes induced by DCs combining cetuximab to kill HNSCC; EGFR and pEGFR in each group were anlysised by Western blot. RESULT: It is successful to induce DCs in vitro. Mature DCs (mDCs) expressed the suface mark such as CD83, CD86 higher compared with immature DCs (imDCs). Compared with other groups, cetuximab combined with DCs significantly enhanced the cytotoxicty and apoptosis to HNSCC (P < 0.05). pEGFR were gradually reduced as the concenetration of cetuximab increasing (P < 0.05). However, comparing with the group of cetuximab, the group of cetuximab combined with DC has no significant difference at the same concentration of cetuximab. In each group EGFR also has no significant diference (P > 0.05). CONCLUSION Cetuximab and DCs have synergistic effects, which can significantly enhance the killing effect of HNSCC.
Antibodies, Monoclonal, Humanized ; pharmacology ; Apoptosis ; drug effects ; Carcinoma, Squamous Cell ; pathology ; Cetuximab ; Dendritic Cells ; immunology ; Head and Neck Neoplasms ; pathology ; Humans ; Squamous Cell Carcinoma of Head and Neck ; Tumor Cells, Cultured

OBJECTIVE: To examine the expression of soluble major histocompatibility complex class I-related chain A (sMICA) in the serum in patients with oral squamous cell carcinoma (OSCC) and to explore its clinicopathological significance. METHODS: Seventy-eight OSCC patients were selected as an experiment group, and 19 healthy persons as a control group. The sMICA in the serum in the experiment group and the control group was detected by enzyme-linked immunosorbent assay. RESULTS: The detection rate of sMICA in the serum in the experiment group was 98.7% (77/78), with the 95% confidence interval 74.30-93.95 pg/mL and the median 82.17 pg/mL, The detection rate in the control group was 94.7% (18/19), with the 95% confidence interval 29.48-50.30 pg/mL and the median 37.54 pg/mL. The sMICA in the serum in the experiment group was higher than that in the control group (P<0.01). There was statistic difference in the serum sMICA in the experiment group among the different clinicopathological parameters such as tumor size, disease stage and regional lymph node status (P<0.05), but no difference was found in gender, age, and tumor differentiation (P>0.05). CONCLUSION The sMICA in the serum in the OSCC patients increases, and is related with the tumor size, disease stage and regional lymph node status. Determination of sMICA in the serum may provide useful information to evaluate the immune state of OSCC patients.
Aged ; Carcinoma, Squamous Cell ; blood ; immunology ; pathology ; Case-Control Studies ; Female ; Histocompatibility Antigens Class I ; blood ; Humans ; Lymphatic Metastasis ; Male ; Middle Aged ; Mouth Neoplasms ; blood ; immunology ; pathology ; Solubility

Aged, 80 and over ; Antibodies, Monoclonal ; metabolism ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Diagnosis, Differential ; Granuloma ; metabolism ; pathology ; Humans ; Keratins ; immunology ; Male ; Mucin-1 ; metabolism ; Skin Neoplasms ; metabolism ; pathology

OBJECTIVE: To observe the immunity changes of patients after CIK cells being transfused back, and then to discuss the effects of CIK cells on curing laryngeal cancers. METHOD: Forty eight laryngeal cancer patients with low immune function were collected. The immunity index in the peripheral blood of patients before/after radiotherapy and after CIK cells therapy were measured and compared with normal one. RESULT: After radiotherapy, the percentage of CD3+, CD4+ cells declined, the percentage of CD8+ cells increased; the rate of CD4+ /CD8+ declined and the rate of Th1/Th2 reversed. There were no significant difference between the immunity indexes before and after radiotherapy (P < 0.05). After CIK cell therapy, the above indexes were improved (P < 0.05), but the values didn't returned to normal. After radiotherapy and after CIK therapy, the value of B cell didn't changed obviously (P > 0.05), while the percentage of NK cells changed obviously (P < 0.05). CONCLUSION Radiotherapy can restrain the immune function of the patients with laryngeal cancers. CIK therapy is safe and might improve the recent immune function of the patients.
Aged ; Carcinoma, Squamous Cell ; immunology ; pathology ; radiotherapy ; Combined Modality Therapy ; Cytokine-Induced Killer Cells ; immunology ; Humans ; Immunotherapy, Adoptive ; Killer Cells, Natural ; immunology ; Laryngeal Neoplasms ; immunology ; pathology ; radiotherapy ; Male ; Middle Aged ; Neoplasm Staging

OBJECTIVETo investigate the effect of anti-ICAm-1/LAF-1 McAb on the cytotoxicity of tumor infiltrating lymphocytes from oral squamous cell carcinoma.

METHODSTIL were isolated from fresh tumor tissues in 15 patients with oral squamous cell carcinoma and cultured in vitro. The expression of ICAM-1/LAF-1 in fresh and activated TIL was examined by FACS. The cytotoxicity of TIL against auto tumor cells and the level of TNF-alpha produced by TIL were compared before and after interrupted with anti-ICAM-1/LAF-1 McAb.

RESULTSThe expression of ICAM-1 and LAF-1 in the activated TIL significantly increased comparing with fresh TIL (P < 0.05). Interrupted by anti-ICAM-1 McAb, the cytotoxicity of TIL against auto tumor cell was not significantly changed. Interrupted by anti-ICAM-1 and anti-LAF-1 McAb, the level of TNF-alpha produced by TIL showed no differences comparing with the control (P < 0.05), but the cytotoxicity of TIL against auto tumor cells significantly decreased (P < 0.05).

CONCLUSIONThe effect of anti-LAF-1 McAb may result from steric interference of antigen-nonspecific adhesion process.

Antibodies, Monoclonal ; pharmacology ; Carcinoma, Squamous Cell ; immunology ; pathology ; Cells, Cultured ; Cytotoxicity, Immunologic ; Female ; Humans ; Intercellular Adhesion Molecule-1 ; biosynthesis ; genetics ; Lymphocyte Function-Associated Antigen-1 ; biosynthesis ; genetics ; Lymphocytes, Tumor-Infiltrating ; immunology ; pathology ; Male ; Mouth Neoplasms ; immunology ; pathology

OBJECTIVETo analyze the expression of co-stimulatory molecules PD-1/PD-L1 in peripheral blood mononuclear cells in lung cancer patients, and to explore its biological significance.

METHODSOne hundred and thirty-three lung cancer patients, 25 lung infection patients and 23 healthy donors were enrolled in this study. 100 µl of whole blood from these subjects were collected. Multi-color immunofluorescence staining and flow cytometry were used to detect PD-1/PD-L1 expression. The results were statistically analyzed.

RESULTSThe expression level of CD3⁺CD8⁺ T cells in the lung cancer patients was (38.83 ± 1.74)%, significantly lower than that in the control group [(43.25 ± 3.35)%, P < 0.05]. CD8⁺CD28⁺ T cell subset in the peripheral blood of lung cancer patients was (17.73 ± 1.21)% significantly lower than that of the healthy donors [(27.96 ± 2.72)%, P < 0.01]. The CD8⁺CD28⁻ T cell subset was (21.19 ± 1.92)% in the lung cancer patients, significantly higher than that of the healthy control group [(15.18 ± 2.93)%, P < 0.05]. The expression level of PD-1 on the surface of CD8⁺CD28⁺ T cells was (10.67 ± 1.12)% in the group of lung cancer patients, significantly higher than that of the control group [(5.32 ± 1.58)%, P < 0.01]. It was also found that the expression of PD-1 on CD8⁺CD28⁻ T cells was up-regulated in the group of lung cancer patients (7.46 ± 1.25)%, significantly higher than that of the healthy control group [(2.68+1.07)%, P < 0.01]. The expression level of PD-L1 on CD68⁺ cells in the lung cancer patients was (16.03 ± 2.06)%, significantly higher than that of the healthy control group [(9.32 ± 2.00)%, P < 0.05].

CONCLUSIONUp-regulation of PD-1/PD-L1 on peripheral blood cells in lung cancer patients negatively regulates the lymphocytes, inhibits the immune response for killing tumor cells, and promotes tumor development and immune escape.

Adenocarcinoma ; blood ; pathology ; B7-H1 Antigen ; metabolism ; CD28 Antigens ; metabolism ; CD3 Complex ; metabolism ; CD8 Antigens ; metabolism ; Carcinoma, Large Cell ; blood ; pathology ; Carcinoma, Squamous Cell ; blood ; pathology ; Case-Control Studies ; Female ; Humans ; Lung Neoplasms ; blood ; pathology ; Male ; Middle Aged ; Programmed Cell Death 1 Receptor ; metabolism ; Small Cell Lung Carcinoma ; blood ; pathology ; T-Lymphocytes ; immunology ; metabolism ; Up-Regulation

OBJECTIVE: To study the relationship between lymphatic vessel density and clinicopathological features of laryngeal squamous cell carcinoma. METHOD: The lymph vessels, 40 specimens of LSCC and normal mucosa, were quantitated by SABC immunohistochemistry staining with lymphatic endothelial marker oncofetal antigen M2A Monoclonal antibody (D2-40). RESULT: The density of peritumoral D2-40(+) vessels in the LSCC group was higher than that of normal mucosa one (P<0.01). The density of peritumoral D2-40(+) vessels in T3/4 group was higher than that of T1/2 group (P<0.05). The density of peritumoral D2-40(+) vessels in the neck lymph nodal metastasis group was higher than that without neck lymph nodal metastasis group (P<0.05). The density of peritumoral D2-40(+) vessels in the supraglottic group was higher than that of glottic group (P<0.01). The density of peritumoral D2-40(+) vessels among high, middle, poor differentiation group were no significant difference (P>0.05). CONCLUSION Tumor lymph vessel mainly means peritumoral lymph vessel. The density of peritumoral lymph vessel marked with D2-40 in laryngeal carcinoma tissues was significantly correlated with progression and invasion of tumor.
Adult ; Aged ; Antibodies, Monoclonal ; Carcinoembryonic Antigen ; immunology ; Carcinoma, Squamous Cell ; pathology ; Female ; Humans ; Immunohistochemistry ; methods ; Laryngeal Neoplasms ; pathology ; Lymph Nodes ; pathology ; Lymphangiogenesis ; Lymphatic Metastasis ; Lymphatic Vessels ; pathology ; Male ; Middle Aged

OBJECTIVETo clone the variable region genes of the monoclonal antibody (McAb) against human heterogeneous nuclear ribonucleoprotein A2/B1 (HnRNPA2/B1), ligate them to assemble single chain Fv (ScFv) gene and express in Escherichia coli.

METHODSThe specificity of the anti-HnRNPA2/B1 McAb 3E8 to synthetic HnRNPA2/B1 peptide, HnRNPA2/B1 protein in lung cancer cells were examined by dot-immunobinding assay, Western blot and immunohistochemistry. The variable region genes of heavy chain (VH) and light chain (VL) were amplified from hybridoma cell by reverse transcription-polymerase chain reaction(RT-PCR), and then were linked by a linker peptide using SOE-PCR (splicing by overlap extension-PCR) to construct recombination ScFv gene. The latter was cloned into the expression vector pET28 (a+) and expressed in E coli BL21. The expressed product was identified by SDS-PAGE and competitive ELISA inhibition test.

RESULTSIt was shown that the McAb combined specifically with synthetic HnRNPA2/B1 peptide and HnRNPA2/B1 protein in three lung cancer cells. The cloned VH gene and VL gene were 345 bp and 309 bp respectively and were linked successfully to obtain ScFv gene. The ScFv protein was expressed in the form of inclusion body, with molecular weight of 28,000 and immunoreactivity to HnRNPA2/B1.

CONCLUSIONVH gene, VL gene and ScFv gene of anti-HnRNPA2/B1 antibody were cloned, constructed and functionally expressed in E coli. These results provide the experimental basis for elucidating the role of HnRNPA2/B1 in lung cancer.

Adenocarcinoma ; metabolism ; pathology ; Antibodies, Monoclonal ; genetics ; immunology ; metabolism ; Carcinoma, Small Cell ; metabolism ; pathology ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Cell Line, Tumor ; Cloning, Molecular ; Escherichia coli ; metabolism ; Heterogeneous-Nuclear Ribonucleoprotein Group A-B ; immunology ; Humans ; Immunoglobulin Fragments ; genetics ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Light Chains ; genetics ; Immunoglobulin Variable Region ; genetics ; Lung Neoplasms ; metabolism ; pathology