细胞因子基因联合放射治疗小鼠头颈部鳞状细胞癌的实验研究

目的观察白细胞介素-2(interleukin-2，IL-2)基因与白细胞介素-12(interleukin-12，IL-12)基因联合放射治疗小鼠头颈鳞状细胞癌(鳞癌)的疗效。方法模拟临床头颈肿瘤治疗方案，利用鳞癌SCCⅦ细胞株在C3H／HeJ小鼠口底建立头颈鳞癌的荷瘤动物模型，在荷瘤部位直接注射多价阳离子脂质体包裹的表达IL-2基因和IL-12基因的真核表达质粒，对照组分别注射无目的基因的空载体和磷酸盐缓冲液(phosphatic buffered saline，PBS)。次日给予2Gy直线加速器的局部肿瘤放射治疗(简称放疗)。4d后重复IL-2基因和IL-12基因局部治疗。观察肿瘤治疗前后大小变化，检测肿瘤组织中CD4^ 、CD8^ 的表达、IL-2和IL-12的表达水平及自然杀伤(naturalkiller，NK)细胞和细胞毒T淋巴细胞(cytotoxic T—lymphocyte，CTL)的活性。结果IL-2基因和IL-12基因联合加放疗治疗组肿瘤生长明显受抑制，疗效显著优于单基因加放疗治疗组、单基因治疗组和各对照组。在注射有IL-2基因及IL-12基因的治疗组中，IL-2与IL-12表达水平明显升高，小鼠脾细胞NK细胞活性和CTL杀伤活性增强，肿瘤组织内可见大片坏死，并含有大量的CD4^ ，CD8^ 淋巴细胞浸润。结论．多价阳离子脂质体携带的IL-2基因和IL-12基因治疗可提高肿瘤局部和机体全身的抗肿瘤免疫应答，能加强放疗的抗肿瘤效果。     

白细胞介素-2基因联合放疗对小鼠头颈鳞癌治疗作用的研究

目的：观察白细胞介素-2（IL-2）基因联合放疗对小鼠头颈鳞癌的抗肿瘤作用。方法：将25只小鼠平分为5组。利用SCCⅦ细胞株在C3H/HeJ小鼠口底建立头颈鳞癌荷瘤动物模型,IL-2组和联合组在荷瘤部位直接注射IL-2基因;EP组和对照组分别注射无目的基因的空载体和PBS,次日联合组与放疗组给予2Gy直线加速器的局部肿瘤放疗。4d后重复IL-2基因治疗,观察肿瘤治疗前后大小变化;检测肿瘤组织中CD4^ ,CD8^ 的表达,IL-2的分泌水平及自然杀伤细胞（NK）和细胞毒T淋巴细胞（CTL）的活性。结果：联合组肿瘤生长明显受抑制,疗效显著优于IL-2组,放疗组,EP组和对照组,IL-2组中,小鼠IL-2分泌水平明显升高,脾细胞NK活性和CTL杀伤活性增强,肿瘤组织内可见大片坏死,并含有大量的CD4^ ,CD2^ 淋巴细胞浸润,结论：IL-2基因治疗可提高肿瘤局部和全身的抗肿瘤免疫应答。能加强放疗的抗肿瘤效果。     

Combination nonviral cytokine gene therapy for head and neck cancer

OBJECTIVE: To establish the feasibility and efficacy of combination nonviral murine interferon-alpha (mIFN-alpha)and murine interleukin-2 (mIL-2) or murine interleukin-12 (mIL-12) gene therapy for head and neck squamous cell carcinoma in a murine model. STUDY DESIGN: Randomized controlled studies in a murine head and neck cancer model were performed to assess antitumor responses, secondary cytokine expression, and both natural killer (NK) cell and cytolytic T-cell (CTL) activity. METHODS: Tumors were established in the floor of mouth in C3H/HeJ immunocompetent mice. Established tumors were directly injected with polymer-formulated murine interferon-alpha (mIFN-alpha), lipid-formulated mIL-2, and polymer-formulated mIL-12 alone or in combination. Primary and secondary cytokine expression,NK cell activity, and CTL activity were assayed. RESULTS: The use of mIFN-alpha gene therapy in combination with either mIL-2 or mIL-12 resulted in significant antitumor effects as compared with each of the single cytokine and control treatment groups (P = .002).Increased levels of NK cell activity and tumor specific CD8+ cytotoxic T-lymphocyte activity were found in the combination mIFN-alpha and mIL-2 or mIL-12 groups. Augmented immune responses correlated with clinical antitumor effects. CONCLUSIONS: The present study demonstrates that mIL-2 or mIL-12 augments tumor inhibition from mIFN-alpha and increases activation of NK and CD8+ T cells. These data support further investigation of polymer and lipid mediated delivery of cytokine genes for head and neck cancer.     

白细胞介素—2基因与单纯疱疹病毒胸苷激酶基因联合治疗小鼠头颈鳞状细胞癌的研究

OBJECTIVE: To assess the efficacy of combined interleukin-2 (IL-2) and herpes simplex virus thymidine kinase (HSV-TK) gene therapy for murine head and neck squamous cell carcinoma (HNSCC). METHODS: Ad IL-2 or/and Ad HSV-TK were injected into the tumor tissues directly after the murine HNSCC model was established. DL312 or PBS was used as control and ganciclovir (GCV) was used at 25 mg/kg for 7 days in Ad HSV-TK gene treatment groups. Tumor size was measured before and after treatment to evaluate the response to treatment. Cytotoxic T-lymphocyte (CTL) and natural killer (NK) assays were performed and IL-2 expressions were also measured after IL-2 gene transfection. RESULTS: HNSCC tumor growth was significantly inhibited following combined IL-2 and HSV-TK gene therapy as compared to other groups (P < 0.05). Increased levels of IL-2 protein expression was found in combined and single IL-2 treated groups. The combination and IL-2 treated groups produced greater activities of CTL and NK than that of the controls. CONCLUSION: IL-2 gene therapy can efficiently induce antitumor immunity of the host and enhance antitumor effects of HSV-TK. Combined IL-2 and HSV-TK gene therapy could significantly inhibit HNSCC tumor growth in the murine model.     

Combination nonviral interleukin-2 gene immunotherapy for head and neck cancer: from bench top to bedside

OBJECTIVE/HYPOTHESIS: Intralesional delivery of cytokine genes has emerged as a promising therapeutic strategy for the treatment of cancer. In addition to the therapeutic effect of the delivered cytokine gene, the components of the gene delivery system also have been shown to induce beneficial immune responses. On the basis of these principles, we hypothesized that a molecular therapy could be developed that would provide synergistic antitumor activity by way of intralesional expression of interleukin (IL)-2 from a recombinant plasmid combined with induction of endogenous interferon (IFN)-gamma and IL-12 cytokines by immunostimulatory DNA. Our objective in these studies was to create and optimize a novel formulation of cationic lipid and DNA that generates local production of IL-2 protein within a targeted tumor environment with concomitant induction of the antitumor cytokines IFN-gamma and IL-12. STUDY DESIGN: Prospective laboratory drug development plan that would produce human clinical trials. MATERIALS AND METHODS: Engineered bacterial plasmids containing a cytomegalovirus promoter (CMV)-IL-2 expression cassette were specifically formulated with cationic lipids and optimized for antitumor effect in a floor of mouth murine tumor model. The treated tumors were assayed for local expression of IL-2 and concurrent expression of secondary cytokines IFN-gamma and IL-12. Established tumors in C3H/HeJ mice were treated with various IL-2 gene formulations, and clinical and immunologic responses were evaluated. Immunologic studies were performed and included cytolytic T-cell assays and cytokine expression profiles. For human clinical trials, a phase I 10 patient formulated IL-2 gene therapy study was completed. Subsequently, two large scale, phase II multi-institutional and multi-international studies were initiated comparing non-viral IL-2 gene therapy to palliative methotrexate chemotherapy or in combination with cisplatin. RESULTS: In the preclinical stage, maximum tumor inhibition in animal models was obtained using IL-2 plasmid formulated with 1,2-dioleyloxypropyl-3-trimethyl ammonium chloride (DOTMA):cholesterol (1:1 mol:mol) at a plasmid:lipid charge ratio of 1:0.5 (-/+). Cationic lipid formulated IL-2 plasmid significantly inhibited tumor growth compared with formulated control plasmid (P < .01) or vehicle (lactose; P < .01). Consistent with previously reported studies of the immunostimulatory activity of DNA of bacterial origin, treatment of tumors with control plasmid in cationic lipid formulation induced production of endogenous IFN-gamma and IL-12 but not IL-2. Treatment of tumors with formulated IL-2 plasmid produced IL-2 protein levels that were 5-fold over background and increased IFN-gamma by 32-fold (P < .001) and IL-12 by 5.5-fold (P < .001) compared with control plasmid formulations. The phase I human trial demonstrated dose escalation safety, which was its primary objective, and there was one anecdotal reduction in tumor size. The phase II studies have been initiated and focus on either comparing the novel nonviral IL-2 gene immunotherapy formulation alone to methotrexate or comparing IL-2 gene therapy in combination with cisplatin in recurrent or unresectable patients with head and neck squamous cell carcinoma. CONCLUSIONS: The preclinical data provided proof of principle for matching a delivered IL-2 transgene with an immunostimulatory nonviral formulation to enhance intralesional production of therapeutic cytokines for the maximization of antitumor response. Human clinical trials have demonstrated this novel therapy to be safe in the human clinical setting. Phase II trials have been initiated to assess efficacy and feasibility as a single or combination therapy for head and neck cancer.     

Interactions and biological mechanisms of action of molecular signal peptides. II. Interleukin 2 (IL-2)

Interleukin 2 (IL-2) is predominantly produced by T-helper cells (TH1) having the phenotype CD4+, and by subpopulations of thymocytes after antigenic or mitogenic stimulation. IL-2 causes an indefinite growth of T-cells, and its function depends on binding to IL-2 receptors (IL-2R alpha and IL-2R beta). Thus the immune response of T cells is controlled through the expression of the IL-2 receptors and the IL-2 binding. IL-2 receptors are expressed not only by T-cells but also by B-cells, NK cells, monocytes, thymocytes, thymic stroma cells, oligodendrocytes and endothelial cells. This explains the various functions of IL-2, such as increased immunoglobulin production, growth of certain B-cell subpopulations, macrophage-dependent cytotoxicity, growth and differentiation of oligodendrocytes and proliferation of lymphokine activated killer (LAK) cells. Abnormal production of IL-2 may lead to autoimmune diseases, immunodeficiencies and, under certain circumstances, to T-cell leukemia. With antibodies against the IL-2 receptors the binding of IL-2 may be blocked to avoid auto-aggressive destruction in autoimmune diseases. LAK cells increase the growth of NK cells and T-cell cytotoxicity against transformed cells. LAK cells, especially those from tumor infiltrating lymphocytes, in conjunction with IL-2 have already been used with promising initial results in the treatment of distant metastases. In the future LAK cell therapy with IL-2 may be adopted to prevent metastases and second primary tumors in high-risk patients with head and neck cancer.     

白细胞介素-2基因与单纯疱疹病毒胸苷激酶基因联合治疗小鼠头颈鳞状细胞癌的研究

目的观察白细胞介素-2(interleukin-2,IL-2)基因与单纯疱疹病毒胸苷激酶(herpes simplex virus thymidine kinase,HSV-TK)基因联合治疗小鼠头颈鳞状细胞癌的疗效.方法建立小鼠头颈鳞状细胞癌动物模型后在荷瘤部位分别注射表达小鼠IL-2基因的重组腺病毒(recombinantadenovirus, Ad)和表达HSV-TK基因的重组腺病毒Ad HSV-TK及联合注射组,对照组分别注射不带目的基因的腺病毒或磷酸盐缓冲液(phosphatic buffered solution,PBS).对注射有Ad HSV-TK基因组和联合治疗组每日腹腔注射抗病毒药物更西罗韦(ganciclovir,GCV) 25 mg/kg体重,每日2次连续7 d.观察肿瘤大小变化并检测脾脏自然杀伤细胞和颈淋巴结细胞毒性T淋巴细胞的活性,探讨其抗肿瘤机制.结果 Ad IL-2和Ad HSV-TK联合治疗组,肿瘤生长明显受抑制,疗效显著优于单独治疗组和对照组(P<0.05),在注射有Ad IL-2基因的治疗组中,IL-2蛋白水平明显升高,自然杀伤细胞活性和细胞毒性T淋巴细胞杀伤活性增强,肿瘤组织中可见大片坏死,并含有大量的CD+4、CD+8淋巴细胞浸润.结论 Ad IL-2基因治疗可提高肿瘤局部和全身的抗肿瘤免疫应答,能加强自杀基因AdHSV-TK的抗肿瘤效果,两者联合应用能显著抑制小鼠头颈鳞状细胞癌的生长.     

Effects of combined therapy with interleukin 2 and interleukin 12 gene-transfected tumor vaccine for head and neck carcinoma

BACKGROUND: The biological effects of cytokines are coming to be understood. The therapeutic effects of interleukin (IL) 2, IL-12, and interferon gamma (IFN-gamma) in cancer treatment have been reported, but there are problems when these cytokines are systemically used as therapeutic agents. OBJECTIVE: To examine the efficacy of IL-2 and IL-12 gene-transfected tumor cell vaccines for head and neck squamous cell carcinoma (SCC). METHODS: Homozygous mice with the autosomal recessive nude gene (BALB/c nu/nu mice) were inoculated subcutaneously in the right flank with cells from a human oral floor SCC cell line (KB cells). The mice were then injected with IL-2 and IL-12 gene-transfected KB cells (KB/IL-2 and KB/IL-12 cells, respectively) irradiated with 2000 rad (20 Gy). RESULTS: No mice died soon after the injection of the gene immunotherapy. The treatment with either KB/human IL-2 (hIL-2) or KB/murine IL-12 (mIL-12) was not very effective. However, the treatment with both KB/hIL-2 and KB/mIL-12 cells significantly and safely inhibited the growth of established tumors (P =.04). There was no significant difference in antitumor effect between once-weekly and twice weekly injections of both KB/hIL-2 and KB/mIL-12 cells. CONCLUSION: Double gene immunotherapy is safe and effective treatment for SCC in mice.     

Cytotoxic T lymphocytes in peripheral blood and regional lymph nodes in laryngeal cancer patients

Cytotoxic T cells is an unique lymphocyte subpopulation able to recognize in specific manner and kill tumor cells. Therefore they constitute an important cells engaged in anti-tumor defense. The aim of the study was to determinate cytotoxic T cells frequency in peripheral blood and among lymphocytes isolated from regional lymph nodes. The study group consisted of twenty patients diagnosed with laryngeal cancer subjected surgical treatement. Cytotoxic T cells were estimated using three color flow cytometry based on CD3(+)CD8(+)CD28(-) GranzymeB(+) phenotype. Additionally TCR zeta chain expression and spontaneous apoptosis considered as a potential markers of immunosupressive effect exert by tumor were determined. In patients with laryngeal cancer significant increase of CD3(+)CD8(+)CD28(-) lymphocytes in peripheral blood in comparison to healthy control was observed. In lymph nodes the content of those cells was much lower, less than 10%, however in a group bearing metastases to regional lymph nodes higher than in a group without metastases. Cytotoxic T cells were also the main population subjected spontaneous apoptosis. The role and specificity of cytotoxic T cells in laryngeal cancer patients still remain to be elucidated, especially in respect to specificity of recognition tumor cell. Understanding details of this process may rise significant progress in an approach to diagnosis and therapy in laryngeal cancer patients.     

Limitations of adenovirus-mediated interleukin-2 gene therapy for oral cancer

OBJECTIVE/HYPOTHESIS: Adenoviral interleukin-2 (AdV-IL-2) gene therapy has previously not proven effective in treating established murine oral cancer. We hypothesize that the intratumoral level of IL-2 expression is a major limiting factor in treatment outcome. METHODS: A microscopic disease and established oral cancer murine model was used to test this hypothesis. IL-2 gene transfer was performed with a recombinant adenovirus vector. RESULTS: Tumor cells were transduced in vitro with AdV-IL-2 and subsequently implanted into the floor of the mouth in C3H/HeJ mice. IL-2 expression in vitro ranged from 990 to 1,050 pg/10(6) tumor cells. This microscopic disease treatment resulted in either complete tumor regression or a dramatic decrease in tumor progression. Cytolytic T-cell (CTL) assays demonstrated a predominance of CD8-specific, T-cell-mediated tumor killing. Reducing IL-2 expression by half with a mixture of 1:1 transduced to nontransduced tumor cells eliminated the antitumor effect and decreased the CTL response. These findings support the presence of a critical "threshold" of IL-2 expression. Adenovirus repurification and amplification allowed isolation of a twofold-higher-titer AdV-IL-2 vector. Treatment of established tumors with the higher-titer AdV-IL-2 at a new maximal dose of 1.4 x 10(9) plaque-forming units (pfu) increased in vivo IL-2 expression to 1,127 pg/10(6) cells and generated a significant antitumor response. Complete regression of established tumors, however, could not be achieved, and we noted a decrease in IL-2 expression well below the threshold at 1 week after treatment. Upon repeat maximal AdV-IL-2 injection in vivo, a greater antitumor effect and increased CTL response was seen, but also, 28% of the animals died of IL-2 toxicity. CONCLUSION: Although limited by expression and toxicity as a single-treatment strategy for established tumors, AdV-IL-2 gene therapy should be considered a potential component of combination therapy strategies.     

Combination nonviral interleukin 2 and interleukin 12 gene therapy for head and neck squamous cell carcinoma.

OBJECTIVE: To determine the feasibility and efficacy of combination nonviral lipid-formulated murine interleukin 2 (mIL-2) and polymer-formulated murine interleukin 12 (mIL-12) gene therapy for head and neck squamous cell carcinoma (HNSCC) in a murine model. METHODS: Randomized, controlled studies in a murine HNSCC model. Tumors were established in the floor of mouth in C3H/HeJ immunocompetent mice. Established tumors were directly injected with lipid-formulated mIL-2 and polymer-formulated mIL-12 alone and in combination. Antitumor responses, cytokine expression, and natural killer cell and cytolytic T-lymphocyte activity were assayed. RESULTS: The use of combined mIL-2 and mIL-12 gene therapy resulted in significant antitumor effects, compared with each of the single-cytokine and no-treatment (control) groups (P =.01 to P =.02). Tumors treated with the formulated cytokine genes showed an increased level of the corresponding proteins and decreased level of transforming growth factor beta (TGF-beta) expression. Combined mIL-2 and mIL-12 treatment consistently produced the greater activation of cytolytic T-lymphocyte and natural killer cells than did single-cytokine treatment or other controls at all concentrations tested. Augmented immune responses correlated with clinical antitumor effects. CONCLUSIONS: The nonviral gene delivery system was well tolerated, and combined mIL-2 and mIL-12 gene transfer generated potent antitumor immune responses against HNSCC in our murine model. Combined nonviral IL-2 and IL-12 gene therapy may have great potential as a primary or adjuvant treatment for HNSCC in humans.     

CD4+ T cells induce productions of IL-5 and IL-13 through MHCII on ILC2s in a murine model of allergic rhinitis

ObjectiveCD4⁺ T cells play an important role not only in the induction of allergy but also in allergic inflammation. Group 2 innate lymphoid cells (ILC2s) also mediate type 2 immune responses in allergic rhinitis (AR). However, the relationships between CD4⁺ T cells and ILC2s in allergic condition are currently not well defined. The study aimed to evaluate the potential influences of CD4⁺ T cells on ILC2s in the murine model of AR.MethodsA murine model of AR was established using ovalbumin (OVA), and OVA-induced ILC2s were sorted and purified from the mouse nasal-associated lymphoid tissue (NALT), and cultured in vitro. Then, the expression of major histocompatibility complex class II (MHCII) on ILC2s was examined. CD4⁺ T cells were separated from AR mice peripheral blood mononuclear cells (PBMCs). After that, productions of IL-5 and IL-13 on ILC2s cultures were assessed when CD4⁺ T cells or plus anti-MHCII antibody or anti-CD4 antibody were administered into the cultures. Finally, we adoptively transferred ILC2s alone or ILC2s plus anti-MHCII antibody to the murine model of AR to investigate their roles in the nasal allergic inflammation.ResultsWe showed that ILC2s could be induced by OVA in the mouse NALT. The number and percentage of ILC2s in AR mice were increased. MHCII was expressed on ILC2s, and its protein and mRNA were all enhanced in allergic condition. IL-5 and IL-13 proteins and mRNAs were elevated after CD4⁺ T cells administration, and were reduced after these cells plus anti-MHCII antibody or anti-CD4 antibody application. Numbers of sneezing and nasal rubbing as well as counts of eosinophils in nasal lavage fluid (NLF) were all enhanced after the adoptive transfer of ILC2s when compared to AR mice. IL-5 and IL-13 in the NLF of allergic mice were also increased in comparison with AR group. However, above parameters were all decreased after the transfer of ILC2s plus anti-MHCII antibody versus AR mice or ILC2s-treated ones.ConclusionThese findings show that CD4⁺ T cells induce productions of IL-5 and IL-13 through MHCII on ILC2s in AR mice models.     

Anti-CD3/anti-CD28 bead stimulation overcomes CD3 unresponsiveness in patients with head and neck squamous cell carcinoma

OBJECTIVES: To test whether T-cell CD3 responses are altered in patients with advanced-stage head and neck squamous cell carcinoma (HNSCC) and whether anti-CD3/anti-CD28 (alphaCD3/alphaCD28) bead stimulation could reverse CD3 unresponsiveness. DESIGN: Anti-CD3 (alphaCD3) monoclonal antibody immobilized on tissue culture plastic was used to stimulate lymph node mononuclear cells (LNMCs) and peripheral blood mononuclear cells (PBMCs) from patients with advanced-stage HNSCC. Proliferation, T-cell phenotype, and cytokines were measured during 8-day in vitro stimulation. Immune-enhancing properties of alphaCD3/ alphaCD28 beads were also tested on LNMCs and PBMCs. Cytotoxicity of bead-activated T cells (ATCs) was measured against autologous and allogeneic HNSCC. RESULTS: Six patients were nonresponders to alphaCD3 stimulation defined by tritium (3H) incorporation of less than 3500 cpm, whereas 11 patients were responders with 3H incorporation of 3500 cpm or more. Responders produced higher levels of interleukin (IL)-12 and interferon gamma (IFN-gamma) after alphaCD3 stimulation than nonresponders. No phenotypic or clinical differences were identified between groups. Stimulation with alphaCD3/alphaCD28 beads enhanced IFN-gamma and IL-2 produced by both groups. Bead ATCs were generated from PBMCs of patient 11 in the responder group and lysed (+/- SD) 100% +/-1% of autologous tumor and 49% +/-1% of allogeneic tumor. Bead ATCs from LNMCs of this patient lysed 58%+/-1% of autologous tumor and 63%+/-1% of allogeneic tumor. CONCLUSIONS: A subpopulation of patients with HNSCC who are nonresponders to alphaCD3 stimulation has been identified, showing reduced proliferation and IL-12 and IFN-gamma secretion. Nonresponders stimulated with alphaCD3/alphaCD28 beads reversed immune unresponsiveness and induced a type 1 cytokine response. Bead-generated ATCs from patient 11 in the responder group lysed autologous and allogeneic HNSCC in vitro, suggesting a possible effective immunotherapeutic modality in the treatment of HNSCC.     

Effects of intranasal immunization on protective immunity against otitis media

It has been reported that intranasal immunization can induce mucosal immune responses. However, the efficacy of intranasal immunization on otitis media caused by non-typeable Haemophilus influenzae (NTHi) is not yet elucidated. Mice were intranasally, orally, intratracheally or intraperitoneally immunized with outer membrane protein (OMP) isolated from NTHi, and antigen-specific immune responses were determined by enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immuno-spot assay (ELISPOT). Cytokine production from splenic CD4+ T cells was examined by ELISA. Following the immunization, the clearance of NTHi from the nasal and nasopharyngeal cavity was examined. OMP-specific IgA antibody titers in nasal washes and the numbers of specific IgA-producing cells in nasal passages were significantly increased in intranasally immunized mice. Cytokine analysis showed that interferon-gamma (IFN-gamma) and interleukins IL-6 and IL-10 were predominantly produced from CD4+ T cells. The clearance of NTHi was significantly enhanced in the intranasal immunization group. Intranasal immunization is an effective vaccination regimen for the induction of OMP-specific mucosal immune responses.     

T lymphocytes from tonsil and peripheral blood show different cytolytic and helper activities.

T lymphocytes from tonsil (To) and peripheral blood (PB) of 4 tonsillectomized children were subjected to clonal expansion with PHA in order to analyze at single cell level their cytolytic activity and their ability to produce interleukins such as IL-2, IFN-gamma and IL-4. Analyzing all T-cell clones (CD4+ and CD8+) obtained from To in comparison with those from PB, a reduced proportion of cells with lectin-dependent cytolytic activity (LDCC) (25% vs. 42%, p less than 0.01) and natural killer (NK) activity (18% vs. 31%, p less than 0.02) was found. These differences were proportionally related to the lower number of CD8+ T-cells in To than in PB. The proportion of CD4+ clones able to produce IL-2 and/or IL-4 were higher in To (75% and 61%) than in PB (52% and 25%, p less than 0.001 and p less than 0.001, respectively). In contrast, the proportion of CD4+ clones able to produce IFN-gamma was similar (53% and 58%) in both series of clones. According to the patterns of lymphokine synthesis, tonsillar T-cells differed from PB T-cells as follows: 1) the number of Th1-like CD4+ clones producing IL-2 and/or IFN-gamma (but not IL-4) were 23% vs. 44% in PB (p less than 0.001); 2) there was no difference between To and PB in the proportion of CD4+ clones producing IL-4 alone (Th2 clones: 9% vs. 8%); 3) CD4+ clones synthesizing IL-2, IL-4 and IFN-gamma at the same time were more frequent in To than in PB (Th3 clones: 53% vs. 17%, p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

变应性鼻炎患者外周血嗜酸粒细胞-骨髓干细胞通路指标检测在激素治疗评价中的意义

目的 检测健康人及变应性鼻炎(AR)患者治疗前后外周血嗜酸粒细胞(eosinophils,EOS)-骨髓干细胞通路相关指标CD34+、白细胞介素5(IL-5)、EOS,探讨外周血-骨髓通路在变应性鼻炎发病机制中的作用以及糖皮质激素对此通路的影响.方法 实验分2组:①试验组:常年性持续性变应性鼻炎患者44例,男24例,女20例,年龄7～68岁;给予糖皮质激素治疗4周;②健康对照组:健康体检者30例.分别检测试验组治疗前后和对照组外周血EOS计数,血清IL-5水平及CD34+细胞数,并分析各指标间的相关性.结果 试验组治疗前血清IL-5含量、CD34+数分别为(88.25±33.47)ng/L、(9.24±2.15)个/105,显著高于治疗后[(44.34±16.32)ng/L、(6.31±1.83)个/103]及健康对照组[(31.24±8.43)ng/L、(3.47±1.32)个/105].试验组治疗前后血清IL-5水平与其CD34+数呈显著正相关(r值分别为0.64、0.61,P值均＜0.01).患者血清IL-5水平与其EOS数呈显著正相关(r=0.64,P＜0.01).结论 外周血EOS、IL-5及CD34+细胞参与AR发病过程,提示AR患者病变局部组织和骨髓造血之间有相关通路存在.通过检测外周血IL-5及CD34+可评价治疗效果.     

Efficacy of local use of recombinant interferon alpha-2 preparations in combined treatment of recurrent respiratory papillomatosis

Clinical-immunological examination of 41 patients with recurrent respiratory papillomatosis (RRP) included determination of phenotype CD3, CD4, CD8, CD16, CD25, CD56, HLA-DR in peripheral blood by flow cytofluorimetry, the levels of IFN-gamma, TNF-alpha, GM-CSF, IL-2, IL-4, IL-5, IL-10, IL-12, IL-13 in the laryngeal secretion by multiplex immunoassay. Interferon inhalation therapy was conducted to prevent recurrence in 23 patients after surgical treatment and in 18 patients as monotherapy. The efficacy of the monotherapy was 45.5%. Treatment with IFN-alpha raised the levels of cytokines modulating an immune response by Th1-type (IFN-gamma, IL-12, IL-2) and GM-CSF, and reduced the levels of IL-4, IL-10 and IL-13. Local treatment with recombinant IFN-alpha is effective in aggressive RRP. As prognostic markers of the treatment efficacy may serve baseline high levels of TNF-alpha and IL-4/IFN-gamma index in laryngeal secretion. Treatment efficacy can be assessed by raise of IFN-gamma, IL-2 and IL-12 in combination with reduction of IL-4/IFN-gamma index.     

Combination nonviral murine interleukin 2 and interleukin 12 gene therapy and radiotherapy for head and neck squamous cell carcinoma

OBJECTIVES: To demonstrate that the combination of nonviral murine interleukin (mIL) 2 and mIL-12 gene therapy and external beam radiation therapy (XRT) have an enhanced therapeutic effect for the treatment of head and neck squamous cell carcinoma (HNSCC) in an orthotopic murine model and to elucidate the mechanism of action. DESIGN: A randomized, controlled study in a murine HNSCC model. INTERVENTIONS: Tumors were established in the floor of the mouth in C3H/HeJ immunocompetent mice with the SCC VII cell line. These tumors were directly injected with single lipid-formulated mIL-2 or single polymer-formulated mIL-12 or a combination of them and with phosphate-buffered saline or vector without mIL-2 and mIL-12 gene as controls. Then the local tumor was radiated twice with a dose of 1 Gy the next day and injected again 4 days later. Antitumor responses, cytokine expression, and natural killer cell and cytolytic T-lymphocyte activity were assayed. Meanwhile, tumor sizes were measured before and after treatment and compared among the different treatment groups and the controls. RESULTS: The combination mIL-2 + mIL-12 + XRT demonstrated a significant increase in antitumor effects compared with single therapy or controls. Increased expression levels of primary and secondary cytokines were found in the group treated with mIL-2 + mIL-12, and this effect was preserved when mIL-2 and mIL-12 treatments were combined with XRT. Combination therapy significantly increased antitumor effects, T-lymphocyte infiltration of CD4(+)and CD8(+), and the numerous necroses compared with monotherapy. CONCLUSIONS: Combination mIL-2 and mIL-12 gene therapy and XRT generates potent antitumor immune responses against HNSCC and significantly increases necrosis (apoptosis) in an orthotopic murine model of HNSCC. The nonviral mIL-2 and mIL-12 gene delivery system was well tolerated. Further optimization of treatment strategy for patients with HNSCC is warranted as well as consideration for human clinical trials.     

白细胞介素12基因治疗小鼠变应性鼻炎的实验研究

目的探讨鼻腔局部应用EB病毒(Epstein-Barrvirus,EBV)质粒载体介导的白细胞介素12(interleukin-12,IL-12)基因治疗对变应性鼻炎炎症反应的调节作用。方法将36只6～8周雄BALB/C实验小鼠随机分为变应性鼻炎组、IL-12基因治疗组和正常对照组,每组12只。以BALB/c小鼠经卵清蛋白(ovalbumin,OVA)免疫建立变应性鼻炎模型,用阳离子脂质体包裹EBV质粒载体介导的IL-12表达质粒(pGEG.mI-L12)形成混合物EBV/lipoplex,于激发前鼻腔局部滴入后,观察小鼠变应性症状的改善情况,并检测该基因在3组实验鼠鼻黏膜局部的表达情况以及对鼻黏膜炎性细胞和Th2细胞因子的影响。结果基因治疗组小鼠鼻黏膜中IL-12mRNA阳性细胞数量明显高于变应性鼻炎组,差异有统计学意义(P<0.01);基因治疗组小鼠鼻黏膜中IL12阳性细胞数量明显高于变应性鼻炎组(P<0.05);变应性鼻炎组鼻黏膜中嗜酸粒细胞、肥大细胞和IL5阳性细胞比例显著高于基因治疗组和正常对照组(P<0.01);变应性鼻炎组外周血中总IgE含量显著高于正常对照组和基因治疗组(F=1216.21,P<0.01)。结论鼻腔局部应用EBV/lipoplex后,pGEG.mIL-12能够在鼻黏膜中高效地表达,能明显抑制鼻腔的变应性反应。EBV/lipoplex有望成为变应性鼻炎免疫治疗一种新的方法。