The effect of altered Toll-like receptor 4 signaling on cancer cachexia

OBJECTIVE: To determine whether mice unable to mount an intact inflammatory response because of a Toll-like receptor (TLR) pathway defect will develop less severe cancer cachexia. DESIGN: Prospective animal study. SETTING: Academic research center. SUBJECTS: Six- to eight-week-old, female C3H/HeJ mice (17-18 g) and age-, weight-, and sex-matched wild-type C3H/HeN mice, differing in that the HeJ mice have nonfunctional TLR4 due to a TLR4 double mutation (TLR4(d/d)). INTERVENTION: The mice were inoculated with equal numbers of SCCF-VII cells and housed in individual cages. MAIN OUTCOME MEASURES: Food intake, body weight, pretumor and posttumor body composition, circulating cytokines, and levels of a marker of muscle atrophy were analyzed. RESULTS: The wild-type HeN mice weighed less on average than the TLR4(d/d) mice (2.6 g vs 4.9 g) (P = .01). They consumed more food, had smaller tumors, and had less lean body mass and fat mass than the TLR4(d/d) mice. Interleukin 1beta level was significantly elevated in the tumor-bearing HeN mice (mean gain of 259 pg/mL) but not in the TLR4(d/d) mice (P = .03). Both mouse strains had evidence of muscle atrophy. CONCLUSIONS: In spite of increased food intake and smaller tumors, the wild-type HeN mice had more severe cachexia than the TLR4(d/d) mice. The impaired ability to secrete proinflammatory cytokines such as interleukin 1beta may protect these animals from developing severe cancer cachexia. This animal model represents a novel system in which the host contributions to cachexia may be further studied.     

The role of Toll-like receptor 4 in eliciting acquired immune responses against nontypeable Haemophilus influenzae following intranasal immunization with outer membrane protein

Objective

Acute otitis media (AOM) is one of the most common infectious diseases in children. Nontypeable Haemophilus influenzae (NTHi) is considered a major pathogen in AOM. Current treatment options depend mainly on the use of antibiotics, thus developing vaccines to prevent this disease is an urgent goal for public health. Outer membrane proteins (OMPs) are promising candidate targets for vaccination against NTHi.

Methods

We used C3H/HeJ (Toll-like receptor 4 [TLR4]-mutant) mice, which arose spontaneously and have a non-functional TLR4 protein, and normal wild-type (WT) C3H/HeN mice. These mice were immunized intranasally with OMP from NTHi to investigate the mechanism of acquired immunity via TLR4. We examined the kinetics of mucosal and systemic antibody secretion and the migration of antibody producing lymphocytes to the mucosa in both strains during the course of intranasal immunization.

Results

The mucosal and systemic immune responses against OMP from NTHi were elicited in both TLR4-mutant and WT mice. However, the mucosal IgA, and systemic IgG, and Th1 immune responses in WT mice were stronger than those in TLR4-mutant mice.

Conclusions

TLR4 plays an important role in relation to Th1 function for optimal development of the acquired immune responses to OMP administered intranasally. The variety of immune responses via TLR4 expression needs to be taken into consideration of individual vaccinations to prevent AOM.     

C3H/HeJ mouse model for spontaneous chronic otitis media

OBJECTIVES/HYPOTHESIS: Chronic otitis media is a significant clinical problem. Understanding the mechanisms of chronic otitis media is critical for its control. However, little is known of these processes as a result of lack of animal models of spontaneous otitis media. The C3H/HeJ mouse has a single amino acid substitution in its toll-like receptor 4 (TLR4), making it insensitive to endotoxin. As a result, these mice cannot clear Gram-negative bacteria. The chronically inflamed middle ear in this animal provides us the opportunity to study spontaneous chronic otitis media. STUDY DESIGN AND METHODS: Otoscopy and auditory brain response (ABR) evaluation of C3H/HeJ mice at 3, 5, 7, 9, and 11 months were carried out under sedation. At 12 months of age, mice were killed and histologic analysis of the middle ear, inner ear, and eustachian tube was carried out. RESULTS: Tympanic membrane visualization and ABR thresholds in 7- to 8-month-old C3H/HeJ mice showed that approximately half developed middle and inner ear disease spontaneously. The significant elevation of thresholds suggested a sensorineural component in addition to the conductive loss. Middle and inner ear histology showed some degree of middle and inner ear inflammation in half the mice, paralleling the ABR data. CONCLUSIONS: The histopathologic changes reported here in the C3H/HeJ mouse model of chronic otitis media have been reported in human chronic otitis media. This spontaneous model of chronic otitis media will be valuable for the characterization of middle and inner ear inflammatory disease processes that are induced by middle ear infections.     

Gram-negative pathogen Klebsiella oxytoca is associated with spontaneous chronic otitis media in Toll-like receptor 4-deficient C3H/HeJ mice

CONCLUSION: This report confirms the presence of gram-negative Klebsiella bacteria in the middle ear of the C3H/HeJ mouse by culture, polymerase chain reaction (PCR), and electron microscopy. Identification of the bacterial pathogen supports the C3H/HeJ mouse as an excellent model for spontaneous chronic otitis media and its effects on the middle and inner ear. OBJECTIVES: The C3H/HeJ mouse has a single amino acid substitution in its Toll-like receptor 4, making it insensitive to endotoxin and suppressing initiation of the innate immune system. This study explored the bacteriology of the resultant middle ear infection by culture, PCR, histology, and electron microscopy. MATERIALS AND METHODS: Twelve-month-old C3H/ HeJ mice were screened positive for spontaneous otitis media. Tympanocentesis and blood cultures of mice were carried out under sedation. Middle ear aspirate material and blood samples were then sent for culture and PCR. Mice were then sacrificed for bright-field and electron microscopy analysis. RESULTS: All tympanocentesis and blood specimens grew gram-negative Klebsiella oxytoca, which was confirmed by PCR. Histopathology confirmed an intense inflammatory reaction and gram-negative bacteria in the middle and inner ears. Electron microscopy of the middle ears revealed abundant rod-shaped Klebsiella bacteria, both free and being engulfed by neutrophils.     

Toll样受体2和4在小鼠肺炎链球菌中耳急性感染中的作用

目的 探讨Toll样受体2(Toll-like receptor 2,TLR2)和Toll样受体4(Toll-like receptor 4,TLR4)在小鼠肺炎链球菌中耳感染中的作用.方法 野生型(wild type,WT)C57BL/6J小鼠、TLR2缺陷(TLR2-/-)和TLR4缺陷(TLR4-/-)小鼠分别通过中耳鼓膜接种肺炎链球菌悬液[1×106菌落形成单位(colony forming unit,CFU)].在细菌接种前、接种后第3天和第7天分别进行听性脑干反应(ABR)和鼓室声导抗测试,血液及中耳积液中细菌滴度测定,颞骨病理切片形态学观察,反转录聚合酶链反应(RT-PCR)检测不同基因型小鼠炎性细胞因子IκB激酶β(IκB kinase β,IKKβ)、核因子κB(NF-κB)、肿瘤坏死因子α(TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)、巨噬细胞炎性蛋白1α(MIP-1α)、黏蛋白/黏液素5AC和5B(MUC5AC和MUC5B)mRNA的表达.结果 中耳接种肺炎链球菌3d后,TLR2-/-小鼠死亡率为58.8％(40/68),TLR4-/-小鼠死亡率为35.6％(21/59),而WT小鼠的死亡率仅为17.3％(9/52).接种7d后,TUR2-/-小鼠死亡率为82.4％(54/68),TLR4-/-小鼠死亡率为54.2％(32/59),而WT小鼠的死亡率仅为23％(12/52),三组之间差异具有统计学意义(P＜0.01).ABR测试结果显示,接种后存活3d及7d的TLR2-/-和TLR4-/-小鼠ABR阈值高于WT小鼠,三组之间的差异具有统计学意义(P＜0.01).颞骨病理发现,TLR2-/-和TLR4-/-小鼠接种肺炎链球菌后第3天中耳出现积液和组织破坏,接种后第7天感染极为严重.TLR2-/-鼠出现严重的耳蜗炎性细胞浸润,内外毛细胞破坏、盖膜肿大和血管纹退变,以及螺旋神经节细胞的损伤;TLR4-/-鼠可见耳蜗内外毛细胞和螺旋神经节细胞的损伤;而WT鼠的耳蜗未见炎性细胞浸润和组织破坏,内外毛细胞基本正常.在接种细菌3d和7d的TLR2-/-鼠中耳黏膜未发现肥大细胞,但在TLR4-/-和WT鼠中耳黏膜发现较多的肥大细胞并有脱颗粒现象.接种肺炎链球菌后3d和7d的TLR2-/-和TLR4-/-小鼠血液中细菌滴度均高于WT小鼠,差异具有统计学意义(P值均＜0.05).接种肺炎链球菌3d后,TLR2-/-小鼠听泡组织中NF-κB、TNFα、IL-1β、MIP-1α、MUC5AC、MUC5B等因子的mRNA表达水平低于TLR4-/-和WT小鼠,差异具有统计学意义(P值均＜0.05).结论 TLR2-/-鼠在肺炎链球菌激惹后产生相对低水平的致炎性细胞因子,中耳清除细菌能力下降,引发脓毒血症,导致高死亡率.TLR2和TLR4是2种重要的小鼠中耳炎性反应的受体和信号转导分子.     

Glucocorticoid impact on cochlear function and systemic side effects in autoimmune C3.MRL-Faslpr and normal C3H/HeJ mice

Glucocorticoids are effective in reversing hearing loss, but their severe side effects limit long term management of many ear disorders. A clearer understanding of these side effects is critical for prolonged therapeutic control of hearing and vestibular dysfunction. Therefore, this study characterized the impact of the glucocorticoid prednisolone on cochlear dysfunction and systemic organ systems in C3.MRL-Fas(lpr) autoimmune mice and their normal C3H/HeJ parent strain. Following 3 months of treatment, autoimmune mice had better auditory thresholds and improved hematocrits, anti-nuclear antibodies, and immune complexes. Steroid treatment also lowered body and spleen weights, both of which rise with systemic autoimmune disease. Steroid treatment of the normal C3H/HeJ mice significantly elevated their blood hematocrits and lowered their body and spleen weights to abnormal levels. Thus, systemic autoimmune disease and its related hearing loss in C3.MRL-Fas(lpr) mice are steroid-responsive, but normal hemopoiesis and organ functions can be significantly compromised. This mouse model may be useful for studies of the detrimental side effects of steroid treatments for hearing loss.     

Cochlear IgG in the C3H/lpr autoimmune strain mouse.

The inner ear of the C3H/lpr autoimmune strain mouse was evaluated to identify potential mechanisms by which systemic autoimmune disease interferes with auditory function. The inner ears were immunohistochemically stained for IgG at ages before (2 months) and after (6-10 months) autoimmune disease onset and compared to age-matched nonautoimmune C3H/HeJ controls. Immunoreactivity for IgG was not seen in the 2 month C3H/lpr autoimmune mice or in either age group of the C3H/HeJ controls. On the other hand, all older C3H/lpr mice showed reaction product in the vessels of the cochlea, particularly the stria vascularis and bony capsule. Less frequent sites of staining were the geniculate ganglion, marrow cavities of the bony capsule, tensor tympani muscle, and on one occasion, a hair cell of the organ of Corti. These findings indicate that IgG is widespread within the cochlea and its vessels during systemic autoimmune disease and not directed against any specific sensorineural structure. This suggests a generalized or indirect mechanism whereby such systemic disease affects the inner ear.     

Immunolocalization of dendritic cells and pattern recognition receptors in chronic rhinosinusitis

BACKGROUND: Dendritic cell (DC) activation and antigen presentation to T cells are critical to innate and adaptive immunity. Toll-like receptors (TLRs) are known to bind pathogen-associated molecular patterns in addition to sinonasally secreted surfactant proteins (SP) such as SP-A and SP-D. TLR binding is known to activate DCs. Based on these observations, we sought to establish the presence, in sinonasal mucosa, of DC and the pattern recognition receptors (PRRs), CD14, TLR2, and TLR4. METHODS: Sinonasal biopsy specimens were taken from patients with eosinophilic nonatopic nasal polyposis (n = 4), allergic fungal sinusitis (n = 1), and nondiseased patients undergoing cerebrospinal fluid leak repair or pituitary tumor resection (n = 2). Tissue samples were stained immunohistochemically for PRR (CD14, TLR2, and TLR4), mature DC marker (CD208), iDC marker (CD209), or isotype controls. RESULTS: Immature and mature DC were immunolocalized to the subepithelial stroma and ciliated epithelial surface, respectively. Diffuse staining of CD14 was observed throughout the stroma with additional staining in the ciliated epithelium. The TLR markers showed no staining in the ciliated epithelium. TLR2 primarily localized in stroma immediately deep to the ciliated epithelial surface. TLR4 immunolocalized to submucosal seromucinous gland ductal epithelium. Data from nondiseased patients were mixed, with one patient showing minimal staining of any of the tested cellular markers. CONCLUSION: This study indicates progressive DC activation and emigration of mature antigen-presenting cells from the epithelial surfaces of sinonasal mucosa. The presence of TLR known to bind SP-A and SP-D suggests a link between SP expression and immune response in sinonasal mucosa.     

C57Bl/6 and BALB/c mice have similar neutrophil response to acute Streptococcus pneumoniae sinus infections

BACKGROUND: Previous investigations have shown that mice with a tendency toward a T(H)1 or T(H)2 lymphocyte response manifest different reactions to inoculation with the parasite Leishmania major. BALB/c mice (with a tendency for a T(H)2 response) showed evidence of systemic infection, whereas C57Bl/6 mice (with a tendency for a T(H)1 response) showed only a local reaction. OBJECTIVE: To investigate whether BALB/c and C57Bl/6 mice respond differently to acute bacterial infection of the sinuses. METHODS: We inoculated the nasal cavities of C57Bl/6 and BALB/c mice with Streptococcus pneumoniae (type ATCC59), or with broth as a control. The mice were humanely killed 2, 5, 10, and 14 days after inoculation. Their heads were fixed, decalcified, and embedded in paraffin blocks. Sections were stained with hematoxylin and eosin, and the degree of inflammation was quantified by the number of neutrophils per square millimeter of the sinus mucosa and the percentage of the sinus cavity occupied by neutrophil clusters. RESULTS: Both groups of mice showed evidence of inflammation that was significantly greater than controls (P =.01), with no difference between groups. There was a correlation between the number of neutrophils per square millimeter in the sinus mucosa and the percentage of neutrophil clusters (C57Bl/6 mice, r = 0.37, P<.001; BALB/c mice, r = 0.20, P<.001). In the infected mice, the number of infiltrating neutrophils was significantly greater (P<.001) in anatomically lower (dependent) areas of the sinuses compared with the upper areas. CONCLUSION: Unlike leishmaniasis, acute bacterial sinusitis is not affected by the tendency of the host to favor either a T(H)1 or T(H)2 response.     

Acute otitis media in older children and adults treated with phenoxymethyl penicillin or erythromycin stearate. Bacteriological and immunological aspects

Seventy-eight patients, all over 10 years of age, with clinical signs of acute otitis media, received either phenoxymethyl penicillin or erythromycin stearate, in a randomized manner, and the clinical, bacteriological and immunological effects were studied. Haemophilus influenzae and Streptococcus pneumoniae were the major pathogens isolated from the nasopharynx in 30 and 28 patients, respectively. Increased levels of C-reactive protein (CRP) were detected in 53 (68%) of the patients. There was no statistical difference in the CRP-levels depending on species of bacteria isolated. The highest incidence was observed in cases with Branhamella catarrhalis and H. influenzae. Persistence of H. influenzae during antibiotic therapy was demonstrated in 70% and after therapy in 63% compared to 4% and 11% persistence of S. pneumoniae. The type of antibiotic treatment did not influence persistence. An immune response to H. influenzae and S. pneumoniae was detected significantly more often in patients treated with erythromycin stearate than with phenoxymethyl penicillin.     

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。     

Bacterial antigens and neutrophil granule proteins in middle ear effusions

Otitis media with effusion is a significant cause of hearing loss in young children. We hypothesized that persistent bacterial antigens in middle ear effusions (MEEs) might act as chronic inflammatory stimuli causing release of neutrophil proteins. Concentrations of neutrophil lactoferrin and a 37-kd cationic bactericidal protein (CAP 37) were measured in 47 MEEs collected from 27 children at the time of tympanostomy tube placement. Antigens of Streptococcus pneumoniae were detected by latex particle agglutination and those of Haemophilus influenzae by dot-blot assay. Bacterial antigens were detectable in 24 (51%) of MEEs: S pneumoniae in 10 (21%), H influenzae in 12 (26%), and both antigens in 2 (4%). Concentrations of lactoferrin and CAP 37 in H influenzae antigen-positive MEEs were significantly higher than in either S pneumoniae antigen-positive or antigen-negative MEEs. We conclude that H influenzae antigen causes a greater middle-ear inflammatory response, as judged by neutrophil products, than does S pneumoniae antigen.     

Signals through CD40 play a critical role in the pathophysiology of Schistosoma mansoni egg antigen--induced allergic rhinitis in mice

BACKGROUND: Interaction between CD40 and CD40L is thought to regulate immune responses in several allergic diseases. However, little is known about its in vivo role in the pathophysiology of allergic rhinitis. We sought to determine whether the lack of signals through CD40 affects the pathophysiology of allergic rhinitis using a murine model. METHODS: Wild type (WT) and CD40-deficient BALB/c (CD40-/-) mice were sensitized intranasally to Schistosoma mansoni egg antigen (SEA). After repeated sensitization, histamine responsiveness, serum antibody titer including immunoglobulin E (IgE), nasal eosinophilia, and cytokine production by nasal mononuclear cells were determined in each group. RESULTS: Intranasal sensitization with SEA in WT mice elicited a strong Th2 response including SEA-specific IgE production, nasal eosinophilia, and interleukin (IL)-4, and IL-5 production by nasal mononuclear cells after antigen challenge. Production of SEA-specific IgE and IgG1 was abolished in SEA-sensitized CD40-/- mice. These mice showed impaired nasal eosinophilia and displayed markedly reduced histamine-induced nasal hyperresponsiveness as compared with WT mice. Furthermore, reduced production of IL-4 and IL-5 by nasal mononuclear cells was seen in CD40-/- mice. CONCLUSION: These results show that signals through CD40 play a critical role in not only IgE production but also pathophysiology of allergic rhinitis such as nasal hyperresponsiveness and nasal eosinophilia.     

Cure of an established nonimmunogenic tumor,SCC VII,with a novel interleukin 12-based immunotherapy regimen in C3H mice

OBJECTIVE: To develop a murine model of effective treatment with immunotherapy for established head and neck squamous cell carcinoma. DESIGN: Prospective animal study.Subjects Female C3H mice, 8 to 12 weeks old. INTERVENTIONS: A subcutaneous inoculation of 2 x 10(5) SCC VII cells in C3H mice was established for 7 to 12 days. Tests for concomitant immunity were performed, with and without interleukin 12 modification. Tumors were also tested for responsiveness to interleukin 12 (5 mice) and to cyclophosphamide followed by interleukin 12 (5 mice). SCC VII tumors in 24 mice were treated with interleukin 12 followed by cyclophosphamide and interleukin 12. Five mice with tumors treated with isotonic sodium chloride solution served as controls. Tumors were measured 3 to 4 times weekly, and cure was defined as complete regression of the tumor for at least 60 days. Cured mice were rechallenged with 2 x 10(5) SCC VII cells to verify antitumor immunity. Immunohistochemistry of regressing tumors was performed for CD4+ and CD8+ T cells. RESULTS: Tumor-bearing mice easily developed second tumors when challenged with 2 x 10(5) tumor cells in the opposite flank. However, interleukin 12 treatment provided immunity to second tumors in 8 (100%) of 8 mice when started at day 4 and in 2 (40%) of 5 when treated from day 7. SCC VII did not respond to standard interleukin 12 or cyclophosphamide plus interleukin 12 therapy. Seventy-five percent of animals (18/24) treated with interleukin 12 followed by cyclophosphamide plus interleukin 12 were successfully cured, and all cured mice resisted subsequent challenge with SCC VII. Immunohistochemistry of regressed tumors showed an intense CD4+ and CD8+ infiltrate that was absent in the untreated and nonresponding tumors. CONCLUSIONS: Nonimmunogenic SCC VII is a nonimmunogenic tumor that can be converted into an immunogenic tumor with interleukin 12 treatment. Additional treatment with cyclophosphamide plus interleukin 12 leads to complete regression in 75% of mice.     

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.     

Presence of Branhamella catarrhalis alters the survival of Streptococcus pneumoniae and Haemophilus influenzae in middle ear effusion: an in vitro study

Viable and non-viable B. catarrhalis were mixed together with S. pneumoniae and H. influenzae and injected into non-bacterial mucoid effusion material collected from the middle ear of patients with a present secretory otitis media. The samples were incubated at 37 degrees C. Presence of viable B. catarrhalis could evidently prolong the survival of both S. pneumoniae and H. influenzae. Presence of non-viable B. catarrhalis could also enhance the growth of S. pneumoniae, but not H. influenzae. In contrast both S. pneumoniae and H. influenzae suppressed the growth of B. catarrhalis.     

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。     

Anti-CD3/anti-CD28 monoclonal antibody-coated suture enhances the immune response of patients with head and neck squamous cell carcinoma.

OBJECTIVE: To test whether anti-CD3/anti-CD28 (alphaCD3/alphaCD28) monoclonal antibodies could be coated on surgical suture and used to enhance T-cell immune function in patients with advanced-stage head and neck squamous cell carcinoma (HNSCC). DESIGN: AlphaCD3/alphaCD28 monoclonal antibodies at varying concentrations and ratios were coated on surgical sutures and tested on peripheral blood mononuclear cells from normal donors to identify the optimal stimulating condition. Immune-enhancing properties of alphaCD3/alphaCD28 monoclonal antibody suture were tested on peripheral blood mononuclear cells and regional lymph node mononuclear cells isolated from patients with advanced HNSCC and on normal donor peripheral blood mononuclear cells. Proliferation, T-cell phenotype, and cytokines were measured during 8-day in vitro stimulation with alphaCD3/alphaCD28 suture and compared with alphaCD3/alphaCD28-coated tissue culture plastic, a previously recognized carrier. RESULTS: Optimal stimulation was observed with monofilament nylon incubated with alphaCD3/alphaCD28, 2 microg/mL, at a 1:1 ratio for 18 hours at 37 degrees C. Strong proliferation of peripheral blood mononuclear cells and lymph node mononuclear cells in patients with HNSCC was induced by alphaCD3/alphaCD28 suture. There was no difference in maximal proliferation between alphaCD3/alphaCD28 plastic and suture. On day 6 after alphaCD3/alphaCD28 suture stimulation, T-cell subpopulations expressing CD3, CD4, CD8, CD28, and CD45RO were enhanced. Suture stimulation significantly enhanced interleukin 2 secretion when compared with plastic stimulation (P = .01). Both alphaCD3/alphaCD28 suture and plastic stimulated interferon gamma secretion. CONCLUSIONS: To our knowledge, this study is the first to report the modification of surgical suture to create an immunomodulant. AlphaCD3/alphaCD28-coated suture expanded T cells from patients with HNSCC and induced a T(H)1 immune response, which may be a useful therapeutic tool in the treatment of HNSCC and other diseases.     

Effect of genetic background on the response to bacterial sinusitis in mice

OBJECTIVE: To study the importance of ongoing allergen exposure and TH1/TH2 genetic background in augmented bacterial and inflammatory responses in allergic and infected mice. DESIGN: BALB/c and C57BL/6 mice were made allergic to ovalbumin. After 1 day of intranasal allergen exposure, they were inoculated intranasally with Streptococcus pneumoniae. The numbers of bacteria and inflammatory cells in the sinuses were determined, and nasal responsiveness to histamine was assessed. RESULTS: Infected BALB/c and C57BL/6 mice that received ongoing ovalbumin challenge following intraperitoneal sensitization showed significantly greater bacterial load and phagocyte level compared with the infected-only mice. Differences were diminished after the allergen challenge was stopped. Allergic and infected C57BL/6 mice showed fewer bacteria and phagocytes compared with the allergic and infected BALB/c mice. Surprisingly, in contrast to the nonallergenic C57BL/6 mice, the infected BALB/c mice showed a larger number of bacteria 28 days after infection. CONCLUSIONS: Ongoing allergic reaction augments bacterial load in both BALB/c and C57BL/6 mice and induces nasal hyperreactivity to histamine. Allergic and infected C57BL/6 mice show less allergic inflammation and bacterial load compared with allergic and infected BALB/c mice. Stopping allergen exposure reduces the response. Infected BALB/c mice, which favor a TH2 response, were less able to clear infection than C57BL/6 mice, which favor a TH1 response. Inflammation and bacterial load are affected by genetic background of mice and ongoing allergen stimulation.