Peripheral blood mononuclear cell (PBMC) responsiveness in patients with head and neck cancer in relation to tumour stage and prognosis.

We have previously shown an increased T lymphocyte and monocyte responsiveness in peripheral blood mononuclear cells (PBMC) from patients with head and neck squamous cell carcinoma (HNSCC) compared with PBMC from control patients. This study reports T lymphocyte function of PBMC of 81 patients with HNSCC dependent on disease stage and prognosis. Males with HNSCC under 80 years of age without cachexia, with no auto-immune disease or previous cancer and on no immuno-active medication were included at the time of diagnosis of disease. The follow-up was for at least 18 months. When cells from patients with early vs late stage disease according to the T, N or T + N stage of HNSCC were compared, decreased in vitro mitogen-stimulated and spontaneous T cell proliferation was seen with increasing tumour stage. When patients were studied according to disease-specific survival, a decreased T lymphocyte mitogen-stimulated proliferation was observed to be associated with a poorer prognosis. No changes in prognosis were noticed related to decreased gamma-IFN, IL-2 or IL-4 level of the supernatants of the T lymphocyte-stimulated PBMC in vitro cultures. With stratification for disease stage, we determined that PBMC in vitro T lymphocyte-stimulated proliferation predicted outcome for the HNSCC patients. The results were similar for both laryngeal and oral cavity/pharyngeal cancers. The present investigation provides evidence to support the idea that the relationship between HNSCC and the immune system of the host may provide clinically useful information about prognosis.     

EB病毒潜伏膜蛋白2A诱导细胞毒性T细胞抗鼻咽癌的体内外实验

目的观察EB病毒（Epstein—Barr virus，EBV）-潜伏膜蛋白2A（1atent membrane protein2A，LMP2A）转染人树突状细胞（dendritic cell，DC）诱导特异性细胞毒性T细胞（cytotoxicity T lymphocyte，CTL）的体外生物学特性；建立表达EBV—LMP2A的裸鼠鼻咽癌动物模型，探讨LMP2A特异性CTL在荷瘤鼠体内的抗肿瘤效应。方法分离人外周血单核细胞（pripheral blood mononuclear cell，PBMC），以粒细胞-巨噬细胞集落刺激因子（granulocyte—monocyte colony stimulating factor，GM—CSF）、白细胞介素4（interleukin-4，IL4）及肿瘤坏死因子（tumor necrosis factor-α，TNF—α）诱导培养获得DC，荧光激活细胞分选仪（fluorescence activated cell sorter，FACS）检测成熟DC的表面分子表达。用LMP2A重组腺病毒转染成熟DC，将转染DC与自体PBMC混合培养，在白细胞介素2（interleukin-2，IL-2）作用下诱导针对LMP2A的特异性CTL，FACS检测CTL群体中阳性细胞的组成。将鼻咽癌CNE细胞接种BALB／c裸鼠，建立鼻咽癌动物模型；在裸鼠肿瘤局部注射LMP2A特异性CTL，观察治疗后移植瘤生长情况及病理变化。结果人外周血PBMC体外在GM—CSF、IL4、TNF—α诱导下获得典型形态及表型特征的成熟DC。FACS检测表明，以LMP2A重组腺病毒转染DC诱导的CTL细胞群体以CIM、CD8阳性细胞组成为主。在接种CNE细胞3周后建立表达EBV—LMP2A的裸鼠鼻咽癌动物模型；动物体内实验表明注射CTL的裸鼠肿瘤生长缓慢，体积明显小于对照组（P〈0．01）；病理检查显示肿瘤局部发生液化性坏死并有淋巴细胞浸润。结论人外周血单核细胞体外经细胞因子诱导，可生成具典型特征的成熟DC。利用LMP2A重组腺病毒将LMP2A基因转入DC，可在同一个体体外诱导出针对LMP2A的特异性CTL。CNE细胞接种裸鼠，可成功构建鼻咽癌动物模型；LMP2A特异性CTL在动物体内可明显抑制鼻咽癌肿瘤的生长，发挥有效的抗肿瘤作用。     

Ex vivo interleukin (IL)-1β, IL-6, IL-12 and tumor necrosis factor-α responsiveness with monocytes from patients with head and neck carcinoma

Seventy newly diagnosed Caucasian male patients with head and neck squamous cell carcinomas (HNSCC) were included in the study. All patients were less than 80 years of age, with no cachexia or auto-immune disease, and they were not taking immuno-active medications. Monocytes from these patients were cultured in vitro and supplemented with autologous serum under ex vivo conditions or cultured with serum-free medium. Comparison was made to monocytes from 59 patients with benign HN diseases. Similar physical activity levels prior to testing as well as a minimum stress load were ensured in both groups. Increased monocyte supernatant levels were determined under ex vivo conditions of interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor (TNF)-α, but not of interleukin-12 (IL-12) with endotoxin stimulated monocytes of HNSCC patients compared to control conditions. Increased monokine levels were not present with mononuclear cell cultures stimulated with a T-cell general stimulatory agent or with purified monocytes not specifically stimulated. With endotoxin-stimulated monocytes under in vitro conditions, an increased supernatant was shown for TNF-α, but not IL-6. With serum from the different patients cultured with monocytes employed from a healthy control, no difference between the groups was shown in the IL-6 and TNF-α response to endotoxin stimulation. The differences in IL-1β and TNF-α, but not IL-6 levels were differentiated statistically from the smoking and alcohol histories of the patients. These findings suggest that the function of monocytes in general, and thus possibly all mononuclear phagocyte system cells in HNSCC patients, are altered. Received: 14 November 1997 / Accepted: 19 October 1998     

The kinetics of CD80 and CD86 expression on antigen-presenting cells in cedar pollinosis subjects

CD80 and CD86, which are costimulatory molecules in T-cell activation, play important roles in the differentiation of Th1- or Th2-phenotypes. The results of blocking studies using neutralizing antibodies have suggested that CD80 and CD86 also play important roles in sensitization to cedar pollen antigen, but very few studies have examined the kinetics of CD80 and CD86 expression on antigen-presenting cells (APC). We studied the kinetics of CD80 and CD86 expression on APC after allergen-stimulation in cedar pollinosis subjects. A skin-prick test was performed in nine subjects with pollinosis and seven control subjects. Peripheral blood mononuclear cells (PBMC) were isolated and stimulated with Japanese cedar pollen extract. Zero to 8 days following in vitro stimulation, the expression of CD80 and CD86 in either CD14+ or CD19+ cells was analyzed by two-color flow cytometry. The expression of CD28, CTLA-4 and CD40L on CD4+ cells was analyzed by two-color flow cytometry after eliminating either CD14+ or CD19+ cells. After in vitro stimulation, the expression of both CD80 and CD86 was significantly upregulated in pollinosis subjects compared to control subjects. However, the difference was observed in the kinetics of CD80 and CD86 expression following allergen stimulation. The expression of CD86 was upregulated earlier than that of CD80 after in vitro stimulation. In the absence of CD19+ cells, the expression of CD28, CTLA-4, and CD40L in CD4+ cells was significantly lower than that in the absence of CD14+ cells. These results indicate that CD19+ cells of pollinosis subjects expressed higher CD80 and CD86 than that of control subjects, and that the kinetics of CD80 and CD86 expression following stimulation differed. In pollinosis subjects, CD19+ cells may thus function as APC in allergen-induced activation of PBMC.     

Peripheral Blood Mononuclear Cell (PBMC) Responsiveness in Patients with Head and Neck Cancer in Relation to Tumour Stage and Prognosis

We have previously shown an increased T lymphocyte and monocyte responsiveness in peripheral blood mononuclear cells (PBMC) from patients with head and neck squamous cell carcinoma (HNSCC) compared with PBMC from control patients. This study reports T lymphocyte function of PBMC of 81 patients with HNSCC dependent on disease stage and prognosis. Males with HNSCC under 80 years of age without cachexia, with no auto-immune disease or previous cancer and on no immuno-active medication were included at the time of diagnosis of disease. The follow-up was for at least 18 months. When cells from patients with early vs late stage disease according to the T, N or T+N stage of HNSCC were compared, decreased     

Depressed PMNC blastogenic response in patients with cancer of the head and neck: a study of IL-2 production, IL-2 consumption, and IL-2 receptor expression

Approximately two thirds of patients with head and neck cancer have been shown to have peripheral mononuclear cells that exhibit a lowered blastogenic response to the T-cell mitogens, concanavalin A and phytohemagglutinin. To investigate the possible mechanisms of this phenomenon, we measured the amount of activated T-cell lymphokine interleukin-2 present in the supernatant of concanavalin A- or phytohemagglutinin-stimulated peripheral mononuclear cells taken from patients with squamous cell carcinoma of the upper aerodigestive tract. Concentrations were found that were similar to those of healthy subjects. The rate of interleukin-2 consumption and the degree of interleukin-2 receptor expression also were similar for patients and controls. In the course of these experiments, it was noted that differences in blastogenic response between patients and controls were abolished when, 24 hours after the beginning of either concanavalin A or phytohemagglutinin stimulation, the culture supernatant was removed and replaced by fresh medium, containing recombinant interleukin-2 to further sustain cell growth. This suggests that the lower blastogenic response found in patients with head and neck cancer is not due to global immune unresponsiveness, but instead, is caused by selective cell dysfunction(s), which may include the production of a suppressor factor following concanavalin A or phytohemagglutinin stimulation.     

变应性鼻炎红细胞和淋巴细胞免疫粘附功能和2种细胞因子的检测

目的 探讨红、白细胞免疫功能及白细胞介素－４（ｉｎｔｅｒｌｅｕｋｉｎ－４,ＩＬ－４）和干扰素γ（ｉｎｔｅｒｆｅｒｏｎ－γ,ＩＦＮ－γ）在变应性鼻炎发病中所起的作用。方法 采用红细胞、淋巴细胞免疫吸附法和ＢＡＥＬＩＳＡ法对外周血单核细胞分泌的ＩＬ－４和ＩＦＮ－γ进行了测定。结果 ４３例常年性变应性鼻炎患者的红细胞、淋巴细胞免疫功能和ＩＦＮ－γ水平均低于健康对照组（分别为Ｐ〈０．０５和Ｐ〈０．０１）,而ＩＬ－４的表达高于对照组（Ｐ〈０．０１）;结论 提示红细胞、淋巴细胞免疫功能、ＩＬ－４和ＩＦＮ－γ的异常在变应性鼻炎的发生发展起重要作用。     

Immune activation by epidermal growth factor receptor specific monoclonal antibody therapy for head and neck cancer

OBJECTIVE: To determine if the epidermal growth factor receptor (EGFR)-specific monoclonal antibodies (mAbs) cetuximab or panitumumab mediate in vitro immune activation against squamous cell carcinoma of the head and neck (SCCHN) cell lines. DESIGN: In vitro study. SETTING: Basic science research laboratory. INTERVENTION: Squamous cell carcinoma of the head and neck cell lines were treated with the Food and Drug Administration-approved EGFR-specific mAbs cetuximab or panitumumab in the presence or absence of peripheral blood mononuclear cells from healthy donors. MAIN OUTCOME MEASURES: Cetuximab and panitumumab were compared in terms of their cytotoxic effects, ability to induce apoptosis, bind to EGFR, and block phosphorylation of this receptor in SCCHN cell lines. RESULTS: We demonstrate that both cetuximab and panitumumab have similar levels of EGFR binding, induction of apoptosis, cell lysis, and inhibition of phospho-EGFR in SCCHN cell lines, suggesting similar direct effects. However, neither of these mAbs demonstrated in vitro antitumor activity when used alone. In contrast, in the presence of peripheral blood lymphocytes, either of them can mediate antibody-dependent cell cytotoxicity in vitro when used in doses similar to those found in patients receiving them clinically. CONCLUSION: We propose that antibody-dependent cell cytotoxicity may constitute an important antitumor mechanism that could contribute to overall clinical effectiveness of EGFR-specific antibodies.     

Production of lymphokine-activated lymphocytes. Lysis of human head and neck squamous cell carcinoma cell lines

Lymphokine-activated killer cells are thought to be important mediators of host tumor defense. In the present study, the cytotoxic potential of lymphokine-activated lymphocytes against different head and neck squamous cell carcinoma cell lines was investigated. Lymphokine-activated killer cells were derived from peripheral blood lymphocytes. Effector peripheral blood lymphocyte cell suspensions were incubated in the presence or absence of recombinant interleukin-2. Cytotoxicity of incubated cells or fresh peripheral blood lymphocytes was determined in a 3-hour chromium 51 release assay. Target cell lines included K562 (a natural killer-sensitive target) and the following head and neck squamous cell carcinoma cell lines: Cal 27, UMSCC-1, UMSCC-8, UMSCC-16, UMSCC-19, and UMSCC-22a. Fresh peripheral blood lymphocytes and peripheral blood lymphocytes cultured in the absence of added interleukin-2 demonstrated minimal cytotoxic effects against the squamous cell carcinoma targets. In contrast, these fresh and incubated lymphocytes showed significant cytotoxic effects against K562. Cells preincubated in the presence of interleukin-2 demonstrated a statistically significant increase in cytotoxic effects against K562 and all squamous cell carcinoma targets. These investigations support the possible role of lymphokine-activated killer cells in host defense against squamous cell carcinoma. In vitro natural killer cell activity against head and neck squamous cell carcinoma cell lines is low; however, significant lymphokine-activated killer cell cytotoxicity is present.     

An autologous system for culturing head and neck squamous cell carcinomas for the assessment of cellular therapies on the chorioallantois membrane

The evaluation of novel immunotherapeutic anti-cancer strategies requires target and effector cells from the autologous host to avoid false positive results because of allo-reactivity. Head and neck squamous cell carcinoma cells (HNSCC) do not proliferate under standard culture conditions in plastic ware. To create an autologous model, the chorioallantois membrane (CAM) of chicken embryos was employed to culture HNSCC and peripheral blood mononuclear cells (PBMC). Separated tumor cells were co-incubated with a cytostatic agent, PBMC, or mere cell culture medium as a control. Tumor cell lysis was assessed by acridine orange staining or by flow cytometry analysis of iodide marked cells after 24 and 48 h of co-incubation. Incubation with cisplatin resulted in a decrease of viable cells by 49% after 24 h and 48 h. In contrast, incubation with mere culture medium resulted in an increase of viable tumor cells by 5% after 24 h and a decrease of 4% after 48 h; the incubation of tumor cells and PBMC led to a non-significant decrease by 14% after 24 h and 16% after 48 h. Taken together, the CAM supports a favorable environment for the co-culture of HNSCC and PBMC, thus providing an approximated in vivo autologous model to assess the efficacy of new cell-therapeutical approaches.     

In vitro effects of preservatives in nasal sprays on human nasal epithelial cells

BACKGROUND: The preservatives benzalkonium chloride and potassium sorbate are widely used in nasal drops and sprays. Recently, side effects resulting from mucosal damage caused by benzalkonium chloride and potassium sorbate were reported. METHODS: We investigated the toxicity of benzalkonium chloride and potassium sorbate on human nasal epithelial cells in vitro. Using primary human nasal epithelial cells, different concentrations of benzalkonium chloride, potassium sorbate, or phosphate-buffered saline (PBS; control group) solutions were cocultured with nasal epithelial cells for 15 minutes. Then, the viability of the cells and the cell morphology were assessed. RESULTS: Nasal epithelial cells were more severely damaged with use of clinical preparations or higher concentrations of benzalkonium chloride than in the control group. In addition, nasal epithelial cell membrane lysis was seen on electronic microscopy in the benzalkonium chloride groups. In contrast, there was no significant cell damage seen in the potassium sorbate groups compared with the control group, even with higher concentrations than clinically used. CONCLUSION: Potassium sorbate appears to be a relatively safer preservative than benzalkonium chloride for use in nasal sprays and drops in vitro study.     

变应原和糖皮质激素对变应性鼻炎患者外周血Th17细胞及其转录因子RORγt的作用

目的 了解变应原和糖皮质激素对变应性鼻炎(allergic rhiaitis,AR)成人患者外周血新型辅助性T细胞17(T help 17,Th17)及其转录因子孤独受体(retinoid-related orphan nuclear receptor,RORγt)mRNA表达水平以及白细胞介素(inter leukin,IL)23、IL-6和IL-17水平的影响.方法 40例AR患者(A组)随机分为2组,每组20例.第1组(A1组)给予经鼻吸入糖皮质激素(intranasal glucocorticoid,INGS)治疗4周,第2组(A2组)给予特异性免疫治疗(special immunotherapy,SIT)1年.分别取治疗前后2组患者和另10例健康对照者(B组)外周血,用流式细胞术检测Th17细胞阳性率;用反转录聚合酶链反应(RT-PCR)法检测RORγt mRNA表达水平;用酶联免疫吸附(ELISA)法检测培养上清液中IL-23、IL-6和IL-17水平.结果 A组患者外周血中Th17细胞阳性率、RORγt mRNA的相对表达量(x-±s,以下同)分别为(18.97±1.05)%和(0.604±0.027),高于B组的(15.12±1.09)%和(0.447±0.024),差异有统计学意义(t值分别为-10.056和-17.986,P值均＜0.01).A组患者IL-6、IL-17和IL-23水平均高于B组,差异有统计学意义(t值分别为-41.149、-17.618和-26.824,P值均＜0.01).A1组治疗前后Th17细胞阳性率、RORγt mRNA的相对表达量、IL-6、IL-17和IL-23水平5项指标差异均无统计学意义(t值分别为0.298、0.240、-1.136、0.283和-1.670,P值均＞0.05).A2组SIT治疗前外周血中Th17细胞阳性率、RORγt mRNA的相对表达量分别为(18.99±1.14)%和(0.603±0.027),高于治疗后的(16.30±1.63)%和(0.429±0.023),差异有统计学意义(t值分别为6.035和22.015,P值均＜0.01).A2组SIT治疗后IL-6、IL-17和IL-23水平显著下降,与治疗前相比差异有统计学意义(t值分别为9.235、11.289、7.267,P值均＜0.01).结论 AR患者Th17细胞数量、RORγt mRNA的表达量以及相应的细胞因子IL-23、IL-6和IL-17水平上调,SIT治疗可以使以上5项指标下降,而INGS治疗对以上5项指标没有影响.     

Selective, histamine-mediated immunosuppression in laryngeal cancer

Immunosuppression observed even in the earliest laryngeal cancers can be, in part, reversed with H2 histamine antagonists. In an effort to explain this, we tested whether histamine could evoke changes in endogenous antitumor lymphokine production using tonsil lymphocytes as test cells. We have observed that lymphocytes, treated in vitro with histamine, display a significant suppression of lymphokine production and mixed lymphocyte proliferation response following galactose oxidase treatment. Interferon gamma production was reduced to less than 2% of control value in the presence of histamine. In contrast, the lymphokine-activated killer cell activity induced by purified, natural interleukin-2 was not affected by similar concentrations of histamine. Histamine appears to exert its inhibitory effect by stimulating the production of a substance released by suppressor T cells. The suppressive effects observed could be reversed by concomitant or sequential treatment with cimetidine or ranitidine and not by H1 histamine antagonists, indicating that it occurs through an H2 histamine receptor. These experiments suggest that histamine may have a profound suppressive effect on the lymphocyte population studied.     

Stereocilia bundle stiffness: effects of neomycin sulphate, A23187 and concanavalin A

The effects that the aminoglycoside antibiotic neomycin sulphate, the ionophore A23187 and the lectin Concanavalin A (Con A) have on the steady state stiffness of the hair-cell stereocilia bundle have been studied using organotypic cultures of the early postnatal mouse cochlea. In normal saline, stereocilia bundle stiffness is increased 1.5-2.0 fold by neomycin sulphate, 1.3-1.7 fold by the ionophore A23187 and 3.0-5.0 fold by Con A. In low-calcium saline neither neomycin sulphate nor A23187 cause increases in stiffness, and the stiffness increases elicited by these two agents in normal saline are reversed on washout with low-calcium saline. In normal saline neomycin sulphate has two independent effects on hair cells; one effect is a reversible inhibition of transduction and the other effect is to cause an irreversible increase in bundle stiffness. Neither Con A nor the ionophore A23187 block transduction. No obvious changes in stereocilia bundle morphology are associated with the increases in stiffness caused by neomycin sulphate, A23187 and Con A. Succinylated Con A binds to stereocilia bundles but does not cause an increase in stiffness, suggesting that the stiffness increase caused by native Con A results from receptor cross-linking. The effects of Con A and neomycin are non-additive, saturating concentrations of neomycin sulphate block the effects of low doses of Con A, and pretreatment of cells with succinylated Con A prevents subsequent neomycin sulphate treatment from causing an increase in stiffness suggesting that neomycin sulphate and Con A are acting via a similar mechanism at the cell surface. Ionophore treatment prevents the subsequent application of neomycin sulphate from causing a further increase in stiffness, but when cells are treated with neomycin sulphate followed by ionophore the effects of the two drugs are additive indicating that they are operating via different mechanisms. The possible nature of these mechanisms and their role in the control of steady state stereocilia bundle stiffness are discussed.     

慢性鼻窦炎鼻息肉合并支气管哮喘患者鼻窦内镜术后对支气管哮喘疗效的影响

目的观察鼻窦内镜术(endoscopic sinu ssurgery，ESS)对慢性鼻窦炎伴支气管哮喘患者哮喘发作的影响。方法对210例慢性鼻窦炎患者施行ESS术，其中伴有支气管哮喘病史者42例(20．0％)。210例患者均于术前、术后采用酶联免疫吸附测定法(ELISA)检测外周血单个核细胞(PBMC)培养上清液中的白细胞介素4(IL-4)，干扰素γ(IFN-γ)，可溶性白细胞介素2受体(sIL-2R)和可溶性IgE低亲和力受体(solube CD23，sCD23)的含量，并与20例正常对照组进行比较。通过主观和客观标准评定42例患者哮喘发作及对皮质类固醇的耐受状况，并对术后患者进行为期1年(10例)和3年(32例)的随访。结果鼻窦炎合并支气管哮喘患者术前PBMC培养上清液中IL-4，sIL-2R、sCD23含量较对照组显著升高，而IFN-γ含量较对照组显著减少。术后IL-4、sIL-2R、sCD23含量较对照组显著降低，而IFN-γ含量显著增高。术后哮喘改善水平由随访术后1年的45％提高到术后3年的70％。42例中32例(76％)哮喘发作次数明显减少，术前长期服用类固醇的2l例中，14例(67％)减少了对口服类固醇的使用。结论ESS对慢性鼻窦炎伴支气管哮喘患者的治疗有较满意的远期疗效。ESS能调节IL-4、IFN-γ sIL-2R、sCD23水平，降低哮喘的发作频率和对类固醇的依赖。     

Permanent in vitro growth is associated with poor prognosis in head and neck cancer

OBJECTIVE: The propensity of head and neck carcinomas to grow in vitro and to form a permanent cell line varies. It is not known whether the outcome of patients whose cancer gives rise to permanent in vitro growth differs from that of patients whose cancer cells fail to grow in vitro. The purpose of this study was to find out whether tumor cell capability for in vitro growth is associated with prognosis in head and neck cancer. MATERIAL AND METHODS: The study group consisted of 30 patients treated for head and neck cancer at the University Central Hospital of Turku between 1987 and 1994, and whose tumor samples had produced a permanent cell line in our laboratory. A control group was selected from patients treated during the same time period and with the same protocols in the same department. The controls were selected on the basis of similar tumor localization, TNM status, histological grade, age, gender and general condition. Tumor samples from 14 of the 30 control patients were also cultured, but did not result in a permanent cell line. The median follow-up time was 54 months in the study group and 52 months in the control group. RESULTS: The 3-year survival rate of the patients whose cancer gave rise to in vitro growth was only 19%, compared to 68% among the controls (p = 0.001). In a multivariate analysis the propensity of cancer cells to grow in vitro had independent prognostic value, the relative risk of death (RR) being 1.95 (95% CI 1.11-3.42) when compared to cancers that did not produce a cell line. Of the other factors tested, only the primary tumor size (RR 1.75; 95% CI 0.97-3.16) and the blood hemoglobin level at diagnosis (RR 0.97; 95% CI 0.95-1.01) were possibly independently associated with survival. CONCLUSIONS: The results suggest that the capability of cancer cells for in vitro growth has prognostic significance in head and neck cancer, and that cancer cells that are able to survive and grow in in vitro conditions behave aggressively in vivo. The independence of cancer cells from the paracrine signals produced by the neighboring host cells may enhance cancer cell survival and the metastatic potential in vivo.     

Calcium regulation of antigen expression on normal and malignant human squamous cells in vitro

In vitro, normal keratinocytes exhibit undifferentiated morphologic features and proliferate for multiple passages in low-calcium medium (less than or equal to 0.3 mmol/L) whereas, in high-calcium medium (greater than or equal to 1.0 mmol/L), these cells assume differentiation characteristics, begin to stratify, and eventually cease proliferating. In contrast, malignant keratinocytes grow well in high-calcium medium. Expression of pemphigus vulgaris antigen, a squamous cell marker, is altered on cultured normal keratinocytes by calcium. In this study we compared the effects of calcium levels on expression of cell surface antigens by UM-SCC-38, a human squamous carcinoma cell line, and normal keratinocytes cultured from newborn foreskin. Pemphigus, pemphigoid, beta 2-microglobulin antigens, as well as the epidermal growth factor receptor and the A9 germinal epithelial cell basement membrane squamous carcinoma antigen were examined. Pemphigus antigen was strongly expressed on normal and malignant cells in high-calcium but not low-calcium medium. Calcium concentration did not affect the expression of any of the other antigens tested. Thus, although calcium induces differentiation and eventual loss of proliferative capacity in normal but not malignant keratinocytes in vitro, we were unable to demonstrate differences in pemphigus vulgaris antigen expression that might be linked to the growth inhibitory effects induced by high calcium levels in nontransformed epithelial cells in culture.     

Peripheral Blood T‐Lymphocyte and Monocyte Function and Survival in Patients With Head and Neck Carcinoma

Objectives/Hypothesis To determine if the T‐lymphocyte and monocyte functions of peripheral blood mononuclear cells (PBMCs) from patients with head and neck squamous cell carcinomas (HNSCC) are predictive factors for outcome. Study Design A prospective study describing the outcome, as to total survival and death by disease after at least 40 months observation, of 81 previously untreated male HNSCC patients in relation to PBMC T‐lymphocyte and monocyte function. Methods T‐lymphocyte mitogenesis and the cytokine level in culture supernatants of PBMC as well as monocytes were analyzed. These parameters were related to survival by Cox regression and Kaplan‐Meier survival analysis. Results When patients with high versus low T‐lymphocyte mitogen–stimulated proliferations were compared, a decreased proliferation was seen to be related to worse outcome. The predictive value of T‐lymphocyte proliferation was shown to be an independent prognostic factor when adjusted for stage and stratified for anatomic location in survival analysis. The predictive value was also retained when the serum values of the major serum proteins and hormones and scores based on the smoking and alcohol history were added to the survival analysis with lymphocyte proliferation. Supernatant levels of γ‐interferon, interleukin (IL)‐2, or IL‐4 in PBMC cultures were not related to outcome. Monocyte function measured by endotoxin‐stimulated IL‐1β, IL‐6, IL‐12, and tumor necrosis factor‐α secretion did not relate to outcome of the patients. Conclusion The PBMC T‐lymphocyte–stimulated proliferation is an independent prognostic factor for male HNSCC patients.     

Costimulatory molecule expression on allergen-stimulated PBMC in cedar pollinosis subjects

B7-1 (CD80) and B7-2 (CD86) play important roles in the differentiation of Th1 and Th2 phenotypes. CD40-CD40L interaction supports the expression of CD80 and CD86 on antigen-presenting cells. Blocking studies suggest these molecules also play important roles in sensitization to a cedar pollen antigen by, but very few studies have concerned their effects on subsequent induction by antigens. We investigated the roles of CD80, CD86, and CD40 in the differentiation of Th1 and Th2 subsets after stimulation with the antigen in subjects with cedar pollinosis. Peripheral blood mononuclear cells (PBMC) were isolated from 12 subjects with pollinosis and 11 healthy controls and stimulated with cedar pollen extract. After in vitro stimulation, CD80, CD86, and CD40 expression on CD19+ cells were analyzed by flow cytometry. The production of type 1 and type 2 cytokines in culture supernatants was measured by FLISA. Proliferation studies were conducted in the presence or absence of anti-CD40 or CD86 mAbs. After in vitro stimulation, CD86 and CD40 were significantly up-regulated following stimulation in pollinosis subjects (p = 0.02 and p = 0.04). A significantly higher level of IL-5 (p = 0.02) was produced by PBMC of pollinosis subjects than by those of controls. Allergen-induced proliferation and IL-5 production of PBMC of pollinosis subjects were inhibited by anti-CD86 mAb but not CD40 mAb. These results indicate that the Th2 response predominated in pollinosis subjects and that CD86 rather than CD80 may be the costimulatory molecule involved in the allergen-induced activation of PBMC.     

Effect of antimicrobial therapy with amoxicillin and cefprozil on bacterial interference and beta-lactamase production in the adenoids

To compare the effects on the bacterial flora of the adenoids of antimicrobial therapy with amoxicillin or cefprozil, we enrolled in a prospective randomized study 60 children scheduled for elective adenoidectomy because of recurrent otitis media. They were randomized before surgery into 3 groups of 20 patients each: a no-therapy group, and groups with 10 days of either amoxicillin or cefprozil therapy. Core adenoid materials were quantitatively cultured for aerobic and facultative bacteria. The in vitro ability of alpha-hemolytic streptococci (AHS) to inhibit the growth of non-type B Haemophilus influenzae and Streptococcus pneumoniae was determined. The number of organisms in adenoids obtained from patients treated with either antibiotic was reduced as compared to that in adenoids from controls. However, in patients treated with amoxicillin, a significant decline in the number of AHS, and an increase (in Staphylococcus aureus) or no change in the number of other beta-lactamase-producing bacteria (BLPB) was noted. In contrast, in those treated with cefprozil, no change was noted in the frequency of recovery of AHS, and the number of BLPB decreased. Interfering AHS were more often recovered in patients treated with cefprozil. We conclude that the adenoids contain more interfering AHS after therapy with a second-generation oral cephalosporin (cefprozil) than after amoxicillin therapy. This study suggests a potential beneficial effect of using an antimicrobial that selectively spares interfering AHS while inhibiting BLPB.