CC6 Development of Asthma-Specific Outcomes Measures for Children

To facilitate the evaluation of a new pediatric asthma management program, HUFF and PUFFSM The Children's Asthma Program, a set of children's outcomes surveys, including measures of quality of life (QoL) and self-efficacy, were prepared. Each instrument was pretested through individual cognitive interviews with asthmatic children ranging in age from four to eight years old.
QUESTIONNAIRE 1: The QoL measure, the Childhood asthma Questionnaire–Form A, was developed in England by Davina French and colleagues. Although it was designed to measure both global and asthma-specific QoL in young children, the questionnaire needed to be adapted for use in the U.S. The most interesting cognitive findings pertain to children's ability to select appropriate responses and relate to item content. For example, the youngest children had difficulty distinguishing between certain response categories, and some items were not optimally worded for older children.
QUESTIONNAIRE 2: The original version of the self-efficacy measure was developed by Thomas Creer for use with adults. Because this program is targeted for young children, we initially modified the scale by reducing the number of items and adding graphics to clarify the response categories. Cognitive findings indicated that children had difficulty answering items about situations that did not trigger their asthma. Therefore, the survey was limited to triggers most commonly acknowledged by children. Gate questions were also added to ensure that children would only be asked to provide self-confidence evaluations about their personal triggers. Additional modifications were aimed at making the questions and response categories as concrete as possible.
PSYCHOMETRIC EVALUATION: Psychometric properties of these instruments will be evaluated during the pilot program which is currently under way.     

外周血Th细胞因子水平与褪黑素变化在自身免疫性肝炎大鼠的表现

目的：褪黑素是机体神经内分泌免疫调节网络中的重要成员，已证实其对四氯化碳致大鼠肝损伤具有保护作用。实验观察褪黑素对自身免疫性肝炎大鼠外周血Th细胞因子水平的影响。方法：实验于2004—10／2006—10在解放军南京军区肝病中心实验室完成。①实验动物：雄性3月龄Wistar大鼠80只，随机数字表法分为4组：正常对照组、模型对照组、褪黑素治疗组、猪促肝细胞生长素治疗组，20只，组。另取同系Wjstar大鼠10只用于提取肝细胞特异性脂蛋白抗原。（爹实验方法：除正常对照组外，其余各组均采用弗氏完全佐剂＋肝细胞特异性脂蛋白建立自身免疫性肝炎模型。褪黑素用含0．01％乙醇的生理盐水配制成1g／L的溶液，褪黑素治疗组腹腔注射2mg／kg；猪促肝细胞生长素用含0．01％乙醇的生理盐水配制成2g／L的溶液，猪促肝细胞生长素治疗组腹腔注射2mg／kg；正常对照组和模型对照组均腹腔注射含0．01％乙醇的生理盐水，1次，d，连续60d。（彭实验评估：检测各组大鼠外周血Th细胞因子的水平。结果：80只大鼠均进入结果分析。①褪黑素对Th1细胞因子水平的影响：与模型对照组比较，褪黑素治疗组、猪促肝细胞生长素治疗组白细胞介素2和干扰素γ水平均显著升高（P〈0．05），且褪黑素治疗组调节Thl细胞因子水平的作用强于猪促肝细胞生长素治疗组（P〈0．01）。②褪黑素对Th2细胞因子水平的影响：与模型对照组比较，褪黑素治疗组、猪促肝细胞生长素治疗组白细胞介素4，6水平均显著降低（P〈0．05），且褪黑素治疗组调节Th1细胞因子水平的作用强于猪促肝细胞生长素治疗组（P〈0．01）。结论：褪黑素能够刺激自身免疫性肝炎大鼠外周血Th1细胞因子表达，并抑制Th2细胞因子水平，从而改善肝组织病变程度。     

大鼠黑质多巴胺能神经元衰老性变化的多项参数测量

目的:探讨大鼠中脑黑质多巴胺能神经元的衰老性变化规律,为进一步揭示黑质病变的病因提供客观依据。方法:实验于2005-07/2006-07在贵阳医学院解剖学教研室完成。选择健康SD大鼠32只,雌雄各半,按随机数字表法分为幼年组(1～2个月龄)、青年组(4～5个月龄)、中年组(11～12个月龄)、老年组(≥24个月龄)4组,每组8只。取中脑组织常规石蜡包埋,行中脑黑质连续冠状切片,焦油紫染色、酪氨酸羟化酶免疫组织化学染色,图像分析仪测量酪氨酸羟化酶免疫阳性产物的吸光度值和酪氨酸羟化酶免疫反应阳性神经元的胞面积、体密度、数密度、圆球度。结果:32只大鼠均进入结果分析。①中脑黑质焦油紫染色形态学观察:幼年组与青年组大鼠黑质神经元胞体大,细胞排列密集,形状规则,细胞成椭圆形或锥体形,胞浆丰富,每个细胞有清晰的胞核,核仁清楚可见,尼氏体粗大而染色深,中年组和老年组大鼠神经元胞浆染色较淡,尼氏体较分散,细胞散在,数量少,可见软化灶形成。幼年组、青年组、中年组、老年组计数单位面积焦油紫染色细胞数分别为(48.00±9.10),(65.00±8.73),(20.00±4.10),(13.25±1.83)个/40倍视野,各组间比较差异有显著性意义(F=3.79,P<0.05)。②中脑黑质酪氨酸羟化酶免疫反应阳性神经元:光镜下,酪氨酸羟化酶免疫反应阳性神经元成群分布,胞浆内阳性产物以幼年组和青年组为显著,中年组和老年组胞浆内酪氨酸羟化酶阳性反应颗粒明显减少,部分神经元丧失大多角形或锥体形形状,胞体变大,排列不规则,数量减少。幼年组、青年组、中年组、老年组胞浆内酪氨酸羟化酶免疫反应阳性产物吸光度值分别为0.1993±0.0711,0.2428±0.1729,0.1978±0.0687,0.1671±0.1018,各组间比较差异有显著性意义(F=1.87,P<0.05)。中年组、老年组酪氨酸羟化酶免疫反应阳性神经元体密度、数密度低于青年组,差异有显著性意义[体密度分别为(2.57±0.02),(2.36±0.01),(3.22±0.01)×10-2μm-3,t=0.66,1.78,P<0.05;数密度分别为(0.91±0.04),(0.59±0.03),(1.20±0.09)×10-5μm-3,t=7.02,2.25,P<0.05]。幼年组平均截面积低于青年组,差异有显著性意义[分别为(27.30±5.56),(30.40±1.08)×101μm2,t=1.47,P<0.05]。幼年组、中年组、老年组平均体积低于青年组,差异有显著性意义[分别为(9.67±0.40),(5.85±0.42),(5.20±0.33),(11.53±0.90)×102μm3,t=1.60,2.93,0.18,P<0.05]。老年组神经元圆球度低于幼年组、青年组、中年组,差异有显著性意义(分别为0.74±0.18,0.91±0.01,0.92±0.05,0.90±0.03,t=0.68,0.99,1.02,P<0.05,0.001)。结论:黑质多巴胺能神经元随年龄增长而出现衰老性变化,可能是黑质病变的原因之一。     

Induction of protein tyrosine phosphorylation in human natural killer cells by triggering via alpha 4 beta 1 or alpha 5 beta 1 integrins

Recent studies have shown that cell-surface integrins expressed on platelets, fibroblasts, or carcinoma cell lines serve not only as adhesion receptors that connect the extracellular matrix to the cytoskeleton, but also as signal-transducing molecules involved in altering cellular patterns of tyrosine phosphorylation. In this present report we provide evidence that adhesion of freshly purified human natural killer (NK) cells to fibronectin (FN) induces tyrosine phosphorylation of intracellular proteins of approximate molecular mass of 60, 70, and 120 kD. Increases in phosphorylation induced by NK cell binding to immobilized FN were partially blocked by EILDV- (CS-1) or RGD-containing peptides, which compete specifically for a distinct binding site for either alpha 4 beta 1 or alpha 5 beta 1 integrins, respectively, within the FN molecule. The presence of either one of the inhibitory peptides alone inhibited tyrosine phosphorylation primarily during short-term (30 minutes) and, to a lesser extent, during long- term (2 to 3 hours) periods of adhesion. These observations indicate that triggering either via alpha 4 beta 1 or alpha 5 beta 1 integrins, which are constitutively expressed on NK cells, induces protein tyrosine phosphorylation. Moreover, FN fragments of 40 or 120 kD, known to contain the binding sites for alpha 4 beta 1 or alpha 5 beta 1 integrins, respectively, used as immobilized substrates for NK cell adhesion, were able to initiate tyrosine kinase activity. The induced tyrosine phosphorylation was observed mainly on intracellular proteins of greater than 50 kD molecular weight. We have identified a 70-kD tyrosine phosphoprotein as paxillin, a cytoskeletal-associated tyrosine kinase substrate previously identified in fibroblasts and shown to localize to focal adhesions. Thus, interaction of NK cells with immobilized extracellular matrix glycoproteins required for migration and extravasation of these cells involves activation of intracellular protein tyrosine kinases and tyrosine phosphorylation of cytoskeleton- associated protein, paxillin, which may play a role in signaling between beta 1 integrins and the underlying cytoskeleton.     

Partial spectrin deficiency in hereditary pyropoikilocytosis

Hereditary pyropoikilocytosis (HPP) is a severe hemolytic anemia in which an instability of the red cell membrane skeleton has been correlated with structural and functional defects of spectrin. We now report that 13 unrelated HPP subjects have approximately 30% less spectrin than normal as evidenced by a decreased spectrin/band 3 ratio. We also examine the role of spectrin degradation as an underlying cause of this partial spectrin deficiency. Our studies demonstrate that the reduced spectrin content of HPP red cells remains constant during in vivo aging of the cells in the peripheral blood, as well as during in vitro incubation. Furthermore, immunoblotting experiments using an affinity-purified antispectrin antibody indicate that there is no loss of spectrin during membrane preparation and also that neither whole HPP red cells nor ghosts nor cytosol contains any abnormal spectrin degradation products. These data suggest that spectrin is not degraded and that it is stable on the membrane of the circulating HPP red cell. In contrast, however, incubation of free spectrin with a lysate of nucleated erythroid precursor cells indicates that HPP alpha I/46 spectrin, but not HPP alpha I/74 spectrin, is more susceptible to proteolytic degradation than a control. These data imply that the decreased spectrin content of HPP is not due to a single defect but that a more complex mechanism is involved. In HPP Sp alpha I/46 subjects, an increased proteolytic degradation in bone marrow erythroid precursors of cytosolic spectrin, prior to its assembly on the membrane, could contribute toward the partial spectrin deficiency.     

Efficacy of quadruple therapy for Helicobacter pylori eradication after failure of proton pump inhibitor‐based triple therapy in Singapore

BACKGROUND: Helicobacter pylori eradication is the mainstay in the treatment of H. pylori‐associated peptic ulcer disease. Current standard eradication therapy consists of 1 week of treatment with a proton pump inhibitor (PPI) and two antibiotics selected from amoxicillin, metronidazole and clarithromycin. In this study we aimed to assess the efficacy of quadruple therapy consisting of a PPI, bismuth, tetra‐cycline and metronidazole in patients for whom initial H. pylori eradication using a triple therapy regimen consisting of a PPI, amoxicillin and clarithromycin was unsuccessful. METHODS: Consecutive patients with H. pylori‐associated peptic ulcer disease, in whom H. pylori with triple therapy had been unsuccessful, were included in the study. These patients had been treated with a regimen that included a PPI (standard dose twice daily), amoxicillin (1 g twice daily) and clarithro­mycin (500 mg twice daily) for 1 week during 1997?2001. Diagnosis of peptic ulcer disease was made at esophagogastroduodenoscopy. Helicobacter pylori infection was considered to be present on the basis of either a positive rapid urease test, positive histo­logical identification of H. pylori or both. Failure of initial H. pylori eradication was established with either a rapid urease test, a ¹³C urea breath test or histology. Quadruple therapy consisted of a PPI (standard dose twice daily), metronidazole (400 mg three times daily), tetracycline (500 mg four times daily) and bismuth subcitrate (240 mg twice daily). Failure of quadruple therapy was diagnosed on the basis of a positive ¹³C urea breath test. RESULTS: Fifty‐three patients received quadruple therapy. The median age was 52 years (range 20?74) and the male to female ratio was 42 : 11. On an intent‐to‐treat basis, the eradication rate was 69.8%, whereas on a per‐protocol basis, the eradication rate was 82.2%. CONCLUSION: We conclude that a 1‐week quadruple therapy regime consisting of a PPI, bismuth, tetracycline and metronidazole was effective in 82.2% of patients who experienced an unsuccessful initial H. pylori eradication attempt with PPI, amoxicillin and clarithromycin.     

In vivo survival studies of 51Cr-labeled methyl acetimidate treated erythrocytes in patients with sickle cell disease

Studies of the survival time of 51Cr labeled erythrocytes treated in vitro with methyl acetimidate (MAI) were conducted in 13 patients with sickle cell disease in order to assess the suitability of this antisickling agent for more extensive clinical testing. In comparison with previously measured control values (average t1/2 8.4 +/- 1.1 days a), the survival time of the treated erythrocytes in 10 of the patients who were not transfused was initially prolonged (average t1/2 24.4 +/- 4.6 days). However, 5 of the 13 patients studied developed circulating antibody against the MAI treated erythrocytes, markedly reducing the survival time of MAI treated erythrocytes in subsequent studies. Two patients, each challenged 3 times with infused MAI treated erythrocytes, failed to show evidence of antibody production, suggesting that not all subjects become immunized even after repeated exposure. In spite of many other promising properties of MAI as an antisickling agent of potential value, consideration of its use in further clinical testing must depend on successful avoidance of immunization in patients receiving infusions of treated erythrocytes.     

Sources of discrepancy in patient and physician global assessments of rheumatoid arthritis disease activity

OBJECTIVE: To investigate discrepancy in the perception of rheumatoid arthritis (RA) disease activity between patient and physician, and its possible sources. METHODS: Eighty patients with RA rated their level of disease activity on a visual analog scale (VAS). Physician global assessment (MDGA) of disease activity was performed blinded to the patient evaluation except for the results of laboratory tests. A discrepancy score (DS) was calculated by subtracting MDGA from patient global assessment (PTGA), leading to definition of 3 groups of patients: (1) no discrepancy when PTGA and MDGA were within 1.0 or 3.0 cm of each other; (2) negative discrepancy when PTGA was under-rated relative to the physician; and (3) positive discrepancy when PTGA was over-rated relative to the physician. Age, sex, disease duration, education, income, residence area, employment, use of antirheumatic drugs, comorbidity, pain score, Health Assessment Questionnaire (HAQ) rating, tender (TJC) and swollen (SJC) joint count, and Disease Activity Score (DAS28) were recorded. RESULTS: Negative discrepancy was found in 27.5% (VAS 1 cm) and 8.7% (VAS 3 cm) of patients, positive discrepancy in 43.7% (VAS 1 cm) and 23.7% (VAS 3 cm), and no discrepancy in 28.7% (VAS 1 cm) and 67.5% (VAS 3 cm). Patients were predominantly older (mean age near 50 yrs), female, with long disease duration and low income. The negative discrepancy group had a lower level of education and higher C-reactive protein (p < 0.05). The positive discrepancy group presented a higher pain score, HAQ score, and TJC (p < 0.0001). The no-discrepancy group had lower SJC (p < 0.05). CONCLUSION: Our results indicate that for disease activity in patients with RA assessed on pain score, HAQ, and TJC, the only important feature that determined perception of their RA disease activity was education.     

Detection of chromosome 20q deletions in bone marrow metaphases but not peripheral blood granulocytes in patients with myeloproliferative disorders or myelodysplastic syndromes

Myeloproliferative disorders and myelodysplastic syndromes arise in multipotent progenitors and may be associated with chromosomal deletions that can be detected in peripheral blood granulocytes. We present here seven patients with myeloproliferative disorders or myelodysplastic syndromes in whom a deletion of the long arm of chromosome 20 was detectable by G-banding and/or fluorescence in situ hybridization in most or all bone marrow metaphases. However, in each case, microsatellite polymerase chain reaction (PCR) using 15 primer pairs spanning the common deleted region on 20q showed that the deletion was absent from most peripheral blood granulocytes. The human androgen receptor clonality assay was used to show that the vast majority of peripheral blood granulocytes were clonal in all four female patients. This represents the first demonstration that the 20q deletion can arise as a second event in patients with pre-existing clonal granulopoiesis. Microsatellite PCR analysis of whole bone marrow from two patients was consistent with cytogenetic studies, a result that suggests that cytogenetic analysis was not merely selecting for a minor subclone of cells carrying the deletion. Furthermore, in one patient, the deletion was present in both erythroid and granulocyte/monocyte colonies. This implies that the absence of the deletion in most peripheral blood granulocytes did not reflect lineage restriction of the progenitors carrying the deletion but may instead result from other selective influences such as preferential retention/destruction within the bone marrow of granulocytes carrying the deletion.