Discrepancy between skin reactivity and blastogenic activity of fractionated PHA-P.

Firstly, it was reported that phytohemagglutinin P (PHA-P) can induce the immediate skin reaction besides the delayed one. 70 out of 80 cases (87.5%) gave positive immediate reactions with 1 mug of PHA-P. There was a close correlation between the radioallergosorbent test (RAST) counts against PHA-P and the total IgE value or erythematous diameter of the immediate skin reactions. Absorption with anti-IgE or PHA-P reduced the RAST counts against PHA-P to 79 and 56% of their initial values, respectively, but did not effect the RAST counts against steptokinase-streptodornase (SK-SD). These results suggest that the immediate skin reaction might be induced by specific IgE antibody against PHA-P. Secondly, using three fractions of PHA-P obtained by Sephadex G-200 gel filtration, skin test and RAST in humans, ACA in rats, and 3H-thymidine incorporation into human leukocytes, were carried out and data obtained that showed some discrepancy between them. That is, on skin testing, it was shown that all of the three fractions (fr.) had the ability to elicit the immediate and delayed skin reaction in humans and rats with the order of fr. 1, 2 and 3, and it was confirmed by RAST. On the other hand, the leukocyte-stimulating activity resided in fr. 1 and 2, while fr. 3, which seemed to be a group of subunits with a molecular weight of around 35,000, had almost no activity. These findings have been discussed in view of the earlier studies on the structure of PHA-P     

Effect of thymosin and lipopolysaccharide on murine lymphocyte cyclic AMP

The effects of thymosin and lipopolysaccharide (LPS) on cyclic AMP levels in lymphocytes were evaluated using three independent assays which included adenine prelabeling, protein kinase binding, and radioimmunoassay. All three assays proved to be both sensitive and accurate in assessing relative changes in lymphocytes after incubation in vitro with various agents. The assays confirmed that basal and stimulated levels of cyclic AMP depended on the origin of the lymphocyte population. Each of the three techniques demonstrated that pyrogen-free bovine thymosin fraction 5 did not elevate thymocyte cAMP levels. In contrast, it was found that lipopolysaccharide (LPS) significantly elevated cAMP levels in both spleen and thymus lymphocytes. These studies indicate that assays for measuring the activity of thymic extracts in which the intracellular levels of cyclic nucleotides are a criterion for activity are only valid if the preparations are not contaminated with endotoxins.     

MicroELISA method for the determination of thymosin beta 9 discriminating between thymosin beta 9 and the structurally closely related thymosin beta 4

In order to obtain specific antibodies against thymosin beta 9 showing minimal cross-reactivity with the highly homologous peptide thymosin beta 4, the N-terminal fragment 1-14 of thymosin beta 9 was used for immunization. These antibodies have been tested in a competitive ELISA and show less than 1% cross-reactivity with thymosin beta 4. On the other hand, antibodies raised against the native thymosin beta 9 (1-14) cross-react 35% with thymosin beta 4. Specific antibodies against thymosin beta 9 are important for studying the concentration and localization of thymosin beta 9 in thymus and other bovine tissues because thymosin beta 9 is always accompanied by thymosin beta 4. Using N-terminal fragments of thymosin beta 4-like peptides may be a general approach for obtaining specific antibodies since this part of sequence is less conserved in thymosin beta 4-like peptides.     

胸腺素α1的免疫调节活性及临床应用进展

胸腺素α1(Tα1)是从胸腺第五组分(TF5)中提取并纯化具有生物活性的酸性多肽,作为免疫增强剂,可以调节免疫系统的许多环节,包括活化NK细胞,T细胞,单核和巨噬细胞,还可以刺激骨髓造血细胞的增殖分化;其中最主要的是对T细胞分化的调节。现在主要用于治疗:乙肝、丙肝等感染性疾病;非小细胞肺癌、黑素瘤、肝癌、胃癌等恶性肿瘤;自身免疫性疾病,如类风湿关节炎、重症无力、系统性红斑狼疮等;以及免疫缺陷病的治疗。在广泛的临床应用中,使用Tα1不良反应非常少见,但在极少数病例也有不良反应的发生。因此在临床应用中,应严格监控,一旦发生不良反应,应立即停药。     

Discrepancy in CD3-transmembrane peptide activity between in vitro and in vivo T-cell inhibition

Electrostatic amino acid interactions between receptor subunits within the T-cell antigen receptor (TCR) transmembrane domain are critical for the formation of the TCR-CD3 complex. Core peptide, a short peptide corresponding to the TCR-alpha transmembrane region, containing two positively charged amino acids, is known to inhibit T-cell function in vitro and in vivo. The aim of this study was to examine peptides corresponding to the syntactic transmembrane CD3 region binding to TCR-alpha for their ability to inhibit T-cell activation in vitro and in vivo. Three peptides matching the transmembrane sequence of CD3-delta, -epsilon and -gamma were synthesized and tested in different biological in vitro and in vivo systems for their effect on T-cell activity. The CD3-peptides had no impact on T-cell function in vitro, but surprisingly, decreased signs of inflammation in the adjuvant arthritis rat model in vivo. Preliminary evidence suggests that peptides with CD3 transmembrane-derived sequences can inhibit an immune response as assessed by adjuvant-induced arthritis. The lack of in vitro activity may lead to a wasteful disregard of active compounds in the process of drug discovery and development.     

HBV颗粒的纯化及生物学活性鉴定

目的优化纯化HBV患者血清中HBV颗粒的技术方法。方法在蔗糖介质中,分别以100 000 g和70 000 g进行不连续蔗糖梯度(60%,45%,35%,25%,15%)离心5 h,离心后,分段吸取6份收集液,PCR荧光定量检测各组分HBV滴度;收集前5份收集液进行超滤,去蔗糖,PCR荧光定量检测HBV超滤液;电镜观察70 000g纯化后的HBV超微形态;70000 g纯化后的HBV颗粒感染树鼩肝细胞,检验70 000 g纯化后的HBV感染活性。结果100 000 g离心造成大量HBV在离心过程中损失,收获率只有21%,70 000g离心不仅降低了离心中的病毒损失,而且可有效对HBV和血清进行分离,收获率达到78%以上,较100 000g还提高3.7倍多。电镜可观察到纯化后病毒液中有大量的病毒颗粒;纯化后的HBV颗粒感染活性良好,可有效感染原代树鼩肝细胞。结论70 000 g不连续蔗糖梯度离心可有效分离纯化HBV病毒,较100 000 g收获率明显提高,纯化后的HBV不但具有典型的HBV特征,还具有良好的生物学感染活性。     

mOX40-Ig的表达及其生物学活性的初步研究

目的:构建pcDNA3.1-mOX40-Ig真核表达载体,转染CHO细胞进行稳定表达,获得有生物学活性的mOX40-Ig融合蛋白.方法:RT-PCR法扩增获得hIgG1Fc段基因,构建pcDNA3.1-hIgG1Fc重组质粒并经测序证实.从本室保存的pIRES2-EGFP-mOX40重组质粒中PCR扩增mOX40胞外段,将其插入pcDNA3.1-hIgG1Fc重组质粒中构建pcDNA3.1-mOX40-Ig真核表达载体.脂质体法转染CHO细胞获得稳定表达,用RT-PCR与ELISA法检测其表达,蛋白A亲和纯化后,SDS-PAGE进行鉴定.3H-TdR掺入法研究OX40信号对B细胞的体外促增殖作用.结果:测序证实hIgG1Fc段、mOX40胞外段及mOX40-Ig基因序列正确,RT-PCR与ELISA证实mOX40-Ig的表达,SDS-PAGE证实其为mOX40-Ig融合蛋白,3H-TdR掺入法显示mOX40-Ig在体外能有效地促进B细胞增殖.结论:成功地构建pcDNA3.1-mOX40-Ig真核表达载体,并获得有生物学活性的mOX40-Ig融合蛋白的稳定表达,为进一步开展OX40相关领域的横向应用研究奠定了基础.     

CIITA锤头状核酶的克隆及其体外活性

通过CIITA核酶抑制HeLa细胞表面MHCⅡ类分子的表达。设计并合成针对人类CIITA的核酶Rz464,通过体外转录和切割实验鉴定其活性。将Rz464亚克隆到真核表达载体pIRES2-EGFP(pRz464),并稳定转染HeLa细胞株,流式细胞术检测MHCⅡ类抗原表达,RT-PCR检测CIITA mRNA水平。结果表明,Rz464与CIITA靶序列体外切割产物电泳见预期切割条带。pRz464~+HeLa细胞与对照组比较,HLA-DR、DP、DQ抗原诱导型表达分别降低了79.21%、90.31%及48.30%;同时CIITA的诱导型mRNA含量明显减少。Rz464通过切割CIITA mRNA,进而阻止了后者调控的MHCⅡ类分子的表达。     

Effect of thymosin on glucocorticoid receptor activity and glucocorticoid sensitivity of human thymocytes

Incubation with thymosin fraction 5, (TMS F5 at 300 micrograms/ml) a partially purified thymic factor, reduced the steroid binding activity of human infant thymocytes from 9.6 +/- 2.1 fmole/ml to 5.0 +/- 2.0 fmole/ml. The glucocorticoid receptor activity in normal infant thymocytes was found to be 2,146 +/- 726 (s.d.) sites per cell with dissociation constant of 1.4 +/- 0.6 X 10(-8)M. TMS F5 also increased the resistance of human thymocytes to the cytolytic effect of dexamethasone (2.5 X 10(-8)M) to 168.6 +/- 30.2% of control (P less than 0.01). In animals, medullary and peripheral blood T cells are more resistant to glucocorticoids than immature thymic T cells. The results show that thymosin can induce changes consistent with differentiation in human thymocytes. These in vitro results are consistent with a physiological role of thymosin in intrathymic T cell maturation in man. Incubation of a human malignant thymus derived T cell line (MOLT 3) with TMS F5 also resulted in a significant reduction of the number of steroid binding sites to 44.2 +/- 15.3% of control (P less than 0.05), but TMS F5 did not significantly reduce the glucocorticoid sensitivity of MOLT 3 cells.     

Immune-neuroendocrine interactions during aging: age-dependent thyrotropin-inhibiting activity of thymosin peptides

Thymosin fraction 5 (TF-5), a partially purified thymic preparation, has been previously shown to have luteinizing hormone-releasing hormone (LH-RH)-releasing activity in perfused rat hypothalamus as well as in vivo stimulatory effect on the pituitary-adrenal axis in prepubertal monkeys. We report here the effect of TF-5 on the TSH-thyroid axis in young (3 months) and old (25 months) Sprague-Dawley male rats. Conscious free-moving animals carrying an indwelling atrial cannula received a single dose of 5 mg/kg body wt. of either bovine serum albumin (BSA) or TF-5 via the cannula. In the young rats, TF-5 induced a marked reduction of plasma thyrotropin (TSH) which was significantly greater than the normal circadian decline observed in the BSA-treated controls. The old males displayed high basal levels of TSH which showed no circadian rhythmicity, and did not respond to TF-5. Thyroxine (T4), triiodothyronine (T3), corticosterone, and prolactin levels were not affected by TF-5 at the dose levels tested. The old rats had significantly lower basal levels of T4, but not T3, than their young counterparts. The synthetic peptides thymosin alpha-1 and serum thymic factor, which are components of TF-5, had no effect on the above hormones when injected in doses up to 5 micrograms/kg body wt. Acute thymectomy in 3-month-old males induced a significant increase in basal levels of TSH without affecting plasma T4 or T3. These results suggest that the thymus has an inhibitory action on TSH in the rat, which is not mediated by the thyroid gland.(ABSTRACT TRUNCATED AT 250 WORDS)     

Discrepancy between estimated energy intake and requirement in female dancers

Dietary intake of energy, protein, fat and carbohydrates in female dance students compared to female music students was estimated by a dietary history method. Energy requirement was estimated from body weight and physical activity both by a standard formula and from the measured work intensity during dance training. Meal patterns showed significant differences in four types of meals on week-days and in one type on weekends. The intake of protein, fat and carbohydrates in absolute amounts and the percentage of energy derived from protein were not significantly different between the two groups. The fraction of energy derived from carbohydrates was higher and that from fat was lower in the dancers than in the musicians. Energy intake was of similar magnitude for the dancers and the musicians (8.3 +/- 1.9 MJ vs. 8.3 +/- 1.7 MJ), while the estimated energy requirement was greater for the dancers (10.3 +/- 0.7 MJ vs. 8.9 +/- 0.4 MJ). The reason for the apparent discrepancy between energy intake and energy requirement of approximately 2 MJ in the dancers is not obvious. The energy intake may have been underestimated. Alternatively, the energy requirement may have been overestimated. The resting metabolic rate may have been depressed by years of low energy intake. In addition, as suggested by some authors, training as such may lower the resting metabolic rate as well as the diet-induced thermogenesis.     

Discrepancy between subjective symptomatology and objective neuropsychological performance in insomnia

STUDY OBJECTIVES: While daytime impairment is a defining feature of primary insomnia (PI), prior research using objective measures has not yielded clear and reliable evidence of global or specific deficits. In this investigation subjective and neuropsychological measures of daytime impairment were concurrently evaluated in subjects with primary insomnia (PIs) and in healthy good sleeper subjects (GSs). The goals for the study were to assess (1) whether PIs differ from GSs on subjective and/or objective measures and (2) the extent to which subjective and objective measures provide discordant information. DESIGN: Subjects were evaluated on multiple self-report measures of sleep and daytime performance and were administered a comprehensive set of neuropsychological tests. SETTING: The University of Rochester Sleep and Neurophysiology Research Laboratory (Rochester, NY). PATIENTS OR PARTICIPANTS: Forty-nine subjects (32 PIs and 17 GSs). Seventy-one percent of the sample was female; average age 39 +/- 11 yrs. RESULTS: Overall, PIs reported worse sleep, diminished activity levels, and a greater number and severity of daytime complaints. However, PIs did not show deficits on neuropsychological tests. Additionally, neuropsychological measures were not associated with severity of daytime complaints. Objectively measured sleep was found to be associated with performance (motor speed), while prospective and objective sleep measures were associated with level of daytime complaint. CONCLUSIONS: The discrepancy between subjective daytime complaints and objective performance in individuals with insomnia is common, but poorly understood. This discordance may suggest that daytime impairment corresponds less to "output" and more to attentional bias or to the realistic appraisal that "effort" is required to maintain normal performance.