Determination of monoclonal antibody specificity by immunoadsorption and western blotting.

A rapid and simple method has been developed for identifying the specificity of monoclonal antibodies at an early stage in the production of hybridomas. The technique is a micro-method utilizing biotinylated crude antigen and the surface of microtiter plates as an immunoaffinity matrix. The monoclonal antibodies to be tested are adsorbed to the microtiter wells and incubated with the labeled antigen preparation. Non-specific binding can be reduced by blocking and repeated washing steps. The specific antigen is then eluted by SDS-containing buffers, subjected to SDS-PAGE, blotted onto nitrocellulose and detected by enzyme-labeled avidin in a Western blot assay. The amount of bound and removed antigen can be quantitated by developing eluted and non-eluted control wells by ELISA techniques. Since this ELISA can be performed rapidly, only samples which contain sufficient specific material can be selected for electrophoresis and blotting. The major advantages of the technique are (i) the use of a non-radioactive label resulting in an easy and time-saving procedure, (ii) the possibility of quantitating the amount of captured and detached antigen by ELISA, (iii) the need for only a minimal amount of antigen, (iv) the use of unpurified antibodies of all isotypes, (v) a high signal-to-noise ratio, and (vi) as with all immunoprecipitation techniques, the possibility of detecting SDS-sensitive epitopes and of using crude antigen preparations. Using this method we were able to characterize monoclonal antibodies against both soluble proteins (mouse and human C1q) and membrane determinants (human pan T cell CD5 and CD7).     

Pneumonien bei akuten Leukämien

Summary Acute leukaemia was complicated by pneumonia in 38 (34.8%) of 109 patients treated between 1979 and 1983; in 39.5% of the patients pneumonia occurred more than once. In 23 patients (60.5%) pneumonia occurred during cytostatic therapy, and 25 patients (65.8%) had less than 1000 mm² granulocytes. Antibiotic therapy had no or only little effect in 70%. A total of 21 patients (55.3%) died of pneumonia. In 15 patients a direct relationship could be seen between pneumonia and the bacterial spectrum in the sputum. A prevalence of gram-negative bacteria was found (24 of 40 bacteria isolated, especially Enterobacteriaceae (19). Fungi were cultivated in 10 cases. Each of the typical pneumonia bacteria was only seen once respectively. It is most important that therapy begin immediately, even before the bacteria have been identified. Only then is there hope that the survival time of patients with acute leukaemia can be influenced.

     

Characterization of the in vivo function of TNF-alpha-related apoptosis-inducing ligand,TRAIL/Apo2L,using TRAIL/Apo2L gene-deficient mice

To define the normal physiological role for the TRAIL/Apo2L in vivo, we generated TRAIL/Apo2L gene-targeted mice. These mice develop normally and show no defects in lymphoid or myeloid cell homeostasis or function. Although TRAIL/Apo2L kills transformed cells in vitro, TRAIL/Apo2L(-/-) mice do not spontaneously develop overt tumors at an early age. However, in the A20 B cell lymphoma-transferred tumor model, TRAIL/Apo2L(-/-) mice are clearly more susceptible to death from overwhelming tumor burden, due to increased lymphoma load in the liver. A20 tumors are susceptible to TRAIL/Apo2L killing in vitro, indicating that TRAIL/Apo2L may act directly to control A20 cells in vivo. Despite the fact that TRAIL binds osteoprotegerin and osteoprotegerin-transgenic mice are osteopetrotic, TRAIL/Apo2L(-/-) mice show no evidence of altered gross bone density, and no alterations in frequency or in vitro differentiation of bone marrow precursor osteoclasts. Moreover, leucine zipper TRAIL has no toxicity when repeatedly administered to osteoprotegerin(-/-) mice. Thus, TRAIL/Apo2L is important in controlling tumors in vivo, but is not an essential regulator of osteoprotegerin-mediated biology, under normal physiological conditions.     

The therapeutic potential in targeting CCR5 and CXCR4 receptors in infectious and allergic pulmonary disease

Targeting chemokines and chemokine receptors in various acute and chronic pulmonary diseases remains a vibrant area of basic and clinical research despite major hurdles including cross-species barriers, toxicity, and redundancy. In this review, we draw upon our basic research with a murine model in which innate and acquired immunity are linked in the development and maintenance of chronic asthma due to Aspergillus fumigatus. Using intact and genetically altered mice, studies have also been undertaken to elucidate safe and effective therapeutic strategies that interrupt the initiation and amplification of inflammatory and immune events that follow the intrapulmonary introduction of Aspergillus into A. fumigatus-sensitized mice. These events include resident immune cell activation, immune and inflammatory cell recruitment to the airways, changes in lung physiology, and profound changes in the architecture of the airway due to the activation of lung resident cells. The expression of 2 major chemokine receptors, namely, CC chemokine receptor (CCR) 5 and CXC chemokine receptor (CXCR) 4, has been identified and their roles in innate and acquired immune events during fungal asthma have been explored. CCR5 and CXCR4 are best known for their roles in human immunodeficiency virus-1 (HIV-1) infection, but both are attractive targets in the context of overt inflammatory and remodeling responses in the lung. This avenue of research is markedly enhanced by the existence of numerous small molecule antagonists that are available to selectively target these receptors.     

Differentiation of Babesia bigemina, B. bovis, B. divergens and B. major by Western blotting—first report of B. bovis in Austrian cattle

To establish an assay for the serological differentiation of bovine Babesia species (B. bigemina, B. bovis, B. divergens and B. major), antigens from experimentally infected cattle were Western blotted and probed with homologous and heterologous sera. Varying antigen patterns for each species allowed the determination of species-specific diagnostic antigens. Blood samples from 36 naturally infected cattle from the province of Styria were tested by indirect immunofluorescence antibody test (IFAT) against B. divergens, as well as by Western blotting against B. bigemina, B. bovis, B. divergens and B. major, 3 weeks after clinical babesiosis was diagnosed by blood smears. All 36 cattle were B. divergens-positive when tested by IFAT. In four cases (11%), an infection with both B. bovis and B. divergens and in two cases a single infection with B. bovis were diagnosed when tested by Western blot. B. bigemina and B. major infections were not detected. These are the first serologically confirmed cases of B. bovis in Austrian cattle.     

In vitro hematopoietic and endothelial potential of flk-1(-/-) embryonic stem cells and embryos

Mice deficient in the Flk-1 receptor tyrosine kinase are known to die in utero because of defective vascular and hematopoietic development. Here, we show that flk-1(-/-) embryonic stem cells are nevertheless able to differentiate into hematopoietic and endothelial cells in vitro, although they give rise to a greatly reduced number of blast colonies, a measure of hemangioblast potential. Furthermore, normal numbers of hematopoietic progenitors are found in 7.5-day postcoitum flk-1(-/-) embryos, even though 8. 5-day postcoitum flk-1(-/-) embryos are known to be deficient in such cells. Our results suggest that hematopoietic/endothelial progenitors arise independently of Flk-1, but that their subsequent migration and expansion require a Flk-1-mediated signal.     

猪胸腰段脊髓火器贯通伤后p53基因的早期表达

目的:建立家猪胸腰段脊髓火器贯通伤模型和改良Allen's打击伤后全瘫模型,观察伤后促凋亡基因p53基因的早期表达。方法:实验于2005-05/08在解放军第一七五医院实验室完成。取健康雄性家猪20只,单纯随机分为3组:①火器伤组:9只,在全麻状态下制作胸腰段(L1～L2)脊髓火器伤模型,分为伤后1,3,6h3个时间处死。②打击伤组:9只,L1节段脊髓行改良Allen’s打击,致伤力为500g·cm,处死时间同前。③空白对照组:2只,只麻醉,不造模,伤后6h处死。伤后不同时间点(伤后1,3,6h)和不同节段(伤点、近伤点、中伤点及远伤点)取材,采用SP法进行P53蛋白免疫组化染色,用TJTY-300型全自动图像分析仪测量P53免疫组织化学染色阳性物质吸光度。结果:经补充后20只猪进入结果分析。①脊髓损伤后3h打击伤组伤点,火器伤组近伤段脊髓P53蛋白的表达高于空白对照组(P<0.001),随着时间推移,打击伤组和火器伤组P53蛋白的表达呈升高趋势(P<0.001),且火器伤组要高于打击伤组(P<0.0001)。②在脊髓损伤后6h,打击伤组仅在伤点和近伤段P53蛋白的表达高于空白对照组(5.57±0.82,3.21±0.43,P<0.05),而火器伤组近伤段、中伤段及远段伤均高于空白对照组(6.46±0.66,4.27±0.39,1.16±0.17,P<0.05)。结论:①细胞凋亡基因p53在脊髓损伤中的表达有一定的时空性,在脊髓损伤后3h出现P53蛋白表达量的增加。②脊髓火器伤的波及范围较打击伤更为广泛。