Enzyme-linked immunosorbent assay and flow cytometric methods to screen hybridoma culture supernatants for antibodies to bovine neutrophil surface antigens, and monoclonal antibody production and characterization

Enzyme-linked immunosorbent assay and flow cytometric methods to screen hybridoma culture supernatants for antibodies to bovine neutrophils (surface antigen-specific) were optimized. Sensitivity of the 2 methods was compared. A panel of 14 murine monoclonal antibodies (MAB) to surface antigens of bovine polymorphonuclear neutrophilic leukocytes (neutrophils) was produced by hybridoma technology, and their isotypes were determined by whole-cell ELISA. Monoclonal antibody reactivity with neutrophils, eosinophils, and lymphocytes isolated on phosphate-buffered saline solution and on Ficoll-sodium diatrizoate were compared. Biochemical characterization of antigens recognized by MAB was performed by immunoblot analysis. Neutrophil plasma membranes were isolated on sucrose gradients (20, 32, and 50%) and purified for polypeptide characterization. Neutrophil surface proteins were characterized by external labeling with 125I. The flow cytometric method was proven to be more sensitive and rapid than ELISA to screen hybridoma supernatants. This method allowed light-scatter gating of live neutrophil populations for analysis, which eliminated nonspecific binding of antibodies to contaminating cells and dead neutrophils. The optimal conditions for flow cytometric analyses were 5 x 10(5) neutrophils and 1 micrograms of fluorescein-labeled F(ab')2/assay as the second antibody. The optimal conditions for hybridoma screening by ELISA were neutrophil concentration of 2.5 x 10(5)/well, using a 96-well polystyrene microtitration plate as solid support, and 2,2'-azino-di[3-ethyl-benzthiazoline sulfonate (6)] with H2O2 as the chromatogenic substrate. Tissue culture plates as solid support and 3,3', 5,5'-tetramethyl benzidine, with H2O2 as the chromogenic substrate, were equally as sensitive. Panel MAB reacted differently with neutrophils, eosinophils, and lymphocytes. Isolation of these cells from blood on Ficoll-sodium diatrizoate generally did not alter MAB reactivity. Coomassie blue-stained gels of neutrophil plasma membrane proteins contained about 25 polypeptide bands, 13 of which were major bands. Autoradiography revealed about 11 surface proteins, 5 of which were heavily labeled with 125I. Monoclonal antibody S7G8 identified a 65-kd protein and MAB S8G10 identified 65- and 70-kd proteins. On the basis of molecular weight, MAB S7G8 and S8G10 are comparable to human CD15, CD16, and CD64 molecules. The MAB generated in this study are potential candidates to discern bovine neutrophil function and heterogeneity.     

Methods used in dentin bonding tests: an analysis of 50 investigations on bond strength

Researchers have carried out a variety of investigations to determine the properties of available dentin bonding systems. The test methods and variables used in 50 published investigations of bond strength were analyzed. Among the studies analyzed, shear tests predominated, being used in 80% of the studies; dentin from human molar teeth was used most often (in 88% of investigations); and the most prevalent time of testing was 24 hours postcuring. However, a number of potential variables were not recorded in a high proportion of articles, among these the film thickness, the type of dentin tested, and the surface condition (moist or dry). The mode of failure was recorded in only 42% of the investigations assessed. It was found that there is little standardization of test methods in studies of dentin bond strength and that a number of variables are often either not recorded or reported.     

Micromanipulation and cloning studies on buffalo oocytes and embryos using nucleus transfer

1. Nine male volunteers were exposed to the pyrethroid insecticide cyfluthrin. The study was performed in an exposure room, where an aerosol containing cyfluthrin was sprayed to obtain atmospheres with mean cyfluthrin concentrations of 160 and 40 micrograms/m3. Four volunteers were exposed for 10, 30 and 60 min at 160 micrograms/m3 and another five volunteers were exposed for 60 min at 40 micrograms/m3. For 160 micrograms/m3 exposure urine samples were collected before and immediately after exposure as well as for the periods 1-2, 2-3, 3-4, 4-5, 5-6, 6-12 and 12-24 h after exposure. For 40 micrograms/m3 exposure urine samples were collected before and 2 h after exposure. 2. The main urinary cyfluthrin metabolites, cis-/trans-3-(2,2-dichlorovinyl)-2,2-dimethylycyclopropane carboxylic acid (DCCA) and 4-fluoro-3-phenoxybenzoic acid (FPBA), were determined. The limit of detection (LOD) for all metabolites was 0.0025 microgram in an urine sample of 5 ml (0.5 microgram/l). After inhalative exposure of 40 micrograms cyfluthrin/m3 air for 60 min, the amount of metabolites in urine collected in the first 2 h after exposure was less than the LOD, namely 0.14 microgram for cis-DCCA, 0.15-0.28 microgram for trans-DCCA and 0.12-0.23 microgram for FPBA. 3. Of the metabolites, 93% was excreted within the first 24 h (peak excretion rates between 0.5 and 3 h) after inhalative exposure of 160 micrograms/m3. The mean half-lives were 6.9 h for cis-DCCA, 6.2 h for trans-DCCA and 5.3 h for FPBA. 4. The mean trans-:cis-DCCA ratio was 1.9 for the time course as well as for each subject. 5. The amount of metabolites in urine depends on the applied dose, on the exposure time and shows interindividual differences.     

Induction of superoxide dismutase isozymes by tumor necrosis factor-alpha and lipopolysaccharide in cultured normal and hyperplastic gingival fibroblasts

We studied 722 reexcision scars of benign and malignant lesions (except melanocytic lesions) excised over a 24-month period. The formalin-fixed, paraffin-embedded tissue sections were examined histologically and immunohistochemically. The histological features of melanocytic hyperplasia were present in 59 cases (8%), 56 from the sun-exposed skin of the face and neck and three from the trunk [p < 0.00001]. The most common sites were the nose and lower eyelids, but the forehead was also frequently involved. Of the 59 patients, 41 were women (p < 0.0001). Basal cell carcinoma was the most frequent original lesion in both sexes (80%). No melanocytic hyperplasia was found in 663 cases (298 on the trunk and extremities and 365 on the head and neck). We have seen this reaction pattern following reexcision of melanocytic lesions as well. Thus, interpreting reexcision margins when lentigo maligna or similar lesions are reexcised may be fraught with difficulty. It is important for pathologists and dermatopathologists to recognize this phenomenon because histologically the presence of increased numbers of large melanocytes could be misinterpreted as melanoma in situ.     

Protein-energy malnutrition and oxidative injury in growing rats

1. Weaning rats were fed ad libitum isocaloric diets containing 5% and 20% casein based proteins. 5% protein diet was protein deficient diet. Pair fed rats with the 5% protein group were maintained simultaneously on 20% protein diet but the amount restricted to the amount taken up by PEM group. 2. Glutathione, antioxidative enzymes, lipid peroxidation and histopathological studies in liver and only glutathione and antioxidative enzymes in blood were carried out. 3. Rats fed the 5% protein diet developed a severe protein energy malnutrition (PEM) whereas those on pair-fed diet developed mild to moderate PEM. 4. Glutathione related thiols superoxide dismutase, glutathione peroxidase, catalase and glutathione-Stransferase with (1 Chloro 2,4-dinitro benzene (CDNB) substrate) were decreased in liver with concomitant increase of lipid peroxidation in severe PEM. In blood glutathione, glutathione peroxidase and catalase were decreased while superoxide dismutase was increased in severe PEM group. 5. Mild to moderate PEM (pair-fed group) also resulted in similar changes in liver except glutathione peroxidase, lipid peroxidation in liver and superoxide dismutase in blood. 6. Hepatic injury was detectable only in the severe PEM group. 7. Oxidative-stress and hepatic injury occurred in severe PEM and to a lesser degree in mild to moderate PEM.     

Effect of immunosuppression and splenectomy in transplantation sarcomas in mice

After spontaneous regression of transplanted tumours, marked reduction in number of tumours was found when challenged with isogenic tumour cells. The ALS abrogates this effect. Tumour removal by surgical excision of limb and subsequent time scheduled challenge by tumour cells maximally suppress on the 10th day and continues up to the 42nd day the tumorogenic effect. Splenectomy has no effect if done before a day or 3 days after challenge but marked decrease in tumour development was seen when challenged on the 8th day after splenectomy. Amputation and splenectomy together potentiates tumour formation. Only in tumour extrication, does resistance develop up to the 42nd day from surgery. Challenging at a different site in mice with tumours, resulted in prolongation of the intervals of tumour formation. Challenge after surgical removal of tumour after a time lapse, results in marked reduction in number and size of tumours. Surgical tumour extrication after splenectomy and subsequent challenge on 11th day inhibited tumour formation. Whereas splenectomized tumour bearing mice when challenged at a heterosite did not develop resistance.     

Spatial memory deficits following stimulation of hippocampal 5-HT1B receptors in the rat

The authors review the principles of reconstructive surgery for lower limb salvage after severe lower limb trauma to determine factors that have been used as decision-making criteria for limb salvage or amputation in severe lower extremity injuries and the methods of reconstruction and their outcome. The use of scoring systems and their value in acute decision making (primary amputation or limb salvage) are described. Soft-tissue reconstructive techniques, with emphasis on the use of flaps and the importance of selecting the best technique and time for the reconstruction are reviewed. Skeletal reconstructive techniques are described, including available options and currently held views on indications and use of the best contemporary methods. It is essential for the physician to make a good initial decision on the need for primary amputation or limb salvage. A multidisciplinary approach is fundamental to successful salvage.     

Silicon bipolar chipset for SONET/SDH 10 Gb/s fiber-opticcommunication links

High-speed multiplexers, demultiplexers, frequency dividers, mixers, and amplifiers are key electronic components in high-speed fiber-optic communications systems such as SONET/SDH. In this paper, we present several important digital and analog integrated circuits (IC) which have been developed for use in SONET/SDH 10 Gb/s optical communication links. The circuits have been fabricated in MOSAIC 5E, an advanced silicon bipolar technology (f_T=26 GHz). The resulting chipset which amounts to a total of 10 IC's consists of multiplexers, demultiplexers, a regenerative frequency divider (2:1), a dual output limiting amplifier, and two different types of mixers for clock extraction. Specifically, the design and performance of these IC's and a hybrid clock recovery module are discussed. The high performance and potential low cost of this research chipset show that advanced silicon bipolar circuit technology can play an important role in future multigigabit fiber-optic communication systems