Characterization of monoclonal antibodies to ovine tumor necrosis factor-alpha and development of a sensitive immunoassay

Monoclonal antibodies (mAbs) and a polyclonal rabbit antiserum were raised against recombinant ovine tumor necrosis factor-alpha (rovTNF alpha). Ten mAbs specific for rovTNF alpha were isolated and designated TNF1-10. All mAbs were of the IgG1 isotype and reacted with rovTNF alpha in Western blot analysis. Eight of the ten mAbs, TNF1, TNF3-7 and TNF9 and 10, completely blocked the activity of rovTNF alpha and macrophage derived native ovTNF alpha, as measured by their ability to inhibit TNF alpha-mediated lysis of WEHI-164 or L929 cells. In addition, TNF3, -7, -9 and -10 blocked the cytolytic activity of recombinant human TNF alpha (rhuTNF alpha). However, when tested for the ability to inhibit TNF alpha induced thymocyte proliferation, only mAbs TNF1, -3, -5, -7, -9 and -10 could completely block activity. Competitive binding analysis using unlabelled and horseradish peroxidase (HRPO) labelled mAbs indicated that the mAbs could be divided into five groups based on their reactivity with rovTNF alpha. The mAbs were used to develop a sensitive sandwich immunoassay for the detection of ovTNF alpha. All combinations of mAbs and the polyclonal antiserum were tested to determine which pair of antibodies gave the most sensitive assay. The combination of TNF5 as the capture antibody and the polyclonal antiserum gave the most sensitive result, detecting less than 0.24 ng rovTNF alpha ml-1. A similar sensitivity was obtained when TNF4 was used as the capture antibody and TNF10 HRPO labelled mAb as the second antibody. The immunoassay was more sensitive than the WEHI-164 bioassay which had a detection limit of 1 ng ml-1 for rovTNF alpha. This immunoassay also detected glycosylated ovTNF alpha in the supernatant of COS-7 cells which had been transfected with an ovTNF alpha cDNA.     

Monotremes and the evolution of rapid eye movement sleep

Early studies of the echidna led to the conclusion that this monotreme did not have rapid eye movement (REM) sleep. Because the monotremes had diverged from the placental and marsupial lines very early in mammalian evolution, this finding was used to support the hypothesis that REM sleep evolved after the start of the mammalian line. The current paper summarizes our recent work on sleep in the echidna and platypus and leads to a very different interpretation. By using neuronal recording from mesopontine regions in the echidna, we found that despite the presence of a high-voltage cortical electroencephalogram (EEG), brainstem units fire in irregular bursts intermediate in intensity between the regular non-REM sleep pattern and the highly irregular REM sleep pattern seen in placentals. Thus the echidna displays brainstem activation during sleep with high-voltage cortical EEG. This work encouraged us to do the first study of sleep, to our knowledge, in the platypus. In the platypus we saw sleep with vigorous rapid eye, bill and head twitching, identical in behaviour to that which defines REM sleep in placental mammals. Recording of the EEG in the platypus during natural sleep and waking states revealed that it had moderate and high-voltage cortical EEGs during this REM sleep state. The platypus not only has REM sleep, but it had more of it than any other animal. The lack of EEG voltage reduction during REM sleep in the platypus, and during the REM sleep-like state of the echidna, has some similarity to the sleep seen in neonatal sleep in placentals. The very high amounts of REM sleep seen in the platypus also fit with the increased REM sleep duration seen in altricial mammals. Our findings suggest that REM sleep originated earlier in mammalian evolution than had previously been thought and is consistent with the hypothesis that REM sleep, or a precursor state with aspects of REM sleep, may have had its origin in reptilian species.     

Immunosuppressive therapy with microemulsion cyclosporine A shortens the hospitalization of pediatric liver transplant recipients

Lipopolysaccharide (LPS) from S. typhimurium on exposure to gamma-radiation resulted in decrease in toxicity and was less mitogenic, Silver stained profiles of irradiated LPS on polyacrylamide gels revealed complete loss of its heteropolysaccharides which was confirmed further by analysing lipid A and LPS from Salmonella minnesota Re mutants on SDS-PAGE. Glucosamine and 2-keto 3-deoxy-octonate(Kdo) contents were significantly decreased on treatment. Lipid A obtained by removal of heteropolysaccharides from LPS was less toxic on exposure to gamma radiations.     

Endodontic applications of guided tissue regeneration in endodontic surgery

This paper is intended to provide an overview of different endodontic applications in guided tissue regeneration. It is our expectation that using these techniques, where applicable, will increase endodontic surgical success. We have described six different clinical conditions including external resorption, root perforation, dehiscence, palatal grooves, oblique root fractures, and large periapical lesions. We believe the use of guided tissue regeneration techniques will allow us to predictably treat teeth that otherwise might be extracted.     

Prediction of functional outcome after myocardial infarction using BMIPP and sestamibi scintigraphy

We determined the predictive value of combined beta-methyl iodophenyl pentadecanoic acid (BMIPP) and sestamibi scintigraphy for the functional outcome after myocardial infarction and compared the value of this approach with dobutamine echocardiography. METHODS: Rest BMIPP, rest sestamibi and low-dose dobutamine echocardiographic studies were obtained in 18 patients 4 to 10 days after infarction (mean 6.7 +/- 2.0 days). Six months later, a rest echocardiographic study was performed to assess functional outcome. RESULTS: Wall motion improved in 27/33 segments (82%) which showed mismatching but not in 19/21 segments (90%) with matched defects (p < 0.001). The accuracy of combined BMIPP and sestamibi SPECT in predicting segmental functional outcome was higher (85%) than that of sestamibi uptake alone (77%). Wall motion improved in 16/20 segments (80%) showing contractile reserve and not in 21/34 segments (63%) with the negative dobutamine test, giving an accuracy of 69% for dobutamine echocardiography. Combination of the two techniques resulted in higher positive (94%) and negative predictive values (94%). CONCLUSION: Mismatching of BMIPP and sestamibi uptake is predictive for long-term functional recovery after acute myocardial infarction. In contrast, segments with matched defects contain only scar tissue. Combined BMIPP and sestamibi scintigraphy offers increased accuracy compared to dobutamine echocardiography.     

Use of inverse thin layer chromatography with amino acids to characterize molecular interactions on conducting polymers

A new method for characterizing conducting polymers, based on thin layer chromatography, is evaluated. Polypyrrole and polyaniline were used as the stationary phase and molecular probes were used to characterize favourable interactions on the polymers. Amino acids were chosen as the molecular probes because of the range of properties they exhibit and because there are numerous methods for their detection. Some difficulties were experienced in preparing stationary phases with suitable physical properties. However, once this was overcome, useful information was obtained with inverse TLC. Polypyrrole and polyaniline were both found to behave predominantly as anion-exchange materials.