The fate of pro-TNF-alpha following inhibition of metalloprotease-dependent processing to soluble TNF-alpha in human monocytes

Human monocytes rapidly produce TNF-alpha following activation by bacterial LPS. The message for TNF-alpha encodes a 26-kDa protein that is proteolytically processed to the secreted 17-kDa form. Sequencing of the N terminus of the protein secreted by monocytes shows processing of the 26-kDa pro-TNF-alpha to a mature form at the projected metalloprotease cleavage site to generate 17-kDa TNF-alpha with the N terminus VRSSSR-. The addition of hydroxamic acid-based metalloprotease inhibitors to the cell culture is capable of blocking >95% of the production of soluble TNF-alpha and leads to a transient, but reproducible, increase in cell surface TNF-alpha as measured by FACS analysis. The cell surface TNF-alpha was demonstrated to increase the cell's ability to kill L929 tumor targets and induce PG production from human gingival fibroblasts. The buildup of cell surface TNF-alpha is unable to account for the TNF-alpha that is not secreted when inhibitor is present. Pulse-chase analysis of the cells demonstrates rapid degradation of the pro-TNF-alpha that remains unprocessed in the monocytes. Through inhibition of processing and secretion by brefeldin A, processing was shown to occur at a postendoplasmic reticulum site and is closely associated with movement to the cell surface.     

Results of a search for missed cases of reportable communicable diseases using hospital discharge data

To assess how reliably hospitals report serious, uncommon communicable diseases to the Department for Public Health, we searched the 1995 hospital discharge data set (HDDS) collected by the Kentucky Health Policy Board for cases of 11 diseases. Of 17 case records found, 4 represented disease occurrences that had been reported to the Department; 6 represented coding errors in the HDDS; 4 were instances where a reportable disease had been suspected but not confirmed by subsequent workup; 1 case was a resident of another state; and 2 were cases of invasive Hemophilus influenzae infection in adults that should have been reported to the Department. The study found no evidence that hospitals failed to report vaccine-preventable diseases. There was evidence that the HDDS needs improved accuracy to maximize its usefulness for public health purposes.     

Seizures involving the supplementary sensorimotor area in children: a video-EEG analysis

Most reports of supplementary sensorimotor seizures have been of adults with medically refractory epilepsy. Typically, supplementary sensorimotor seizures have onset in childhood. We describe the electroclinical features in 12 children. Cases were selected from an EEG laboratory population in whom video-EEG was performed routinely on all children. Supplementary sensorimotor seizures were diagnosed when there was bilateral tonic posturing of the upper or lower extremities, preserved consciousness, and lack of postictal confusion. Sensory auras were reported by 8 of 9 children aged > 3 years who had daytime seizures. Speech arrest occurred in all patients in whom it could be assessed, and abnormal vocalization was observed in 7 children. Interictal EEGs were often normal (49% of recordings), and ictal EEG changes could be subtle. An etiology was demonstrated in only 2 children, and brain imaging studies were normal in the other 10 patients. Seizures were controlled with antiepileptic drugs (AEDs) in 6 of the 12 children. The clinical manifestations of supplementary sensorimotor seizures in children are similar to those reported in adults; misdiagnosis is common at all ages.     

Susceptibility to hepatotoxicity in transgenic mice that express a dominant-negative human keratin 18 mutant

Keratins 8 and 18 (K8/18) are intermediate filament phosphoglycoproteins that are expressed preferentially in simple-type epithelia. We recently described transgenic mice that express point-mutant human K18 (Ku, N.-O., S. Michie, R.G. Oshima, and M.B. Omary. 1995. J. Cell Biol. 131:1303-1314) and develop chronic hepatitis and hepatocyte fragility in association with hepatocyte keratin filament disruption. Here we show that mutant K18 expressing transgenic mice are highly susceptible to hepatotoxicity after acute administration of acetaminophen (400 mg/Kg) or chronic ingestion of griseofulvin (1.25% wt/wt of diet). The predisposition to hepatotoxicity results directly from the keratin mutation since nontransgenic or transgenic mice that express normal human K18 are more resistant. Hepatotoxicity was manifested by a significant difference in lethality, liver histopathology, and biochemical serum testing. Keratin glycosylation decreased in all griseofulvin-fed mice, whereas keratin phosphorylation increased dramatically preferentially in mice expressing normal K18. The phosphorylation increase in normal K18 after griseofulvin feeding appears to involve sites that are different to those that increase after partial hepatectomy. Our results indicate that hepatocyte intermediate filament disruption renders mice highly susceptible to hepatotoxicity, and raises the possibility that K18 mutations may predispose to drug hepatotoxicity. The dramatic phosphorylation increase in nonmutant keratins could provide survival advantage to hepatocytes.     

Membrane currents induced in Xenopus oocytes by the C-terminal fragment of the beta-amyloid precursor protein

There is mounting evidence that at least some of the neurotoxicity associated with Alzheimer's disease (AD) is due to proteolytic fragments of the beta-amyloid precursor protein (beta APP). Most research has focused on the amyloid beta protein (A beta), which has been shown to possess ion channel activity. However, the possible role of other cleaved products of the beta APP is less clear. We have investigated the ability of various products of beta APP to induce membrane ion currents by applying them to Xenopus oocytes, a model system used extensively for investigating electrophysiological aspects of cellular, including neuronal, signalling. We focussed on the 105-amino-acid C-terminal fragment (CT105) (containing the full sequence A beta), which has previously been found to be toxic to cells, although little is known about its mode of action. We have found that CT105 is exceedingly potent, with a threshold concentration of 100-200 nM, in inducing nonselective ion currents when applied from either outside or inside the oocyte and is more effective than either beta APP or the A beta fragments, beta 25-35 or beta 1-40. The ion channel activity of CT105 was concentration dependent and blocked by a monoclonal antibody to A beta. These results suggest the possible involvement of CT105 in inducing the neural toxicity characteristic of AD.     

An in vitro model for infection with Leishmania major that mimics the immune response in mice

By using a primary in vitro response specific for Leishmania major, normal T cells from resistant CBA/CaH-T6J and susceptible BALB/c mice commit to a Th1 and a Th2 response, respectively. Since commitment occurred, we measured the production of gamma interferon (IFN-gamma), interleukin-1 (IL-1), IL-2, IL-4, IL-5, IL-10, and IL-12, prostaglandin E2 (PGE2), transforming growth factor beta (TGF-beta), and nitric oxide in the first 7 days of the response to identify factors that are critical for Th1 and Th2 development. While cells from resistant CBA mice produced more IFN-gamma, IL-10, and nitric oxide, cells from susceptible BALB/c mice produced more IL-1alpha, IL-5, PGE2, and TGF-beta. Although substantial amounts of IL-12 were detected, IL-12 did not associate with either Th1 or Th2 development. We did not anticipate that cells from resistant CBA mice would make more IL-10 in vitro. However, this also occurred in vivo since CBA mice produced substantial amounts of IL-10 following infection with L. major. Moreover, adding anti-IL-10 to primary in vitro responses enhanced production of IFN-gamma and nitric oxide by cells from CBA and BALB/c mice. Therefore, IL-10 cannot be regarded as a cytokine that associates with susceptibility to infection with L. major. Finally, the data presented here suggest that a collection of factors that can be produced by accessory cells influence Th commitment (e.g., IL-1, PGE2, and TGF-beta favor Th2 development).     

Nutritional support of critically ill patients

Severe depletion of body protein stores can result from prolonged starvation or from hormonal and cytokine-mediated effects during critical illness. Recent advances in the understanding of cytokine actions have substantially refined the interpretation of the nutritional assessment of critically ill patients. In addition, the design of nutritional programs for hospitalized patients has changed considerably during the past decade. Although nutritional support of critically ill patients will not lead to positive nitrogen balance, nutrition can increase protein synthesis, enhance immune function, and beneficially modify the body's response to an illness.     

Nonsense suppression in thymine-requiring strains of Escherichia coli is a consequence of altered folate metabolism

Thymine-requiring strains of Escherichia coli suppress nonsense and frameshift mutants of T4 phage. We proposed that these mutants make errors during translation because of an imbalance in folate metabolism. A thymine-requiring strain grown under suppressing conditions has elevated levels of reduced folates. We tested the effect of either mutational blocks or the inhibition of various steps in folate biosynthesis on suppression. Conditions which prevent the accumulation of 5-methyl tetrahydrofolate inhibit suppression, suggesting that elevated levels of this folate are required for suppression. Furthermore, conditions that result in an accumulation in dihydrofolate inhibit suppression.