The Role of Neurotrophins in Inflammation and Allergy.

Allergic inflammation is the result of a specific pattern of cellular and humoral responses leading to the activation of the innate and adaptive immune system, which, in turn, results in physiological and structural changes affecting target tissues such as the airways and the skin. Eosinophil activation and the production of soluble mediators such as IgE antibodies are pivotal features in the pathophysiology of allergic diseases. In the past few years, however, convincing evidence has shown that neurons and other neurosensory structures are not only a target of the inflammatory process but also participate in the regulation of immune responses by actively releasing soluble mediators. The main products of these activated sensory neurons are a family of protein growth factors called neurotrophins. They were first isolated in the central nervous system and identified as important factors for the survival and differentiation of neurons during fetal and postnatal development as well as neuronal maintenance later in life. Four members of this family have been identified and well defined: nerve growth factor, brain-derived neurotrophic factor, neurotrophin 3, and neurotrophin 4/5. Neurotrophins play a critical role in the bidirectional signaling mechanisms between immune cells and the neurosensory network structures in the airways and the skin. Pruritus and airway hyperresponsiveness, two major features of atopic dermatitis and asthma, respectively, are associated with the disruption of the neurosensory network activities. In this chapter, we provide a comprehensive description of the neuroimmune interactions underlying the pathophysiological mechanisms of allergic and inflammatory diseases.

Ataxin-3 with an altered conformation that exposes the polyglutamine domain is associated with the nuclear matrix.

Spinocerebellar ataxia type-3 or Machado-Joseph disease (SCA3/MJD) is a member of the CAG/polyglutamine repeat disease family. In this family of disorders, a normally polymorphic CAG repeat becomes expanded, resulting in expression of an expanded polyglutamine domain in the disease gene product. Experimental models of polyglutamine disease implicate the nucleus in pathogenesis; however, the link between intranuclear expression of expanded polyglutamine and neuronal dysfunction remains unclear. Here we demonstrate that ataxin-3, the disease protein in SCA3/MJD, adopts a unique conformation when expressed within the nucleus of transfected cells. The monoclonal antibody 1C2 is known preferentially to bind expanded polyglutamine, but we find that it also binds a fragment of ataxin-3 containing a normal glutamine repeat. In addition, expression of ataxin-3 within the nucleus exposes the glutamine domain of the full-length non-pathological protein, allowing it to bind the monoclonal antibody 1C2. Fractionation and immunochemical experiments indicate that this novel conformation of intranuclear ataxin-3 is not due to proteolysis, suggesting instead that association with nuclear protein(s) alters the structure of full-length ataxin-3 which exposes the polyglutamine domain. This conformationally altered ataxin-3 is bound to the nuclear matrix. The pathological form of ataxin-3 with an expanded polyglutamine domain also associates with the nuclear matrix. These data suggest that an early event in the pathogenesis of SCA3/MJD may be an altered conformation of ataxin-3 within the nucleus that exposes the polyglutamine domain.

Evidence for proteasome involvement in polyglutamine disease: localization to nuclear inclusions in SCA3/MJD and suppression of polyglutamine aggregation in vitro.

Spinocerebellar ataxia type 3, also known as Machado-Joseph disease (SCA3/MJD), is one of at least eight inherited neurodegenerative diseases caused by expansion of a polyglutamine tract in the disease protein. Here we present two lines of evidence implicating the ubiquitin-proteasome pathway in SCA3/MJD pathogenesis. First, studies of both human disease tissue and in vitro models showed redistribution of the 26S proteasome complex into polyglutamine aggregates. In neurons from SCA3/MJD brain, the proteasome localized to intranuclear inclusions containing the mutant protein, ataxin-3. In transfected cells, the proteasome redistributed into inclusions formed by three expanded polyglutamine proteins: a pathologic ataxin-3 fragment, full-length mutant ataxin-3 and an unrelated GFP-polyglutamine fusion protein. Inclusion formation by the full-length mutant ataxin-3 required nuclear localization of the protein and occurred within specific subnuclear structures recently implicated in the regulation of cell death, promyelocytic leukemia antigen oncogenic domains. In a second set of experiments, inhibitors of the proteasome caused a repeat length-dependent increase in aggregate formation, implying that the proteasome plays a direct role in suppressing polyglutamine aggregation in disease. These results support a central role for protein misfolding in the pathogenesis of SCA3/MJD and suggest that modulating proteasome activity is a potential approach to altering the progression of this and other polyglutamine diseases.

Recruitment and the role of nuclear localization in polyglutamine-mediated aggregation.

The inherited neurodegenerative diseases caused by an expanded glutamine repeat share the pathologic feature of intranuclear aggregates or inclusions (NI). Here in cell-based studies of the spinocerebellar ataxia type-3 disease protein, ataxin-3, we address two issues central to aggregation: the role of polyglutamine in recruiting proteins into NI and the role of nuclear localization in promoting aggregation. We demonstrate that full-length ataxin-3 is readily recruited from the cytoplasm into NI seeded either by a pathologic ataxin-3 fragment or by a second unrelated glutamine-repeat disease protein, ataxin-1. Experiments with green fluorescence protein/polyglutamine fusion proteins show that a glutamine repeat is sufficient to recruit an otherwise irrelevant protein into NI, and studies of human disease tissue and a Drosophila transgenic model provide evidence that specific glutamine-repeat-containing proteins, including TATA-binding protein and Eyes Absent protein, are recruited into NI in vivo. Finally, we show that nuclear localization promotes aggregation: an ataxin-3 fragment containing a nonpathologic repeat of 27 glutamines forms inclusions only when targeted to the nucleus. Our findings establish the importance of the polyglutamine domain in mediating recruitment and suggest that pathogenesis may be linked in part to the sequestering of glutamine-containing cellular proteins. In addition, we demonstrate that the nuclear environment may be critical for seeding polyglutamine aggregates.

Intranuclear inclusions of expanded polyglutamine protein in spinocerebellar ataxia type 3.

The mechanism of neurodegeneration in CAG/polyglutamine repeat expansion diseases is unknown but is thought to occur at the protein level. Here, in studies of spinocerebellar ataxia type 3, also known as Machado-Joseph disease (SCA3/MJD), we show that the disease protein ataxin-3 accumulates in ubiquitinated intranuclear inclusions selectively in neurons of affected brain regions. We further provide evidence in vitro for a model of disease in which an expanded polyglutamine-containing fragment recruits full-length protein into insoluble aggregates. Together with recent findings from transgenic models, our results suggest that intranuclear aggregation of the expanded protein is a unifying feature of CAG/polyglutamine diseases and may be initiated or catalyzed by a glutamine-containing fragment of the disease protein.

Machado-Joseph disease gene product is a cytoplasmic protein widely expressed in brain.

Machado-Joseph disease (MJD) is one of at least six neurodegenerative diseases caused by expansion of a CAG repeat encoding a polyglutamine tract in the disease protein. To study the molecular mechanism of disease, we isolated both normal and expanded repeat MJD1 cDNAs, and generated antiserum against the recombinant gene product, called ataxin-3. Using this antiserum, we demonstrate that in disease tissue, both the normal and mutant ataxin-3 protein are expressed throughout the body and in all regions of the brain examined, including areas generally spared by disease. In brain, certain regions (the striatum, for example) express ataxin-3 in only a limited subset of neurons. Immunolocalization studies in normal and disease brain, and in transfected cells, indicate that ataxin-3 is predominantly a cytoplasmic protein that localizes to neuronal processes as well. We conclude that in MJD, as in other polyglutamine repeat diseases, cellular expression of the disease gene is not itself sufficient to cause neuronal degeneration; other cell-specific factors must be invoked to explain the restricted neuropathology seen in MJD. The restricted expression of ataxin-3 in certain regions, however, may influence the pattern of neurodegeneration and provide clues to the protein's function.

High performance thin-layer chromatographic analysis of sugars in Biomphalaria glabrata (Gastropoda) infected with Echinostoma caproni (Trematoda).

High performance thin-layer chromatographic analysis was done on the hemolymph and digestive gland-gonad complex (DGG) of Biomphalaria glabrata snails experimentally infected with the intramolluscan stages of Echinostoma caproni. The major sugars detected in both the DGG and hemolymph of infected and uninfected snails were glucose and trehalose. Quantitative analysis by scanning densitometry showed a significant reduction in glucose in both the hemolymph and DGG of infected snails at 4, 6, and 8 wk postinfection. A similar analysis on trehalose showed that this sugar was significantly reduced at 6 wk postinfection in the hemolymph and DGG of infected snails and could not be detected from these sites in infected snails by 8 wk postinfection. Findings from this study were compared with information on sugars in B. glabrata infected with larval stages of Schistosoma mansoni.

Analytical methodology and the interface with animal drug approval.

FDA is engaged in a systematic review of approved animal drugs to determine whether upgrading of safety data is required to provide continuing assurance that the approved uses for these drugs are safe. These reviews will be undertaken on the basis of a priority of anticipated human health significance.

Human safety data collection and evaluation for the approval of new animal drugs.

Before a new drug is approved for use in food-producing animals, data are required which demonstrate that food derived from the animal does not contain unsafe residues. Also, an analytical method for residues must be provided which is practicable for government surveillance and enforcement activities. Residue information is derived by using radiolabeled drugs to study metabolism in the animal for which the drug is intended. Residues in the edible tissues are characterized, and the amount of residues and their rates of depletion from the different tissues after cessation of drug treatment are determined. Bioassays with laboratory animals are used to study the toxicity of the drug and its important metabolites and to establish tolerance limitations. From this information, the conditions for the analytical method are established--that is, the compound(s) measured (the marker), the tissue (target tissue) monitored to ensure control of "total residue", and the required sensitivity. The method is subjected to validation in government laboratories and must meet the Food and Drug Administration's standards of precision, accuracy, specificity, and practicability. Finally, the method is used in simulated field trials to establish the required withdrawal time after drug treatment before the animals can be marketed for their milk or for slaughter for food.

Human safety considerations from the use of anabolic agents in foodproducing animals.

The various anabolic agents used in food-producing animals may differ in terms of toxicological considerations related to evaluating human safety. Aside from initial toxicological testing, after chemical characterization of the compound to be administered and its related metabolites expected to occur as residues in food, most synthetic anabolic agents are subjected to chronic/carcinogenicity testing because of usage pattern likely to lead to the occurrence of residues in derived edible products. Initial testing requirements include acute and subchronic studies in appropriate rodent species including a reproduction test with the first generation offspring tested for 90 days post-weaning. This subchronic study serves to indicate potential problems with reproductive performance, foetal toxicity, birth deformities, and other chronic or preneoplastic conditions. The Food and Drug Administration (FDA) may grant approval for certain usages of specific compounds using a 2.000-fold safety margin in relation to a "no deleterious effect" level from the subchronic studies, with upper residue limits of 0.1 ppm in tissue and 0.01 ppm in milk or eggs if there are no indications that further testing should be required. If higher residue limits are requested, the petitioner must perform lifetime testing in two rodent species including in utero exposure and a minimum of three dose levels. Other rodent offspring should be carried for a total of three generations. A six to twelve month study in a non-rodent population is required. Teratology studies might be rquired in at least two species. If no carcinogenic potential is observed, a 100-fold safety margin in relation to the no effect level is generally accepted as the safe exposure level for residues. Should a statistically significant increase in tumors be observed in the test animals as compared to controls, the compound will be classified as a carcinogen or a suspect carcinogen depending on histopathological observations...