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Analysis of the humoral immune response to infectious diseases has played, and will to continue to play, a key role in their diagnosis and immune surveillance. Although rapid genome detection methodologies, such as PCR, are beginning to replace immune assays for disease diagnosis, they are not suitable for all applications, especially the surveillance of the immune status of human populations. Here we review the limitations of current conventional tools for measuring immune responses and outline principles for the design and production of novel diagnostic reagents. Methods for the production of viral diagnostic antigens by a variety of recombinant systems are described and their relative merits and disadvantages discussed. Protocols for the production of viral diagnostic antigens in eukaryotic, insect and mammalian systems are described using measles nucleocapsid antigen as a model. Indirect ELISA protocols which can differentiate immunoglobulin classes and subclasses are also described. Examples of the use of these analyses in research and surveillance are given.
Methods Mol Med 2004
PMID:Design and preparation of recombinant antigens as diagnostic reagents in solid-phase immunosorbent assays. 1495 40

Recombinant measles virus nucleoprotein (N) was produced in insect cells where it bound to cellular RNA to form helical N-RNA structures. These structures were observed by electron microscopy but were too flexible for high-resolution image analysis. Removal of the C-terminal tail of N by trypsin treatment resulted in structures that were much more rigid and seemed more regular. Several methods of image analysis were employed in order to make a helical reconstruction of the digested N-RNA. During this analysis, it became clear that the apparently regular coils of digested N-RNA consisted of a series of closely related helical states. The iterative helical real space reconstruction method allowed the identification of two helical states for which a reconstruction could be calculated. The model with the highest resolution shows N monomers that consist of three domains and that are connected to their neighbours by two narrow connections, one close to the helical axis and another toward the middle of the monomers. There are no connections between N molecules in subsequent helical turns. After labelling the RNA in the structure with cis-platinum, the connection closest to the helical axis increased in density, suggesting the position of the RNA. The shapes of the monomers of the nucleoproteins of influenza virus, rabies virus (both determined before) and that of measles virus (determined here) are all similar, whereas the overall shapes of their respective N-RNAs (nucleocapsids) is very different. This is probably due to the position and number of the connections between the N subunits in the N-RNA, one for influenza virus allowing much flexibility, two for rabies virus at either end of the N molecules leading to ribbons and two for measles virus leading to the typical paramyxovirus helical nucleocapsid.
J Mol Biol 2004 May 28
PMID:The 12 A structure of trypsin-treated measles virus N-RNA. 1513 34

Respiratory tract infections are among the bacterial infections that affect humans with higher frequency. Those produced by Streptococcus pneumoniae are reported to have the highest incidence in the world, affecting both children and old people. As a 2001 report from the World Health Organization expressed it, the basic fight of children under 5 yr old is to survive. Five different conditions (acute respiratory infections, diarrhea, measles, palludism, and undernutrition) directly produce more than 50% of the deaths in this age group. Respiratory tract infections in the developing countries in the Americas are among the first three causes of death in children under 1 yr and between the first and second cause in children between 1 and 4 yr old. Pneumonia is responsible for 85 and 90% of deaths in children under 5 yr old (approx 150,000 annually), 95% of them occurring in the developing countries in the Americas. There is an increased worldwide tendency to use preventive measures and to consume products that help to maintain the health status of the individual. Thus the use of probiotics has increased systematically during the last decade, and the scientific literature trying to demonstrate the positive effect of such preparations has also increased. The term probiotic has been applied to products that (1) contain live microorganisms, freeze-dried or included in fermented products or (2) improve the health status of humans and animals, exerting effects in the mouth or gastrointestinal tract (included in foods or capsules), in the respiratory tract (as aerosols), or in the urogenital tract (by local application)Having in mind the high incidence of respiratory tract infections, and looking for preventive measures as well as the possible applications of probiotics, the aim of this chapter was to use mice as experimental models to determine whether members of the genus Lactobacillus were able to colonize and give protection from infections after inoculation by the intranasal route. To this end, the following procedures were carried out: 1. Screening of the predominant bacterial species in respiratory organs. 2. Study of the kinetics of colonization of the different groups of microorganisms from 15 d up to adult (2 mo). 3. Screening of the probiotic characteristics of all the isolated strains.
Methods Mol Biol 2004
PMID:Colonization capability of lactobacilli and pathogens in the respiratory tract of mice: microbiological, cytological, structural, and ultrastructural studies. 1515 48

Measles virus is a highly contagious virus that, despite the existence of an effective vaccine, is a major cause of illness and mortality worldwide. The virus has a negative-sense, single-stranded RNA genome that is encapsidated by the nucleocapsid protein (N) to form a helical ribonucleoprotein complex known as the nucleocapsid. This structure serves as the template for both transcription and replication. Paramyxovirus nucleocapsids are flexible structures, a trait that has hitherto hampered structural analysis even at low resolution. We have investigated the extent of this structural plasticity, using real-space methods to calculate three-dimensional reconstructions of recombinant nucleocapsids from cryo-negative stain transmission electron micrographs. Images of short sections of helix were sorted according to both pitch (the axial rise per turn) and twist (the number of subunits per turn). Our analysis indicates that there is extensive conformational flexibility within these structures, ranging in pitch from 50 Angstrom to 66 Angstrom, while twist varies from at least 13.04 to 13.44 with a greater number of helices comprising around 13.1 subunits per turn. We have also investigated the influence of the C terminus of N on helix conformation, analysing nucleocapsids after having removed this domain by trypsin digestion. We have found that this causes a marked change in both pitch and twist, such that the pitch becomes shorter, ranging from 46 Angstrom to 52 Angstrom, while more helices have a twist of approximately 13.3 subunits per turn. Our findings lead us to propose a mechanism whereby changes in conformation, influenced by interactions between viral or host proteins and the C terminus of N, might have a role in regulating the balance of transcription and replication during virus infection.
J Mol Biol 2004 Jul 02
PMID:Conformational flexibility in recombinant measles virus nucleocapsids visualised by cryo-negative stain electron microscopy and real-space helical reconstruction. 1520 Oct 55

(23S)-25-Dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone (TEI-9647) functions an antagonist of the 1alpha,25-dihydroxyvitamin D(3) (1alpha,25-(OH)(2)D(3)) nuclear receptor (VDR)-mediated differentiation of human leukemia (HL-60) cells [J. Biol. Chem. 274 (1999) 16392]. We examined the effect of vitamin D antagonist, TEI-9647, on osteoclast formation induced by 1alpha,25-(OH)(2)D(3) from bone marrow cells of patients with Paget's disease. TEI-9647 itself never induced osteoclast formation even at 10(-6)M, but dose-dependently (10(-10) to 10(-6)M) inhibited osteoclast formation induced by physiologic concentrations of 1alpha,25-(OH)(2)D(3) (41 pg/ml, 10(-10)M) from bone marrow cells of patients with Paget's disease. At the same time, 10(-8)M of TEI-9647 alone did not cause 1alpha,25-(OH)(2)D(3) dependent gene expression, but almost completely suppressed TAF(II)-17, a potential coactivator of VDR and 25-hydroxyvitamin D(3)-24-hydroxylase (25-OH-D(3)-24-hydroxylase) gene expression induced by 10(-10)M 1alpha,25-(OH)(2)D(3) in bone marrow cells of patients with Paget's disease. Moreover, TEI-9647 dose-dependently inhibited bone resorption induced by 10(-9)M 1alpha,25-(OH)(2)D(3) by osteoclasts produced by RANKL and M-CSF treatment of measles virus nucleocapsid gene transduced bone marrow cells. These results suggest that TEI-9647 acts directly on osteoclast precursors and osteoclasts, and that TEI-9647 may be a novel agent to suppress the excessive bone resorption and osteoclast formation in patients with Paget's disease.
J Steroid Biochem Mol Biol 2004 May
PMID:Vitamin D antagonist, TEI-9647, inhibits osteoclast formation induced by 1alpha,25-dihydroxyvitamin D3 from pagetic bone marrow cells. 1522 95

The phosphoprotein (P) of rabies virus binds the viral polymerase to the nucleoprotein (N)-RNA template for transcription and replication. By limited protease digestion we defined a monomeric C-terminal domain of P that can bind to N-RNA. The atomic structure of this domain was determined and previously described mutations that interfere with binding of P to N-RNA could now be interpreted. There appears to be two features involved in this activity situated at opposite surfaces of the molecule: a positively charged patch and a hydrophobic pocket with an exposed tryptophan side-chain. Other previously published work suggests a conformational change in P when it binds to N-RNA, which may imply the repositioning of two helices that would expose a hydrophobic groove for interaction with N. This domain of rabies virus P is structurally unrelated to the N-RNA binding domains of the phosphoproteins of Sendai and measles virus that are members of the same order of viruses, the non-segmented negative strand RNA viruses. The implications of this finding for the evolution of this virus group are discussed.
J Mol Biol 2004 Oct 29
PMID:Structure and function of the C-terminal domain of the polymerase cofactor of rabies virus. 1547 3

The purpose of this study is to document three cases of fatal measles infection in children who ranged in age from 1 to 6 years old. In each case, there was a rapidly progressive illness marked by severe respiratory and central nervous system disease; in two cases, tonsillar herniation occurred. The lung tissues showed marked interstitial pneumonitis with diffuse endothelial cell and pneumocyte degeneration; occasional multinucleated giant cells were observed. Brain sections showed a paucicellular inflammatory infiltrate with diffuse neuronal damage. Measles nucleoprotein and measles RNA were detected in each case by immunohistochemistry and reverse transcriptase (RT) in situ PCR, respectively. In the lung tissues, the viral protein and RNA localized primarily to pneumocytes and macrophages; infected endothelial cells were also evident. In the brain sections, the virus-infected cells cytologically had the appearance of neurons and microglial cells. The viral load, defined by the percentage of cells infected in a given field, was very high in the lung, spleen, and brain. Viral infection was associated with a marked increase in the number of cells expressing tumor necrosis factor alpha and concomitant reduction in the cells expressing suppressors of cytokine signaling (SOCS). It is concluded that measles infection should be in the differential diagnosis of a rapidly progressive illness in young children in the United States and that the pathogenesis is based, in part, on massive viral infection with up-regulation of cytokine expression that likely reflects, in part, down-regulation of inhibitors of cytokine mRNA receptor synthesis.
Diagn Mol Pathol 2005 Jun
PMID:Histologic and molecular correlates of fatal measles infection in children. 1590 93

The enzyme-linked immunospot (ELISPOT) assay is a highly sensitive tool used to measure the frequency of antigen-specific T-cells in vitro. Among its many applications, this assay is useful for the characterization of cellular immune responses after immunization against measles and other viral pathogens. A description of the measles virus-specific interferon (IFN)-gamma ELISPOT assay optimized in our laboratory is described in detail in this chapter. Procedures for the preparation of measles virus, infection of peripheral blood mononuclear cells with measles virus, and the IFN-gamma ELISPOT assay are also outlined. These methods can also be broadly adapted to measure activated T-cells to other viral pathogens and/or pathogen-derived peptides. Therefore, the ELISPOT assay can be used for the design, development, and evaluation of new vaccines.
Methods Mol Biol 2005
PMID:Detection of measles virus-specific interferon-gamma-secreting T-cells by ELISPOT. 1593 54

For measles viruses, fusion on the cell membrane is an important initial step in the entry into the infected cells. The recent research indicated that hemagglutinin firstly leads the conformational changes in the fusion protein then co-mediates the membrane fusion. In the work, we use the co-immunoprecipitation and pull-down techniques to identify the interactions among fusion protein, hemagglutinin and signaling lymphocyte activation molecule (SLAM), which reveal that the three proteins can form a functional complex to mediate the SLAM-dependent fusion. Moreover, under the confocal microscope, fusion protein and hemagglutinin protein can show the cocapping mediated by the SLAM. So fusion protein not only is involved in the fusion but also might directly interact with the SLAM to be a new fusion-trimer model, which might account for the infection mechanism of measles virus.
J Biochem Mol Biol 2005 Jul 31
PMID:Interactions among measles virus hemagglutinin, fusion protein and cell receptor signaling lymphocyte activation molecule (SLAM) indicating a new fusion-trimer model. 1605 2

Regulation, recognition and cell signaling involve the coordinated actions of many players. To achieve this coordination, each participant must have a valid identification (ID) that is easily recognized by the others. For proteins, these IDs are often within intrinsically disordered (also ID) regions. The functions of a set of well-characterized ID regions from a diversity of proteins are presented herein to support this view. These examples include both more recently described signaling proteins, such as p53, alpha-synuclein, HMGA, the Rieske protein, estrogen receptor alpha, chaperones, GCN4, Arf, Hdm2, FlgM, measles virus nucleoprotein, RNase E, glycogen synthase kinase 3beta, p21(Waf1/Cip1/Sdi1), caldesmon, calmodulin, BRCA1 and several other intriguing proteins, as well as historical prototypes for signaling, regulation, control and molecular recognition, such as the lac repressor, the voltage gated potassium channel, RNA polymerase and the S15 peptide associating with the RNA polymerase S-protein. The frequent occurrence and the common use of ID regions in important protein functions raise the possibility that the relationship between amino acid sequence, disordered ensemble and function might be the dominant paradigm for the molecular recognition that serves as the basis for signaling and regulation by protein molecules.
J Mol Recognit
PMID:Showing your ID: intrinsic disorder as an ID for recognition, regulation and cell signaling. 1609 5


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