Molecular characterization of infectious bursal disease virus (IBDV) isolated in Argentina indicates a regional lineage.

In Argentina, classical vaccines are used to control infectious bursal disease virus (IBDV); however, outbreaks of IBDV are frequently observed. This could be due to failures in the vaccination programs or to the emergence of new strains, which would be able to break through the protection given by vaccines. Hence, genetic characterization of the viruses responsible for the outbreaks that occurred in recent years is crucial for the evaluation of the control programs and the understanding of the epidemiology and evolution of IBDV. In this study, we characterized 51 field samples collected in Argentina (previously identified as IBDV positive) through the analysis of previously identified apomorphic sequences. Phylogenetic analysis of regVP2 showed that 42 samples formed a unique cluster (Argentinean lineage), seven samples were typical classical strains (one of them was a vaccine strain), and two belonged to the very virulent lineage (vvIBDV). Interestingly, when the analysis was performed on the regVP1 sequences, the field samples segregated similarly to regVP2; thus, we observed no evidence of a reassortment event in the Argentinean samples. Amino acid sequence analysis of regVP2 showed a particular pattern of residues in the Argentinean lineage, particularly the presence of T272, P289 and F296, which had not been reported before as signature sequences for any IBDV phenotype. Notably, the residue S254, characteristic of the antigenic variant, was not present in any of the Argentinean samples.

Detection and kinetic analysis of Epinotia aporema granulovirus in its lepidopteran host by real-time PCR.

Epinotia aporema granulovirus (EpapGV) has attracted interest as a potential biocontrol agent of the soybean pest Epinotia aporema in Argentina. Studies on virus/host interactions conducted so far have lacked an accurate method to assess the progress of virus load during the infection process. The present paper reports the development of a real-time PCR for EpapGV and its application to describe viral kinetics following ingestion of two different virus doses by last-instar E. aporema larvae. Real-time PCR was shown to be a reliable method to detect and quantify the presence of EpapGV in the analyzed samples. The increase in virus titer (log) exhibited a sigmoidal pattern, with an exponential growth phase between 24 and 48 h postinfection for both initial doses tested.

Molecular characterization of chicken infectious anemia virus circulating in Argentina during 2007.

Chicken infectious anemia virus (CAV) is a worldwide-distributed infectious agent that affects commercial poultry. Although this agent was first detected in Argentina in 1994, no further studies on CAV in this country were reported after that. The recent increased occurrence of clinical cases of immunosuppression that could be caused by CAV has prompted this study. Our results confirmed that CAV is still circulating in commercial flocks in Argentina. Phylogenetic analysis focusing on the VP1 nucleotide sequence showed that all Argentinean isolates grouped together in a cluster, sharing a high similarity (> 97%) with genotype B reference strains. However, Argentinean isolates were distantly related to other strains commonly used for vaccination in this country, such as Del-Ros and Cux-1. Sequence analysis of predicted VP1 peptides showed that most of the Argentinean isolates have a glutamine residue at positions 139 and 144, suggesting that these isolates might have a reduced spread in cell culture compared with Cux-1. In addition, a particular amino acid substitution at position 290 is present in all studied Argentinean isolates, as well as in several VP1 sequences from Malaysia, Australia, and Japan isolates. Our results indicate that it is possible to typify CAV strains by comparison of VPI nucleotide sequences alone because the same tree topology was obtained when using the whole genome sequence. The molecular analysis of native strains sheds light into the epidemiology of CAV in Argentinean flocks. In addition, this analysis could be considered in future control strategies focused not only on breeders but on broilers and layer flocks.

Detection of bovine viral diarrhoea virus (BVDV) nucleic acid and antigen in different organs of water buffaloes (Bubalus bubalis).

Bovine viral diarrhoea virus (BVDV) is a pestivirus that infects mainly bovine cattle. Nevertheless, there are several reports about infections in other members of the Artiodactyla order including serological studies, that indicate infection of BVDV in buffaloes. The aim of this article is to study the presence of BVDV in three young water buffaloes, displaying nonspecific clinical signs, compatible with the BVDV infection. Both immunohistochemistry and RT-PCR confirmed the presence of BVDV in the animals. The sequence analysis on RT-PCR amplicons revealed high identity with reference strains of genotypes 1a and 1b. Although BVDV was unequivocally identified in the sick animals, it has not been proved it is responsible for the clinical signs. Further studies are needed to clarify the pathogenic role of BVDV infection in this animal species, and the role of buffaloes in the epidemiology of BVDV infection.

Molecular epidemiology of foot-and-mouth disease virus types A and O isolated in Argentina during the 2000-2002 epizootic.

During 2000-2002 a foot-and-mouth disease (FMD) epizootic affected Argentina and spread across the country resulting in more than 2500 outbreaks. In order to study the evolution of the FMD viruses (FMDV) and help with disease control measures, a genetic characterization and phylogenetic analysis was performed of 43 field isolates representative of the epizootic. The nucleotide sequence of the VP1-coding region was determined for the viruses and used in this study. Two serotype A lineages, A/Arg/00 and A/Arg/01 (1000/1000 bootstrap value) and two different serotype O/Arg/00 lineages (848/1000 bootstrap value) were identified. Phylogenetic analysis showed that viruses A/Arg/01 and O/Arg/00 could be related with former South American isolates, while the origin of A Argentina 2000 viruses remains unclear. Comparison of the amino acid sequences with vaccine reference strains revealed differences at critical antigenic sites for emergent strains A/Arg/00 and A/Arg/01, leading to a change in the current vaccine formulation.

Retinoic acid induced differentiated neuroblastoma cells show increased expression of the beta A4 amyloid gene of Alzheimer's disease and an altered splicing pattern.

Retinoic acid (RA) induced differentiation of SH-SY5Y neuroblastoma cells is associated with more than a tenfold induction of total Alzheimer's disease beta A4 amyloid protein precursor (APP) mRNA as analyzed by Northern blot hybridisation. S1 nuclease protection experiments reveal that the splicing pattern of these differentiated cells is altered in favor of APP695 mRNA, coding for the shortest amyloidogenic beta A4 amyloid precursor protein. Induction of differentiation of SH-SY5Y cells with NGF leads to a fivefold increase of total APP mRNA without change in the splicing pattern. This suggests that RA but not NGF induces factor(s) which are responsible for an APP hnRNA splicing favoring APP695 mRNA.

Synthesis and secretion of Alzheimer amyloid beta A4 precursor protein by stimulated human peripheral blood leucocytes.

Alzheimer amyloid precursor proteins (APP) are actively secreted by stimulated human peripheral mononuclear blood leucocytes (PMBLs). Induction of APP transcription, translation and secretion was observed with several T cell mitogens but was highest with phytohemagglutinin. The time course of induction is similar to that reported for IL-2 and IL-2 receptor. We suggest that APP may play an important role in the construction of the immunological network and the differentiation of T cells.

In-vitro matured human macrophages express Alzheimer's beta A4-amyloid precursor protein indicating synthesis in microglial cells.

Microglia which are consistently associated with Alzheimer's disease (AD) senile plaques are part of the mononuclear phagocyte system. In-vitro matured human monocyte-derived macrophages feature many immunological characteristics of microglia. We found strong constitutive expression of Alzheimer's beta A4-amyloid precursor protein (APP) in human mononuclear phagocytes after terminal in-vitro maturation from monocytes to macrophages. Amyloid has previously been found to be associated with microglia in AD brains, however, it remained unclear whether the material was synthesized in or had been phagocytosed by the cells. The findings presented here support the assumption that brain microglia may contribute to APP synthesis in AD brain.

Inhibition of heme detoxification processes underlies the antimalarial activity of terpene isonitrile compounds from marine sponges.

A series of terpene isonitriles, isolated from marine sponges, have previously been shown to exhibit antimalarial activities. Molecular modeling studies employing 3D-QSAR with receptor modeling methodologies performed with these isonitriles showed that the modeled molecules could be used to generate a pharmacophore hypothesis consistent with the experimentally derived biological activities. It was also shown that one of the modeled compounds, diisocyanoadociane (4), as well as axisonitrile-3 (2), both of which have potent antimalarial activity, interacts with heme (FP) by forming a coordination complex with the FP iron. Furthermore, these compounds were shown to inhibit sequestration of FP into beta-hematin and to prevent both the peroxidative and glutathione-mediated destruction of FP under conditions designed to mimic the environment within the malaria parasite. By contrast, two of the modeled diterpene isonitriles, 7-isocyanoamphilecta-11(20),15-diene (12) and 7-isocyano-15-isothiocyanatoamphilecta-11(20)-ene (13), that displayed little antimalarial activity also showed little inhibitory activity in these FP detoxification assays. These studies suggest that the active isonitrile compounds, like the quinoline antimalarials, exert their antiplasmodial activity by preventing FP detoxification. Molecular dynamics simulations performed with diisocyanoadociane (4) and axisonitrile-3 (2) allowed their different binding to FP to be distinguished.

Central cholinergic functions in human amyloid precursor protein knock-in/presenilin-1 transgenic mice.

Alzheimer's disease is characterized by amyloid peptide formation and deposition, neurofibrillary tangles, central cholinergic dysfunction, and dementia; however, the relationship between these parameters is not well understood. We studied the effect of amyloid peptide formation and deposition on central cholinergic function in knock-in mice carrying the human amyloid precursor protein (APP) gene with the Swedish/London double mutation (APP-SL mice) which were crossbred with transgenic mice overexpressing normal (PS1wt) or mutated (M146L; PS1mut) human presenilin-1. APP-SLxPS1mut mice had increased levels of Abeta peptides at 10 months of age and amyloid plaques at 14 months of age while APP-SLxPS1wt mice did not have increased peptide levels and did not develop amyloid plaques. We used microdialysis in 15-27 months old mice to compare hippocampal acetylcholine (ACh) levels in the two mouse lines and found that extracellular ACh levels were slightly but significantly reduced in the APP-SLxPS1mut mice (-26%; P=0.044). Exploratory activity in the open field increased hippocampal ACh release by two-fold in both mouse lines; total and relative increases were not significantly different for the two strains under study. Similarly, infusion of scopolamine (1 microM) increased hippocampal ACh release to a similar extent (3-5-fold) in both groups. High-affinity choline uptake, a measure of the ACh turnover rate, was identical in both mouse lines. Neurons expressing choline acetyltransferase were increased in the septum of APP-SLxPS1mut mice (+26%; P=0.046). We conclude that amyloid peptide production causes a small decrease of extracellular ACh levels. The deposition of amyloid plaques, however, does not impair stimulated ACh release and proceeds without major changes of central cholinergic function.

Pulmonary involvement in zinc fume fever.

A patient with the clinical history of recurring zinc fume fever underwent an experimental welding exposure; this resulted in a systemic reaction and a distinct self-limiting response in the periphery of the lung, demonstrated by pulmonary function tests and bronchoalveolar lavage. These pulmonary changes observed for the first time in man were reproducible.

Implantation of in vitro endothelialized polytetrafluoroethylene grafts in human beings. A preliminary report.

To assess the impact of in vitro endothelialization on prosthetic graft patency, we performed femorotibial reconstruction in four patients. Polytetrafluoroethylene grafts (6 mm), lined with cultivated autologous endothelial cells, harvested from the veins of the forearm, were used. Autologous endothelial cells were harvested enzymatically and characterized by morphology and factor VII staining. After a cultivation period of 17 to 23 days, the cell count increased from 27 +/- 3 x 10(4) endothelial cells to 5.4 +/- 1.1 x 10(6). Endothelial cell seeding on polytetrafluoroethylene prostheses was then performed. To improve endothelial cell attachment to the graft surface, polytetrafluoroethylene grafts (60 to 70 cm; 6 mm diameter) were precoated with fibrin glue containing fibrin and fibronectin and the fibrinolysis inhibitor aprotinin. Seeding density of 49 +/- 10 x 10(3) endothelial cells per square centimeter yielded a preconfluent monolayer immediately after seeding, as demonstrated by scanning electron microscopy. A second cultivation period of 6 days, after seeding and before implantation, was necessary for establishment of a confluent monolayer and to allow for maturation of the endothelial cell cytoskeleton as well as production and excretion of extracellular matrix. Grafts endothelialized in vitro were implanted in four patients requiring femorotibial reconstruction. Scintigraphic studies with indium 111-labeled platelets demonstrated little or no platelet deposition, indicating persistent endothelialization. All grafts remained patent at 3 months after implantation.

Lung involvement in scleroderma.

Lung involvement (LI) was studied by lung function (LF) in 101 scleroderma patients (circumscribed scleroderma, n = 17; progressive systemic scleroderma [PSS], n = 84; with the subtypes I, acroscleroderma [n = 19]; 2, proximal ascending scleroderma [n = 61]; 3, trunk scleroderma [n = 4]). Eighteen percent of morphea, 32 percent of type 1, 56 percent of type 2, and 75 percent of type 3 patients had impaired LF. The LI was more frequent (57 percent vs 45 percent) and more severe (20 percent vs 3 percent) in PSS with systemic inflammation (form A) compared to those without (form B). Elevated lymphocytes/neutrophils in bronchoalveolar lavage (BAL) were found associated with form A and severe LI. The LF of patients showing an inflammatory cell pattern in initial BAL (n = 3) worsened, whereas those with normal BAL findings (n = 4) did not. Collagenase activity in BAL was significantly elevated in those with elevated lymphocytes/neutrophils in lavage. Patients with type 2 or 3 of PSS, especially form A, carry a higher risk of developing severe LI than circumscribed scleroderma, type 1, or form B patients. Differential cell count and collagenase activity in BAL is correlated with active disease and provides prognostic information.

Alternative splicing of the beta A4 amyloid gene of Alzheimer's disease in cortex of control and Alzheimer's disease patients.

An S1 nuclease protection assay was designed to study the splicing pattern of the alternatively spliced beta A4 amyloid gene (APP gene) of Alzheimer's disease (AD). We determined the splicing pattern of the APP gene in fetal, adult, aged adult and AD human cortex. The results suggest that alternative splicing of the APP gene in AD is not significantly different from age-matched controls, but distinct from the developing fetal brain.

Expression of L-APP mRNA in brain cells.

Several reports addressed the issue of how the alternative splicing of exon 7 and 8 in the APP pre-mRNA is regulated in different tissues. Of special interest here was the potential involvement of exon 7 containing APP splice isoforms, since this exon codes for a serine protease inhibitor and is therefore of putative relevance for amyloidogenic catabolism of the precursor protein. The recent identification of a third alternative splice site in close proximity to the beta A4-amyloid portion in the APP gene which may also increase APP amyloidogenicity, allowed us to investigate its regulation in cells of the central nervous system. With our assay, we were able to resolve six different APP isoforms of the eight potential isoforms which can be generated from the three alternatively spliced exons 7, 8, and 15. We demonstrate here that, in addition to rat brain microglia cells, astrocyte-enriched cultures also skip the novel alternative 3'-splice site in front of exon 15, generating L-APP mRNA. Neurons are the only cells in the central nervous system which seem to use the 3'-splice site of intron 14 nearly 100%. Interestingly, this very 3'-splice site is the only one present in the APP gene that completely matches the consensus sequence for the branchpoint sequence proposed for introns. We would therefore suggest that neurons lack a specific splicing factor which inhibits the use of the rather strong 3'-splice site in front of exon 15. It remains to be shown whether this is also the case for neurons in Alzheimer's disease.

Development and characterization of a monoclonal antibody 369.2B specific for the carboxyl-terminus of the beta A4 peptide.

The beta-amyloid deposits in Alzheimer's diseased (AD) brain are made up mainly of beta A4 peptides which show both N- and C-terminal heterogeneity. The predominant C-terminal species, identified by peptide sequencing of purified beta-amyloid, end either at position 40 or 42 of the beta A4 peptide. The distribution of these two beta A4 species in postmortem tissue as well as their generation in vitro could not be addressed previously due to the lack of specific antibodies that could differentiate them. This report describes the generation of a highly specific monoclonal antibody, MAb 369.2B which was raised against a synthetic peptide consisting of the C-terminus of the 1-42 beta A4 species. MAb 369.2B does not recognize the shorter beta A4 species 1-40 in solution or in solid phase. Furthermore, both beta A4 1-40 and 1-43 were unable to absorb out the antibody when used immunocytochemically. The regional distribution of MAb 369.2B immunoreactivity in AD and non-AD brain samples were compared to MAb 286.8A, an antibody raised against the N-terminal region of beta A4. Overall, the staining patterns were very similar. In AD cases with extreme vascular involvement, the N-terminal (MAb 286.8A) antibody was more likely to label the vascular basement membrane of affected vessels, and to label them more uniformly. In addition, preliminary quantitative analyses revealed that the C-terminal antibody labeled fewer, larger plaques than the N-terminal antibody. Qualitative analyses revealed that these smaller plaques were typically subregions of the larger plaques. The data corroborate the biochemical findings of N-terminal raggedness in beta A4 plaques. Further studies are required to explain this differential pattern of deposition.

Regulation and expression of the Alzheimer's beta/A4 amyloid protein precursor in health, disease, and Down's syndrome.

A four- to fivefold overexpression of the gene for the Alzheimer beta/A4 amyloid precursor protein (APP) in individuals with Down's Syndrome (DS) appears to be responsible for the fifty year earlier onset of Alzheimer's disease (AD) pathology in DS compared to the normal population. It is therefore likely that a deregulated overexpression of the APP gene is a risk factor for the beta/A4 amyloid formation. To test this hypothesis and to get a better understanding of how APP expression is regulated, we studied the 5' control region of the human APP gene, alternative splicing of the 19 APP exons, and APP biogenesis, metabolism and function. The analysis of the APP promoter revealed its similarity with those of housekeeping genes by the presence of a GC-rich region around the transcription start site and the lack of a TATA box. Gene transfer experiments showed this GC-rich region to contain overlapping binding sites for different transcription factors whose binding is mutually excluded. An imbalance between these factors may cause APP overexpression and predispose to AD pathology. Another putative risk factor for AD is regulation of splicing of exon 7 in APP mRNA's which changes in brain during aging. This is relevant for APP processing since exon 7 codes for a Kunitz protease inhibitory domain. Investigation of further splicing adjacent to the beta/A4 exons 16 and 17 which might also interfere with APP processing led to the identification of the leukocyte-derived (L-APP) splice forms which lack exon 15. In brain this splicing occurs in activated astrocytes and microglia. The localization of APP at synaptic sites in brain suggests that APP regulation and expression are critical determinants of a potential and early impairment of central synapses. This may be the case during pathological evolution of AD and DS when beta/A4 derived from synaptic APP is converted to beta/A4 amyloid by radical generation.

Mechanisms of amyloid deposition in Alzheimer's disease.

At the cellular level, Alzheimer's disease (AD) must be the result of neuronal dysfunction and degeneration leading to a reduction in synaptic density. Filamentous deposits of amyloid, which define the disease at the molecular level, occur within perikarya, axons, dendrites, and terminals of neurons as neurofibrillary tangles (NFT), in the extracellular neuropil as amyloid plaques (APC), and around blood vessels as amyloid congophilic angiopathy (ACA). These fibrillar amyloid protein aggregates are also found in the brain of all individuals with Down's syndrome after the age of 30 years. The amyloid deposits apparently occur in the terminal zones of neurons that develop NFT. It is suggested that amyloid deposition is of fundamental significance in AD and that a thorough understanding of amyloid formation will eventually lead to successful therapeutic intervention in AD. As elucidation of the reasons behind amyloid deposition must shed some light on the pathogenesis of AD, we review the current state of knowledge on the nature of the AD amyloid protein, its origin, and its formation. Although there is yet no agreement about the chemical nature of the amyloid protein of NFT, the major constituent of both APC and ACA has been shown to be a 4.5-kD amyloid protein originally termed "beta-protein" or "amyloid A4" which we now denote as "beta A4." Amyloid beta A4 protein is proteolytically derived from a transmembrane protein termed amyloid precursor protein (APP) which is encoded by a widely expressed gene on chromosome 21. Our present results are consistent with the possibility that amyloid formation requires membrane damage or APP molecules that are not or are incorrectly integrated into membranes. To allow the generation of the C-terminus of beta A4, one proteolytic cleavage step has to occur in the sequence that normally forms the transmembrane domain of the APP proteins. This cleavage is crucial for amyloid formation because we could show that the ability of synthetic beta A4 to form amyloid depositions is mainly based on hydrophobic parts of the sequence that have to interact with each other and build up large aggregates under physiologic conditions. Membrane association of APP is expected to interfere with this cleavage and the process of aggregation.

Association of progressive systemic scleroderma to several HLA-B and HLA-DR alleles.

The HLA-A, B, C, and DR loci of 136 patients with progressive systemic scleroderma have been determined. The patients were classified according to the extent of their skin affection and into groups with or without immunologic and inflammatory signs of the disease. The antigens of the A locus did not show any significant deviations in frequency of occurrence. An increase of HLA-B8 and HLA-DR3 was only proved in the male patient group. Furthermore, in the HLA-DR gene locus, an increase in frequency of HLA-DR1, 2, 3, and 5 could be found. However, in the total set of patients, only the correlation of HLA-DR5 with progressive systemic scleroderma reached significance. Patients suffering from the CREST (calcinosis, Raynaud's phenomenon, esophagus, sclerodactyly, and telangiectasia) syndrome showed an increase of HLA-DR1. Patients with inflammatory signs of the scleroderma showed an accumulation of HLA-DR2. Several HLA-linked genes control the susceptibility to scleroderma.

Preventable causative factors leading to hospital admission with decompensated heart failure.

OBJECTIVE: To determine the distribution and importance of various factors, especially the preventable ones, that contribute to cardiac decompensation and subsequent hospital admission for heart failure. METHODS: During a one year period patients were prospectively recruited and evaluated during their hospital stay by means of a structured personal interview by trained medical staff and through clinical examination and laboratory investigation. SETTING: The cardiological department at a teaching affiliated general community hospital in Berlin, Germany. PATIENTS: Consecutive sample of 179 patients admitted to hospital with acute decompensation of pre-existing heart failure. MAIN OUTCOME MEASURES: Proportional distribution of causative factors leading to hospital admission for heart failure; relative importance of preventable factors; details of patient compliance with diet and medication, and knowledge about medication. RESULTS: Mean (SD) age was 75.4 (9.9) years. Potential causative factors for decompensated heart failure were identified in 85.5% of patients. Lack of adherence to the medical regimen was the most commonly identified factor and was regarded as the cause of the cardiac decompensation in 41.9% of cases. Non-compliance with drugs was found in 23.5% of patients. Other factors related to hospital admission were coronary ischaemia (13.4%), cardiac arrhythmias (6.1%), uncontrolled hypertension (5.6%), and inadequate preadmission treatment (12.3%). In all, 54.2% of admissions could be regarded as preventable. CONCLUSIONS: Many hospital admissions for decompensation of chronic heart failure in patients at a district hospital in Berlin are preventable. Measures are necessary to improve this situation and evaluation of programmes that include patient education, patient follow up, and physician training is needed.