Serum concentrations of lectin-pathway components in healthy neonates, children and adults: mannan-binding lectin (MBL), M-, L-, and H-ficolin, and MBL-associated serine protease-2 (MASP-2)

This study aimed to measure serum concentrations of five lectin-pathway components, mannan-binding lectin (MBL), M-ficolin, L-ficolin, H-ficolin, and MBL-associated serine protease-2 (MASP-2), in healthy neonates and children, to determine if they change with age and to compare them with serum concentrations in healthy adults. Concentrations were measured in 141 preterm and 30 term neonates, in 120 children including infants and adolescents, and in 350 adults (97 for L-ficolin) by inhouse time-resolved immunofluorometric assays or commercially available enzyme-linked immunosorbent assays. The adjacent categories method applying Wilcoxon-Mann-Whitney tests was used to determine age categories where concentrations differed significantly. Displaying serum concentration vs. age, an inverted-U shape (higher concentrations in children than in neonates and adults) was found for MBL and the ficolins, and an S-shape for MASP-2. Serum concentrations of all five lectin-pathway components were significantly lower in preterm neonates <32-wk gestational age compared to older neonates, infants, and children. Only M-ficolin in children >1 yr and H-ficolin in term neonates and in children were found to be comparable with adult values. MBL, M-, L-, and H-ficolin, and MASP-2 serum concentrations show important changes with age. The respective normal ranges for adults should not be used in the pediatric population. The age-specific pediatric ranges established here may be used instead.

Serodiagnosis of Echinococcus spp. infection: explorative selection of diagnostic antigens by peptide microarray

BACKGROUND: Production of native antigens for serodiagnosis of helminthic infections is laborious and hampered by batch-to-batch variation. For serodiagnosis of echinococcosis, especially cystic disease, most screening tests rely on crude or purified Echinococcus granulosus hydatid cyst fluid. To resolve limitations associated with native antigens in serological tests, the use of standardized and highly pure antigens produced by chemical synthesis offers considerable advantages, provided appropriate diagnostic sensitivity and specificity is achieved. METHODOLOGY/PRINCIPAL FINDINGS: Making use of the growing collection of genomic and proteomic data, we applied a set of bioinformatic selection criteria to a collection of protein sequences including conceptually translated nucleotide sequence data of two related tapeworms, Echinococcus multilocularis and Echinococcus granulosus. Our approach targeted alpha-helical coiled-coils and intrinsically unstructured regions of parasite proteins potentially exposed to the host immune system. From 6 proteins of E. multilocularis and 5 proteins of E. granulosus, 45 peptides between 24 and 30 amino acids in length were designed. These peptides were chemically synthesized, spotted on microarrays and screened for reactivity with sera from infected humans. Peptides reacting above the cut-off were validated in enzyme-linked immunosorbent assays (ELISA). Peptides identified failed to differentiate between E. multilocularis and E. granulosus infection. The peptide performing best reached 57% sensitivity and 94% specificity. This candidate derived from Echinococcus multilocularis antigen B8/1 and showed strong reactivity to sera from patients infected either with E. multilocularis or E. granulosus. CONCLUSIONS/SIGNIFICANCE: This study provides proof of principle for the discovery of diagnostically relevant peptides by bioinformatic selection complemented with screening on a high-throughput microarray platform. Our data showed that a single peptide cannot provide sufficient diagnostic sensitivity whereas pooling several peptide antigens improved sensitivity; thus combinations of several peptides may lead the way to new diagnostic tests that replace, or at least complement conventional immunodiagnosis of echinococcosis. Our strategy could prove useful for diagnostic developments in other pathogens.

Bullous pemphigoid: from the clinic to the bench

Bullous pemphigoid (BP) constitutes the most frequent autoimmune subepidermal blistering disease. It is associated with autoantibodies directed against the BP antigens 180 (BP180, BPAG2) and BP230 (BPAG1-e). The pathogenicity of anti-BP180 antibodies has been convincingly demonstrated in animal models. The clinical features of BP are extremely polymorphous. The diagnosis of BP critically relies on immunopathologic findings. The recent development of novel enzyme-linked immunosorbent assays has allowed the detection of circulating autoantibodies with relatively high sensitivity and specificity. Although potent topical steroids have emerged in the past decade as first-line treatment of BP, management of the disease may be challenging.

The systemic angiogenic response during bone healing

INTRODUCTION: Angiogenesis is known to be a critical and closely regulated step during bone formation and fracture healing driven by a complex interaction of various cytokines. Delays in bone healing or even nonunion might therefore be associated with altered concentrations of specific angiogenic factors. These alterations might in turn be reflected by changes in serum concentrations. METHOD: To determine physiological time courses of angiogenic cytokines during fracture healing as well as possible changes associated with failed consolidation, we prospectively collected serum samples from patients who had sustained surgical treatment for a long bone fracture. Fifteen patients without fracture healing 4 months after surgery (nonunion group) were matched to a collective of 15 patients with successful healing (union group). Serum concentrations of angiogenin (ANG), angiopoietin 2 (Ang-2), basic fibroblast growth factor (bFGF), platelet derived growth factor AB (PDGF-AB), pleiotrophin (PTN) and vascular endothelial growth factor (VEGF) were measured using enzyme linked immunosorbent assays over a period of 24 weeks. RESULTS: Compared to reference values of healthy uninjured controls serum concentrations of VEGF, bFGF and PDGF were increased in both groups. Peak concentrations of these cytokines were reached during early fracture healing. Serum concentrations of bFGF and PDGF-AB were significantly higher in the union group at 2 and 4 weeks after the injury when compared to the nonunion group. Serum concentrations of ANG and Ang-2 declined steadily from the first measurement in normal healing fractures, while no significant changes over time could be detected for serum concentrations of these factures in nonunion patients. PTN serum levels increased asymptotically over the entire investigation in timely fracture healing while no such increase could be detected during delayed healing. CONCLUSION: We conclude that fracture healing in human subjects is accompanied by distinct changes in systemic levels of specific angiogenic factors. Significant alterations of these physiologic changes in patients developing a fracture nonunion over time could be detected as early as 2 (bFGF) and 4 weeks (PDGF-AB) after initial trauma surgery.

MOUSE MAMMARY TUMOR VIRUS EPITOPES EXPRESSED ON TUMOR SPECIFIC TRANSPLANTATION ANTIGENS OF CHEMICALLY INDUCED C3H/HEJ SARCOMAS (TSTA, FIBROSARCOMA, CARCINOGENESIS, MMTV, METHYLCHOLANTHRONE)

This dissertation addressed the hypothesis that the unique tumor specific transplantation antigens (TSTA) of chemically induced sarcomas express epitopes encoded by endogenous viral genes. TSTA from two 3-methylcholanthrene-induced, C3H/HeJ fibrosarcomas (MCA-F and MCA-D) were serologically assessed for viral epitopes in an enzyme-linked immunospecific assay (ELISA) and by immunoaffinity chromatography. Initial evidence with an anti-TSTA antiserum suggested that TSTA were associated with mouse mammary tumor virus (MMTV) peptides, but not peptides from murine leukemia virus (MuLV). TSTA extracted from MCA-F, was assessed with specific anti viral antibodies at three levels of purification for its association with MuLV peptides (gp 70 and p 15E) and MMTV peptides (gp52, gp36 and p27). The results demonstrate that purified preparations enriched for TSTA activity are devoid of MuLV epitopes, but enriched for a subset of MMTV epitopes. Immunoaffinity supports constructed with anti-MMTV antibodies retained TSTA from partially purified MCA-F or MCA-D extracts. Immunoaffinity chromatography with antibodies against individual MMTV peptides demonstrated that the MCA-F TSTA was specifically retained by anti-gp36 and anti-p27 supports, but not by anti-gp52 supports nor a support made with bovine serum albumin. Analysis of the affinity purified TSTA preparations by HPGPC and SDS-PAGE revealed only a few components. Application of the anti-gp36 and anti-p27 retained materials to HPGPC and subsequent in vivo analysis demonstrated that the TSTA migrated in a low and a high molecular weight region. These results suggest that TSTA specificity in C3H/HeJ mice, results from MMTV recombinant proteins. ^

Comparative assessment of ELISAs using recombinant saposin-like protein 2 and recombinant cathepsin L-1 from Fasciola hepatica for the serodiagnosis of human Fasciolosis.

Two recombinant Fasciola hepatica antigens, saposin-like protein-2 (recSAP2) and cathepsin L-1 (recCL1), were assessed individually and in combination in enzyme-linked immunosorbent assays (ELISA) for the specific serodiagnosis of human fasciolosis in areas of low endemicity as encountered in Central Europe. Antibody detection was conducted using ProteinA/ProteinG (PAG) conjugated to alkaline phosphatase. Test characteristics as well as agreement with results from an ELISA using excretory-secretory products (FhES) from adult stage liver flukes was assessed by receiver operator characteristic (ROC) analysis, specificity, sensitivity, Youdens J and overall accuracy. Cross-reactivity was assessed using three different groups of serum samples from healthy individuals (n=20), patients with other parasitic infections (n=87) and patients with malignancies (n=121). The best combined diagnostic results for recombinant antigens were obtained using the recSAP2-ELISA (87% sensitivity, 99% specificity and 97% overall accuracy) employing the threshold (cut-off) to discriminate between positive and negative reactions that maximized Youdens J. The findings showed that recSAP2-ELISA can be used for the routine serodiagnosis of chronic fasciolosis in clinical laboratories; the use of the PAG-conjugate offers the opportunity to employ, for example, rabbit hyperimmune serum for the standardization of positive controls.

A screening for anti-West Nile immunoglobulin-G and immunoglobulin-M in the Cameron County Hispanic cohort

This study is designed to be a cross-sectional, retrospective analysis of the seroprevalence of anti-WNV antibodies in 1,006 plasma samples collected from February 2, 2006 to June 18, 2007 originally for The Cameron County Hispanic Cohort: Extreme obesity and uncontrolled diabetes on the U.S.-Mexico border, major concerns for populations with health disparities. The aim of this thesis research is to give a more up-to-date picture of Flavivirus activity in south Texas, which can potentially contribute to the surveillance objective of arboviral control in this area. A West Nile virus (WNV) seroprevalence study in humans in this particular area has never before been completed. Plasma samples were tested using immunoglobulin-G (IgG) and immunoglobulin-M (IgM) WNV enzyme linked immunosorbent assays (ELISA). Estimated seroprevalence for this particular population was 0.98% or 9.8 cases of West Nile disease per 1,000 citizens. After IgG testing, seroprevalence in the study population was found to be 15.4%. Specimens tested for WNV IgG were compared with a subset of specimens (N=803) tested for history of primary dengue virus (DENV) infection. Of the 803 specimens tested for IgG to DENV, 308 were positive. Of the 132 positive WNV IgG specimens in the subset, 131 (99.2%) tested also tested positive for DENV IgG. It would be helpful to use standard plaque reduction neutralization testing to determine if the seroprevalence is in fact lower because of cross-reaction to DENV on testing. Regardless of the possibility of other Flavivirus activity occurring prior to the introduction of WNV into the United States and the potential for cross-reactivity, Texas has ranked in the top 5 states with the highest, laboratory confirmed incidence of infection with WNV since 2003. Indicating that climate factors and the presence of suitable vectors makes Texas a hotspot for WNV activity. ^ A description of the study population by age, gender, and income was done indicating a statistically significant income difference with a mean household income per year being $13,413.55 for a case and $20,268.80 for non-cases (p=0.001). Lower income neighborhoods should be targeted for education and prevention of vector-borne diseases during the summer months in Cameron County. With respect to gender, being male has been noted in the literature to be a risk factor for infection with WNV (25). In this study, females comprised approximately 68% of the study population, they also made up 66.5% of the positive IgG specimens. An odds ratio of 0.91 indicates that women are less likely to be IgG positive for WNV as compared to men; however, this was not found to be significant based on the 95% confidence interval, but is consistent with the literature. When looking at age difference between positive and negative/equivocal cases, there was no statistical difference found between the two groups. ^ We concluded that this study will enable us to understand the epidemiology of WNV transmission since its introduction into the United States and hopefully to maintain or improve the current measures we have in place to prevent infections that are seen annually with WNV.^

Remoção da biomassa algal e determinação da concentração de microcistina pelo Método ELISA em ensaios de coagulação, sedimentação, filtração e adsorção

Nessa pesquisa são relatados os resultados da determinação das concentrações de microcistina e de biomassa algal após as várias etapas de tratamento de amostras de água coletadas junto ao reservatório de Barra Bonita-SP visando obtenção de água potável. O tratamento foi realizado em escala de laboratório com e sem aplicação de carvão ativado em pó (CAP) e as etapas foram: coagulação com aplicação de cloreto férrico, sedimentação, filtração em papel de filtro. Foi possível observar que a pré-clarificação desse tipo de água por coagulação seguida de sedimentação requereu dosagens relativamente elevadas de cloreto férrico (80 mg/L), tendo sido verificada eficiência muito baixa de remoção de microcistina nas etapas de tratamento por sedimentação seguida de filtração, quando não foi aplicado CAP. Apenas com a aplicação de CAP a microcistina foi reduzida à níveis que atendessem os padrões de potabilidade previstos na Portaria 518/04 (concentração menor que 1 μg/L). A determinação de microcistina pelo método que utiliza Imunoadsorventes Ligados à Enzima (ELISA) mostrou-se uma ferramenta útil e confiável para detectar e quantificar essa toxina, embora ainda apresente custo relativamente elevado.

Tetracycline-Inducible packaging cell line for production of Flavivirus replicon particles.

We have previously developed replicon vectors derived from the Australian flavivirus Kunjin that have a unique noncytopathic nature and have been shown to direct prolonged high-level expression of encoded heterologous genes in vitro and in vivo and to induce strong and long-lasting immune responses to encoded immunogens in mice. To facilitate further applications of these vectors in the form of virus-like particles (VLPs), we have now generated a stable BHK packaging cell line, tetKUNCprME, carrying a Kunjin structural gene cassette under the control of a tetracycline-inducible promoter. Withdrawal of tetracycline from the medium resulted in production of Kunjin structural proteins that were capable of packaging transfected and self-amplified Kunjin replicon RNA into the secreted VLPs at titers of up to 1.6 x 10(9) VLPs per ml. Furthermore, secreted KUN replicon VLPs from tetKUNCprME cells could be harvested continuously for as long as 10 days after RNA transfection, producing a total yield of more than 1010 VLPs per 106 transfected cells. Passaging of VLPs on Vero cells or intracerebral injection into 2- to 4-day-old suckling mice illustrated the complete absence of any infectious Kunjin virus. tetKUNCprME cells were also capable of packaging replicon RNA from closely and distantly related flaviviruses, West Nile virus and dengue virus type 2, respectively. The utility of high-titer KUN replicon VLPs was demonstrated by showing increasing CD8(+)-T-cell responses to encoded foreign protein with increasing doses of KUN VLPs. A single dose of 2.5 x 10(7) VLPs carrying the human respiratory syncytial virus M2 gene induced 1,400 CD8 T cells per 10(6) splenocytes in an ex vivo gamma interferon enzyme-linked immunospot assay. The packaging cell line thus represents a significant advance in the development of the noncytopathic Kunjin virus replicon-based gene expression system and may be widely applicable to the basic studies of flavivirus RNA packaging and virus assembly as well as to the development of gene expression systems based on replicons from different flaviviruses.

West Nile and St. Louis encephalitis virus antibody seroconversion, prevalence, and persistence in naturally infected pig-tailed macaques (Macaca nemestrina)

Pig-tailed macaques (Macaca nemestrina) naturally infected with West Nile virus were monitored from 1999 to 2005 to determine virus-specific antibody seroconversion, prevalence, and persistence. Antibodies persisted for up to 36 months, as detected by epitope-blocking enzyme-linked immunosorbent and hemagglutination inhibition assays. Exposure to cocirculating St. Louis encephalitis virus was evaluated by Western blotting and immunofluorescence assays.

A Rapid and Sensitive High-Throughput Screening Method to Identify Compounds Targeting Protein–Nucleic Acids Interactions

DNA-binding and RNA-binding proteins are usually considered ‘undruggable’ partly due to the lack of an efficient method to identify inhibitors from existing small molecule repositories. Here we report a rapid and sensitive high-throughput screening approach to identify compounds targeting protein–nucleic acids interactions based on protein–DNA or protein–RNA interaction enzyme-linked immunosorbent assays (PDI-ELISA or PRI-ELISA). We validated the PDI-ELISA method using the mammalian highmobility- group protein AT-hook 2 (HMGA2) as the protein of interest and netropsin as the inhibitor of HMGA2–DNA interactions. With this method we successfully identified several inhibitors and an activator for HMGA2–DNA interactions from a collection of 29 DNA-binding compounds. Guided by this screening excise, we showed that netropsin, the specific inhibitor of HMGA2–DNA interactions, strongly inhibited the differentiation of the mouse pre-adipocyte 3T3-L1 cells into adipocytes, most likely through a mechanism by which the inhibition is through preventing the binding of HMGA2 to the target DNA sequences. This method should be broadly applicable to identify compounds or proteins modulating many DNA-binding or RNA-binding proteins.

Tonsils of the soft palate do not mediate the response of pigs to oral vaccination with heat-inactivated Mycobacterium bovis

Mycobacterium bovis causes animal tuberculosis (TB) in cattle, humans, and other mammalian species, including pigs. The goal of this study was to experimentally assess the responses of pigs with and without a history of tonsillectomy to oral vaccination with heat-inactivated M. bovis and challenge with a virulent M. bovis field strain, to compare pig and wild boar responses using the same vaccination model as previously used in the Eurasian wild boar (Sus scrofa), to evaluate the use of several enzyme-linked immunosorbent assays (ELISAs) and lateral flow tests for in vivo TB diagnosis in pigs, and to verify if these tests are influenced by oral vaccination with inactivated M. bovis. At necropsy, the lesion and culture scores were 20% to 43% higher in the controls than those in the vaccinated pigs. Massive M. bovis growth from thoracic tissue samples was observed in 4 out of 9 controls but in none of the 10 vaccinated pigs. No effect of the presence or absence of tonsils was observed on these scores, suggesting that tonsils are not involved in the protective response to this vaccine in pigs. The serum antibody levels increased significantly only after challenge. At necropsy, the estimated sensitivities of the ELISAs and dual path platform (DPP) assays ranged from 89% to 94%. In the oral mucosa, no differences in gene expression were observed in the control group between the pigs with and without tonsils. In the vaccinated group, the mRNA levels for chemokine (C-C motif) receptor 7 (CCR7), interferon beta (IFN-β), and methylmalonyl coenzyme A mutase (MUT) were higher in pigs with tonsils. Complement component 3 mRNA levels in peripheral blood mononuclear cells (PBMC) increased with vaccination and decreased after M. bovis challenge. This information is relevant for pig production in regions that are endemic for M. bovis and for TB vaccine research.

Oligonucleotide ligation assay-based DNA chip for multiplex detection of single nucleotide polymorphism

An oligonucleotide ligation assay-based DNA chip has been developed to detect single nucleotide polymorphism. Synthesized nonamers, complementary to the flanking sequences of the mutation sites in target DNA, were immobilized onto glass slides through disulfide bonds on their 5' terminus. Allele-specific pentamers annealed adjacent to the nonamers on the complementary target DNA, containing 5'-phosphate groups and biotin labeled 3'-ends, were mixed with the target DNA in tube. Ligation reactions between nonamers and pentamers were carried out on chips in the presence of T4 DNA ligase. Ligation products were directly visualized on chips through enzyme-linked assay. The effect of G:T mismatch at different positions of pentamers on the ligation were evaluated. The results showed that any mismatch between pentamer and the target DNA could lead to the decrease of ligation, which can be detected easily. The established approach was further used for multiplex detection of mutations in rpoB gene of rifampin-resistant Mycobacterium tuberculosis clinical isolates. (C) 2003 Elsevier B.V. All rights reserved.

In vitro o-antigen ligase assay

WaaL is a membrane enzyme that catalyzes the glycosidic bonding of a sugar at the proximal end of the undecaprenyl-diphosphate (Und-PP)-O-antigen with a terminal sugar of the lipid A-core oligosaccharide (OS). This is a critical step in lipopolysaccharide synthesis. We describe here an assay to perform the ligation reaction in vitro utilizing native substrates.