An enzyme-linked immunosorbent assay (elisa) for the detection of antibodies against babesia boris in cattle

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Aspectos estruturais da membrana eritrocitária

This article describes the structures and functions of the erythrocyte membrane and its importance in transfusional medicine. The erythrocyte membrane is one of the best known membranes in terms of structure, function and genetic disorders. As any other plasma membrane, it mediates transport functions. It also provides the erythrocytes with their resilience and deformability. According to the International Society of Blood Transfusion (ISBT), more than 500 antigens are expressed in the erythrocyte membrane, and around 270 are involved in transfusion reaction cases and hemolytic diseases of the fetus and newborn. In the ISBT classification, the high frequency series is represented by antigens in more than 99% of population (high prevalence antigen). In transfusion, the absence of these antigens determines severe problems as for example, one woman without the P antigen suffered 6 repetitive miscarriages due to placental insufficiency, which was caused by an antibody formed against the absent P antigen. Some important erythrocyte membrane proteins are described here including Band 3, Glycophorins and spectrin. The most abundant integral membrane protein is Band 3 and its main function is to mediate exchange of chloride and bicarbonate anions across the plasma membrane. The second most abundant integral membrane protein in the human erythrocyte is sialoglycoprotein glycophorin A (GPA). With its high sialic acid content, GPA is the main contributor to the net negative cell-surface charge and is thus critical for minimizing cell-cell interactions and preventing red cell aggregation. Glycophorin C (GPC) is the receptor for PfEBP-2 (baebl, EBA-140), the newly identified erythrocyte binding ligand of Plasmodium falciparum. The ternary complex of spectrin, actin and 4.1R defines the nodes of the erythrocyte membrane skeletal network, and is inseparable from membrane stability when under mechanical stress. This erythrocyte membrane review is important for a better understanding of transfusion reactions, where the antibody formation against high prevalence antigens makes compatible transfusions difficult. The study of antigen diversity and biochemical characterization of different proteins will contribute to healthcare, as well as diagnosis, development of technology such as monoclonal antibody production and the therapeutic conduct of many diseases.

Morbus Hunter : Neutralisationskapazität von Antikörpern unter Enzymersatztherapie und deren klinischen Auswirkungen

Morbus Hunter, eine lysosomale Speicherkrankheit, ist eine seltene, progrediente, x-chromosomal vererbte Stoffwechselkrankheit, die durch ein Defizit an Iduronat-2-sulfatase (IDS) hervorgerufen wird. Als Folge daraus erfolgt kein Abbau von Heparan- und Dermatansulfat und die Glykosaminoglykane reichern sich in de Lysosomen der Zelle an. M. Hunter ist eine Multisystemerkrankung und weist ein breites klinisches Spektrum mit interindividuell unterschiedlichem Krankheitsbeginn, Ausprägungen und Progression der Symptome auf. Seit 2007 besteht die Therapieoption einer Enzymersatztherapie (ERT) mit Elaprase®. Einige Patienten entwickeln Antikörper gegen das substituierte Enzym, welche partiell neutralisierende Eigenschaften besitzen. Ziel dieser Untersuchung war es zu klären, ob die Neutralisationskapazität der gebildeten Antikörper mittels einer Bestimmung im Mischserum festgestellt werden kann und ob persistierende Antikörper mit Neutralisationskapazität zu einer Einschränkung der Wirksamkeit der Enzymersatztherapie führen. Es sollte weiterhin untersucht werden, ob sich mittels Messung der neuronenspezifischen Enolase (NSE) und S-100 Rückschlüsse auf eine neuropathische Beteiligung ziehen lassen, da bis jetzt noch keine klinische oder biochemische Messmethode existiert, die für M. Hunter-Patienten eine verlässliche Vorhersage für eine neuropathische Beteiligung bietet. 30 Patienten wurden in die retrospektive/prospektive Kohortenstudie eingeschlossen. Bei der Bestimmung der IDS-Aktivität im Mischserum mit einem gesunden Menschen zeigten fünf der Patienten (17%) in zwölf Mischseren eine um ≥ 40% reduzierte Aktivität. Zwei (7%) der 30 untersuchten Patienten wurden mit dieser Methode als positiv für persistierende neutralisierende Antikörper identifiziert. Zum gleichen Ergebnis bezüglich der persistierenden neutralisierenden Antikörper führten die Anti-Elaprase®-Immunglobulin-Bestimmungen unter Berücksichtigung des Bestimmungszeitpunkts, die bei Shire Pharmaceuticals durchgeführt wurden. Die Untersuchungsergebnisse lassen den Schluss zu, dass die gebildeten Antikörper auch intraindividuell unterschiedlich sind. Zudem interagieren sie mit den verschiedensten Epitopen des Enzyms der ERT und besitzen nicht alle neutralisierende Eigenschaften. Aufgrund der heterogenen Zusammensetzung folgt die Hemmung der Enzymaktivität vermutlich keiner eindeutigen Kinetik. Anti-Elaprase®-Immunglobulin G spielt für die Neutralisationskapazität jedoch eine wichtige Rolle. Die Auswertung und Beurteilung der Einschränkung der Wirksamkeit der Therapie hervorgerufen durch die Antikörper mit Neutralisationskapazität gestaltete sich kompliziert. Im Ergebnis zeigte sich, dass sich die beiden Patienten mit persistierenden neutralisierenden Antikörpern in der Entwicklung der klinischen Parameter interindividuell stark unterschieden. Um einen Zusammenhang zwischen klinischem Verlauf und Antikörperbildung gegen die ERT zu finden, müssen in einem größeren Patientenkollektiv mehr Patienten mit persistierenden neutralisierenden Antikörpern identifiziert werden und der Einfluss der Antikörper untersucht werden. Die Untersuchung der NSE und S-100 ergab, dass weder die Konzentration der NSE noch der S-100 Rückschlüsse auf die neuropathische Beteiligung des Patienten zulässt.

A phase IIa randomized controlled pilot study evaluating the safety and clinical outcomes following the use of rhGDF-5/β-TCP in regenerative periodontal therapy

To present the safety profile, the early healing phase and the clinical outcomes at 24 weeks following treatment of human intrabony defects with open flap debridement (OFD) alone or with OFD and rhGDF-5 adsorbed onto a particulate β-tricalcium phosphate (β-TCP) carrier. Twenty chronic periodontitis patients, each with at least one tooth exhibiting a probing depth ≥6 mm and an associated intrabony defect ≥4 mm entered the study. Ten subjects (one defect/patient) were randomized to receive OFD alone (control) and ten subjects OFD combined with rhGDF-5/β-TCP. Blood samples were collected at screening, and at weeks 2 and 24 to evaluate routine hematology and clinical chemistry, rhGDF-5 plasma levels, and antirhGDF-5 antibody formation. Plaque and gingival indices, bleeding on probing, probing depth, clinical attachment level, and radiographs were recorded pre- and 24 weeks postsurgery. Comparable safety profiles were found in the two treatment groups. Neither antirhGDF-5 antibody formation nor relevant rhGDF-5 plasma levels were detected in any patient. At 6 months, treatment with OFD + rhGDF-5/β-TCP resulted in higher but statistically not significant PD reduction (3.7 ± 1.2 vs. 3.1 ± 1.8 mm; p = 0.26) and CAL gain (3.2 ± 1.7 vs. 1.7 ± 2.2 mm; p = 0.14) compared to OFD alone. In the tested concentration, the use of rhGDF-5/β-TCP appeared to be safe and the material possesses a sound biological rationale. Thus, further adequately powered, randomized controlled clinical trials are warranted to confirm the clinical relevance of this new approach in regenerative periodontal therapy. rhGDF-5/β-TCP may represent a promising new techology in regenerative periodontal therapy.

Antibody deficiency due to a missense mutation in CD19 demonstrates the importance of the conserved tryptophan 41 in immunoglobulin superfamily domain formation.

Immunoglobulin superfamily (IgSF) domains are conserved structures present in many proteins in eukaryotes and prokaryotes. These domains are well-capable of facilitating sequence variation, which is most clearly illustrated by the variable regions in immunoglobulins (Igs) and T cell receptors (TRs). We studied an antibody-deficient patient suffering from recurrent respiratory infections and with impaired antibody responses to vaccinations. Patient's B cells showed impaired Ca(2+) influx upon stimulation with anti-IgM and lacked detectable CD19 membrane expression. CD19 sequence analysis revealed a homozygous missense mutation resulting in a tryptophan to cystein (W52C) amino acid change. The affected tryptophan is CONSERVED-TRP 41 located on the C-strand of the first extracellular IgSF domain of CD19 and was found to be highly conserved, not only in mammalian CD19 proteins, but in nearly all characterized IgSF domains. Furthermore, the tryptophan is present in all variable domains in Ig and TR and was not mutated in 117 Ig class-switched transcripts of B cells from controls, despite an overall 10% amino acid change frequency. In vitro complementation studies and CD19 western blotting of patient's B cells demonstrated that the mutated protein remained immaturely glycosylated. This first missense mutation resulting in a CD19 deficiency demonstrates the crucial role of a highly conserved tryptophan in proper folding or stability of IgSF domains.

Chimeric anti-angiogenin antibody cAb 26–2F inhibits the formation of human breast cancer xenografts in athymic mice

Angiogenin (Ang), an inducer of neovascularization, is secreted by several types of human tumor cells and appears critical for their growth. The murine anti-Ang monoclonal antibody (mAb) 26–2F neutralizes the activities of Ang and dramatically prevents the establishment and metastatic dissemination of human tumor cell xenografts in athymic mice. However, for use clinically, the well-documented problem of the human anti-globulin antibody response known to occur with murine antibodies requires resolution. As a result, chimeric as well as totally humanized antibodies are currently being evaluated as therapeutic agents for the treatment of several pathological conditions, including malignancy. Therefore, we have constructed a chimeric mouse/human antibody based on the structure of mAb 26–2F. Complementary DNAs from the light and heavy chain variable regions of mAb 26–2F were cloned, sequenced, and genetically engineered by PCR for subcloning into expression vectors that contain human constant region sequences. Transfection of these vectors into nonproducing mouse myeloma cells resulted in the secretion of fully assembled tetrameric molecules. The chimeric antibody (cAb 26–2F) binds to Ang and inhibits its ribonucleolytic and angiogenic activities as potently as mAb 26–2F. Furthermore, the capacities of cAb 26–2F and its murine counterpart to suppress the formation of human breast cancer tumors in athymic mice are indistinguishable. Thus cAb 26–2F, with its retained neutralization capability and likely decreased immunogenicity, may be of use clinically for the treatment of human cancer and related disorders where pathological angiogenesis is a component.

Paclitaxel resistance and multicellular spheroid formation are induced by kallikrein-related peptidase 4 in serous ovarian cancer cells in an ascites mimicking microenvironment

High tumor kallikrein-related-peptidase 4 (KLK4) levels are associated with a poor outcome for women with serous epithelial ovarian cancer (EOC), for which peritoneal dissemination and chemoresistance are key events. To determine the role of KLK4 in these events, we examined KLK4-transfected SKOV-3 and endogenous KLK4 expressing OVCA432 cells in 3-dimensional (3D) suspension culture to mimic the ascites microenvironment. KLK4-SKOV-3 cells formed multicellular aggregates (MCAs) as seen in ascites, as did SKOV-3 cells treated with active KLK4. MCA formation was reduced by treatment with a KLK4 blocking antibody or the selective active site KLK4 sunflower trypsin inhibitor (SFTI-FCQR). KLK4-MCAs formed larger cancer cell foci in mesothelial cell monolayers than those formed by vector and native SKOV-3 cells, suggesting KLK4-MCAs are highly invasive in the peritoneal microenvironment. A high level of KLK4 is expressed by ascitic EOC cells compared to matched primary tumor cells, further supporting its role in the ascitic microenvironment. Interestingly, KLK4 transfected SKOV-3 cells expressed high levels of the KLK4 substrate, urokinase plasminogen activator (uPA), particularly in 3D-suspension, and high levels of both KLK4 and uPA were observed in patient cells taken from ascites. Importantly, the KLK4-MCAs were paclitaxel resistant which was reversed by SFTI-FCQR and to a lesser degree by the general serine protease inhibitor, Aprotinin, suggesting that in addition to uPA, other as yet unidentified substrates of KLK4 must be involved. Nonetheless, these data suggest that KLK4 inhibition, in conjunction with paclitaxel, may improve the outcome for women with serous epithelial ovarian cancer and high KLK4 levels in their tumors.

Antibodies mediate formation of neutrophil extracellular traps in the middle ear and facilitate secondary pneumococcal otitis media

Otitis media (OM) (a middle ear infection) is a common childhood illness that can leave some children with permanent hearing loss. OM can arise following infection with a variety of different pathogens, including a coinfection with influenza A virus (IAV) and Streptococcus pneumoniae (the pneumococcus). We and others have demonstrated that coinfection with IAV facilitates the replication of pneumococci in the middle ear. Specifically, we used a mouse model of OM to show that IAV facilitates the outgrowth of S. pneumoniae in the middle ear by inducing middle ear inflammation. Here, we seek to understand how the host inflammatory response facilitates bacterial outgrowth in the middle ear. Using B cell-deficient infant mice, we show that antibodies play a crucial role in facilitating pneumococcal replication. We subsequently show that this is due to antibody-dependent neutrophil extracellular trap (NET) formation in the middle ear, which, instead of clearing the infection, allows the bacteria to replicate. We further demonstrate the importance of these NETs as a potential therapeutic target through the transtympanic administration of a DNase, which effectively reduces the bacterial load in the middle ear. Taken together, these data provide novel insight into how pneumococci are able to replicate in the middle ear cavity and induce disease.

Assembly of physalis mottle virus capsid protein in Escherichia coli and the role of amino and carboxy termini in the formation of the icosahedral particles

The coat protein gene of physalis mottle tymovirus (PhMV) was over expressed in Escherichia coli using pET-3d vector. The recombinant protein was found to self assemble into capsids in vivo. The purified recombinant capsids had an apparent s value of 56.5 S and a diameter of 29(±2) nm. In order to establish the role of amino and carboxy-terminal regions in capsid assembly, two amino-terminal deletions clones lacking the first 11 and 26 amino acid residues and two carboxy-terminal deletions lacking the last five and ten amino acid residues were constructed and overexpressed. The proteins lacking N-terminal 11 (PhCPN1) and 26 (PhCPN2) amino acid residues self assembled into T = 3 capsids in vivo, as evident from electron microscopy, ultracentrifugation and agarose gel electrophoresis. The recombinant, PhCPN1 and PhCPN2 capsids were as stable as the empty capsids formed in vivo and encapsidated a small amount of mRNA. The monoclonal antibody PA3B2, which recognizes the epitope within region 22 to 36, failed to react with PhCPN2 capsids while it recognized the recombinant and PhCPN1 capsids. Disassembly of the capsids upon treatment with urea showed that PhCPN2 capsids were most stable. These results demonstrate that the N-terminal 26 amino acid residues are not essential for T = 3 capsid assembly in PhMV. In contrast, both the proteins lacking the C-terminal five and ten amino acid residues were present only in the insoluble fraction and could not assemble into capsids, suggesting that these residues are crucial for folding and assembly of the particles.

On the relationship of thermodynamic parameters with the buried surface area in protein-ligand complex formation

Prediction of thermodynamic parameters of protein-protein and antigen-antibody complex formation from high resolution structural parameters has recently received much attention, since an understanding of the contributions of different fundamental processes like hydrophobic interactions, hydrogen bonding, salt bridge formation, solvent reorganization etc. to the overall thermodynamic parameters and their relations with the structural parameters would lead to rational drug design. Using the results of the dissolution of hydrocarbons and other model compounds the changes in heat capacity (DeltaCp), enthalpy (DeltaH) and entropy (DeltaS) have been empirically correlated with the polar and apolar surface areas buried during the process of protein folding/unfolding and protein-ligand complex formation. In this regard, the polar and apolar surfaces removed from the solvent in a protein-ligand complex have been calculated from the experimentally observed values of changes in heat capacity (DeltaCp) and enthalpy (DeltaH) for protein-ligand complexes for which accurate thermodynamic and high resolution structural data are available, and the results have been compared with the x-ray crystallographic observations. Analyses of the available results show poor correlation between the thermodynamic and structural parameters. Probable reasons for this discrepancy are mostly related with the reorganization of water accompanying the reaction which is indeed proven by the analyses of the energetics of the binding of the wheat germ agglutinin to oligosaccharides.

Elevated CDCP1 predicts poor patient outcome and mediates ovarian clear cell carcinoma by promoting tumor spheroid formation, cell migration and chemoresistance

Hematogenous metastases are rarely present at diagnosis of ovarian clear cell carcinoma (OCC). Instead dissemination of these tumors is characteristically via direct extension of the primary tumor into nearby organs and the spread of exfoliated tumor cells throughout the peritoneum, initially via the peritoneal fluid, and later via ascites that accumulates as a result of disruption of the lymphatic system. The molecular mechanisms orchestrating these processes are uncertain. In particular, the signaling pathways used by malignant cells to survive the stresses of anchorage-free growth in peritoneal fluid and ascites, and to colonize remote sites, are poorly defined. We demonstrate that the transmembrane glycoprotein CUB-domain-containing protein 1 (CDCP1) has important and inhibitable roles in these processes. In vitro assays indicate that CDCP1 mediates formation and survival of OCC spheroids, as well as cell migration and chemoresistance. Disruption of CDCP1 via silencing and antibody-mediated inhibition markedly reduce the ability of TOV21G OCC cells to form intraperitoneal tumors and induce accumulation of ascites in mice. Mechanistically our data suggest that CDCP1 effects are mediated via a novel mechanism of protein kinase B (Akt) activation. Immunohistochemical analysis also suggested that CDCP1 is functionally important in OCC, with its expression elevated in 90% of 198 OCC tumors and increased CDCP1 expression correlating with poor patient disease-free and overall survival. This analysis also showed that CDCP1 is largely restricted to the surface of malignant cells where it is accessible to therapeutic antibodies. Importantly, antibody-mediated blockade of CDCP1 in vivo significantly increased the anti-tumor efficacy of carboplatin, the chemotherapy most commonly used to treat OCC. In summary, our data indicate that CDCP1 is important in the progression of OCC and that targeting pathways mediated by this protein may be useful for the management of OCC, potentially in combination with chemotherapies and agents targeting the Akt pathway.

A Monoclonal Antibody against Human Notch1 Ligand-Binding Domain Depletes Subpopulation of Putative Breast Cancer Stem-like Cells

Overexpression of Notch receptors and ligands has been associated with various cancers and developmental disorders, making Notch a potential therapeutic target. Here, we report characterization of Notch1 monoclonal antibodies (mAb) with therapeutic potential. The mAbs generated against epidermal growth factor (EGF) repeats 11 to 15 inhibited binding of Jagged1 and Delta-like4 and consequently, signaling in a dose-dependent manner, the antibodies against EGF repeats 11 to 12 being more effective than those against repeats 13 to 15. These data emphasize the role of EGF repeats 11 to 12 in ligand binding. One of the mAbs, 602.101, which specifically recognizes Notch1, inhibited ligand-dependent expression of downstream target genes of Notch such as HES-1, HES-5, and HEY-L in the breast cancer cell line MDA-MB-231. The mAb also decreased cell proliferation and induced apoptotic cell death. Furthermore, exposure to this antibody reduced CD44(Hi)/CD24(Low) subpopulation in MDA-MB-231 cells, suggesting a decrease in the cancer stem-like cell subpopulation. This was confirmed by showing that exposure to the antibody decreased the primary, secondary, and tertiary mammosphere formation efficiency of the cells. Interestingly, effect of the antibody on the putative stem-like cells appeared to be irreversible, because the mammosphere-forming efficiency could not be salvaged even after antibody removal during the secondary sphere formation. The antibody also modulated expression of genes associated with stemness and epithelial-mesenchymal transition. Thus, targeting individual Notch receptors by specific mAbs is a potential therapeutic strategy to reduce the potential breast cancer stem-like cell subpopulation. Mol Cancer Ther; 11(1); 77-86. (C) 2011 AACR.

Computer simulation of human iodothyronine deiodinase single-chain antibody

Iodothyronine plays a major role in growth, basic metabolism and organ formation. It has an extremely limited source in the body. In this thesis, we designed iodothyronine(T4) as hapten. Then a single chain antibody displayed on phange was obtained from a human phage displaying a single chain antibody library. The specific genes of E3 was subcloned in P-5E vector. According to its amino acid sequences, we simulate its three dimention structure by computer. It has never been reported in PDB.