Fast, accurate, and cost-effective poultry sex genotyping using real-time polymerase chain reaction.

According to The Organization for Economic Co-operation and Development (OECD), demand for poultry meat and eggs consumption is growing consistently since poultry meat and eggs are readily available and cheap source for nutritional protein. As such, there is pressing demand from industry for improved protocols to determine chicken sex, especially in layer industry since only females can lay eggs. Extensive efforts are being dedicated to avoiding male chicks culling by developing in-ovo sexing detection methods. Any established in-ovo detection method will need to be validated by embryo genotyping. Therefore, there is a growing demand for fast, inexpensive, and precise method for proper discrimination between males and females in the poultry science community. Our aim with this study was to develop an accurate, high-throughput protocol for sex determination using small volumes of blood. We designed primers targeting the Hint-W gene within the W chromosome clearly distinguishing between males and females. In the interest of establishing an efficient protocol without the need for gel electrophoresis, crude DNA extraction without further purification was coupled with qPCR. We validated the accuracy of our method using established protocols and gonad phenotyping and tested our protocol with four different chicken breeds, day-nine embryos, day-old chicks and adult chicken. In summary, we developed a fast, cost-effective, and accurate method for the genotyping of sex chromosomes in chicken.

mAb14, a Monoclonal Antibody against Cell Surface PCNA: A Potential Tool for Sezary Syndrome Diagnosis and Targeted Immunotherapy.

Mycosis fungoides (MF) and Sézary syndrome (SS) are the most common types of primary cutaneous T-cell lymphoma (CTCL). Proliferating cell nuclear antigen (PCNA) is expressed on the cell surface of cancer cells (csPCNA), but not on normal cells. It functions as an immune checkpoint ligand by interacting with natural killer (NK) cells through the NK inhibitory receptor NKp44, leading to the inhibition of NK cytotoxicity. A monoclonal antibody (mAb14) was established to detect csPCNA on cancer cells and block their interaction with NKp44. In this study, three CTCL cell lines and peripheral blood mononuclear cells (PBMCs) from patients with SS and healthy donors were analyzed for csPCNA using mAb14, compared to monoclonal antibody PC10, against nuclear PCNA (nPCNA). The following assays were used: immunostaining, imaging flow cytometry, flow cytometry, cell sorting, cell cycle analysis, ELISA, and the NK-cell cytotoxic assay. mAb14 successfully detected PCNA on the membrane and in the cytoplasm of viable CTCL cell lines associated with the G2/M phase. In the Sézary PBMCs, csPCNA was expressed on lymphoma cells that had an atypical morphology and not on normal cells. Furthermore, it was not expressed on PBMCs from healthy donors. In the co-culture of peripheral blood NK (pNK) cells with CTCL lines, mAb14 increased the secretion of IFN-γ, indicating the reactivation of pNK activity. However, mAb14 did not enhance the cytotoxic activity of pNK cells against CTCL cell lines. The unique expression of csPCNA detected by mAb14 suggests that csPCNA and mAb14 may serve as a potential biomarker and tool, respectively, for detecting malignant cells in SS and possibly other CTCL variants.

Multiple viral proteins and immune response pathways act to generate robust long-term immunity in Sudan virus survivors.

BACKGROUND: Profiles of immunity developed in filovirus patients and survivors have begun to shed light on antigen-specific cellular immune responses that had been previously under-studied. However, our knowledge of the breadth and length of those responses and the viral targets which mediate long-term memory immunity still lags significantly behind. METHODS: We characterized antigen-specific immune responses in whole blood samples of fifteen years post-infected survivors of the Sudan virus (SUDV) outbreak in Gulu, Uganda (2000-2001). We examined T cell and IgG responses against SUDV complete antigen and four SUDV proteins; glycoprotein (GP), nucleoprotein (NP), and viral protein 30 (VP30), and 40 (VP40). FINDINGS: We found survivors-maintained antigen-specific CD4+ T cell memory immune responses mediated mainly by the viral protein NP. In contrast, activated CD8+ T cell responses were nearly absent in SUDV survivors, regardless of the stimulating antigen used. Analysis of anti-viral humoral immunity revealed antigen-specific IgG antibodies against SUDV and SUDV proteins. Survivor IgGs mediated live SUDV neutralization in vitro and FcγRI and FcγRIII antibody Fc-dependent responses, mainly via antibodies to the viral proteins GP and VP40. INTERPRETATION: We highlight the key role of several proteins, i.e., GP, NP, and VP40, to act as mediators of distinctive and sustained cellular memory immune responses in long-term SUDV survivors. We suggest that the inclusion of these viral proteins in vaccine development may best mimic survivor native memory immune responses with the potential of protecting against viral infection. FUNDS: This research was funded by the Defense Threat Reduction Agency (CB4088) and by the National Institute Of Allergy And Infectious Diseases of the National Institutes of Health under Award Number R01AI111516. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

The Development and Validation of a Novel Nanobody-Based Competitive ELISA for the Detection of Foot and Mouth Disease 3ABC Antibodies in Cattle.

Effective management of foot and mouth disease (FMD) requires diagnostic tests to distinguish between infected and vaccinated animals (DIVA). To address this need, several enzyme-linked immunosorbent assay (ELISA) platforms have been developed, however, these tests vary in their sensitivity and specificity and are very expensive for developing countries. Camelid-derived single-domain antibodies fragments so-called Nanobodies, have demonstrated great efficacy for the development of serological diagnostics. This study describes the development of a novel Nanobody-based FMD 3ABC competitive ELISA, for the serological detection of antibodies against FMD Non-Structural Proteins (NSP) in Uganda cattle herds. This in-house ELISA was validated using more than 600 sera from different Uganda districts, and virus serotype specificities. The evaluation of the performance of the assay demonstrated high diagnostic sensitivity and specificity of 94 % (95 % CI: 88.9-97.2), and 97.67 % (95 % CI: 94.15-99.36) respectively, as well as the capability to detect NSP-specific antibodies against multiple FMD serotype infections. In comparison with the commercial PrioCHECK FMDV NSP-FMD test, there was a strong concordance and high correlation and agreement in the performance of the two tests. This new developed Nanobody based FMD 3ABC competitive ELISA could clearly benefit routine disease diagnosis, the establishment of disease-free zones, and the improvement of FMD management and control in endemically complex environments, such as those found in Africa.

Sudan ebolavirus long recovered survivors produce GP-specific Abs that are of the IgG1 subclass and preferentially bind FcγRI.

Ebolavirus is a highly lethal pathogen, causing a severe hemorrhagic disease with a high fatality rate. To better understand immune correlates of protection by virus specific IgG, we investigated the evolution of the Fcγ receptors (FcγRs)-activating capabilities of antiviral IgG in serum samples of long recovered survivors. To this end, longitudinal serum samples from survivors of Sudan ebolavirus (SUDV) infection, studied over years, were examined for the presence of Ebola-GP specific IgG subclasses, and for their binding to FcγRs. We developed a cell-based reporter system to quantitate pathogen-specific antibody binding to FcγRIIIA, FcγRIIA, FcγRIIB and FcγRI. With this system, we demonstrate that anti-GP-specific stimulation of the FcγRI reporter by survivors' sera was substantially high one year after acute infection, with a slight reduction in activity over a decade post infection. We further demonstrate that GP-specific IgG1 is by far the seroprevalent subclass that retained and even enhanced its presence in the sera, over ten years post infection; the prevalence of other GP-specific IgG subclasses was considerably reduced over time. In accordance, GP-specific FcγRI reporter response and GP-specific total IgG1 subclass correlated in the studied group of Ebola survivors. These observations are important for further informing Ebola vaccine and therapeutic development.

Correspondence of Neutralizing Humoral Immunity and CD4 T Cell Responses in Long Recovered Sudan Virus Survivors.

Robust humoral and cellular immunity are critical for survival in humans during an ebolavirus infection. However, the interplay between these two arms of immunity is poorly understood. To address this, we examined residual immune responses in survivors of the Sudan virus (SUDV) outbreak in Gulu, Uganda (2000-2001). Cytokine and chemokine expression levels in SUDV stimulated whole blood cultures were assessed by multiplex ELISA and flow cytometry. Antibody and corresponding neutralization titers were also determined. Flow cytometry and multiplex ELISA results demonstrated significantly higher levels of cytokine and chemokine responses in survivors with serological neutralizing activity. This correspondence was not detected in survivors with serum reactivity to SUDV but without neutralization activity. This previously undefined relationship between memory CD4 T cell responses and serological neutralizing capacity in SUDV survivors is key for understanding long lasting immunity in survivors of filovirus infections.

Immune memory to Sudan virus: comparison between two separate disease outbreaks.

Recovery from ebolavirus infection in humans is associated with the development of both cell-mediated and humoral immune responses. According to recent studies, individuals that did not survive infection with ebolaviruses appear to have lacked a robust adaptive immune response and the expression of several early innate response markers. However, a comprehensive protective immune profile has yet to be described. Here, we examine cellular memory immune responses among survivors of two separate Ebolavirus outbreaks (EVDs) due to Sudan virus (SUDV) infection in Uganda-Gulu 2000-2001 and Kibaale 2012. Freshly collected blood samples were stimulated with inactivated SUDV, as well as with recombinant SUDV or Ebola virus (EBOV) GP (GP1-649). In addition, ELISA and plaque reduction neutralization assays were performed to determine anti-SUDV IgG titers and neutralization capacity. Cytokine expression was measured in whole blood cultures in response to SUDV and SUDV GP stimulation in both survivor pools, demonstrating recall responses that indicate immune memory. Cytokine responses between groups were similar but had distinct differences. Neutralizing, SUDV-specific IgG activity against irradiated SUDV and SUDV recombinant proteins were detected in both survivor cohorts. Furthermore, humoral and cell-mediated crossreactivity to EBOV and EBOV recombinant GP1-649 was observed in both cohorts. In conclusion, immune responses in both groups of survivors demonstrate persistent recognition of relevant antigens, albeit larger cohorts are required in order to reach greater statistical significance. The differing cytokine responses between Gulu and Kibaale outbreak survivors suggests that each outbreak may not yield identical memory responses and promotes the merits of studying the immune responses among outbreaks of the same virus. Finally, our demonstration of cross-reactive immune recognition suggests that there is potential for developing cross-protective vaccines for ebolaviruses.

Amplified lipid rafts of malignant cells constitute a target for inhibition of aberrantly active NFAT and melanoma tumor growth by the aminobisphosphonate zoledronic acid.

Nuclear factors of activated T cells (NFAT) are critical modulators of cancer cell growth and survival. However, the mechanisms of their oncogenic dysregulation and strategies for targeting in tumors remain elusive. Here, we report coupling of anti- apoptotic NFAT (NFAT2) activation to cholesterol-enriched lipid raft microdomains of malignant melanoma cells and interruption of this pathway by the aminobisphosphonate zoledronic acid (Zol). The pathway was indicated by capability of Zol to promote apoptosis and to retard in vivo outgrowth of tumorigenic melanoma cell variants through inhibition of permanently active NFAT2. NFAT2 inhibition resulted from disintegration of cholesterol-enriched rafts due to reduction of cellular cholesterol by Zol. Mechanistically, raft disruption abolished raft-localized robust store-operated Ca(2+) (SOC) entry, blocking constitutive activation of protein kinase B/Akt (PKB) and thereby reactivating the NFAT repressor glycogen synthase kinase 3ß (GSK3ß). Pro-apoptotic inactivation of NFAT2 also followed reactivation of GSK3ß by direct inhibition of PKB or SOC, whereas GSK3ß blockade prevented Zol-induced NFAT2 inhibition and cell death. The rescuing effect of GSK3ß blockade was reproduced by recovery of entire SOC/PKB/GSK3ß cascade after reconstitution of rafts by cholesterol replenishment of Zol-treated tumorigenic cells. Remarkably, these malignant cells displayed higher cholesterol and lipid raft content than non-tumorigenic cells, which expressed weak SOC, PKB and NFAT2 activities and resisted raft-ablating action of Zol. Together, the results underscore the functional relevance of amplified melanoma rafts for tumor-promoting NFAT2 signaling and reveal these distinctive microdomains as a target for in vitro and in vivo demise of tumorigenic cells through NFAT2 inhibition by the clinical agent Zol.

Lipid rafts couple store-operated Ca2+ entry to constitutive activation of PKB/Akt in a Ca2+/calmodulin-, Src- and PP2A-mediated pathway and promote melanoma tumor growth.

Central role of constitutively active protein kinase B/Akt (PKB) in melanoma drives the search for new targets to abolish its deranged signaling. PKB activation is promoted by cholesterol-enriched lipid rafts and is Ca(2+)-dependent, but the pathway linking rafts and Ca(2+) to deregulation of this enzyme remains poorly understood. Here employing B16BL6 melanoma model, we show that ablation of rafts with methyl-ß-cyclodextrin (MßCD) inactivated PKB by inhibiting Src kinase and reactivating the negative PKB modulator, PP2A phosphatase. Blockade of PP2A with okadaic acid rescued PKB, indicating that raft ablation reactivated PP2A through inhibiting Src. Indeed, direct Src blockade with the Src kinase inhibitor-1 or the dominant-negative Src-mutant was sufficient for PP2A reactivation and downregulation of PKB, whereas reconstitution of rafts in MßCD-treated cells restored PKB, PP2A and Src activities to their basal levels. This pathway was also interrupted by inhibition of the Ca(2+) sensor calmodulin, either by its antagonist N-(6-aminohexyl)-5-chloro-1-naphtalenesulfonamide or the Ca(2+)-insensitive calmodulin-mutant or the intracellular Ca(2+)-chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N#N#-tetraacetic acid tetra-(acetocymethyl)-ester or by diminishing the store-operated Ca(2+) entry with 2-aminoethoxydiphenyl borate or small hairpin RNA against Stim1. Ablation of rafts prevented Stim1-mediated store-operated Ca(2+) entry, aborted Ca(2+) stimulation of raft-residing calmodulin and disrupted its Ca(2+)-dependent binding to Src, abolishing Src activity and entire Src/PP2A/PKB cascade. Most importantly, blockade of this cascade in the tumor site by raft-ablating MßCD, administered to melanoma-bearing mice, robustly retarded tumor growth and extended animal survival. Together, our data suggest that lipid rafts couple store-operated Ca(2+) entry to sustained activation of major tumor-promoting signaling elements in melanoma cells and underscore the potential of raft-targeting agents as effective anticancer drugs.

Coupling of mitochondria to store-operated Ca(2+)-signaling sustains constitutive activation of protein kinase B/Akt and augments survival of malignant melanoma cells.

Mitochondria are emerging as a major hub for cellular Ca(2+)-signaling, though their contribution to Ca(2+)-driven growth- and survival-promoting events in cancer is poorly understood. Here employing flow cytometry to monitor mitochondrial and cytosolic Ca(2+), we assessed trans-mitochondrial Ca(2+)-transport and store-operated Ca(2+)-influx (store-operated channels (SOC)) in malignant vs. non-malignant B16BL6 melanoma clones. Remarkably, mitochondrial Ca(2+)-fluxes measured in whole cells or in isolated mitochondria were accelerated in the malignant clones compared to their non-malignant counterpart clones. This coincided with enhanced SOC-mediated Ca(2+)-influx and high levels of constitutively active protein kinase B/Akt (PKB). Interruption of trans-mitochondrial Ca(2+)-transport in the malignant cells with an antagonist of the mitochondrial Na(+)/Ca(2+) exchanger, CGP-37157, abolsihed SOC-mediated Ca(2+)-influx, inactivated PKB, retarded cell growth and increased vulnerability to apoptosis. Similarly, direct SOC blockade by silencing Stim1 inhibited PKB, indicating that the crosstalk between SOC and mitochondria is essential to preserve PKB in constitutively active state. Finally, the retraction of mitochondria from sub-plasmalemmal micro-domains triggered by Fis1 over-expression inhibited SOC-coupled trans-mitochondrial Ca(2+)-flux, Ca(2+)-entry via SOC and PKB activity. Taken together, our data show that in the malignant melanoma cells, the functional and spatial relationship of up-regulated mitochondrial Ca(2+)-transport to the SOC sustains the robust Ca(2+)-responses and down-stream signaling critical for apoptosis-resistance and proliferation.

Targeting lipid rafts inhibits protein kinase B by disrupting calcium homeostasis and attenuates malignant properties of melanoma cells.

Failure of current therapeutic modalities to treat melanoma remains a challenge for clinical and experimental oncology. The aggressive growth and apoptotic resistance of this tumor are mediated, in part, by aberrantly activated protein kinase B/Akt (PKB). In many cells, PKB signaling depends on integrity of cholesterol-enriched membrane microdomains (rafts). However, it is still unclear if rafts support deregulated PKB activity in melanoma. In this study, ablation of rafts in murine (B16BL6-8, JB/RH1) and human (GA) melanoma lines by cholesterol-chelating methyl-beta-cyclodextrin (MbetaCD) reduced levels of constitutively active PKB in a dose- and time-dependent manner, while reconstitution of microdomains restored PKB activity. PKB was sensitive to the membrane-permeable Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid tetra (acetocymethyl) ester and to the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphtalenesulfonamide (W7) implying the contribution of Ca2+ signaling to PKB deregulation. Indeed, malignant and apoptosis-resistant clone of B16BL6 melanoma (B16BL6-8) displayed significantly higher [Ca2+](i) and store-operated Ca2+ influx (SOC) relative to non-malignant apoptosis-sensitive B16BL6 clone (Kb30) expressing barely detectable basal levels of active PKB. Raft ablation in B16BL6-8 cells robustly inhibited SOC and decreased [Ca2+](i) to levels comparable with those detected in Kb30 cells. Treating cells by PKB-inhibiting doses of M beta CD dramatically impaired their apoptotic resistance and capacity to generate tumors. Furthermore, weekly intraperitoneal injections of M beta CD to mice grafted with melanoma cells at doses of 300 and 800 mg/kg significantly attenuated tumor development. Our data implicate membrane rafts in enhancing the resistance of melanoma to apoptosis and indicate that targeting raft microdomains is a potentially effective strategy to cure this frequently fatal form of cancer.