Transcriptome and proteome analysis of innate immune responses to inactivated Leptospira and bivalent Leptospira vaccines in canine 030-D cells

Mandatory potency testing of Leptospira vaccine batches relies partially on in vivo procedures, requiring large numbers of laboratory animals. Cell-based assays could replace in vivo tests for vaccine quality control if biomarkers indicative of Leptospira vaccine potency are identifed. We investigated innate immune responsiveness induced by inactivated L. interrogans serogroups Canicola and Icterohaemorrhagiae, and two bivalent, non-adjuvanted canine Leptospira vaccines containing the same serogroups. First, the transcriptome and proteome analysis of a canine monocyte/ macrophage 030-D cell line stimulated with Leptospira strains, and vaccine B revealed more than 900 DEGs and 23 DEPs in common to these three stimuli. Second, comparison of responses induced by vaccine B and vaccine D revealed a large overlap in DEGs and DEPs as well, suggesting potential to identify biomarkers indicative of Leptospira vaccine quality. Because not many common DEPs were identifed, we selected seven molecules from the identifed DEGs, associated with pathways related to innate immunity, of which CXCL-10, IL-1β, SAA, and complement C3 showed increased secretion upon stimulation with both Leptospira vaccines. These molecules could be interesting targets for development of biomarker-based assays for Leptospira vaccine quality control in the future. Additionally, this study contributes to the understanding of the mechanisms by which Leptospira vaccines induce innate immune responses in the dog.DATA AVAILABITY STATEMENT : The transcriptomics datasets generated during the current study are available in the GEO repository, (www.ncbi. nlm.nih.gov/geo/) under accession number GSE192945. The proteomics datasets generated are available in the PRIDE repository, [http://www.ebi.ac.uk/pride/archive/projects/PXD031875].The Innovative Medicines Initiative 2 Joint Undertaking.https://www.nature.com/srepVeterinary Tropical Disease

Transcriptome and proteome analysis of innate immune responses to inactivated Leptospira and bivalent Leptospira vaccines in canine 030-D cells

Mandatory potency testing of Leptospira vaccine batches relies partially on in vivo procedures, requiring large numbers of laboratory animals. Cell-based assays could replace in vivo tests for vaccine quality control if biomarkers indicative of Leptospira vaccine potency are identified. We investigated innate immune responsiveness induced by inactivated L. interrogans serogroups Canicola and Icterohaemorrhagiae, and two bivalent, non-adjuvanted canine Leptospira vaccines containing the same serogroups. First, the transcriptome and proteome analysis of a canine monocyte/macrophage 030-D cell line stimulated with Leptospira strains, and vaccine B revealed more than 900 DEGs and 23 DEPs in common to these three stimuli. Second, comparison of responses induced by vaccine B and vaccine D revealed a large overlap in DEGs and DEPs as well, suggesting potential to identify biomarkers indicative of Leptospira vaccine quality. Because not many common DEPs were identified, we selected seven molecules from the identified DEGs, associated with pathways related to innate immunity, of which CXCL-10, IL-1β, SAA, and complement C3 showed increased secretion upon stimulation with both Leptospira vaccines. These molecules could be interesting targets for development of biomarker-based assays for Leptospira vaccine quality control in the future. Additionally, this study contributes to the understanding of the mechanisms by which Leptospira vaccines induce innate immune responses in the dog

An improved method to study NK-independent mechanisms of MTLn3 breast cancer lung metastasis

Toxicolog

Haemoxygenase-1 expression induction by particulate matter extracts: Oxydative stress reactions

No summary availabl

Identification of biomarkers to detect residual pertussis toxin using microarray analysis of dendritic cells

tIn this study we aimed to identify genes that are responsive to pertussis toxin (PTx) and might eventu-ally be used as biological markers in a testing strategy to detect residual PTx in vaccines. By microarrayanalysis we screened six human cell types (bronchial epithelial cell line BEAS-2B, fetal lung fibroblastcell line MRC-5, primary cardiac microvascular endothelial cells, primary pulmonary artery smooth mus-cle cells, hybrid cell line EA.Hy926 of umbilical vein endothelial cells and epithelial cell line A549 andimmature monocyte-derived dendritic cells) for differential gene expression induced by PTx. Imma-ture monocyte-derived dendritic cells (iMoDCs) were the only cells in which PTx induced significantdifferential expression of genes. Results were confirmed using different donors and further extendedby showing specificity for PTx in comparison to Escherichia coli lipopolysaccharide (LPS) and Bordetellapertussis lipo-oligosaccharide (LOS). Statistical analysis indicated 6 genes, namely IFNG, IL2, XCL1, CD69,CSF2 and CXCL10, as significantly upregulated by PTx which was also demonstrated at the protein levelfor genes encoding secreted proteins. IL-2 and IFN- gave the strongest response. The minimal PTx con-centrations that induced production of IL-2 and IFN- in iMoDCs were 12.5 and 25 IU/ml, respectively.High concentrations of LPS slightly induced IFN- but not IL-2, while LOS and detoxified pertussis toxindid not induce production of either cytokine. In conclusion, using microarray analysis we evaluated sixhuman cell lines/types for their responsiveness to PTx and found 6 PTx-responsive genes in iMoDCs ofwhich IL2 is the most promising candidate to be used as a biomarker for the detection of residual PTx

Identification and prevalence of CD8(+) T-cell responses directed against Epstein-Barr virus-encoded latent membrane protein 1 and latent membrane protein 2

Epstein-Barr virus (EBV) is associated with several human malignancies that each show different viral gene expression profiles. In malignancies such as Hodgkin's disease and nasopharyngeal carcinoma only Epstein-Barr nuclear antigen 1 (EBNA1) and varying levels of latent membrane proteins 1 and 2 (LMP1 and -2) are expressed. Since endogenously expressed EBNA1 is protected from CTL recognition, LMP1 and LMP2 are the most likely target antigens for anti-tumor immunotherapy. Therefore, we sought to identify in a systematic way CD8(+) T-cell responses directed against eptitopes derived from LMP1 and LMP2. Using IFNgamma-ELISPOT assays of interferon-gamma release, peripheral blood mononuclear cells (PBMC) of healthy donors were screened with peptide panels (15 mer overlapping by 10) spanning the LMP1 and LMP2 sequences of the prototype EBV strain B95.8. When positive responses were found, CD4(+) or CD8(+) T cells were depleted from donor PBMC to determine the origin of the responder population. We detected CD8(+) T-cell responses to LMP1 in 9/50(18%) donors and to LMP2 in 15/28 (54%) donors. In addition to the already described epitopes, 3 new LMP1- and 5 new LMP2-derived CD8(+) epitopes were identified. In most donors LMP1- and LMP2-specific CD8(+) precursor frequencies were low compared with precursors against immunodominant EBV epitopes from latent (EBNA3A, -3B and -3C) and lytic cycle antigens. These results demonstrate that CD8(+) memory T cell responses to LMP1 and especially to LMP2 do exist in Caucasians, albeit at low levels and could potentially be exploited for therapeutic use

Transcriptome and proteome analysis of innate immune responses to inactivated Leptospira and bivalent Leptospira vaccines in canine 030-D cells.

Mandatory potency testing of Leptospira vaccine batches relies partially on in vivo procedures, requiring large numbers of laboratory animals. Cell-based assays could replace in vivo tests for vaccine quality control if biomarkers indicative of Leptospira vaccine potency are identified. We investigated innate immune responsiveness induced by inactivated L. interrogans serogroups Canicola and Icterohaemorrhagiae, and two bivalent, non-adjuvanted canine Leptospira vaccines containing the same serogroups. First, the transcriptome and proteome analysis of a canine monocyte/macrophage 030-D cell line stimulated with Leptospira strains, and vaccine B revealed more than 900 DEGs and 23 DEPs in common to these three stimuli. Second, comparison of responses induced by vaccine B and vaccine D revealed a large overlap in DEGs and DEPs as well, suggesting potential to identify biomarkers indicative of Leptospira vaccine quality. Because not many common DEPs were identified, we selected seven molecules from the identified DEGs, associated with pathways related to innate immunity, of which CXCL-10, IL-1β, SAA, and complement C3 showed increased secretion upon stimulation with both Leptospira vaccines. These molecules could be interesting targets for development of biomarker-based assays for Leptospira vaccine quality control in the future. Additionally, this study contributes to the understanding of the mechanisms by which Leptospira vaccines induce innate immune responses in the dog

Transcriptome and proteome analysis of innate immune responses to inactivated Leptospira and bivalent Leptospira vaccines in canine 030-D cells

Mandatory potency testing of Leptospira vaccine batches relies partially on in vivo procedures, requiring large numbers of laboratory animals. Cell-based assays could replace in vivo tests for vaccine quality control if biomarkers indicative of Leptospira vaccine potency are identified. We investigated innate immune responsiveness induced by inactivated L. interrogans serogroups Canicola and Icterohaemorrhagiae, and two bivalent, non-adjuvanted canine Leptospira vaccines containing the same serogroups. First, the transcriptome and proteome analysis of a canine monocyte/macrophage 030-D cell line stimulated with Leptospira strains, and vaccine B revealed more than 900 DEGs and 23 DEPs in common to these three stimuli. Second, comparison of responses induced by vaccine B and vaccine D revealed a large overlap in DEGs and DEPs as well, suggesting potential to identify biomarkers indicative of Leptospira vaccine quality. Because not many common DEPs were identified, we selected seven molecules from the identified DEGs, associated with pathways related to innate immunity, of which CXCL-10, IL-1β, SAA, and complement C3 showed increased secretion upon stimulation with both Leptospira vaccines. These molecules could be interesting targets for development of biomarker-based assays for Leptospira vaccine quality control in the future. Additionally, this study contributes to the understanding of the mechanisms by which Leptospira vaccines induce innate immune responses in the dog

Paxillin serine 178 phosphorylation in control of cell migration and metastasis formation through regulation of EGFR expression in breast cancer

Aim: Paxillin is a well-known multidomain scaffold protein that is involved in the regulation of cell-matrix adhesion dynamics, a process required for the tumor cell migration and invasion. Phosphorylation of the serine residue 178 requires c-Jun NH2-terminal kinase (JNK) activation, which occurs downstream of epidermal growth factor receptor (EGFR)-mediated signaling and drives cell migration. In this study, we investigated the significance of paxillin Ser178 phosphorylation in breast cancer progression.Methods: We employed the rat mammary carcinoma MTLn3 cell line with which we established stabile variants of both wild type and mutant GFP-paxillin constructs. With those, we next performed several in vitro assays including cell proliferation, migration and focal adhesion dynamics. Finally, we monitored the metastatic spread of both cell line variants in an othrotopic mouse model for breast cancer.Results: Here we show that expression of the phospho-defective mutant paxillinS178A in the metastatic mammary adenocarcinoma MTLn3 cell-line significantly decreased EGF-induced cell migration, which was correlated with impaired focal adhesion dynamics. Moreover, paxillinS178A attenuated lung metastasis formation in an orthotopic In vivo mammary gland tumor/metastasis model, demonstrating the importance of JNK-mediated paxillin phosphorylation in breast cancer progression. Expression of paxillinS178A caused a decrease in EGFR expression, while re-expression of EGFR in MTLn3-paxillinS178A cells fully restored EGF-driven cell motility and focal adhesion dynamics. Furthermore, re-expression of EGFR in MTLn3-paxillinS178A rescued spontaneous metastasis from breast to lung.Conclusion: Overall our data show an important role for JNK-mediated paxillin Ser178 phosphorylation in the regulation of EGFR expression and thereby, in EGF-driven cell migration and metastasis formation