Project description:The present study describes a novel xenograft-based biomarker discovery platform and proves its usefulness in the discovery of novel serum markers for prostate cancer (PCa). By immunizing immuno-competent mice with serum from nude mice bearing PCa xenografts, an antibody response against xenograft-derived antigens was elicited. By probing protein microarrays with serum from immunized mice, several PCa-derived antigens were identified, of which a subset was successfully retrieved in serum from mice bearing PCa xenografts and validated in human serum samples of PCa patients. In conclusion, this novel method allows for the identification of low abundant cancer-derived serum proteins, circumventing dynamic range and host-response issues in standard patient cohort proteomics comparisons.
Project description:Male Non-Obese Diabetic (NOD) mice develops autoimmune dacryoadenitis beginning 6-8 weeks of age including lymphocytic infiltration of the lacrimal glands, reduced tear production, increased cysteine tear proteases and restructuring of extracellular membrane structural proteins. Autoantigens with high antibody reactivity in serum of disease model mice may have diagnostic potential for Sjögren's Disease. Serum was collected from mice at early (8 weeks,), intermediate (24 weeks) and advanced (33 weeks) stage of autoimmune dacryoadenitis. Serum was also collected from healthy control BALB/c mice at 8 and 28 weeks of age. Approximately 0.8 mL of blood was collected by cardiac puncture into serum separator tubes. Following cold centrifugation, serum was collected. 10 uL of serum per sample was used to determine reactivity against 94 autoantigens using proteinarray. Additionally, effect of age on the level of reactivity was also studied to determine if aging was a confounding the observed up or downregulation of serum autoantibodies.
Project description:Dilated cardiomyopathy (DCM) is the most common form of cardiomyopathy and main indication for heart transplantation in children. Therapies specific to pediatric DCM remains limited due to lack of a disease model. Our previous study showed that treatment of neonatal rat ventricular myocytes (NRVMs) with non-failing or DCM pediatric patient serum activates the fetal gene program (FGP). Here we show that serum treatment with Proteinase K prevents activation of the FGP, whereas RNase treatment exacerbates it, suggesting that circulating proteins, but not circulating microRNAs, promote these pathological changes. Evaluation of the protein secretome showed that midkine (MDK) is up-regulated in DCM serum, and NRVM treatment with MDK activates the FGP. Changes in gene expression in serum-treated NRVMs, evaluated by next-generation RNA sequencing (RNA-Seq), indicates extracellular matrix remodeling and focal adhesion pathways are upregulated in pediatric DCM serum and serum-treated NRVMs, suggesting alterations in cellular stiffness. Cellular stiffness was evaluated by Atomic Force Microscopy, which showed an increase in stiffness in DCM serum-treated NRVMs. Of the proteins increased in DCM sera, secreted frizzled related protein 1 (sFRP1) was a potential candidate for the increase in cellular stiffness, and sFRP1 treatment of NRVMs recapitulated the increase in cellular stiffness observed in response to DCM-serum treatment. Our results show that serum circulating proteins promote pathological changes in gene expression and cellular stiffness, and circulating miRNAs are protective against pathological changes.
Project description:Protein microarray was used to identify proteins with elevated interactions with serum autoantibodies in a responding patient with rhabdomyosarcoma before and after multiple doses of HER2 CAR T cell therapy. Elevated signals were observed for multiple proteins interacting with serum autoantibodies following multiple doses of HER2-CAR T cell treatment when compared to pre-treatment serum.
Project description:The autoantibodies in serum (IgG antibody labeled with Cy3 and IgM antibody labeled with Cy5) were quantified by serum incubation human protein microarray, and the data of normal people and lung cancer patients were compared to find the predictive markers of autoantibodies in lung cancer
Project description:The present study describes a novel xenograft-based biomarker discovery platform and proves its usefulness in the discovery of novel serum markers for prostate cancer (PCa). By immunizing immuno-competent mice with serum from nude mice bearing PCa xenografts, an antibody response against xenograft-derived antigens was elicited. By probing protein microarrays with serum from immunized mice, several PCa-derived antigens were identified, of which a subset was successfully retrieved in serum from mice bearing PCa xenografts and validated in human serum samples of PCa patients. In conclusion, this novel method allows for the identification of low abundant cancer-derived serum proteins, circumventing dynamic range and host-response issues in standard patient cohort proteomics comparisons. To perform a large-scale identification of antibodies generated against human PCa-derived proteins in the serum of immunized mice, sera from mice immunized with either depleted serum, full serum or both from PC346 and PC339-bearing mice as well as preimmune serum and serum from mice immunized with normal mouse serum were incubated onto ProtoArrays. These ProtoArrays contain approximately 8,000 partial and full-length human proteins, expressed as N-terminal glutathione S-transferase (GST) fusion proteins. To detect antibodies bound to spotted proteins, ProtoArrays were developed using a fluorescent labeled secondary antibody. Before being used for immunization, serum from xenografted mice was not treated (full) or depleted for most abundant proteins (depleted). Two arrays were hybridized with pre-immune serum and one array with serum from an immune competent mouse that was immunized with serum from a nude mouse. Six arrays were performed using serum from immune competent mice that were immunized with serum from xenograft-bearing nude mice.
Project description:Male Non-Obese Diabetic (NOD) derived NOR mice develop autoimmune dacryoadenitis by 16 weeks of age including lymphocytic infiltration of the lacrimal glands, reduced tear production, and increased cysteine tear proteases. Unlike the NOD mice, the NOR mice do not develop diabetes and studies designed with the NOR mice are expected to not have diabetes development counfounding the results observed for Sjogren's development. Autoantigens with high levels of reactivity in serum and/or tears of diseased mice may have diagnostic potential. Serum was collected from mice at 16 weeks old male NOR mice along with age matched male healthy BALBc controls. Approximately 0.8 mL of blood was collected by cardiac puncture into serum separator tubes. Following cold centrifugation, serum was collected. Tears were collected following topical stimulation of the lacrimal gland with carbachol. For tear samples, tears were pooled from 1, 2 or 3 mice, respectively to generate three samples. This was done mainly to optimize the number of mice required to obtain sufficient sample for the assay as well as to assess the sensitivity of mouse tears for the proteinarray. 1 to 10 uL of tear or 10 uL of serum per sample was used to determine reactivity against 94 autoantigens using proteinarray.
Project description:Sjögren's Disease (SjD) is an autoimmune disease characterized by lymphocytic infiltration of salivary and lacrimal glands (LG). The LG produces the protein-rich aqueous component of tears, and SjD-associated autoimmune dacryoadenitis (AD) may thus alter tear autoantibody composition. The presence of tertiary lymphoid structures (TLS) in LG from two murine models of SjD-associated AD, male non-obese diabetic (NOD) and male non-obese insulitis resistant (NOR) mice, were evaluated using immunofluorescence. IgG and IgA reactivity in serum and tears from these models were probed in three studies against a panel of 80-120 autoantigens using autoantibody microarrays relative to serum and tears from healthy male BALB/c mice. Sources of Ig in tears were investigated using scRNA-Seq of the LG (GSE132420). Data were analyzed by R package Limma and Seurat. Analysis of immunofluorescence in LG sections from both SjD models showed TLS. Only one autoantibody was significantly elevated in tears and serum in both SjD models across all studies. Three autoantibodies were significantly elevated in serum but not in tears in both SjD models across all studies. Conversely, six IgG and thirteen IgA autoantibodies (6 sharing the same autoantigen) were significantly elevated in tears but not serum in both SjD models. Igha and Ighg2b expressing cells were identified in the plasma cell cluster of NOD.H2b LG. NOD and NOR mice with SjD-associated AD have distinct autoantibody profiles in tears and serum. Tear IgA isotype autoantibodies showed a greater diversity than tear IgG autoantibodies. TLS observed in LG are a likely source of the tear autoantibodies.
Project description:we evaluated cytokine profiling of bone marrow serum samples in AML patients and healthy controls. Protein expression profiling of serum from 9 AML patients and 5 healthy controls was obtained using biotinylated antibody chip. Total 507 cytokines and growth factors were analyzed. Compared with healthy people, AML patients expressed 31 signature proteins, among which, 27 were found significantly higher expressed and 4 proteins were lower.