Vathany Kulasingam

A Third Vaccine Dose in Organ-Transplant Recipients It is known that people receiving immune suppressive therapy, such as recipients of solid-organ transplants, have a suboptimal response to SARS-C...

A “bottom-up” proteomics approach and a two-dimensional (strong cation exchange followed by reversed-phase) LC-MS/MS strategy on a linear ion trap (LTQ) were utilized to identify and compare expressions of extracellular and membrane-bound proteins in the conditioned media of three breast cell lines (MCF-10A, BT474, and MDA-MB-468). Proteomics analysis of the media identified in excess of 600, 500, and 700 proteins in MCF-10A, BT474, and MDA-MB-468, respectively. We successfully identified the internal control proteins, kallikreins 5, 6, and 10 (ranging in concentration from 2 to 50 μg/liter) in MDA-MB-468 conditioned medium as validated by ELISA and confidently identified Her-2/neu in BT474 cells. Subcellular localization was determined based on Genome Ontology terms for all the 1,139 proteins of which 34% were classified as extracellular and membrane-bound. Proteomics analysis of MDA-MB-468 cell lysate demonstrated that only 5% of all identified proteins were extracellular. This confirmed our hypothesis that examining the CM of cell lines, as opposed to the cell lysates, leads to a significant enrichment in secreted proteins. Tissue specificity, functional classifications, and spectral counting were performed. Elafin, a protease inhibitor, identified in the conditioned media of BT474 and MDA-MB-468 and the three kallikreins (KLK5, KLK6, and KLK10) were validated using an immunoassay on various serum and biological samples. Some of the secreted proteins identified have established roles in breast cancer development (cell growth, differentiation, and metastasis) and/or are linked to early onset breast cancer. Our approach to mining for low abundance molecules could identify proteins in various stages of breast cancer development. Many of the identified proteins are potentially useful to investigate as circulating serum breast cancer biomarkers. A “bottom-up” proteomics approach and a two-dimensional (strong cation exchange followed by reversed-phase) LC-MS/MS strategy on a linear ion trap (LTQ) were utilized to identify and compare expressions of extracellular and membrane-bound proteins in the conditioned media of three breast cell lines (MCF-10A, BT474, and MDA-MB-468). Proteomics analysis of the media identified in excess of 600, 500, and 700 proteins in MCF-10A, BT474, and MDA-MB-468, respectively. We successfully identified the internal control proteins, kallikreins 5, 6, and 10 (ranging in concentration from 2 to 50 μg/liter) in MDA-MB-468 conditioned medium as validated by ELISA and confidently identified Her-2/neu in BT474 cells. Subcellular localization was determined based on Genome Ontology terms for all the 1,139 proteins of which 34% were classified as extracellular and membrane-bound. Proteomics analysis of MDA-MB-468 cell lysate demonstrated that only 5% of all identified proteins were extracellular. This confirmed our hypothesis that examining the CM of cell lines, as opposed to the cell lysates, leads to a significant enrichment in secreted proteins. Tissue specificity, functional classifications, and spectral counting were performed. Elafin, a protease inhibitor, identified in the conditioned media of BT474 and MDA-MB-468 and the three kallikreins (KLK5, KLK6, and KLK10) were validated using an immunoassay on various serum and biological samples. Some of the secreted proteins identified have established roles in breast cancer development (cell growth, differentiation, and metastasis) and/or are linked to early onset breast cancer. Our approach to mining for low abundance molecules could identify proteins in various stages of breast cancer development. Many of the identified proteins are potentially useful to investigate as circulating serum breast cancer biomarkers. Breast cancer is a leading cause of death among women with solid tumors in North America (1van Diest P.J. van der W.E. Baak J.P. Prognostic value of proliferation in invasive breast cancer: a review.J. Clin. Pathol. 2004; 57: 675-681Crossref PubMed Scopus (290) Google Scholar). It is a disease of the middle and late ages of life as 75% of breast cancer is diagnosed in women over the age of 50 (2Jemal A. Tiwari R.C. Murray T. Ghafoor A. Samuels A. Ward E. Feuer E.J. Thun M.J. Cancer statistics, 2004.CA Cancer J. Clin. 2004; 54: 8-29Crossref PubMed Scopus (3914) Google Scholar). Although breast cancer is less common at a young age, younger women tend to have a more aggressive form of the disease than older women. The 5-year survival rate is close to 97% when the cancer is confined to the breast (2Jemal A. Tiwari R.C. Murray T. Ghafoor A. Samuels A. Ward E. Feuer E.J. Thun M.J. Cancer statistics, 2004.CA Cancer J. Clin. 2004; 54: 8-29Crossref PubMed Scopus (3914) Google Scholar). However, when breast cancer has metastasized at the time of diagnosis, the 5-year survival rate is ∼23%. Gene expression patterns have been used to classify breast tumors into clinically relevant subgroups (luminal A, luminal B, basal, ERBB2-overexpressing, and normal-like) (3Sorlie T. Tibshirani R. Parker J. Hastie T. Marron J.S. Nobel A. Deng S. Johnsen H. Pesich R. Geisler S. Demeter J. Perou C.M. Lonning P.E. Brown P.O. Borresen-Dale A.L. Botstein D. Repeated observation of breast tumor subtypes in independent gene expression data sets.Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8418-8423Crossref PubMed Scopus (4222) Google Scholar, 4Sorlie T. Perou C.M. Tibshirani R. Aas T. Geisler S. Johnsen H. Hastie T. Eisen M.B. van de R.M. Jeffrey S.S. Thorsen T. Quist H. Matese J.C. Brown P.O. Botstein D. Eystein L.P. Borresen-Dale A.L. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications.Proc. Natl. Acad. Sci. U. S. 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Solid organ transplant recipients are at high risk of severe disease from COVID-19. We assessed the immunogenicity of mRNA-1273 vaccine using a combination of antibody testing, surrogate neutralization assays, and T cell assays. Patients were immunized with two doses of vaccine and immunogenicity assessed after each dose using the above tests. CD4+ and CD8+ T cell responses were assessed in a subset using flow-cytometry. A total of 127 patients were enrolled of which 110 provided serum at all time points. A positive anti-RBD antibody was seen in 5.0% after one dose and 34.5% after two doses. Neutralizing antibody was present in 26.9%. Of note, 28.5% of patients with anti-RBD did not have neutralizing antibody. T cell responses in a sub-cohort of 48 patients showed a positive CD4+ T cell response in 47.9%. Of note, in this sub-cohort, 46.2% of patients with a negative anti-RBD, still had a positive CD4+ T cell response. The vaccine was safe and well-tolerated. In summary, immunogenicity of mRNA-1273 COVID-19 vaccine was modest, but a subset of patients still develop neutralizing antibody and CD4+T- cell responses. Importantly polyfunctional CD4+T cell responses were observed in a significant portion who were antibody negative, further highlighting the importance of vaccination in this patient population.IRB Statement: This study was approved by the University Health Network Research Ethics Board (CAPCR ID 20–6069). Solid organ transplant recipients are at high risk of severe disease from COVID-19. We assessed the immunogenicity of mRNA-1273 vaccine using a combination of antibody testing, surrogate neutralization assays, and T cell assays. Patients were immunized with two doses of vaccine and immunogenicity assessed after each dose using the above tests. CD4+ and CD8+ T cell responses were assessed in a subset using flow-cytometry. A total of 127 patients were enrolled of which 110 provided serum at all time points. A positive anti-RBD antibody was seen in 5.0% after one dose and 34.5% after two doses. Neutralizing antibody was present in 26.9%. Of note, 28.5% of patients with anti-RBD did not have neutralizing antibody. T cell responses in a sub-cohort of 48 patients showed a positive CD4+ T cell response in 47.9%. Of note, in this sub-cohort, 46.2% of patients with a negative anti-RBD, still had a positive CD4+ T cell response. The vaccine was safe and well-tolerated. In summary, immunogenicity of mRNA-1273 COVID-19 vaccine was modest, but a subset of patients still develop neutralizing antibody and CD4+T- cell responses. Importantly polyfunctional CD4+T cell responses were observed in a significant portion who were antibody negative, further highlighting the importance of vaccination in this patient population. IRB Statement: This study was approved by the University Health Network Research Ethics Board (CAPCR ID 20–6069).