Enrichment is essential for phosphoproteome analysis because phosphorylated proteins are usually present in cells in low abundance. Recently, titanium dioxide (TiO2) has been demonstrated to enrich ...phosphopeptides from simple peptide mixtures with high specificity; however, the technology has not been optimized. In the present study, significant non-specific bindings were observed when proteome samples were applied to TiO2 columns. Column wash with an NH4Glu solution after loading peptide mixtures significantly increased the efficiency of TiO2 phosphopeptide enrichment with a recovery of up to 84%. Also, for proteome samples, more than a 2-fold increase in unique phosphopeptide identifications has been achieved. The use of NH4Glu for a TiO2 column wash does not significantly reduce the phosphopeptide recovery. A total of 858 phosphopeptides corresponding to 1034 distinct phosphosites has been identified from HeLa cells using the improved TiO2 enrichment procedure in combination with data-dependent neutral loss nano-RPLC-MS2-MS3 analysis. While 41 and 35% of the phosphopeptides were identified only by MS2 and MS3, respectively, 24% was identified by both MS2 and MS3. Cross-validation of the phosphopeptide assignment by MS2 and MS3 scans resulted in the highest confidence in identification (99.5%). Many phosphosites identified in this study appear to be novel, including sites from antigen Ki-67, nucleolar phosphoprotein p130, and Treacle protein. The study also indicates that evaluation of confidence levels for phosphopeptide identification via the reversed sequence database searching strategy might underestimate the false positive rate.
Background. Women hospitalized with ST-elevation myocardial infarction (STEMI) experience higher risk of early mortality than men. We aimed to investigate the potential impact of risk factors, ...clinical characteristics, and management among gender-related risk differences. Method. We analyzed 5063 STEMI patients prospectively enrolled from 66 hospitals during 2016–2018 and compared sex differences in mortality, death, or treatment withdrawal and main adverse cardiovascular and cerebrovascular events (MACCE) using the generalized linear mixed model, following sequential adjustment for covariates. Results. Women were older and had a higher prevalence of hypertension (53.3% vs. 41.1%, P<0.001) and diabetes (24.5% vs. 15.2%, P<0.001). Eligible women were less likely to receive reperfusion therapy (56.1% vs. 62.4%, P<0.001); the onset to first medical contact (FMC) (255 vs. 190 minutes, P<0.001), onset to fibrinolysis (218 vs. 185 minutes, P<0.001), and onset to percutaneous coronary intervention (PCI) (307 vs. 243 minutes, P<0.001) were significantly delayed in women. The incidence of in-hospital death (6.8% vs. 3.0%, P<0.001), death or treatment withdrawal (14.5% vs. 5.6%, P<0.001), and MACCE (18.5% vs. 9.4%, P<0.001) were notably higher. The gender disparities persist in death (OR: 1.61, 95% CI: 1.12–2.33), death or treatment withdrawal (OR: 1.68, 95% CI: 1.26–2.24), and MACCE (OR: 1.37, 95% CI: 1.08–1.74) after adjustment for covariates. Among possible explanatory factors, age (−58.46%, −59.04%, −62.20%) and cardiovascular risk factors (−40.77%, −39.36%, −41.73%) accounted for most of the gender-associated risk differences. Conclusions. Women experienced worse in-hospital outcomes, and age and cardiovascular risk factors were major factors influencing sex-related differences. The sex disparity stressed the awareness and importance of quality improvement efforts against female patients in clinical practice.
Cardiovascular disease including ST elevation myocardial infarction (STEMI) is increasing and the leading cause of death in China. There has been limited data available to characterize STEMI ...management and outcomes in rural areas of China. The Henan STEMI Registry is a regional STEMI project with the objectives to timely obtain real-world knowledge about STEMI patients in secondary and tertiary hospitals and to provide a platform for care quality improvement efforts in predominantly rural central China.
The Henan STEMI Registry is a multicentre, prospective and observational study for STEMI patients. The registry includes 66 participating hospitals (50 secondary hospitals; 16 tertiary hospitals) that cover 15 prefectures and one city direct-controlled by the province in Henan province. Patients were consecutively enrolled with a primary diagnosis of STEMI within 30 days of symptom onset. Clinical treatments, outcomes and cost are collected by local investigators and captured electronically, with a standardized set of variables and standard definitions, and rigorous data quality control. Post-discharge patient follow-up to 1 year is planned. As of August 2018, the Henan STEMI Registry has enrolled 5479 patients of STEMI.
The Henan STEMI Registry represents the largest Chinese regional platform for clinical research and care quality improvement for STEMI. The board inclusion of secondary hospitals in Henan province will allow for the exploration of STEMI in predominantly rural central China.
NCT02641262 29 December, 2015.
BackgroundChimeric antigen receptor modified (CAR-T) cell therapy has shown tremendous clinical efficacy for hematological malignancies,1 2 and limited efficacy in solid tumor therapy.3 4 However, ...bottlenecks in the ability to manufacture and deliver these engineered cell products, combined with their high cost, have limited patient accessibility of CAR T therapy, especially those in underserved areas or who are uninsured. Here, we describe the development of a flexible, cost-effective CAR T manufacturing process starting with fresh whole blood that generates sufficient CAR T cells numbers with excellent functionality.Methods and ResultsFor T cell Enrichment, a Sepax C-Pro (Cytiva, Inc.) was used to reduce 450ml fresh whole blood volume to 100ml. Concentrated cells were labeled for negative T cell enrichment (RosetteSep, Stemcell Research, Inc.), then fractionated on the Sepax C-Pro by density gradient centrifugation. Purified T cells were harvested, washed, and analyzed by flow cytometry. A cell product with >85% T cell purity, and 200x106 total T cells was routinely recovered. For large-scale T cell transduction with CAR LV expression vector, 1x 108 T cells were suspended in 100ml TexMACSTM medium (Miltenyi Biotec) with 200 IU/ml IL-2 and transferred to a G-Rex cell culture device (Wilson-Wolf), followed by addition of T cell TransActTM reagent and HIV gp120 DuoCAR lenti-vector5 (MOI 40). On Day 2, a complete medium exchange was carried out, cells were re-suspended in 400ml of fresh medium with 200 IU/ml IL-2, and returned to the same G-Rex. The cells were cultured in a tissue culture incubator to Day 8, when the final cell product was harvested. On days 3, 6 and 8, cultures were sampled to monitor T cell growth, phenotype, CAR expression, and function. Evaluating 4 healthy donors, at least 5x108 CAR-T cells could be harvested on Day 6, with ~90% cell viability. Day 6 CAR-T showed similar transduction efficiency and increased Tscm% and Tcm% compared to the standard Day 8 manufacturing time frame.5 6 Comparable target leukemia cell cytolysis was also observed.ConclusionsThis large-scale CAR T production process can: 1) replace the complicated and expensive leukapheresis process using fresh whole blood for starting material; 2) provide the flexibility of earlier culture harvest. We thereby approach our goal to generate an economical process for CAR T production that can be administered to all who would benefit. Future work will include evaluation of this process with patient-derived material.ReferencesAli S, Kjeken R, et al. The European Medicines Agency Review of Kymriah (Tisagenlecleucel) for the Treatment of Acute Lymphoblastic Leukemia and Diffuse Large B-Cell Lymphoma. Oncologist. 2020 Feb;25(2):e321-e327.Neelapu SS, Locke FL, et al. Axicabtagene Ciloleucel CAR T-Cell Therapy in Refractory Large B-Cell Lymphoma. N Engl J Med 2017 Dec;377(26):2531–2544.Vitanza NA, Wilson AL, et al. Intraventricular B7-H3 CAR T Cells for Diffuse Intrinsic Pontine Glioma: Preliminary First-in-Human Bioactivity and Safety. Cancer Discov 2023 Jan;13(1):114–131.Del Bufalo F, De Angelis B, et al. GD2-CART01 for Relapsed or Refractory High-Rish Neuroblastoma. N Engl J Med 2023 Apr;388(14):1284–1295.Anthony-Gonda K, Barhi A, et al. Multispecific anti-HIV duoCAR-T cells display broad in vitro antiviral activity and potent in vivo elimination of HIV-infected cells in a humanized mouse model. Sci Transl Med. 2019 Aug;11(504):eaav5685.Jackson Z, Roe A, et al. Automated Manufacture of Autologous CD19 CAR-T Cells for Treatment of Non-Hodgkin Lymphoma. Front Immunol. 2020 Aug;11:1941.
Currently used antiretroviral therapy is highly successful but there is still a need for new effective and safe prophylactics and therapeutics. We have previously identified and characterized a human ...engineered antibody domain (eAd), m36, which exhibits potent broadly neutralizing activity against HIV-1 by targeting a highly conserved CD4 binding-induced (CD4i) structure on the viral envelope glycoprotein (Env) gp120. m36 has very small size (∼15
kDa) but is highly specific and is likely to be safe in long-term use thus representing a novel class of potentially promising HIV-1 inhibitors. Major problems with the development of m36 as a candidate therapeutic are possible short serum half life and lack of effector functions that could be important for effective protection
in vivo. Fusion of m36 to human IgG1 Fc resulted in dramatically diminished neutralization potency most likely due to the sterically restricted nature of the m36 epitope that limits access of large molecules. To confer effector functions and simultaneously increase the potency, we first matured m36 by panning and screening a mutant library for mutants with increased binding to gp120. We next fused m36 and its mutants with the first two domains (soluble CD4, sCD4) of the human CD4 using a polypeptide linker. Our results showed that the selected m36 mutants and the sCD4 fusion proteins exhibited more potent antiviral activities than m36. The m36-sCD4 fusion proteins with human IgG1 Fc showed even higher potency likely due to their bivalency and increased avidity although with a greater increase in molecular size. Our data suggest that m36 derivatives are promising HIV-1 candidate therapeutics and tools to study highly conserved gp120 structures with implications for understanding mechanisms of entry and design of vaccine immunogens and small-molecule inhibitors.
Mesothelin is a potential new target for cancer immunotherapy because it is present at relatively low levels only in mesothelial cells of pleura, peritoneum, and pericardium of healthy people, but is ...significantly elevated in a number of tumors, including mesothelioma, ovarian, pancreatic, and lung cancers. However, all currently available antibodies against mesothelin are either murine or chimeric, which could limit their use because of increased likelihood of immunogenicity compared with fully human antibodies. Here, we report the identification and characterization of a novel fully human monoclonal antibody, m912, which was isolated from a human Fab library by panning against recombinant mesothelin. This antibody in scFv, Fab, and IgG1 formats bound specifically and with high affinity (equilibrium dissociation constant in the nmol/L range) to cell surface-associated human mesothelin and to recombinant mesothelin. It specifically lysed cancer cells engineered to express mesothelin in the presence of peripheral blood mononuclear cells isolated from healthy donors most likely by antibody-dependent cellular cytotoxicity. M912 is the first reported fully human monoclonal antibody to mesothelin, which has potential for cancer treatment and diagnosis.
Recently, several potently neutralizing fully human monoclonal antibodies (hmAbs) targeting the severe acute respiratory syndrome-associated coronavirus (SARS CoV) S glycoprotein, and the G ...glycoprotein of the paramyxoviruses Hendra virus (HeV) and Nipah virus (NiV) have been discovered corrected.
To examine, compare and contrast the functional characteristics of hmAbs with the potential for prophylaxis and treatment of diseases caused by SARS CoV, HeV and NiV.
A review of relevant literature.
Structural, functional and biochemical analyses corrected have provided insights into the molecular mechanisms of receptor recognition and antibody neutralization, and suggested that these antibodies alone or in combination could fight the viruses' heterogeneity and mutability, which is a major problem in the development of effective therapeutic agents against viruses, including therapeutic antibodies.
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