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Discover how PEPperPRINT Peptide Microarray products have been used in different fields of research.

Influenza‐associated thrombotic thrombocytopenic purpura: A report of two cases and a brief review of the literature

Onkarappa Mangala, Yashvin; Sweeney, Joseph D.
Vox Sanguinis.
Nov 2021
Background and Objectives Thrombotic thrombocytopenic purpura (TTP) is often preceded by a recent history of an acute infection and influenza is the most implicated virus. Materials and Methods We identified two cases of TTP, which were preceded by influenza between 2010 and 2021. In one patient, we epitope mapped the binding specificity of antibodies using an overlapping peptide approach of the stalk protein of Influenza B and the cysteine-rich spacer domain (CRSD) of ADAMTS13. A literature search was performed for reports of influenza-associated TTP over the period 1980–2021. Results Two patients were identified in which TTP was preceded by influenza, one Influenza A and the other Influenza B. Epitope mapping of the latter’s plasma identified target epitopes in both the stalk protein of Influenza B and CRSD of ADAMTS13. The literature review revealed only seven case reports, all but one from Europe or Asia and associated with Influenza A. Severe ADAMTS13 deficiency was demonstrated in only four cases. Conclusion We report the first small case series of influenza-associated TTP. Moreover, it is the first case implicating Influenza B and a mechanism favouring polyclonal B-cell proliferation rather than molecular mimicry as the stimulus to form anti-ADAMTS13 auto-antibodies is suggested.

The Potential Role of Human NME1 in Neuronal Differentiation of Porcine Mesenchymal Stem Cells: Application of NB-hNME1 as a Human NME1 Suppressor

Cho, Jin Hyoung; Ju, Won Seok; Seo, Sang Young; Kim, Bo Hyun; Kim, Ji-Su; Kim, Jong-Geol; Park, Soon Ju; Choo, Young-Kug
Int J Mol Sci.
Nov 2021
This study aimed to investigate the effects of the human macrophage (MP) secretome in cellular xenograft rejection. The role of human nucleoside diphosphate kinase A (hNME1), from the secretome of MPs involved in the neuronal differentiation of miniature pig adipose tissue-derived mesenchymal stem cells (mp AD-MSCs), was evaluated by proteomic analysis. Herein, we first demonstrate that hNME1 strongly binds to porcine ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 1 (pST8SIA1), which is a ganglioside GD3 synthase. When hNME1 binds with pST8SIA1, it induces degradation of pST8SIA1 in mp AD-MSCs, thereby inhibiting the expression of ganglioside GD3 followed by decreased neuronal differentiation of mp AD-MSCs. Therefore, we produced nanobodies (NBs) named NB-hNME1 that bind to hNME1 specifically, and the inhibitory effect of NB-hNME1 was evaluated for blocking the binding between hNME1 and pST8SIA1. Consequently, NB-hNME1 effectively blocked the binding of hNME1 to pST8SIA1, thereby recovering the expression of ganglioside GD3 and neuronal differentiation of mp AD-MSCs. Our findings suggest that mp AD-MSCs could be a potential candidate for use as an additive, such as an immunosuppressant, in stem cell transplantation.

The cellular modifier MOAG-4/SERF drives amyloid formation through charge complementation

Pras, Anita; Houben, Bert; Aprile, Francesco A.; Seinstra, Renée; Gallardo, Rodrigo; Janssen, Leen; Hogewerf, Wytse; Gallrein, Christian; De Vleeschouwer, Matthias; Mata-Cabana, Alejandro; Koopman, Mandy; Stroo, Esther; de Vries, Minke; Louise Edwards, Samantha; Kirstein, Janine; Vendruscolo, Michele; Falsone, Salvatore Fabio; Rousseau, Frederic; Schymkowitz, Joost; Nollen, Ellen A. A.
EMBO J.
Nov 2021
While aggregation-prone proteins are known to accelerate aging and cause age-related diseases, the cellular mechanisms that drive their cytotoxicity remain unresolved. The orthologous proteins MOAG-4, SERF1A, and SERF2 have recently been identified as cellular modifiers of such proteotoxicity. Using a peptide array screening approach on human amyloidogenic proteins, we found that SERF2 interacted with protein segments enriched in negatively charged and hydrophobic, aromatic amino acids. The absence of such segments, or the neutralization of the positive charge in SERF2, prevented these interactions and abolished the amyloid-promoting activity of SERF2. In protein aggregation models in the nematode worm Caenorhabditis elegans, protein aggregation and toxicity were suppressed by mutating the endogenous locus of MOAG-4 to neutralize charge. Our data indicate that MOAG-4 and SERF2 drive protein aggregation and toxicity by interactions with negatively charged segments in aggregation-prone proteins. Such charge interactions might accelerate primary nucleation of amyloid by initiating structural changes and by decreasing colloidal stability. Our study points at charge interactions between cellular modifiers and amyloidogenic proteins as potential targets for interventions to reduce age-related protein toxicity.

Human Antibody Domains and Fragments Targeting Neutrophil Elastase as Candidate Therapeutics for Cancer and Inflammation-Related Diseases

Chu, Xiaojie; Sun, Zehua; Baek, Du-San; Li, Wei; Mellors, John W.; Shapiro, Steven D.; Dimitrov, Dimiter S.
Int J Mol Sci.
Oct 2021
Neutrophil elastase (NE) is a serine protease released during neutrophil maturation. High levels of NE are related to lung tissue damage and poor prognosis in cancer; thus, NE is a potential target for therapeutic immunotherapy for multiple lung diseases and cancers. Here, we isolate and characterize two high-affinity, specific, and noncompetitive anti-NE antibodies Fab 1C10 and VH 1D1.43 from two large phage-displayed human Fab and VH libraries. After fusion with human IgG1 Fc, both of them (VH-Fc 1D1.43 and IgG1 1C10) inhibit NE enzymatic activity with VH-Fc 1D1.43 showing comparable inhibitory effects to that of the small molecule NE inhibitor SPCK and IgG1 1C10 exhibiting even higher (2.6-fold) activity than SPCK. Their epitopes, as mapped by peptide arrays combined with structural modeling, indicate different mechanisms for blocking NE activity. Both VH-Fc and IgG1 antibodies block NE uptake by cancer cells and fibroblast differentiation. VH-Fc 1D1.43 and IgG1 1C10 are promising for the antibody-based immunotherapy of cancer and inflammatory diseases.

HSP70iQ435A to subdue autoimmunity and support anti-tumor responses

Jaishankar, Dinesh; Cosgrove, Cormac; Ramesh, Prathyaya; Mahon, James; Shivde, Rohan; Dellacecca, Emilia R.; Yang, Shiayin F.; Mosenson, Jeffrey; Guevara-Patiño, José A.; Le Poole, I. Caroline
Cell Stress and Chaperones.
Sep 2021
Developing immunosuppressive therapies for autoimmune diseases comes with a caveat that immunosuppression may promote the risk of developing other conditions or diseases. We have previously shown that biolistic delivery of an expression construct encoding inducible HSP70 (HSP70i) with one amino acid modification in the dendritic cell (DC) activating moiety 435–445 (HSP70iQ435A) to mouse skin resulted in significant immunosuppressive activity of autoimmune vitiligo, associated with fewer tissue infiltrating T cells. To prepare HSP70iQ435A as a potential therapeutic for autoimmune vitiligo, in this study we evaluated whether and how biolistic delivery of HSP70iQ435A in mice affects anti-tumor responses. We found that HSP70iQ435A in fact supports anti-tumor responses in melanoma-challenged C57BL/6 mice. Biolistic delivery of the HSP70iQ435A-encoding construct to mice elicited significant anti-HSP70 titers, and anti-HSP70 IgG and IgM antibodies recognize surface-expressed and cytoplasmic HSP70i in human and mouse melanoma cells. A peptide scan revealed that the anti-HSP70 antibodies recognize a specific C-terminal motif within the HSP70i protein. The antibodies elicited surface CD107A expression among mouse NK cells, representative of antibody-mediated cellular cytotoxicity (ADCC), supporting the concept, that HSP70iQ435A-encoding DNA elicits a humoral response to the stress protein expressed selectively on the surface of melanoma cells. Thus, besides limiting autoimmunity and inflammation, HSP70iQ435A elicits humoral responses that limit tumor growth and may be used in conjunction with immune checkpoint inhibitors to not only control tumor but to also limit adverse events following tumor immunotherapy.

Heterotypic Assembly Mechanism Regulates CHIP E3 Ligase Activity

Das, Aniruddha; Thapa, Pankaj; Santiago, Ulises; Shanmugam, Nilesh; Banasiak, Katarzyna; Dabrowska, Katarzyna; Nolte, Hendrik; Szulc, Natalia A.; Gathungu, Rose M.; Cysewski, Dominik; Krüger, Marcus; Dadlez, Michal; Nowotny, Marcin; Camacho, Carlos J.; Hoppe, Thorsten; Pokrzywa, Wojciech
The E3 ubiquitin ligases CHIP/CHN-1 and UFD-2 team up to accelerate ubiquitin chain formation. However, it remained largely unclear how the high processivity of this E3 set is achieved. Here we studied the molecular mechanism and function of the CHN-1/UFD-2 complex in Caenorhabditis elegans. Our data show that UFD-2 binding promotes the cooperation between CHN-1 and ubiquitin-conjugating E2 enzymes by stabilizing the CHN-1 U-box dimer. The HSP-1 chaperone outcompetes UFD-2 for CHN-1 binding and promotes the auto-inhibited CHN-1 state by acting on the conserved position of the U-box domain. The interaction with UFD-2 enables CHN-1 to efficiently ubiquitinate S-Adenosylhomocysteinase (AHCY-1), an enzyme crucial for lipid metabolism. Our results define the molecular mechanism underlying the synergistic cooperation of CHN-1 and UFD-2 in substrate ubiquitylation.

Serum Peptide Immunoglobulin G Autoantibody Response in Patients with Different Central Nervous System Inflammatory Demyelinating Disorders

Lee, Hye Lim; Park, Jin-Woo; Seok, Jin Myoung; Jeon, Mi Young; Kim, Hojin; Lim, Young-Min; Shin, Ha Young; Kang, Sa-Yoon; Kwon, Oh-Hyun; Lee, Sang-Soo; Seok, Hung Youl; Min, Ju-Hong; Lee, Sung-Hyun; Kim, Byung-Jo; Kim, Byoung Joon
Diagnostics.
Jul 2021
Previous efforts to discover new surrogate markers for the central nervous system (CNS) inflammatory demyelinating disorders have shown inconsistent results; moreover, supporting evidence is scarce. The present study investigated the IgG autoantibody responses to various viral and autoantibodies-related peptides proposed to be related to CNS inflammatory demyelinating disorders using the peptide microarray method. We customized a peptide microarray containing more than 2440 immobilized peptides representing human and viral autoantigens. Using this, we tested the sera of patients with neuromyelitis optica spectrum disorders (NMOSD seropositive, n = 6; NMOSD seronegative, n = 5), multiple sclerosis (MS, n = 5), and myelin-oligodendrocyte glycoprotein antibody-associated disease (MOGAD, n = 6), as well as healthy controls (HC, n = 5) and compared various peptide immunoglobulin G (IgG) responses between the groups. Among the statistically significant peptides based on the pairwise comparisons of IgG responses in each disease group to HC, cytomegalovirus (CMV)-related peptides were most clearly distinguishable among the study groups. In particular, the most significant differences in IgG response were observed for HC vs. MS and HC vs. seronegative NMOSD (p = 0.064). Relatively higher IgG responses to CMV-related peptides were observed in patients with MS and NMOSD based on analysis of the customized peptide microarray.

Non-invasive immunoPET imaging of PD-L1 using anti-PD-L1-B11 in breast cancer and melanoma tumor model

Bansal, Aditya; Pandey, Mukesh K.; Barham, Whitney; Liu, Xin; Harrington, Susan M.; Lucien, Fabrice; Dong, Haidong; Park, Sean S.; DeGrado, Timothy R.
Nuclear Medicine and Biology.
May 2021
Introduction Immunotherapy targeting PD-1/PD-L1 immune checkpoint inhibition (ICI) is efficacious in various solid and hematologic malignancies. However, the response rate to PD-1/PD-L1 therapy is only 15–35%. To obtain optimal clinical response to ICI therapies, a reliable assessment of tumor PD-L1 expression is needed to select appropriate patients, and a non-invasive imaging-based assessment of PD-L1 expression is critically needed. Although radiolabeled PET probes based on PD-L1 targeted therapeutic antibodies (e.g. atezolizumab) have shown encouraging results, there is concern that residual therapeutic antibody may compete for binding with the radiotracer thereby compromising imaging studies that follow treatment. Methods and results In this study, we used novel anti-PD-L1-B11 clone antibody known to bind to a different epitope of PD-L1 than the therapeutic antibodies to avoid potential saturation effects. The anti-PD-L1-B11 clone was radiolabeled with zirconium-89 and evaluated to detect PD-L1 expression in various in vitro and in vivo cancer model systems in comparison with [89Zr]Zr-DFO-NCS-atezolizumab. In vitro binding parameters of anti-PD-L1-B11 were like those of atezolizumab. [89Zr]Zr-DFO-NCS-anti-PD-L1-B11 clone showed favorable properties to [89Zr]Zr-DFO-NCS-atezolizumab in an in vivo breast cancer tumor model system with higher uptake in PD-L1 expressing tumors. Conclusion Our data demonstrates that [89Zr]Zr-DFO-NCS-anti-PD-L1-B11 exhibits excellent imaging properties for the assessment PD-L1 expression. The independent binding site of anti-PD-L1-B11 relative to therapeutic anti-PD-L1 antibodies may be advantageous for anti-PD-L1 therapy monitoring.

Analysis of chronic inflammatory lesions of the colon for BMMF Rep antigen expression and CD68 macrophage interactions

Bund, Timo; Nikitina, Ekaterina; Chakraborty, Deblina; Ernst, Claudia; Gunst, Karin; Boneva, Boyana; Tessmer, Claudia; Volk, Nadine; Brobeil, Alexander; Weber, Achim; Heikenwalder, Mathias; Zur Hausen, Harald; de Villiers, Ethel-Michele
Proc Natl Acad Sci U S A.
Mar 2021
Consumption of Eurasian bovine meat and milk has been associated with cancer development, in particular with colorectal cancer (CRC). In addition, zoonotic infectious agents from bovine products were proposed to cause colon cancer (zur Hausen et al., 2009). Bovine meat and milk factors (BMMF) are small episomal DNA molecules frequently isolated from bovine sera and milk products, and recently, also from colon cancer (de Villiers et al., 2019). BMMF are bioactive in human cells and were proposed to induce chronic inflammation in precancerous tissue leading to increased radical formation: for example, reactive oxygen and reactive nitrogen species and elevated levels of DNA mutations in replicating cells, such as cancer progenitor cells (zur Hausen et al., 2018). Mouse monoclonal antibodies against the replication (Rep) protein of H1MSB.1 (BMMF1) were used to analyze BMMF presence in different cohorts of CRC peritumor and tumor tissues and cancer-free individuals by immunohistochemistry and Western blot. BMMF DNA was isolated by laser microdissection from immunohistochemistry-positive tissue regions. We found BMMF Rep protein present specifically in close vicinity of CD68+ macrophages in the interstitial lamina propria adjacent to CRC tissues, suggesting the presence of local chronic inflammation. BMMF1 (modified H1MSB.1) DNA was isolated from the same tissue regions. Rep and CD68+ detection increased significantly in peritumor cancer tissues when compared to tissues of cancer-free individuals. This strengthens previous postulations that BMMF function as indirect carcinogens by inducing chronic inflammation and DNA damage in replicating cells, which represent progress to progenitor cells for adenoma (polyps) formation and cancer.

A new reliable and highly specific monoclonal antibody to detect the C‐terminal region of silencer of cytokine signaling 1

Weissinger, Stephanie E.; Zahn, Malena; Marienfeld, Ralf; Tessmer, Claudia; Moldenhauer, Gerhard; Möller, Peter
Eur J Haematol.
Mar 2021
Introduction SOCS1, a negative regulator of JAK/STAT signaling, is among the most frequently mutated genes in DLBCL and classical Hodgkin lymphoma. The C-terminal SOCS box domain, mediating the degradation of phospho-JAK2, is often affected or even lacking. The analysis of such variants is hampered by the lack of a SOCS1-specific monoclonal antibody recognizing the C-terminus of SOCS1. As this C-terminus is often lost or mutated in B-cell lymphomas, staining with amino-terminal targeting antibodies in a lymphoma setting might be misleading. Methods BALB/c mice were immunized with a truncated SOCS1 C-terminal protein. The supernatant of generated hybridoma cells was screened by ELISA and, immunohistochemically, on formalin-fixed and paraffin-embedded tonsil. After antibody purification by affinity chromatography, epitope mapping and cross-reactivity check followed via substitution scans. SOCS1 protein expression was investigated on cell cultures and cytoblocks of SOCS1WT stably transfected HEK293T cells, lymphoma cell lines and lymphoid tissues. Results Procedures resulted in one monoclonal IgG1 anti-SOCS1 antibody, 424C, that recognizes and strongly binds to the C-terminal region of SOCS1 in immunoblot and immunohistochemistry analyses. Conclusion This new anti-SOCS1 monoclonal antibody is a valuable tool to detect SOCS1 expression dependent on an existing SOCS1 box and, therefore, indicating a full-length SOCS1 protein.

Scoping review of the applications of peptide microarrays on the fight against human infections

Vengesai, Arthur; Kasambala, Maritha; Mutandadzi, Hamlet; Mduluza-Jokonya, Tariro L.; Mduluza, Takafira; Naicker, Thajasvarie
Abstract Introduction This scoping review explores the use of peptide microarrays in the fight against infectious diseases. The research domains explored included the use of peptide microarrays in the mapping of linear B-cell and T cell epitopes, antimicrobial peptide discovery, immunosignature characterisation and disease immunodiagnostics. This review also provides a short overview of peptide microarray synthesis.   Methods Electronic databases were systematically searched to identify relevant studies. The review was conducted using the Joanna Briggs Institute methodology for scoping reviews and data charting was performed using a predefined form. The results were reported by narrative synthesis in line with the Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews guidelines. Results Eighty-six articles from 100 studies were included in the final data charting process. The majority (93%) of the articles were published during 2010–2020 and were mostly from Europe (44%) and North America (34 %). The findings were from the investigation of viral (44%), bacterial (30%), parasitic (25%) and fungal (2%) infections. Out of the serological studies, IgG was the most reported antibody type followed by IgM. The largest portion of the studies (78%) were related to mapping B-cell linear epitopes, 10% were on diagnostics, 9% reported on immunosignature characterisation and 6% reported on viral and bacterial cell binding assays. Two studies reported on T-cell epitope profiling. Conclusion The most important application of peptide microarrays was found to be B-cell epitope mapping or antibody profiling to identify diagnostic and vaccine targets. Immunosignatures identified by random peptide microarrays were found to be applied in the diagnosis of infections and interrogation of vaccine responses. The analysis of the interactions of random peptide microarrays with bacterial and viral cells using binding assays enabled the identification of antimicrobial peptides. Peptide microarray arrays were also used for T-cell linear epitope mapping which may provide more information for the design of peptide-based vaccines and for the development of diagnostic reagents.

Comparison of Monoclonal Gammopathies Linked to Poliovirus or Coxsackievirus vs. Other Infectious Pathogens

Harb, Jean; Mennesson, Nicolas; Lepetit, Cassandra; Fourny, Maeva; Louvois, Margaux; Bosseboeuf, Adrien; Allain-Maillet, Sophie; Decaux, Olivier; Moreau, Caroline; Tallet, Anne; Piver, Eric; Moreau, Philippe; Salle, Valéry; Bigot-Corbel, Edith; Hermouet, Sylvie
Chronic stimulation by infectious pathogens or self-antigen glucosylsphingosine (GlcSph) can lead to monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM). Novel assays such as the multiplex infectious antigen microarray (MIAA) and GlcSph assays, permit identification of targets for >60% purified monoclonal immunoglobulins (Igs). Searching for additional targets, we selected 28 purified monoclonal Igs whose antigen was not represented on the MIAA and GlcSph assays; their specificity of recognition was then analyzed using microarrays consisting of 3760 B-cell epitopes from 196 pathogens. The peptide sequences PALTAVETG and PALTAAETG of the VP1 coat proteins of human poliovirus 1/3 and coxsackievirus B1/B3, respectively, were specifically recognized by 6/28 monoclonal Igs. Re-analysis of patient cohorts showed that purified monoclonal Igs from 10/155 MGUS/SM (6.5%) and 3/147 MM (2.0%) bound to the PALTAVETG or PALTAAETG epitopes. Altogether, PALTAV/AETG-initiated MGUS are not rare and few seem to evolve toward myeloma.

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