Search

Abstract Background Interleukin-6 (IL-6) is a multifunctional cytokine that controls the immune response, and its role has been described in the development of autoimmune diseases. Signaling via its cognate IL-6 receptor (IL-6R) complex is critical in tumor progression and, therefore, IL-6R represents an important therapeutic target. Methods An albumin-binding domain-derived highly complex combinatorial library was used to select IL-6R alpha (IL-6Rα)-targeted small protein binders using ribosome display. Large-scale screening of bacterial lysates of individual clones was performed using ELISA, and their IL-6Rα blocking potential was verified by competition ELISA. The binding of proteins to cells was monitored by flow cytometry and confocal microscopy on HEK293T-transfected cells, and inhibition of signaling function was examined using HEK-Blue IL-6 reporter cells. Protein binding kinetics to living cells was measured by LigandTracer, cell proliferation and toxicity by iCELLigence and Incucyte, cell migration by the scratch wound healing assay, and prediction of binding poses using molecular modeling by docking. Results We demonstrated a collection of protein variants called NEF ligands, selected from an albumin-binding domain scaffold-derived combinatorial library, and showed their binding specificity to human IL-6Rα and antagonistic effect in HEK-Blue IL-6 reporter cells. The three most promising NEF108, NEF163, and NEF172 variants inhibited cell proliferation of malignant melanoma (G361 and A2058) and pancreatic (PaTu and MiaPaCa) cancer cells, and suppressed migration of malignant melanoma (A2058), pancreatic carcinoma (PaTu), and glioblastoma (GAMG) cells in vitro. The NEF binders also recognized maturation-induced IL-6Rα expression and interfered with IL-6-induced differentiation in primary human B cells. Conclusion We report on the generation of small protein blockers of human IL-6Rα using directed evolution. NEF proteins represent a promising class of non-toxic anti-tumor agents with migrastatic potential.

Abstract Background Programmed cell death 1 (PD-1) belongs to immune checkpoint proteins ensuring negative regulation of the immune response. In non-small cell lung cancer (NSCLC), the sensitivity to treatment with anti-PD-1 therapeutics, and its efficacy, mostly correlated with the increase of tumor infiltrating PD-1+ lymphocytes. Due to solid tumor heterogeneity of PD-1+ populations, novel low molecular weight anti-PD-1 high-affinity diagnostic probes can increase the reliability of expression profiling of PD-1+ tumor infiltrating lymphocytes (TILs) in tumor tissue biopsies and in vivo mapping efficiency using immune-PET imaging. Methods We designed a 13 kDa β-sheet Myomedin scaffold combinatorial library by randomization of 12 mutable residues, and in combination with ribosome display, we identified anti-PD-1 Myomedin variants (MBA ligands) that specifically bound to human and murine PD-1-transfected HEK293T cells and human SUP-T1 cells spontaneously overexpressing cell surface PD-1. Results Binding affinity to cell-surface expressed human and murine PD-1 on transfected HEK293T cells was measured by fluorescence with LigandTracer and resulted in the selection of most promising variants MBA066 (hPD-1 KD = 6.9 nM; mPD-1 KD = 40.5 nM), MBA197 (hPD-1 KD = 29.7 nM; mPD-1 KD = 21.4 nM) and MBA414 (hPD-1 KD = 8.6 nM; mPD-1 KD = 2.4 nM). The potential of MBA proteins for imaging of PD-1+ populations in vivo was demonstrated using deferoxamine-conjugated MBA labeled with 68Galium isotope. Radiochemical purity of 68Ga-MBA proteins reached values 94.7–99.3% and in vitro stability in human serum after 120 min was in the range 94.6–98.2%. The distribution of 68Ga-MBA proteins in mice was monitored using whole-body positron emission tomography combined with computerized tomography (PET/CT) imaging up to 90 min post-injection and post mortem examined in 12 mouse organs. The specificity of MBA proteins was proven by co-staining frozen sections of human tonsils and NSCLC tissue biopsies with anti-PD-1 antibody, and demonstrated their potential for mapping PD-1+ populations in solid tumors. Conclusions Using directed evolution, we developed a unique set of small binding proteins that can improve PD-1 diagnostics in vitro as well as in vivo using PET/CT imaging. Graphical Abstract

Introduction: Immunoglobulin A1 (IgA1) with galactose-deficient O-glycans (Gd-IgA1) play a key role in the pathogenesis of IgA nephropathy (IgAN). Mucosal-tissue infections increase IL-6 production and, in patients with IgAN, are often associated with macroscopic hematuria. IgA1-secreting cell lines derived from the circulation of patients with IgAN, compared to those of healthy controls (HCs), produce more IgA1 that has O-glycans with terminal or sialylated N-acetylgalactosamine (GalNAc). GalNAc residues are added to IgA1 hinge region by some of the 20 GalNAc transferases, the O-glycosylation-initiating enzymes. Expression of GALNT2, encoding GalNAc-T2, the main enzyme initiating IgA1 O-glycosylation, is similar in cells derived from patients with IgAN and HCs. In this report, we extend our observations of GALNT14 overexpression in IgA1-producing cell lines from patients with IgAN. Methods: GALNT14 expression was analyzed in peripheral blood mononuclear cells (PBMCs) from patients with IgAN and from HCs. Moreover, the effect of GALNT14 overexpression or knock-down on Gd-IgA1 production in Dakiki cells was assessed. Results: GALNT14 was overexpressed in PBMCs from patients with IgAN. IL-6 increased GALNT14 expression in PBMCs from patients with IgAN and HCs. We used IgA1-producing cell line Dakiki, a previously reported model of Gd-IgA1-producing cells, and showed that overexpression of GalNAc-T14 enhanced galactose deficiency of IgA1, whereas siRNA-mediated GalNAc-T14 knock-down reduced it. GalNAc-T14 was localized in trans-Golgi network, as expected. Conclusions: Overexpression of GALNT14 due to inflammatory signals during mucosal infections may contribute to overproduction of Gd-IgA1 in patients with IgAN.

One of the proposed strategies for the development of a more efficient HIV-1 vaccine is based on the identification of proteins binding to a paratope of chosen broadly neutralizing antibody (bNAb) that will mimic cognate HIV-1 Env (glyco)protein epitope and could be used as potent immunogens for induction of protective virus-neutralizing antibodies in the immunized individuals. To verify this “non-cognate ligand” concept, we developed a highly complex combinatorial library designed on a scaffold of human myomesin-1 protein domain and selected proteins called Myomedins specifically binding to variable regions of HIV-1 broadly neutralizing antibody 10E8. Immunization of mice with these Myomedin variants elicited the production of HIV-1 Env-specific antibodies. Hyperimmune sera bound to Env pseudotyped viruses and weakly/moderately neutralized 54% of tested clade A, B, C, and AE pseudotyped viruses variants in vitro. These results demonstrate that Myomedin variants have the potential to mimic Env epitopes and could be used as potential HIV-1 vaccine components.

IntroductionImprinting broadly neutralizing antibody (bNAb) paratopes by shape complementary protein mimotopes represents a potential alternative for developing vaccine immunogens. This approach, designated as a Non-Cognate Ligand Strategy (NCLS), has recently been used for the identification of protein variants mimicking CD4 binding region epitope or membrane proximal external region (MPER) epitope of HIV-1 envelope (Env) glycoprotein. However, the potential of small binding proteins to mimic viral glycan-containing epitopes has not yet been verified.MethodsIn this work, we employed a highly complex combinatorial Myomedin scaffold library to identify variants recognizing paratopes of super candidate bNAbs, PGT121 and PGT126, specific for HIV-1 V3 loop epitopes.ResultsIn the collection of Myomedins called MLD variants targeted to PGT121, three candidates competed with gp120 for binding to this bNAb in ELISA, thus suggesting an overlapping binding site and epitope-mimicking potential. Myomedins targeted to PGT126 designated MLB also provided variants that competed with gp120. Immunization of mice with MLB or MLD binders resulted in the production of anti-gp120 and -Env serum antibodies. Mouse hyper-immune sera elicited with MLB036, MLB041, MLB049, and MLD108 moderately neutralized 8-to-10 of 22 tested HIV-1-pseudotyped viruses of A, B, and C clades in vitro.DiscussionOur data demonstrate that Myomedin-derived variants can mimic particular V3 glycan epitopes of prominent anti-HIV-1 bNAbs, ascertain the potential of particular glycans controlling neutralizing sensitivity of individual HIV-1 pseudoviruses, and represent promising prophylactic candidates for HIV-1 vaccine development.

Aims: Epstein-Barr virus (EBV) targets predominantly B cells and these cells could acquire new phenotype characteristics. Here we analyzed whether EBV-infected and -uninfected B cells from healthy subjects differ in proportion of dominant phenotypes, maturation stage, and homing receptors expression. Methods: EBV-infected and -uninfected cells were identified by flow cytometry using fluorophore-labeled EBV RNA-specific DNA probes combined with fluorophore-labeled antibody to surface lineage markers, integrins, chemokine receptors, and immunoglobulin isotypes, including intracellular ones. Results: Our results show that the trafficking characteristics of EBERpos B cells are distinct from EBERneg B cells with most dominant differences detected for α4β1 and α4β7 and CCR5 and CCR7. EBV-positive cells are predominantly memory IgM+ B cells or plasmablasts/plasma cells (PB/PC) positive for IgA or less for IgM. In comparison to uninfected B cells, less EBV-positive B cells express α4β7 and almost no cells express α4β1. EBV-positive B cells contained significantly higher proportion of CCR5+ and CCR7+ cells in comparison to EBV-negative cells. In vitro exposure of blood mononuclear cells to pro-inflammatory cytokine IL-6 reduces population of EBV-positive B cell. Conclusion: Although EBV-infected B cells represent only a minor subpopulation, their atypical functions could contribute in predisposed person to development abnormities such as some autoimmune diseases or tumors. Using multi-parameter flow cytometry we characterized differences in migration of EBV-positive and -negative B cells of various maturation stage and isotype of produced antibodies particularly different targeting to mucosal tissues of gastrointestinal and respiratory tracts.

Analysing complex datasets while maintaining the interpretability and explainability of outcomes for clinicians and patients is challenging, not only in viral infections. These datasets often include a variety of heterogeneous clinical, demographic, laboratory, and personal data, and it is not a single factor but a combination of multiple factors that contribute to patient characterisation and host response. Therefore, multivariate approaches are needed to analyse these complex patient datasets, which are impossible to analyse with univariate comparisons (e.g., one immune cell subset versus one clinical factor). Using a SARS-CoV-2 infection as an example, we employed a patient similarity network (PSN) approach to assess the relationship between host immune factors and the clinical course of infection and performed visualisation and data interpretation. A PSN analysis of ~85 immunological (cellular and humoral) and ~70 clinical factors in 250 recruited patients with coronavirus disease (COVID-19) who were sampled four to eight weeks after a PCR-confirmed SARS-CoV-2 infection identified a minimal immune signature, as well as clinical and laboratory factors strongly associated with disease severity. Our study demonstrates the benefits of implementing multivariate network approaches to identify relevant factors and visualise their relationships in a SARS-CoV-2 infection, but the model is generally applicable to any complex dataset.

Background: The development of an effective vaccine preventing HIV-1 infection is hindered by the enormous antigenic variability and unique biochemical and immunological properties of HIV-1 Env glycoprotein, the most promising target for HIV-1 neutralizing antibody. Functional studies of rare elite neutralizers led to the discovery of broadly neutralizing antibodies. Methods: We employed a highly complex combinatorial protein library derived from a 5 kDa albumin-binding domain scaffold, fused with support protein of total 38 kDa, to screen for binders of broadly neutralizing antibody VRC01 paratope. The most specific binders were used for immunization of experimental mice to elicit Env-specific antibodies and to test their neutralization activity using a panel of HIV-1 clade C and B pseudoviruses. Findings: Three most specific binders designated as VRA017, VRA019, and VRA177 exhibited high specificity to VRC01 antibody. Immunized mice produced Env-binding antibodies which neutralize eight of twelve HIV-1 Tier 2 pseudoviruses. Molecular modelling revealed a shape complementarity between VRA proteins and a part of VRC01 gp120 interacting surface. Interpretation: This strategy based on the identification of protein replicas of broadly neutralizing antibody paratope represents a novel approach in HIV-1 vaccine development. This approach is not affected by low immunogenicity of neutralization-sensitive epitopes, variability, and unique biochemical properties of HIV-1 Env used as a crucial antigen in the majority of contemporary tested vaccines. Fund: Czech Health Research Council 15-32198A, Ministry of Health, Czech Republic. Keywords: Neutralizing antibody, Albumin-binding domain scaffold, Combinatorial protein library, Antibody paratope mimetics, Protein docking, HIV-1 vaccine

IgA nephropathy (IgAN) is the dominant type of primary glomerulonephritis worldwide. However, IgAN rarely affects African Blacks and is uncommon in African Americans. Polymeric IgA1 with galactose-deficient hinge-region glycans is recognized as auto-antigen by glycan-specific antibodies, leading to formation of circulating immune complexes with nephritogenic consequences. Because human B cells infected in vitro with Epstein-Barr virus (EBV) secrete galactose-deficient IgA1, we examined peripheral blood B cells from adult IgAN patients, and relevant controls, for the presence of EBV and their phenotypic markers. We found that IgAN patients had more lymphoblasts/plasmablasts that were surface-positive for IgA, infected with EBV, and displayed increased expression of homing receptors for targeting the upper respiratory tract. Upon polyclonal stimulation, these cells produced more galactose-deficient IgA1 than did cells from healthy controls. Unexpectedly, in healthy African Americans, EBV was detected preferentially in surface IgM- and IgD-positive cells. Importantly, most African Blacks and African Americans acquire EBV within 2 years of birth. At that time, the IgA system is naturally deficient, manifested as low serum IgA levels and few IgA-producing cells. Consequently, EBV infects cells secreting immunoglobulins other than IgA. Our novel data implicate Epstein-Barr virus infected IgA+ cells as the source of galactose-deficient IgA1 and basis for expression of relevant homing receptors. Moreover, the temporal sequence of racial-specific differences in Epstein-Barr virus infection as related to the naturally delayed maturation of the IgA system explains the racial disparity in the prevalence of IgAN.

Background/Aims: IgA nephropathy is associated with aberrant O-glycosylation of IgA1, which is recognized by autoantibodies leading to the formation of circulating immune complexes. Some of them, after deposition into kidney mesangium, trigger glomerular injury. In patients with active disease nonresponding to angiotensin-converting enzyme inhibitors or angiotensin II blockers, corticosteroids are recommended. Methods: The relationship between the corticosteroid therapy and serum levels of IgA, aberrantly O-glycosylated IgA1, IgA-containing immune complexes and their mesangioproliferative activity was analyzed in IgA nephropathy patients and disease and healthy controls. Results: Prednisone therapy significantly reduced proteinuria and levels of serum IgA, galactose-deficient IgA1, and IgA-IgG immune complexes in IgA nephropathy patients and thus reduced differences in all of the above parameters between IgAN patients and control groups. A moderate but not significant reduction of mesangioproliferative potential of IgA-IgG immune complexes and IgA sialylation was detected. Conclusion: The prednisone therapy reduces overall aberrancy in IgA1 O-glycosylation in IgA nephropathy patients, but the measurement of IgA1 parameters does not allow us to predict the prednisone therapy outcome in individual patients.

Mdm2 and MdmX are related proteins serving in the form of the Mdm2 homodimer or Mdm2/MdmX heterodimer as an E3 ubiquitin ligase for the tumor suppressor p53. The dimerization is required for the E3 activity and is mediated by the conserved RING domains present in both proteins, but only the RING domain of Mdm2 can form homodimers efficiently. We performed a systematic mutational analysis of human Mdm2, exchanging parts of the RING with the corresponding MdmX sequence, to identify the molecular determinants of this difference. Mdm2 can also promote MdmX degradation, and we identified several mutations blocking it. They were located mainly at the Mdm2/E2 interface and did not disrupt the MdmX-Mdm2 interaction. Surprisingly, some mutations of the Mdm2/E2 interface inhibited MdmX degradation, which is mediated by the Mdm2/MdmX heterodimer, but did not affect p53 degradation, mediated by the Mdm2 homodimer. Only one mutant, replacing a conserved cysteine 449 with asparagine (C449N), disrupted the ability of Mdm2 to dimerize with MdmX. When we introduced the cysteine residue into the corresponding site in MdmX, the RING domain became capable of forming dimers with other MdmX molecules in vivo, suggesting that one conserved amino acid residue in the RINGs of Mdm2 and MdmX could serve as the determinant of the differential ability of these domains to form dimers and their E3 activity. In immunoprecipitations, however, the homodimerization of MdmX could be observed only when the asparagine residue was replaced with cysteine in both RINGs. This result suggested that heterocomplexes consisting of one mutated MdmX RING with cysteine and one wild-type MdmX RING with asparagine might be less stable, despite being readily detectable in the cell-based assay. Moreover, Mdm2 C449N blocked Mdm2-MdmX heterodimerization but did not disrupt the ability of Mdm2 homodimer to promote p53 degradation, suggesting that the effect of the conserved cysteine and asparagine residues on dimerization was context-specific. Collectively, our results indicate that the effects of individual exchanges of conserved residues between Mdm2 and MdmX RING domains might be context-specific, supporting the hypothesis that Mdm2 RING homodimers and Mdm2-MdmX heterodimers may not be entirely structurally equivalent, despite their apparent similarity.

Copyright of Klinická Biochemie a Metabolismus is the property of Czech Medical Association of JE Purkyne and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Williams-Beuren syndrome-associated transcription factor TFII-I plays a critical regulatory role in bone and neural tissue development and in immunity, in part by regulating cell proliferation in response to mitogens. Mdm2, a cellular oncogene responsible for the loss of p53 tumor suppressor activity in a significant proportion of human cancers, was identified in this study as a new binding partner for TFII-I and a negative regulator of TFII-I-mediated transcription. These findings suggest a new p53-independent mechanism by which increased Mdm2 levels found in human tumors could influence cancer cells. In addition to that, we present data indicating that TFII-I is an important cellular regulator of transcription from the immediate-early promoter of human cytomegalovirus, a promoter sequence frequently used in mammalian expression vectors, including vectors for gene therapy. Our observation that Mdm2 over-expression can decrease the ability of TFII-I to activate the CMV promoter might have implications for the efficiency of experimental gene therapy based on CMV promoter-derived vectors in cancers with Mdm2 gene amplification.

Aims. Epstein-Barr virus (EBV) targets predominantly B cells and these cells could acquire new phenotype characteristics. Here we analyzed whether EBV-infected and -uninfected B cells from healthy subjects differ in proportion of dominant phenotypes, maturation stage, and homing receptors expression. Methods. EBV-infected and -uninfected cells were identified by flow cytometry using fluorophore-labeled EBV RNAspecific DNA probes combined with fluorophore-labeled antibody to surface lineage markers, integrins, chemokine receptors, and immunoglobulin isotypes, including intracellular ones. Results. Our results show that the trafficking characteristics of EBERpos B cells are distinct from EBERneg B cells with most dominant differences detected for a4ß1 and a4ß7 and CCR5 and CCR7. EBV-positive cells are predominantly memory IgM+ B cells or plasmablasts/plasma cells (PB/PC) positive for IgA or less for IgM. In comparison to uninfected B cells, less EBV-positive B cells express a4ß7 and almost no cells express a4ß1. EBV-positive B cells contained significantly higher proportion of CCR5+ and CCR7+ cells in comparison to EBV-negative cells. In vitro exposure of blood mononuclear cells to pro-inflammatory cytokine IL-6 reduces population of EBV-positive B cell. Conclusion. Although EBV-infected B cells represent only a minor subpopulation, their atypical functions could contribute in predisposed person to development abnormities such as some autoimmune diseases or tumors. Using multi-parameter flow cytometry we characterized differences in migration of EBV-positive and -negative B cells of various maturation stage and isotype of produced antibodies particularly different targeting to mucosal tissues of gastrointestinal and respiratory tracts. [ABSTRACT FROM AUTHOR]

Liposomes are one of the most important drug delivery vectors, nowadays used in clinics. In general, polyethylene glycol (PEG) is used to ensure the stealth properties of the liposomes. Here, we have employed hydrophilic, biocompatible and highly non-fouling N-(2-hydroxypropyl) methacrylamide (HPMA)-based copolymers containing hydrophobic cholesterol anchors for the surface modification of liposomes, which were prepared by the method of lipid film hydration and extrusion through 100 nm polycarbonate filters. Efficient surface modification of liposomes was confirmed by transmission electron microscopy, atomic force microscopy, and gradient ultracentrifugation. The ability of long-term circulation in the vascular bed was demonstrated in rabbits after i.v. application of fluorescently labelled liposomes. Compared to PEGylated liposomes, HPMA-based copolymer-modified liposomes did not induce specific antibody formation and did not activate murine and human complement. Compared with PEGylated liposomes, HPMA-based copolymer-modified liposomes showed a better long-circulating effect after repeated administration. HPMA-based copolymer-modified liposomes thus represent suitable new candidates for a generation of safer and improved liposomal drug delivery platforms., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Introduction: Immunoglobulin A1 (IgA1) with galactose-deficient O -glycans (Gd-IgA1) play a key role in the pathogenesis of IgA nephropathy (IgAN). Mucosal-tissue infections increase IL-6 production and, in patients with IgAN, are often associated with macroscopic hematuria. IgA1-secreting cell lines derived from the circulation of patients with IgAN, compared to those of healthy controls (HCs), produce more IgA1 that has O - glycans with terminal or sialylated N -acetylgalactosamine (GalNAc). GalNAc residues are added to IgA1 hinge region by some of the 20 GalNAc transferases, the O -glycosylation-initiating enzymes. Expression of GALNT2 , encoding GalNAc-T2, the main enzyme initiating IgA1 O -glycosylation, is similar in cells derived from patients with IgAN and HCs. In this report, we extend our observations of GALNT14 overexpression in IgA1-producing cell lines from patients with IgAN., Methods: GALNT14 expression was analyzed in peripheral blood mononuclear cells (PBMCs) from patients with IgAN and from HCs. Moreover, the effect of GALNT14 overexpression or knock-down on Gd-IgA1 production in Dakiki cells was assessed., Results: GALNT14 was overexpressed in PBMCs from patients with IgAN. IL-6 increased GALNT14 expression in PBMCs from patients with IgAN and HCs. We used IgA1-producing cell line Dakiki, a previously reported model of Gd-IgA1-producing cells, and showed that overexpression of GalNAc-T14 enhanced galactose deficiency of IgA1, whereas siRNA-mediated GalNAc-T14 knock-down reduced it. GalNAc-T14 was localized in trans-Golgi network, as expected., Conclusions: Overexpression of GALNT14 due to inflammatory signals during mucosal infections may contribute to overproduction of Gd-IgA1 in patients with IgAN., (© 2023 Published by Elsevier Inc. on behalf of the International Society of Nephrology.)

Introduction: Imprinting broadly neutralizing antibody (bNAb) paratopes by shape complementary protein mimotopes represents a potential alternative for developing vaccine immunogens. This approach, designated as a Non-Cognate Ligand Strategy (NCLS), has recently been used for the identification of protein variants mimicking CD4 binding region epitope or membrane proximal external region (MPER) epitope of HIV-1 envelope (Env) glycoprotein. However, the potential of small binding proteins to mimic viral glycan-containing epitopes has not yet been verified., Methods: In this work, we employed a highly complex combinatorial Myomedin scaffold library to identify variants recognizing paratopes of super candidate bNAbs, PGT121 and PGT126, specific for HIV-1 V3 loop epitopes., Results: In the collection of Myomedins called MLD variants targeted to PGT121, three candidates competed with gp120 for binding to this bNAb in ELISA, thus suggesting an overlapping binding site and epitope-mimicking potential. Myomedins targeted to PGT126 designated MLB also provided variants that competed with gp120. Immunization of mice with MLB or MLD binders resulted in the production of anti-gp120 and -Env serum antibodies. Mouse hyper-immune sera elicited with MLB036, MLB041, MLB049, and MLD108 moderately neutralized 8-to-10 of 22 tested HIV-1-pseudotyped viruses of A, B, and C clades in vitro ., Discussion: Our data demonstrate that Myomedin-derived variants can mimic particular V3 glycan epitopes of prominent anti-HIV-1 bNAbs, ascertain the potential of particular glycans controlling neutralizing sensitivity of individual HIV-1 pseudoviruses, and represent promising prophylactic candidates for HIV-1 vaccine development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Daniel Lišková, Kosztyu, Kuchař, Černý, Bharadwaj, Petroková, Vroblová, Křupka, Malý, Zosinčuková, Šulc, Rašková Kafková, Raška and Malý.)

Analysing complex datasets while maintaining the interpretability and explainability of outcomes for clinicians and patients is challenging, not only in viral infections. These datasets often include a variety of heterogeneous clinical, demographic, laboratory, and personal data, and it is not a single factor but a combination of multiple factors that contribute to patient characterisation and host response. Therefore, multivariate approaches are needed to analyse these complex patient datasets, which are impossible to analyse with univariate comparisons (e.g., one immune cell subset versus one clinical factor). Using a SARS-CoV-2 infection as an example, we employed a patient similarity network (PSN) approach to assess the relationship between host immune factors and the clinical course of infection and performed visualisation and data interpretation. A PSN analysis of ~85 immunological (cellular and humoral) and ~70 clinical factors in 250 recruited patients with coronavirus disease (COVID-19) who were sampled four to eight weeks after a PCR-confirmed SARS-CoV-2 infection identified a minimal immune signature, as well as clinical and laboratory factors strongly associated with disease severity. Our study demonstrates the benefits of implementing multivariate network approaches to identify relevant factors and visualise their relationships in a SARS-CoV-2 infection, but the model is generally applicable to any complex dataset.

IgA nephropathy (IgAN) is the dominant type of primary glomerulonephritis worldwide. However, IgAN rarely affects African Blacks and is uncommon in African Americans. Polymeric IgA1 with galactose-deficient hinge-region glycans is recognized as auto-antigen by glycan-specific antibodies, leading to formation of circulating immune complexes with nephritogenic consequences. Because human B cells infected in vitro with Epstein-Barr virus (EBV) secrete galactose-deficient IgA1, we examined peripheral blood B cells from adult IgAN patients, and relevant controls, for the presence of EBV and their phenotypic markers. We found that IgAN patients had more lymphoblasts/plasmablasts that were surface-positive for IgA, infected with EBV, and displayed increased expression of homing receptors for targeting the upper respiratory tract. Upon polyclonal stimulation, these cells produced more galactose-deficient IgA1 than did cells from healthy controls. Unexpectedly, in healthy African Americans, EBV was detected preferentially in surface IgM- and IgD-positive cells. Importantly, most African Blacks and African Americans acquire EBV within 2 years of birth. At that time, the IgA system is naturally deficient, manifested as low serum IgA levels and few IgA-producing cells. Consequently, EBV infects cells secreting immunoglobulins other than IgA. Our novel data implicate Epstein-Barr virus infected IgA + cells as the source of galactose-deficient IgA1 and basis for expression of relevant homing receptors. Moreover, the temporal sequence of racial-specific differences in Epstein-Barr virus infection as related to the naturally delayed maturation of the IgA system explains the racial disparity in the prevalence of IgAN., (Copyright © 2020 Zachova, Kosztyu, Zadrazil, Matousovic, Vondrak, Hubacek, Julian, Moldoveanu, Novak, Kostovcikova, Raska and Mestecky.)

The interaction between ABCB1 transporter and its substrates takes place in cell membranes but the available data precludes quantitative analysis of the interaction between transporter and substrate molecules. Further, the amount of transporter is usually expressed as a number of ABCB1 molecules per cell. In contrast, the substrate concentration in cell membranes is estimated by determination of substrate-lipid partition coefficient, as examples. In this study, we demonstrate an approach, which enables us to estimate the concentration of ABCB1 molecules within plasma membranes. For this purpose, human leukemia K562 cells with varying expression levels of ABCB1 were used: drug selected K562/Dox and K562/HHT cells with very high transporter expression, and K562/DoxDR2, K562/DoxDR1, and K562/DoxDR05 cells with gradually decreased expression of ABCB1 derived from K562/Dox cells using RNA interference technology. First, we determined the absolute amount of ABCB1 in cell lysates using immunoblotting and recombinant ABCB1 as a standard. We then determined the relative portion of transporter residing in the plasma membrane using immunohistochemistry in nonpermeabilized and permeabilized cells. These results enabled us to estimate the concentration of ABCB1 in the plasma membrane in resistant cells. The ABCB1 concentrations in the plasma membrane of drug selected K562/Dox and K562/HHT cells containing the highest amount of transporter reached millimolar levels. Concentrations of ABCB1 in the plasma membrane of resistant K562/DoxDR2, K562/DoxDR1, and K562/DoxDR05 cells with lower transporter expression were proportionally decreased., (© 2019 Wiley Periodicals, Inc.)

Background: The development of an effective vaccine preventing HIV-1 infection is hindered by the enormous antigenic variability and unique biochemical and immunological properties of HIV-1 Env glycoprotein, the most promising target for HIV-1 neutralizing antibody. Functional studies of rare elite neutralizers led to the discovery of broadly neutralizing antibodies., Methods: We employed a highly complex combinatorial protein library derived from a 5 kDa albumin-binding domain scaffold, fused with support protein of total 38 kDa, to screen for binders of broadly neutralizing antibody VRC01 paratope. The most specific binders were used for immunization of experimental mice to elicit Env-specific antibodies and to test their neutralization activity using a panel of HIV-1 clade C and B pseudoviruses., Findings: Three most specific binders designated as VRA017, VRA019, and VRA177 exhibited high specificity to VRC01 antibody. Immunized mice produced Env-binding antibodies which neutralize eight of twelve HIV-1 Tier 2 pseudoviruses. Molecular modelling revealed a shape complementarity between VRA proteins and a part of VRC01 gp120 interacting surface., Interpretation: This strategy based on the identification of protein replicas of broadly neutralizing antibody paratope represents a novel approach in HIV-1 vaccine development. This approach is not affected by low immunogenicity of neutralization-sensitive epitopes, variability, and unique biochemical properties of HIV-1 Env used as a crucial antigen in the majority of contemporary tested vaccines. FUND: Czech Health Research Council 15-32198A, Ministry of Health, Czech Republic., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Mdm2 and MdmX are related proteins serving in the form of the Mdm2 homodimer or Mdm2/MdmX heterodimer as an E3 ubiquitin ligase for the tumor suppressor p53. The dimerization is required for the E3 activity and is mediated by the conserved RING domains present in both proteins, but only the RING domain of Mdm2 can form homodimers efficiently. We performed a systematic mutational analysis of human Mdm2, exchanging parts of the RING with the corresponding MdmX sequence, to identify the molecular determinants of this difference. Mdm2 can also promote MdmX degradation, and we identified several mutations blocking it. They were located mainly at the Mdm2/E2 interface and did not disrupt the MdmX-Mdm2 interaction. Surprisingly, some mutations of the Mdm2/E2 interface inhibited MdmX degradation, which is mediated by the Mdm2/MdmX heterodimer, but did not affect p53 degradation, mediated by the Mdm2 homodimer. Only one mutant, replacing a conserved cysteine 449 with asparagine (C449N), disrupted the ability of Mdm2 to dimerize with MdmX. When we introduced the cysteine residue into the corresponding site in MdmX, the RING domain became capable of forming dimers with other MdmX molecules in vivo , suggesting that one conserved amino acid residue in the RINGs of Mdm2 and MdmX could serve as the determinant of the differential ability of these domains to form dimers and their E3 activity. In immunoprecipitations, however, the homodimerization of MdmX could be observed only when the asparagine residue was replaced with cysteine in both RINGs. This result suggested that heterocomplexes consisting of one mutated MdmX RING with cysteine and one wild-type MdmX RING with asparagine might be less stable, despite being readily detectable in the cell-based assay. Moreover, Mdm2 C449N blocked Mdm2-MdmX heterodimerization but did not disrupt the ability of Mdm2 homodimer to promote p53 degradation, suggesting that the effect of the conserved cysteine and asparagine residues on dimerization was context-specific. Collectively, our results indicate that the effects of individual exchanges of conserved residues between Mdm2 and MdmX RING domains might be context-specific, supporting the hypothesis that Mdm2 RING homodimers and Mdm2-MdmX heterodimers may not be entirely structurally equivalent, despite their apparent similarity.

Background/aims: IgA nephropathy is associated with aberrant O-glycosylation of IgA1, which is recognized by autoantibodies leading to the formation of circulating immune complexes. Some of them, after deposition into kidney mesangium, trigger glomerular injury. In patients with active disease nonresponding to angiotensin-converting enzyme inhibitors or angiotensin II blockers, corticosteroids are recommended., Methods: The relationship between the corticosteroid therapy and serum levels of IgA, aberrantly O-glycosylated IgA1, IgA-containing immune complexes and their mesangioproliferative activity was analyzed in IgA nephropathy patients and disease and healthy controls., Results: Prednisone therapy significantly reduced proteinuria and levels of serum IgA, galactose-deficient IgA1, and IgA-IgG immune complexes in IgA nephropathy patients and thus reduced differences in all of the above parameters between IgAN patients and control groups. A moderate but not significant reduction of mesangioproliferative potential of IgA-IgG immune complexes and IgA sialylation was detected., Conclusion: The prednisone therapy reduces overall aberrancy in IgA1 O-glycosylation in IgA nephropathy patients, but the measurement of IgA1 parameters does not allow us to predict the prednisone therapy outcome in individual patients., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

Recently, it has been suggested that imatinib (IM) and nilotinib (NIL) could be studied beyond their original application, as inhibitors of the drug efflux pump ABCB1 (P-glycoprotein, MDR1). Since the reversal of ABCB1-mediated resistance has never been successfully demonstrated in the clinic, we addressed the question of whether IM and NIL may actually serve as efficient inhibitors of ABCB1. Here we define an efficient inhibitor as a compound that achieves full (90-100%) reversal of drug efflux at a concentration that does not exhibit significant off-target toxicity in vitro. In this study, human leukemia K562 cells expressing various levels of ABCB1 were used. We observed that cells expressing higher ABCB1 levels required higher concentrations of IM and NIL to achieve full reversal of drug efflux. Among the well-known ABCB1 inhibitors, a similar effect was found for cyclosporin A (CsA) but not for zosuquidar. IM was efficient only in cells with the low and moderate ABCB1 expression at high concentrations that were cytotoxic in the absence of Bcr-Abl. In contrast, NIL was as efficient an inhibitor of ABCB1 as CsA. Low and moderate expression levels of ABCB1 could be efficiently inhibited by NIL concentrations without cytotoxic effects in the absence of Bcr-Abl. However, high expression levels of ABCB1 required higher NIL concentrations with off-target cytotoxic effects. In conclusion, application of NIL, but not of IM, in clinics is promising, however, only in cells with low ABCB1 expression levels. We hypothesize that some patients may benefit from an inhibitor exhibiting an ABCB1 expression-dependent effect., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Williams-Beuren syndrome-associated transcription factor TFII-I plays a critical regulatory role in bone and neural tissue development and in immunity, in part by regulating cell proliferation in response to mitogens. Mdm2, a cellular oncogene responsible for the loss of p53 tumor suppressor activity in a significant proportion of human cancers, was identified in this study as a new binding partner for TFII-I and a negative regulator of TFII-I-mediated transcription. These findings suggest a new p53-independent mechanism by which increased Mdm2 levels found in human tumors could influence cancer cells. In addition to that, we present data indicating that TFII-I is an important cellular regulator of transcription from the immediate-early promoter of human cytomegalovirus, a promoter sequence frequently used in mammalian expression vectors, including vectors for gene therapy. Our observation that Mdm2 over-expression can decrease the ability of TFII-I to activate the CMV promoter might have implications for the efficiency of experimental gene therapy based on CMV promoter-derived vectors in cancers with Mdm2 gene amplification.

Increased expression of the ABCB1 gene in cancer cells is usually connected with occurrence of multidrug resistance (MDR) and poor prognosis. However, the correlation between ABCB1 expression and MDR phenotype is difficult to prove in clinical samples. Most of the researchers believe that these difficulties are due to the poor reliability and sensitivity of assays for detection of ABCB1 expression in clinical samples. However, the complexity of P-gp mediated resistance cannot be reduced to the methodical difficulties only. Here, we addressed the question how widely used methods for detection of ABCB1 expression levels could predict its functional activity and thus its contribution to drug resistance in defined conditions in vitro. The ABCB1 expression was assessed at the mRNA level by quantitative real-time polymerase chain reaction (qRT-PCR), and at the protein level by flow cytometry using UIC2 antibody. The ABCB1 function was monitored using a calcein AM accumulation assay. We observed that K562 cells have approximately 320 times higher level of ABCB1 mRNA than HL-60 cells without detectable function. In addition, resistant K562/Dox cells exhibited significantly higher ABCB1 mRNA expression than resistant K562/HHT cells. However, the functional tests clearly indicated opposite results. Flow cytometric assessment of P-gp, although suggested as a reliable method, contradicted the functional test in K562/Dox and K562/HHT cells. We further used a set of MDR cells expressing various levels of P-gp. Similarly here, flow cytometry not always corresponded to the functional analysis. Our results strongly suggest that an approach which exclusively relies on a simple correlation between ABCB1 expression, either at the mRNA level or protein level, and overall resistance may fail to predict actual contribution of P-gp to overall resistance as the data indicating transporter expression reflect its function only roughly even in well-defined in vitro conditions., (© 2014 The Authors. European Journal of Haematology Published by John Wiley & Sons Ltd.)

The effect of ABCB1 (P-gp, (P-glycoprotein), MDR1) and ABCG2 (BCRP1, (breast cancer resistance protein 1)) expressions on cell resistance to daunorubicin (DRN), imatinib, and nilotinib was studied in human leukemia cells. We used a set of cells derived from a parental K562 cell line, expressing various levels of ABCB1 and ABCG2, respectively. The function of ABCB1 and ABCG2 was confirmed using calcein AM and pheophorbide A accumulation assays, respectively. These assays indicated distinct differences in activities of ABCB1 and ABCG2 which corresponded to their expression levels. We observed that the resistance to DRN and imatinib was proportional to the expression level of ABCB1. Similarly, the resistance to nilotinib and imatinib was proportional to the expression level of ABCG2. Importantly, K562/DoxDR05 and K562/ABCG2-Z cells with the lowest expressions of ABCB1 and ABCG2, respectively, failed to reduce the intracellular levels of imatinib to provide a significant resistance to this drug. However, the K562/DoxDR05 and K562/ABCG2-Z cells significantly decreased the intracellular levels of DRN and nilotinib, respectively, thereby mediating significant resistances to these drugs. Only cells which expression of ABCB1 or ABCG2 exceeded a certain level exhibited a significantly decreased intracellular level of imatinib, and this effect was accompanied by a significantly increased resistance to this drug. Our results clearly indicated that resistance to anticancer drugs mediated by main ABC transporters, ABCB1 and ABCG2, strongly depends on their expressions at protein levels. Importantly, resistance for one drug might be maintained while resistance for other ones might become undetectable at low transporter expression levels., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

The effect of P-glycoprotein (P-gp, ABCB1, MDR1) expression on cell resistance to nilotinib was studied in human leukaemia cells. We used K562/Dox cells overexpressing P-gp and their variants (subclones) with a gradually decreased P-gp expression. These subclones were established by stable transfection of K562/Dox cells with a plasmid vector expressing shRNA targeting the ABCB1 gene. Functional analysis of P-gp using a specific fluorescent probe indicated gradually decreased dye efflux which was proportional to the P-gp expression. We observed that K562/Dox cells overexpressing P-gp contained a significantly reduced intracellular level of nilotinib when compared to their counter partner K562 cells, which do not express P-gp. This effect was accompanied by a decreased sensitivity of the K562/Dox cells to nilotinib. Importantly, cells with downregulated expression of P-gp gradually lost their ability to decrease the intracellular level of nilotinib although they still significantly decreased the intracellular level of daunorubicin (DNR). Accordingly, cells with the reduced expression of P-gp concomitantly failed to provide resistance to nilotinib, however, they exhibited a significant resistance to DNR. Taken together, we demonstrated that the conclusion as to whether P-gp is involved in nilotinib resistance or not strongly depends on its expression at protein level., (Copyright © 2012. Published by Elsevier Ltd.)

We studied effects of 2-chloro-N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA) on apoptosis induction in the K562/Dox cell line, which overexpressed P-glycoprotein (P-gp, ABCB1, MDR1). We found that the K562/Dox cell line was significantly more resistant to Cl-IB-MECA than the maternal cell line K562, which did not express P-gp. Although both cell lines expressed the A3 adenosine receptor (A3AR), cytotoxic effects of Cl-IB-MECA were not prevented by its selective antagonist MRS1523 (3-propyl-6-ethyl-5-[(ethylthio)carbonyl]-2 phenyl-4-propyl-3-pyridine carboxylate). Analysis of cell extracts revealed that the intracellular level of Cl-IB-MECA was significantly lower in the K562/Dox cell line than in the maternal cell line K562. The downregulation of P-gp expression using shRNA targeting ABCB1 gene led to increased intracellular level of Cl-IB-MECA and restored cell sensitivity to this drug. Similarly, valspodar (PSC-833), a specific inhibitor of P-gp, restored sensitivity of the K562/Dox cell line to Cl-IB-MECA with concomitant increase of intracellular level of Cl-IB-MECA in the resistant cell line, while it affected cytotoxicity of Cl-IB-MECA in the sensitive cell line only marginally. An enzyme based assay provided evidence for interaction of P-gp with Cl-IB-MECA. We further observed that cytotoxic effects of Cl-IB-MECA could be augmented by activation of extrinsic cell death pathway by Apo-2L (TRAIL) but not FasL or TNF-α. Our results revealed that Cl-IB-MECA induced an increase in expression of TRAIL receptors in K562 cells, which could sensitize cells to apoptosis induction via an extrinsic cell death pathway. Importantly, these effects were inversely related to P-gp expression. In addition, MRS1523 did not affect Cl-IB-MECA induced expression of TRAIL receptors., (Copyright © 2011 Wiley Periodicals, Inc.)