In Vitro Immune Responses of Lymphoid Cell Populations to Proteins and Peptides

Abstract Children have lower hospitalisation and mortality rates for coronavirus disease-2019 (COVID-19) than adults; however, younger children (<4 years of age)1 may develop more severe disease than older children. To date, the immune correlates of severe COVID-19 in young children have been poorly characterized. We report the kinetics of immune responses in relation to clinical and virological features in an infant with acute severe COVID-19. Systemic cellular and cytokine profiling showed initial increase in neutrophils and monocytes with depletion of lymphoid cell populations (particularly CD8+ T and NK cells) and elevated inflammatory cytokines. Expansion of memory CD4+T (but not CD8+T) cells occurred over time, with predominant Th2 bias. Marked activation of T cell populations observed during the acute infection gradually resolved as the child recovered. Significant in vitro activation of T-cell populations and robust cytokine production, in response to inactivated SARS-CoV-2 stimulation, was observed 3 months after infection indicating durable, long-lived cellular immune memory.

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Properties and Biotechnological Application of mutant Derivatives of Mini-intein PRP8 from Penicillium chrysogenum with Improved Control of C-terminal Processing

Biotekhnologiya ◽

10.21519/0234-2758-2019-35-6-91-101 ◽

2019 ◽

Vol 35 (6) ◽

pp. 91-101

Author(s):

F.A. Klebanov ◽

S.E. Cheperegin ◽

D.G. Kozlov

Keyword(s):

Penicillium Chrysogenum ◽

Protein Denaturation ◽

Biologically Active ◽

Cultivation Temperature ◽

Target Proteins ◽

E Coli ◽

Complete Inactivation ◽

Target Molecules ◽

Proteins And Peptides

Mutant variants of mini-intein PRP8 from Penicillium chrysogenum (Int4b) with improved control of C-terminal processing were characterized. The presented variants can serve as a basis for self-removed polypeptide tags capable of carrying an affine label and allowing to optimize the process of obtaining target proteins and peptides in E. coli cells. They allow to synthesize target molecules in the composition of soluble and insoluble hybrid proteins (fusions), provide their afnne purification, autocatalytic processing and obtaining mature target products. The presented variants have a number of features in comparison with the known prototypes. In particular the mutant mini-intein Int4bPRO, containing the L93P mutation, has temperature-dependent properties. At cultivation temperature below 30 °C it allows the production of target molecules as part of soluble fusions, but after increasing of cultivation temperature to 37 °C it directs the most of synthesized fusions into insoluble intracellular aggregates. The transition of Int4bPRO into insoluble form is accompanied by complete inactivation of C-terminal processing. Further application of standard protein denaturation-renaturation procedures enable efficiently reactivate Int4bPRO and to carry out processing of its fusions in vitro. Two other variants, Int4b56 and Int4b36, containing a point mutation T62N or combination of mutations D144N and L146T respectively, have a reduced rate of C-terminal processing. Their use in E. coli cells allows to optimize the biosynthesis of biologically active target proteins and peptides in the composition of soluble fusions, suitable for afnne purification and subsequent intein-dependent processing without the use of protein denaturation-renaturation procedures. intein, fusion, processing, processing rate, gelonin The work was supported within the framework of the State Assignment no. 595-00003-19 PR.

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In silico approach for designing a novel recombinant fusion protein as a Candidate Vaccine against HPV

Current Proteomics ◽

10.2174/1570164617999201014162235 ◽

2020 ◽

Vol 17 ◽

Author(s):

Mohsen Sisakht ◽

Amir Mahmoodzadeh ◽

Mohammadsaeid Zahedi ◽

Davood Rostamzadeh ◽

Amin Moradi Hasan-Abad ◽

...

Keyword(s):

Immune Responses ◽

Fusion Protein ◽

In Silico ◽

Precancerous Lesions ◽

Candidate Vaccine ◽

T Cell Epitopes ◽

Binding Interaction ◽

Hpv16 E6 ◽

E7 Proteins

Background: Human papillomavirus (HPV) is the main biological agent causing sexually transmitted diseases (STDs), including precancerous lesions and several types of prevalent cancers. To date, numerous types of vaccines are designed to prevent high-risk HPV. However, their prophylactic effect is not the same and does not clear previous infections. Therefore, there is an urgent need for developing therapeutic vaccines that trigger cell-mediated immune responses for the treatment of HPV. The HPV16 E6 and E7 proteins are ideal targets for vaccine therapy against HPV. Fusion protein vaccines, which include both immunogenic interest protein and an adjuvant for augmenting the immunogenicity effects, are theoretically capable of guarantee the power of the immune system against HPV. Method: A vaccine construct, including HPV16 E6/E7 proteins along with a heat shock protein GP96 (E6/E7-NTGP96 construct), was designed using in silico methods. By the aid of the SWISS-MODEL server, the optimal 3D model of the designed vaccine was selected, followed by physicochemical and molecular parameters were performed using bioinformatics tools. Docking studies were done to evaluate the binding interaction of the vaccine. Allergenicity, immunogenicity, B, and T cell epitopes of the designed construct were predicted. Results: Immunological and structural computational results illustrated that our designed construct is potentially proper for stimulation of cellular and humoral immune responses against HPV. Conclusion: Computational studies showed that the E6/E7-NTGP96 construct is a promising candidate vaccine that needs further in vitro and in vivo evaluations.

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Bone Marrow Niches for Skeletal Progenitor Cells and their Inhabitants in Health and Disease

Current Stem Cell Research & Therapy ◽

10.2174/1574888x14666190123161447 ◽

2019 ◽

Vol 14 (4) ◽

pp. 305-319 ◽

Cited By ~ 4

Author(s):

Marietta Herrmann ◽

Franz Jakob

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Progenitor Cell ◽

Adult Stem Cells ◽

Current Knowledge ◽

Cell Populations ◽

Surface Markers ◽

Bone Marrow Niches

The bone marrow hosts skeletal progenitor cells which have most widely been referred to as Mesenchymal Stem or Stromal Cells (MSCs), a heterogeneous population of adult stem cells possessing the potential for self-renewal and multilineage differentiation. A consensus agreement on minimal criteria has been suggested to define MSCs in vitro, including adhesion to plastic, expression of typical surface markers and the ability to differentiate towards the adipogenic, osteogenic and chondrogenic lineages but they are critically discussed since the differentiation capability of cells could not always be confirmed by stringent assays in vivo. However, these in vitro characteristics have led to the notion that progenitor cell populations, similar to MSCs in bone marrow, reside in various tissues. MSCs are in the focus of numerous (pre)clinical studies on tissue regeneration and repair.Recent advances in terms of genetic animal models enabled a couple of studies targeting skeletal progenitor cells in vivo. Accordingly, different skeletal progenitor cell populations could be identified by the expression of surface markers including nestin and leptin receptor. While there are still issues with the identity of, and the overlap between different cell populations, these studies suggested that specific microenvironments, referred to as niches, host and maintain skeletal progenitor cells in the bone marrow. Dynamic mutual interactions through biological and physical cues between niche constituting cells and niche inhabitants control dormancy, symmetric and asymmetric cell division and lineage commitment. Niche constituting cells, inhabitant cells and their extracellular matrix are subject to influences of aging and disease e.g. via cellular modulators. Protective niches can be hijacked and abused by metastasizing tumor cells, and may even be adapted via mutual education. Here, we summarize the current knowledge on bone marrow skeletal progenitor cell niches in physiology and pathophysiology. We discuss the plasticity and dynamics of bone marrow niches as well as future perspectives of targeting niches for therapeutic strategies.

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Lysosomotropic agents including azithromycin, chloroquine and hydroxychloroquine activate the integrated stress response

Cell Death and Disease ◽

10.1038/s41419-020-03324-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Ai-Ling Tian ◽

Qi Wu ◽

Peng Liu ◽

Liwei Zhao ◽

Isabelle Martins ◽

...

Keyword(s):

Stress Response ◽

Immune Responses ◽

Initiation Factor ◽

Integrated Stress Response ◽

Serine Residue ◽

Eif2α Phosphorylation ◽

Lysosomotropic Agents ◽

Cultured Human Cells

AbstractThe integrated stress response manifests with the phosphorylation of eukaryotic initiation factor 2α (eIF2α) on serine residue 51 and plays a major role in the adaptation of cells to endoplasmic reticulum stress in the initiation of autophagy and in the ignition of immune responses. Here, we report that lysosomotropic agents, including azithromycin, chloroquine, and hydroxychloroquine, can trigger eIF2α phosphorylation in vitro (in cultured human cells) and, as validated for hydroxychloroquine, in vivo (in mice). Cells bearing a non-phosphorylatable eIF2α mutant (S51A) failed to accumulate autophagic puncta in response to azithromycin, chloroquine, and hydroxychloroquine. Conversely, two inhibitors of eIF2α dephosphorylation, nelfinavir and salubrinal, enhanced the induction of such autophagic puncta. Altogether, these results point to the unexpected capacity of azithromycin, chloroquine, and hydroxychloroquine to elicit the integrated stress response.

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Intestinal mesenchymal cells regulate immune responses and promote epithelial regeneration in vitro and in dextran sulfate sodium‐induced experimental colitis in mice

Acta Physiologica ◽

10.1111/apha.13699 ◽

2021 ◽

Author(s):

Laura Hidalgo‐Garcia ◽

José Alberto Molina‐Tijeras ◽

Francisco Huertas‐Peña ◽

Antonio Jesús Ruiz‐Malagón ◽

Patricia Diez‐Echave ◽

...

Keyword(s):

Immune Responses ◽

Dextran Sulfate ◽

Dextran Sulfate Sodium ◽

Experimental Colitis ◽

Mesenchymal Cells ◽

Epithelial Regeneration ◽

Regeneration In Vitro

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Clinical and immunological effects of mRNA vaccines in malignant diseases

Molecular Cancer ◽

10.1186/s12943-021-01339-1 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Annkristin Heine ◽

Stefan Juranek ◽

Peter Brossart

Keyword(s):

Immune Responses ◽

Messenger Rna ◽

Immune Escape ◽

Special Focus ◽

Potential Efficacy ◽

Protective Antibodies ◽

Technological Developments ◽

Broad Variety ◽

Antigen Loss

AbstractIn vitro-transcribed messenger RNA-based therapeutics represent a relatively novel and highly efficient class of drugs. Several recently published studies emphasize the potential efficacy of mRNA vaccines in treating different types of malignant and infectious diseases where conventional vaccine strategies and platforms fail to elicit protective immune responses. mRNA vaccines have lately raised high interest as potent vaccines against SARS-CoV2. Direct application of mRNA or its electroporation into dendritic cells was shown to induce polyclonal CD4+ and CD8+ mediated antigen-specific T cell responses as well as the production of protective antibodies with the ability to eliminate transformed or infected cells. More importantly, the vaccine composition may include two or more mRNAs coding for different proteins or long peptides. This enables the induction of polyclonal immune responses against a broad variety of epitopes within the encoded antigens that are presented on various MHC complexes, thus avoiding the restriction to a certain HLA molecule or possible immune escape due to antigen-loss. The development and design of mRNA therapies was recently boosted by several critical innovations including the development of technologies for the production and delivery of high quality and stable mRNA. Several technical obstacles such as stability, delivery and immunogenicity were addressed in the past and gradually solved in the recent years.This review will summarize the most recent technological developments and application of mRNA vaccines in clinical trials and discusses the results, challenges and future directions with a special focus on the induced innate and adaptive immune responses.

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Human fetal liver MSCs are more effective than adult bone marrow MSCs for their immunosuppressive, immunomodulatory, and Foxp3+ T reg induction capacity

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02176-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Yi Yu ◽

Alejandra Vargas Valderrama ◽

Zhongchao Han ◽

Georges Uzan ◽

Sina Naserian ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Immune Responses ◽

Molecular Mechanisms ◽

Fetal Liver ◽

Cytotoxic T Cells ◽

Cell Contact ◽

Adult Bone Marrow ◽

Adult Bone

Abstract Background Mesenchymal stem cells (MSCs) exhibit active abilities to suppress or modulate deleterious immune responses by various molecular mechanisms. These cells are the subject of major translational efforts as cellular therapies for immune-related diseases and transplantations. Plenty of preclinical studies and clinical trials employing MSCs have shown promising safety and efficacy outcomes and also shed light on the modifications in the frequency and function of regulatory T cells (T regs). Nevertheless, the mechanisms underlying these observations are not well known. Direct cell contact, soluble factor production, and turning antigen-presenting cells into tolerogenic phenotypes, have been proposed to be among possible mechanisms by which MSCs produce an immunomodulatory environment for T reg expansion and activity. We and others demonstrated that adult bone marrow (BM)-MSCs suppress adaptive immune responses directly by inhibiting the proliferation of CD4+ helper and CD8+ cytotoxic T cells but also indirectly through the induction of T regs. In parallel, we demonstrated that fetal liver (FL)-MSCs demonstrates much longer-lasting immunomodulatory properties compared to BM-MSCs, by inhibiting directly the proliferation and activation of CD4+ and CD8+ T cells. Therefore, we investigated if FL-MSCs exert their strong immunosuppressive effect also indirectly through induction of T regs. Methods MSCs were obtained from FL and adult BM and characterized according to their surface antigen expression, their multilineage differentiation, and their proliferation potential. Using different in vitro combinations, we performed co-cultures of FL- or BM-MSCs and murine CD3+CD25−T cells to investigate immunosuppressive effects of MSCs on T cells and to quantify their capacity to induce functional T regs. Results We demonstrated that although both types of MSC display similar cell surface phenotypic profile and differentiation capacity, FL-MSCs have significantly higher proliferative capacity and ability to suppress both CD4+ and CD8+ murine T cell proliferation and to modulate them towards less active phenotypes than adult BM-MSCs. Moreover, their substantial suppressive effect was associated with an outstanding increase of functional CD4+CD25+Foxp3+ T regs compared to BM-MSCs. Conclusions These results highlight the immunosuppressive activity of FL-MSCs on T cells and show for the first time that one of the main immunoregulatory mechanisms of FL-MSCs passes through active and functional T reg induction.

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