Nanobodies as powerful pulmonary targeted biotherapeutics against SARS-CoV-2, pharmaceutical point of view.

Background Since December 2019, the newly emerged SARS-CoV-2 virus continues to infect humans and many people died from severe Covid-19 during the last 2 years worldwide. Different approaches are being used for treatment of this infection and its consequences, but limited results have been achieved and new therapeutics are still needed. One of the most interesting biotherapeutics in this era are Nanobodies which have shown very promising results in recent researches. Scope of review Here, we have reviewed the potentials of Nanobodies in Covid-19 treatment. We have also discussed the properties of these biotherapeutics that make them very suitable for pulmonary drug delivery, which seems to be very important route of administration in this disease. Major conclusion Nanobodies with their special biological and biophysical characteristics and their resistance against harsh manufacturing condition, can be considered as promising, targeted biotherapeutics which can be administered by pulmonary delivery pharmaceutical systems against Covid-19. General significance Covid-19 has become a global problem during the last two years and with emerging mutant strains, prophylactic and therapeutic approaches are still highly needed. Nanobodies with their specific properties can be considered as valuable and promising candidates in Covid-19 therapy.

A novel anti-human IL-1R7 antibody reduces IL-18-mediated inflammatory signaling.

Unchecked inflammation can result in severe diseases with high mortality, such as macrophage activation syndrome (MAS). MAS and associated cytokine storms have been observed in COVID-19 patients exhibiting systemic hyperinflammation. Interleukin-18 (IL-18), a proinflammatory cytokine belonging to the IL-1 family, is elevated in both MAS and COVID-19 patients, and its level is known to correlate with the severity of COVID-19 symptoms. IL-18 binds its specific receptor IL-1 receptor 5 (IL-1R5, also known as IL-18 receptor alpha chain), leading to the recruitment of the coreceptor, IL-1 receptor 7 (IL-1R7, also known as IL-18 receptor beta chain). This heterotrimeric complex then initiates downstream signaling, resulting in systemic and local inflammation. Here, we developed a novel humanized monoclonal anti-IL-1R7 antibody to specifically block the activity of IL-18 and its inflammatory signaling. We characterized the function of this antibody in human cell lines, in freshly obtained peripheral blood mononuclear cells (PBMCs) and in human whole blood cultures. We found that the anti-IL-1R7 antibody significantly suppressed IL-18-mediated NFκB activation, reduced IL-18-stimulated IFNγ and IL-6 production in human cell lines, and reduced IL-18-induced IFNγ, IL-6, and TNFα production in PBMCs. Moreover, the anti-IL-1R7 antibody significantly inhibited LPS- and Candida albicans-induced IFNγ production in PBMCs, as well as LPS-induced IFNγ production in whole blood cultures. Our data suggest that blocking IL-1R7 could represent a potential therapeutic strategy to specifically modulate IL-18 signaling and may warrant further investigation into its clinical potential for treating IL-18-mediated diseases, including MAS and COVID-19.

Virulence-attenuated Salmonella engineered to secrete immunomodulators reduce tumour growth and increase survival in an autochthonous mouse model of breast cancer.

The ultimate goal of bacterial based cancer therapy is to achieve non-toxic penetration and colonisation of the tumour microenvironment. To overcome this efficacy-limiting toxicity of anticancer immunotherapy, we have tested a therapy comprised of systemic delivery of a vascular disrupting agent to induce intratumoral necrotic space, cannabidiol to temporarily inhibit angiogenesis and acute inflammation, and a strain of Salmonella Typhimurium that was engineered for non-toxic colonisation and expression of immunomodulators within the tumour microenvironment. This combination treatment strategy was administered to transgenic mice burdened with autochthonous mammary gland tumours and demonstrated a statistically significant 64% slower tumour growth and a 25% increase in mean survival time compared to control animals without treatment. These experiments were accomplished with minimal toxicity as measured by less than 7% weight loss and a return to normal weight gain within three days following intravenous administration of the bacteria. Thus, non-toxic, robust colonisation of the microenvironment was achieved to produce a significant antitumor effect.

Murine Model of Thermal Burn Injury for Evaluating Protein Therapeutics Derived from Viruses.

In vivo wound healing models are predictive preclinical tests for therapeutics that enhance skin repair or limit scarring. Large animals, such as swine, heal in a manner similar to humans, but testing is impractical and expensive. Experiments in mice are more economic, but may be less translatable as this species heals primarily through contraction, not by the processes of epithelialization and granulation tissue formation as seen in human wounds. Here, we describe a murine model of thermal burn injury that closely mimics human healing, resulting in a large, hypertrophic-like scar. This practical, reproducible model is ideal for testing promising wound-healing therapies, such as virus-derived growth factors and immune-modulatory proteins.

Kidney Subcapsular Allograft Transplants as a Model to Test Virus-Derived Chemokine-Modulating Proteins as Therapeutics.

Solid tissue transplant is a growing medical need that is further complicated by a limited donor organ supply. Acute and chronic rejection occurs in nearly all transplants and reduces long-term graft survival, thus increasing the need for repeat transplantation. Viruses have evolved highly adapted responses designed to evade the host's immune defenses. Immunomodulatory proteins derived from viruses represent a novel class of potential therapeutics that are under investigation as biologics to attenuate immune-mediated rejection and damage. These immune-modulating proteins have the potential to reduce the need for traditional posttransplant immune suppressants and improve graft survival. The myxoma virus-derived protein M-T7 is a promising biologic that targets chemokine and glycosaminoglycan pathways central to kidney transplant rejection. Orthotopic transplantations in mice are prohibitively difficult and costly and require a highly trained microsurgeon to successfully perform the procedure. Here we describe a kidney-to-kidney subcapsular transplant model as a practical and simple method for studying transplant rejection, a model that requires fewer mice. One kidney can be used as a donor for transplants into six or more recipient mice. Using this model there is lower morbidity, pain, and mortality for the mice. Subcapsular kidney transplantation provides a first step approach to testing virus-derived proteins as new potential immune-modulating therapeutics to reduce transplant rejection and inflammation.

A Mouse Model of Acute Liver Injury by Warm, Partial Ischemia-Reperfusion for Testing the Efficacy of Virus-Derived Therapeutics.

Ischemia-reperfusion injury (IRI) drives early and long-term damage to organs as well as compounding damage from acute transplant rejection and surgical trauma. IRI initiates an aggressive and prolonged inflammation leading to tissue injury, organ failure, and death. However, there are few effective therapeutic interventions for IRI. The destructive inflammatory cell activity in IRI is part of an aberrant innate immune response that triggers multiple pathways. Hence, immune-modulating treatments to control pathways triggered by IRI hold great therapeutic potential. Viruses, especially large DNA viruses, have evolved highly effective immune-modulating proteins for the purpose of immune evasion and to protect the virus from the host immune defenses. A number of these immune-modulating proteins have proven therapeutically effective in preclinical models, many with function targeting pathways known to be involved in IRI. The use of virus-derived immune-modulating proteins thus represents a promising source for new treatments to target ischemia-reperfusion injury. Laboratory small animal models of IRI are well established and are able to reproduce many aspects of ischemia-reperfusion injury seen in humans. This chapter will discuss the methods used to perform the IRI procedure in mice, as well as clinically relevant diagnostic tests to evaluate liver injury and approaches for assessing histological damage while testing novel immune modulating protein treatments.

The Structural Characteristics and Biological Activities of Intracellular Polysaccharide Derived from Mutagenic Sanghuangporous sanghuang Strain.

Sanghuangporous sanghuang is a rare medicinal fungus which contains polysaccharide as the main active substance and was used to treat gynecological diseases in ancient China. The intracellular polysaccharide yield of S. sanghuang was enhanced by the strain A130 which was screened from mutant strains via atmospheric and room temperature plasma (ARTP) mutagenesis. The objective of this research was to investigate the effects of ARTP mutagenesis on structural characteristics and biological activities of intracellular polysaccharides from S. sanghuang. Six intracellular polysaccharide components were obtained from S. sanghuang mycelia cultivated by the mutagenic strain (A130) and original strain (SH1), respectively. The results revealed that the yields of polysaccharide fractions A130-20, A130-50 and A130-70 isolated from the mutagenic strain fermentation mycelia were significantly higher than those of the original ones by 1.5-, 1.3- and 1.2-fold, and the clear physicochemical differences were found in polysaccharide fractions precipitated by 20% ethanol. A130-20 showed a relatively expanded branching chain with higher molecular weight and better in vitro macrophage activation activities and the IL-6, IL-1, and TNF-α production activities of macrophages were improved by stimulation of A130-20 from the mutagenic strain. This study demonstrates that ARTP is a novel and powerful tool to breed a high polysaccharide yield strain of S. sanghuang and may, therefore, contribute to the large-scale utilization of rare medicinal fungi.

Enhanced production and immunomodulatory activity of levan from the acetic acid bacterium, Tanticharoenia sakaeratensis.

Levan is a fructose polymer with ß-(2 â†’ 6) glycosidic linkages. It is produced by several microorganisms, and due to its potential biotechnological and industrial applications, various levan-producing bacteria with different levels of production efficiencies have been reported. We investigated the levan-producing ability of the acetic acid bacterium, Tanticharoenia sakaeratensis. The exopolysaccharides produced by the bacterium under a sucrose environment were characterized as levan by FT-IR, and 1H and 13C NMR. The molecular weight of levan thus produced range from 1.0 × 105-6.8 × 105 Da. The maximum yield of levan from T. sakaeratensis is 24.7 g·L-1 in a liquid medium containing 20% (w/v) sucrose and incubated at 37 °C, 250 RPM for 35 h. The levan produced by T. sakaeratensis can promote nitric oxide production in RAW264.7 macrophage cells in a concentration-dependent manner, suggesting it has immunomodulatory effects. Our study reveals that T. sakaeratensis can be potentially employed as a new source of levan for industrial applications.

Structural characterization and immunomodulatory activity of an exopolysaccharide produced by Lactobacillus helveticus LZ-R-5.

Exopolysaccharide (R-5-EPS) was isolated from the fermented milk of Lactobacillus helveticus LZ-R-5 and purified by DEAE-52 cellulose anion-exchange column, and characterization of the structure was conducted. Results showed that R-5-EPS was a heteropolysaccharide containing linear repeating units of →6)-ß-D-Galp-(1→3)-ß-D-Glcp-(1→3)-ß-D-Glcp-(1→3)-ß-D-Glcp-(1→3)-ß-D-Glcp-(1→ with an average Mw of 5.41 × 105 Da. Furthermore, at a cellular level, R-5-EPS showed immunostimulatory activity due to its strong effect on increasing proliferation of RAW264.7 macrophages and enhancing phagocytosis, acid phosphatase activity, nitric oxide production and cytokines production in macrophages. These results suggest that R-5-EPS have a potent immunostimulatory activity and may be explored as a potential immunomodulatory agent.

Immunopathogenesis of Progressive Scarring Trachoma: Results of a 4-Year Longitudinal Study in Tanzanian Children.

Trachoma is initiated during childhood following repeated conjunctival infection with Chlamydia trachomatis, which causes a chronic inflammatory response in some individuals that leads to scarring and in-turning of the eyelids in later life. There is currently no treatment to halt the progression of scarring trachoma due to an incomplete understanding of disease pathogenesis. A cohort study was performed in northern Tanzania in 616 children aged 6 to 10 years at enrollment. Every 3 months for 4 years, children were examined for clinical signs of trachoma, and conjunctival swabs were collected for C. trachomatis detection and to analyze the expression of 46 immunofibrogenic genes. Data were analyzed in relation to progressive scarring status between baseline and the final time point. Genes that were significantly associated with scarring progression included those encoding proinflammatory chemokines (CXCL5, CCL20, CXCL13, and CCL18), cytokines (IL23A, IL19, and IL1B), matrix modifiers (MMP12 and SPARCL1), immune regulators (IDO1, SOCS3, and IL10), and a proinflammatory antimicrobial peptide (S100A7). In response to C. trachomatis infection, IL23A and PDGF were significantly upregulated in scarring progressors relative to in nonprogressors. Our findings highlight the importance of innate proinflammatory signals from the epithelium and implicate interleukin 23A (IL-23A)-responsive cells in driving trachomatous scarring, with potential key mechanistic roles for PDGFB, MMP12, and SPARCL1 in orchestrating fibrosis.

Tumor cells educate mesenchymal stromal cells to release chemoprotective and immunomodulatory factors.

Factors released by surrounding cells such as cancer-associated mesenchymal stromal cells (CA-MSCs) are involved in tumor progression and chemoresistance. In this study, we characterize the mechanisms by which naïve mesenchymal stromal cells (MSCs) can acquire a CA-MSCs phenotype. Ovarian tumor cells trigger the transformation of MSCs to CA-MSCs by expressing pro-tumoral genes implicated in the chemoresistance of cancer cells, resulting in the secretion of high levels of CXC chemokine receptors 1 and 2 (CXCR1/2) ligands such as chemokine (C-X-C motif) ligand 1 (CXCL1), CXCL2, and interleukin 8 (IL-8). CXCR1/2 ligands can also inhibit the immune response against ovarian tumor cells. Indeed, through their released factors, CA-MSCs promote the differentiation of monocytes towards M2 macrophages, which favors tumor progression. When CXCR1/2 receptors are inhibited, these CA-MSC-activated macrophages lose their M2 properties and acquire an anti-tumoral phenotype. Both ex vivo and in vivo, we used a CXCR1/2 inhibitor to sensitize ovarian tumor cells to carboplatin and circumvent the pro-tumoral effects of CA-MSCs. Since high concentrations of CXCR1/2 ligands in patients' blood are associated with chemoresistance, CXCR1/2 inhibition could be a potential therapeutic strategy to revert carboplatin resistance.

Discovery of IL-6 and Development of Anti-IL-6R Antibody.

A series of our studies on IL-6 have revealed that it has a pleiotropic activity in various tissues and cells and its deregulated expression is responsible for several chronic inflammations and hemopoietic malignancies.Humanized antibody against 80kd IL-6R (Tocilizumab) has shown significant therapeutic effect in RA, JIA, Castleman's diseases and several other autoimmune inflammatory diseases, such as, giant cell arteritis, reactive arthritis, polymyalgia rheumatica and adult still's disease. Cytokine storm induced by CAR-T cell therapy has been shown to be controlled by Tocilizumab.Therapeutic effect of Tocilizumab confirmed that over and constitutive-production of IL-6 is responsible for the pathogenesis of autoimmune diseases.Then, the question to be asked is how is IL-6 production regulated. We identified a novel molecule called Arid5a which binds with the 3'-UTR of IL-6 mRNA and protects its degradation by competing with Regnase-1. Interestingly, this molecule is present in nuclei and inflammatory stimulation induced translocation of Arid5a from nuclei into cytoplasm and it competes with Regnase-1 for the protection of mRNA of IL-6.Our study indicates that Arid5a is one of the key molecules for inflammation as well as the development of septic shock.The results also suggest the therapeutic potential of anti-agonistic agents for Arid5a in the prevention of various inflammatory diseases and septic shock.

Effects of starter feeding on caecal mucosal bacterial composition and expression of genes involved in immune and tight junctions in pre-weaned twin lambs.

The objective of this study was to explore the effects of starter feeding on caecal mucosal bacterial composition and the expression of genes involved in immune and tight junctions in pre-weaned lambs. Six pairs of new-born twin lambs were selected. From 10 days of age, one lamb of each pair received ewe's milk only (M group, n = 6), while the other one was fed ewe's milk plus starter feed (M + S group, n = 6). At 56 days of age, the lambs were sacrificed, and then cecum digesta was collected to measure pH values and concentrations of volatile fatty acid (VFA), and caecal mucosa were collected to determine the changes in bacterial communities and the mRNA expression of cytokines, toll-like receptors (TLRs) and tight junction proteins. The results showed the body weight and average daily gain were not significantly different between both groups. Starter feeding significantly (P < 0.05) increased the concentrations of propionate and butyrate; the proportions of acetate, propionate and butyrate to total concentrations of VFA; and decreased the ratio of acetate to propionate in caecal contents. Principal coordinate analysis showed that samples from the M + S group could be distinguished from those from the M group; starter feeding also increased the diversity of caecal mucosal bacteria. At the genus level, starter feeding significantly (FDR < 0.05) increased the relative abundance of Alistipes, Parabacteroides, Parasutterella and Butyricimonas, and caused a decreasing trend (FDR < 0.10) in the relative abundance of Campylobacter and Helicobacter. The real-time PCR results showed that starter feeding significantly (FDR < 0.05) decreased the relative mRNA expression level of IL-12, TNF-α and TLR4 and increased the relative mRNA expression level of claudin-4. These results indicate that starter feeding altered caecal mucosal bacterial communities and decreased the expression of inflammatory factors, which may be beneficial in alleviating the weaning stress of lambs.

Structure and immunomodulatory activity of polysaccharides from Fusarium solani DO7 by solid-state fermentation.

Two polysaccharides, DGS1 and DGS2, were obtained by solid-state fermentation (SSF) of Fusarium solani DO7, an endophytic fungus isolated from the orchid Dendrobium officinale. Structural characterizations revealed that DGS1 consisted of arabinose, glucose, mannose and galactose with a molar ratio of 2.9:13.4:3.0:1, respectively, and contained (1 → 5)-Araf, (1 → 4)-Glcp, (1 → 6)-Glcp, (1 → 3)-Manp, (1 → 2,6)-Manp and (1 → 6)-Galp glycosidic linkages, while DGS2 was composed of arabinose, glucose, mannose and galactose in a molar ratio of 3.5:8.1:2.1:1, respectively, and contained (1 → 5)-Araf, (1 → 4)-Glcp, (1 → 6)-Glcp, (1 → 3)-Manp and (1 → 6)-Galp glycosidic linkages. Neither polysaccharide was toxic to human embryonic kidney cells or mouse RAW 264.7 macrophage cells. An immunomodulatory activity assay indicated that both polysaccharides could significantly enhance the levels of TNF-α, IL-6 and NO by activating TNF-α, IL-6 and iNOs gene expression, respectively, especially DGS2. Interestingly, DGS2 also possessed relatively high antioxidant activity. These results illustrate that, due to its cost-effectiveness and environmentally friendly features, SSF has significant potential as a commercially competitive source of natural products, including fungal polysaccharides, with immunomodulatory activity.

Host Immune Response to ZIKV in an Immunocompetent Embryonic Mouse Model of Intravaginal Infection.

Zika virus (ZIKV) only induces mild symptoms in adults; however, it can cause congenital Zika syndrome (CZS), including microcephaly. Most of the knowledge on ZIKV pathogenesis was gained using immunocompromised mouse models, which do not fully recapitulate human pathology. Moreover, the study of the host immune response to ZIKV becomes challenging in these animals. Thus, the main goal of this study was to develop an immunocompetent mouse model to study the ZIKV spread and teratogeny. FVB/NJ immune competent dams were infected intravaginally with ZIKV during the early stage of pregnancy. We found that the placentae of most fetuses were positive for ZIKV, while the virus was detected in the brain of only about 42% of the embryos. To investigate the host immune response, we measured the expression of several inflammatory factors. Embryos from ZIKV-infected dams had an increased level of inflammatory factors, as compared to Mock. Next, we compared the gene expression levels in embryos from ZIKV-infected dams that were either negative or positive for ZIKV in the brain. The mRNA levels of viral response genes and cytokines were increased in both ZIKV-positive and negative brains. Interestingly, the levels of chemokines associated with microcephaly in humans, including CCL2 and CXCL10, specifically increased in embryos harboring ZIKV in the embryo brains.

Early Interaction of Alternaria infectoria Conidia with Macrophages.

Fungi of the genus Alternaria are ubiquitous indoor and outdoor airborne agents, and individuals are daily exposed to their spores. Although its importance in human infections and, particularly in respiratory allergies, there are no studies of how Alternaria spp. spores interact with host cells. Our aim was to study the early interaction of Alternaria infectoria spores with macrophages, the first line of immune defense. RAW 264.7 macrophages were infected with A. infectoria conidia, and the internalization and viability of conidia once inside the macrophages were quantified during the first 6 h of interaction. Live cell imaging was used to study the dynamics of this interaction. TNF-α production was quantified by relative gene expression, and the concentration of other cytokines (IL-1α, IL-1ß, IL-6, IL-4, IL-10, IL-17, GM-CSF and INF-γ) and a chemokine, MIP-1α, was quantified by ELISA. Conidia were rapidly internalized by macrophages, with approximately half internalized after 30 min of interaction. During the first 6 h of interaction, macrophages retained the ability to mitotically divide while containing internalized conidia. The classical macrophage-activated morphology was absent in macrophages infected with conidia, and TNF-α and other cytokines and chemokines failed to be produced. Thus, macrophages are able to efficiently phagocyte A. infectoria conidia, but, during the first 6 h, no effective antifungal response is triggered, therefore promoting the residence of these fungal conidia inside the macrophages.

Partial characterization of a levan type exopolysaccharide (EPS) produced by Leuconostoc mesenteroides showing immunostimulatory and antioxidant activities.

Leuconostoc mesenteroides S81 was isolated from traditional sourdough as an exopolysaccharide (EPS) producer strain. The monosaccharide composition of the EPS from strain S81 was characterized by HPLC analysis and only fructose was found in the repeating unit structure. The NMR spectroscopy analysis revealed that EPS was a levan type EPS as a ß-(2 → 6)-linked fructan. The FTIR analysis further confirmed the presence of the furanoid rings in the EPS structure. The levan S81 showed high level of thermal stability determined by DSC and TGA analysis. The lyophilised levan S81 showed a sheet-like compact morphology and its aqueous solution formed spheroidal lumps with a compact structure detected by SEM and AFM analysis, respectively. Importantly the levan S81 showed a high level of immunomodulatory role, induced the anti-inflammatory cytokine IL-4, and exhibited a strong antioxidant capacity with EC50 value 1.7 mg mL-1 obtained by hydroxyl radical scavenging activity test under in vitro conditions. These findings reveal potential of levan S81 for technological purposes and as a potential natural immunomodulatory and antioxidant.

The hallmarks of CMV-specific CD8 T-cell differentiation.

Upon cytomegalovirus (CMV) infection, large T-cell responses are elicited that remain high or even increase over time, a phenomenon named memory T-cell inflation. Besides, the maintained robust T-cell response, CMV-specific T cells seem to have a distinctive phenotype, characterized by an advanced differentiation state. Here, we will review this "special" differentiation status by discussing the cellular phenotype based on the expression of CD45 isoforms, costimulatory, inhibitory and natural killer receptors, adhesion and lymphocyte homing molecules, transcription factors, cytokines and cytotoxic molecules. In addition, we focus on whether the differentiation state of CMV-specific CD8 T cells is unique in comparison with other chronic viruses and we will discuss the possible impact of factors such as antigen exposure and aging on the advanced differentiation status of CMV-specific CD8 T cells.

RNA regulation of the antiviral protein 2'-5'-oligoadenylate synthetase.

The innate immune system is a broad collection of critical intra- and extra-cellular processes that limit the infectivity of diverse pathogens. The 2'-5'-oligoadenylate synthetase (OAS) family of enzymes are important sensors of cytosolic double-stranded RNA (dsRNA) that play a critical role in limiting viral infection by activating the latent ribonuclease (RNase L) to halt viral replication and establish an antiviral state. Attesting to the importance of the OAS/RNase L pathway, diverse viruses have developed numerous distinct strategies to evade the effects of OAS activation. How OAS proteins are regulated by viral or cellular RNAs is not fully understood but several recent studies have provided important new insights into the molecular mechanisms of OAS activation by dsRNA. Other studies have revealed unanticipated features of RNA sequence and structure that strongly enhance activation of at least one OAS family member. While these discoveries represent important advances, they also underscore the fact that much remains to be learned about RNA-mediated regulation of the OAS/RNase L pathway. In particular, defining the full complement of RNA molecular signatures that activate OAS is essential to our understanding of how these proteins maximize their protective role against pathogens while still accurately discriminating host molecules to avoid inadvertent activation by cellular RNAs. A more complete knowledge of OAS regulation may also serve as a foundation for the development of novel antiviral therapeutic strategies and lead the way to a deeper understanding of currently unappreciated cellular functions of the OAS/RNase L pathway in the absence of infection. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications Translation > Translation Regulation.