Clonally expanded EOMES+ Tr1-like cells in primary and metastatic tumors are associated with disease progression.

Regulatory T (Treg) cells are a barrier for tumor immunity and a target for immunotherapy. Using single-cell transcriptomics, we found that CD4+ T cells infiltrating primary and metastatic colorectal cancer and non-small-cell lung cancer are highly enriched for two subsets of comparable size and suppressor function comprising forkhead box protein P3+ Treg and eomesodermin homolog (EOMES)+ type 1 regulatory T (Tr1)-like cells also expressing granzyme K and chitinase-3-like protein 2. EOMES+ Tr1-like cells, but not Treg cells, were clonally related to effector T cells and were clonally expanded in primary and metastatic tumors, which is consistent with their proliferation and differentiation in situ. Using chitinase-3-like protein 2 as a subset signature, we found that the EOMES+ Tr1-like subset correlates with disease progression but is also associated with response to programmed cell death protein 1-targeted immunotherapy. Collectively, these findings highlight the heterogeneity of Treg cells that accumulate in primary tumors and metastases and identify a new prospective target for cancer immunotherapy.

Tracking the immune response profiles elicited by the BNT162b2 vaccine in COVID-19 unexperienced and experienced individuals.

Multiple vaccines have been approved to control COVID-19 pandemic, with Pfizer/BioNTech (BNT162b2) being widely used. We conducted a longitudinal analysis of the immune response elicited after three doses of the BNT162b2 vaccine in individuals who have previously experienced SARS-CoV-2 infection and in unexperienced ones. We conducted immunological analyses and single-cell transcriptomics of circulating T and B lymphocytes, combined to CITE-seq or LIBRA-seq, and VDJ-seq. We found that antibody levels against SARS-CoV-2 Spike, NTD and RBD from wild-type, delta and omicron VoCs show comparable dynamics in both vaccination groups, with a peak after the second dose, a decline after six months and a restoration after the booster dose. The antibody neutralization activity was maintained, with lower titers against the omicron variant. Spike-specific memory B cell response was sustained over the vaccination schedule. Clonal analysis revealed that Spike-specific B cells were polyclonal, with a partial clone conservation from natural infection to vaccination. Spike-specific T cell responses were oriented towards effector and effector memory phenotypes, with similar trends in unexperienced and experienced individuals. The CD8 T cell compartment showed a higher clonal expansion and persistence than CD4 T cells. The first two vaccinations doses tended to induce new clones rather than promoting expansion of pre-existing clones. However, we identified a fraction of Spike-specific CD8 T cell clones persisting from natural infection that were boosted by vaccination and clones specifically induced by vaccination. Collectively, our observations revealed a moderate effect of the second dose in enhancing the immune responses elicited after the first vaccination. Differently, we found that a third dose was necessary to restore comparable levels of neutralizing antibodies and Spike-specific T and B cell responses in individuals who experienced a natural SARS-CoV-2 infection.

Plasma Proteomic Variables Related to COVID-19 Severity: An Untargeted nLC-MS/MS Investigation.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection leads to a wide range of clinical manifestations and determines the need for personalized and precision medicine. To better understand the biological determinants of this heterogeneity, we explored the plasma proteome of 43 COVID-19 patients with different outcomes by an untargeted liquid chromatography-mass spectrometry approach. The comparison between asymptomatic or pauci-symptomatic subjects (MILDs), and hospitalised patients in need of oxygen support therapy (SEVEREs) highlighted 29 proteins emerged as differentially expressed: 12 overexpressed in MILDs and 17 in SEVEREs. Moreover, a supervised analysis based on a decision-tree recognised three proteins (Fetuin-A, Ig lambda-2chain-C-region, Vitronectin) that are able to robustly discriminate between the two classes independently from the infection stage. In silico functional annotation of the 29 deregulated proteins pinpointed several functions possibly related to the severity; no pathway was associated exclusively to MILDs, while several only to SEVEREs, and some associated to both MILDs and SEVEREs; SARS-CoV-2 signalling pathway was significantly enriched by proteins up-expressed in SEVEREs (SAA1/2, CRP, HP, LRG1) and in MILDs (GSN, HRG). In conclusion, our analysis could provide key information for 'proteomically' defining possible upstream mechanisms and mediators triggering or limiting the domino effect of the immune-related response and characterizing severe exacerbations.

OligoMinerApp: a web-server application for the design of genome-scale oligonucleotide in situ hybridization probes through the flexible OligoMiner environment.

Fluorescence in situ hybridization (FISH) is a powerful single-cell technique that harnesses nucleic acid base pairing to detect the abundance and positioning of cellular RNA and DNA molecules in fixed samples. Recent technology development has paved the way to the construction of FISH probes entirely from synthetic oligonucleotides (oligos), allowing the optimization of thermodynamic properties together with the opportunity to design probes against any sequenced genome. However, comparatively little progress has been made in the development of computational tools to facilitate the oligos design, and even less has been done to extend their accessibility. OligoMiner is an open-source and modular pipeline written in Python that introduces a novel method of assessing probe specificity that employs supervised machine learning to predict probe binding specificity from genome-scale sequence alignment information. However, its use is restricted to only those people who are confident with command line interfaces because it lacks a Graphical User Interface (GUI), potentially cutting out many researchers from this technology. Here, we present OligoMinerApp (http://oligominerapp.org), a web-based application that aims to extend the OligoMiner framework through the implementation of a smart and easy-to-use GUI and the introduction of new functionalities specially designed to make effective probe mining available to everyone.

Eomesodermin controls a unique differentiation program in human IL-10 and IFN-γ coproducing regulatory T cells.

Whether human IL-10-producing regulatory T cells ("Tr1") represent a distinct differentiation lineage or an unstable activation stage remains a key unsolved issue. Here, we report that Eomesodermin (Eomes) acted as a lineage-defining transcription factor in human IFN-γ/IL-10 coproducing Tr1-like cells. In vivo occurring Tr1-like cells expressed Eomes, and were clearly distinct from all other CD4+ T-cell subsets, including conventional cytotoxic CD4+ T cells. They expressed Granzyme (Gzm) K, but had lost CD40L and IL-7R expression. Eomes antagonized the Th17 fate, and directly controlled IFN-γ and GzmK expression. However, Eomes binding to the IL-10 promoter was not detectable in human CD4+ T cells, presumably because critical Tbox binding sites of the mouse were not conserved. A precommitment to a Tr1-like fate, i.e. concominant induction of Eomes, GzmK, and IFN-γ, was promoted by IL-4 and IL-12-secreting myeloid dendritic cells. Consistently, Th1 effector memory cells contained precommitted Eomes+ GzmK+ T cells. Stimulation with T-cell receptor (TCR) agonists and IL-27 promoted the generation of Tr1-like effector cells by inducing switching from CD40L to IL-10. Importantly, CD4+ Eomes+ T-cell subsets were present in lymphoid and nonlymphoid tissues, and their frequencies varied systemically in patients with inflammatory bowel disease and graft-versus-host disease. We propose that Eomes+ Tr1-like cells are effector cells of a unique GzmK-expressing CD4+ T-cell subset.

Composite chitosan hydrogels as advanced wound dressings with sustained ibuprofen release and suitable application characteristics.

The physical chitosan hydrogel, obtained by ionic gelation in lactic acid solution, was combined with biocompatible oil-in-water microemulsion with ibuprofen, to prepare composite hydrogels with 0.25-1% of the polymer and 5% of the drug. The electrical conductivity measurement, photon correlation spectroscopy (PCS), and rheological analysis showed that the composite hydrogels comprise oil nanodroplets (16.21-22.56 nm) embedded within pseudoplastic chitosan hydrogel. In vitro ibuprofen release was sustained for 12 h and followed zero-order kinetics. pH values of the composite hydrogels were in the range of 4.80-5.27, thus physiologically acceptable. The formulation containing 0.5% chitosan enabled the maximum drug release rate of 239.25 µgh-1cm-2 as well as cohesiveness (154.958 ± 0.731 g*s) higher than hardness (13.546 ± 0.065 g) and adhesiveness (-12.042 ± 1.161 g*s), so textural properties were suitable for application along skin surface, without spillage, and for easy removal. This is the first study in which the composite chitosan hydrogels with ibuprofen were formulated by combining the chitosan hydrogel prepared without harmful chemical crosslinkers and low viscosity oil-in-water microemulsion, and the preclinical characterization of their biopharmaceutical aspect and textural characterization, that is of key importance in improving the patient's compliance, were performed.

Corrigendum: The Impact of Anti-rheumatic Drugs on the Seroprevalence of Anti-SARS-CoV-2 Antibodies in a Cohort of Patients With Inflammatory Arthritis: The MAINSTREAM Study.

[This corrects the article DOI: 10.3389/fmed.2022.850858.].

The Impact of Anti-rheumatic Drugs on the Seroprevalence of Anti-SARS-CoV-2 Antibodies in a Cohort of Patients With Inflammatory Arthritis: The MAINSTREAM Study.

Objectives: Given the high occurrence of asymptomatic subsets, the true prevalence of SARS-CoV-2 infection in rheumatic patients is still underestimated. This study aims to evaluate the seroprevalence of SARS-CoV-2 antibodies in rheumatic musculoskeletal diseases (RMD) patients receiving immunomodulatory drugs. Methods: All consecutive patients with rheumatoid arthritis or spondyloarthritis receiving disease-modifying antirheumatic drugs (DMARDs) evaluated between 4th May and 16th June 2020 were included. All participants were tested for anti-SARS-CoV-2 antibodies (IgG, IgM, IgA) by ELISA and were questioned about previous COVID-19 symptoms and clinical course. Results were compared with healthy population from the same region and with a control group of healthy subjects diagnosed with confirmed COVID-19. Results: The study population includes 358 patients. The overall prevalence of anti-SARS-CoV-2 antibodies (18.4%) was higher than prevalence rate based on swab-positivity (1.12%) or clinically suspected cases (10.6%), but consistent with seroprevalence observed in the healthy population. Among seropositive patients 58% were asymptomatic. Mean anti-SARS-CoV-2 titer was comparable with the control group. No differences in seroprevalence were observed according to age, sex, rheumatic disease and treatment with conventional, biologic or targeted synthetic DMARDs, whereas glucocorticoids and comorbidities resulted in higher seroprevalence rate. Conclusions: The results of this study are reassuring about the low impact of RMDs and immunomodulatory therapies on the risk and clinical course of COVID-19 and on humoral immune response to SARS-CoV-2 infection.

Immunosuppressant Treatment in Rheumatic Musculoskeletal Diseases Does Not Inhibit Elicitation of Humoral Response to SARS-CoV-2 Infection and Preserves Effector Immune Cell Populations.

COVID-19 has proven to be particularly serious and life-threatening for patients presenting with pre-existing pathologies. Patients affected by rheumatic musculoskeletal disease (RMD) are likely to have impaired immune responses against SARS-CoV-2 infection due to their compromised immune system and the prolonged use of disease-modifying anti-rheumatic drugs (DMARDs), which include conventional synthetic (cs) DMARDs or biologic and targeted synthetic (b/ts) DMARDs. To provide an integrated analysis of the immune response following SARS-CoV-2 infection in RMD patients treated with different classes of DMARDs we carried out an immunological analysis of the antibody responses toward SARS-CoV-2 nucleocapsid and RBD proteins and an extensive immunophenotypic analysis of the major immune cell populations. We showed that RMD individuals under most DMARD treatments mount a sustained antibody response to the virus, with neutralizing activity. In addition, they displayed a sizable percentage of effector T and B lymphocytes. Among b-DMARDs, we found that anti-TNFα treatments are more favorable drugs to elicit humoral and cellular immune responses as compared to CTLA4-Ig and anti-IL6R inhibitors. This study provides a whole picture of the humoral and cellular immune responses in RMD patients by reassuring the use of DMARD treatments during COVID-19. The study points to TNF-α inhibitors as those DMARDs permitting elicitation of functional antibodies to SARS-CoV-2 and adaptive effector populations available to counteract possible re-infections.

Integrated longitudinal immunophenotypic, transcriptional and repertoire analyses delineate immune responses in COVID-19 patients.

To understand how a protective immune response against SARS-CoV-2 develops over time, we integrated phenotypic, transcriptional and repertoire analyses on PBMCs from mild and severe COVID-19 patients during and after infection, and compared them to healthy donors (HD). A type I IFN-response signature marked all the immune populations from severe patients during the infection. Humoral immunity was dominated by IgG production primarily against the RBD and N proteins, with neutralizing antibody titers increasing post infection and with disease severity. Memory B cells, including an atypical FCRL5+ T-BET+ memory subset, increased during the infection, especially in patients with mild disease. A significant reduction of effector memory, CD8+ T cells frequency characterized patients with severe disease. Despite such impairment, we observed robust clonal expansion of CD8+ T lymphocytes, while CD4+ T cells were less expanded and skewed toward TCM and TH2-like phenotypes. MAIT cells were also expanded, but only in patients with mild disease. Terminally differentiated CD8+ GZMB+ effector cells were clonally expanded both during the infection and post-infection, while CD8+ GZMK+ lymphocytes were more expanded post-infection and represented bona fide memory precursor effector cells. TCR repertoire analysis revealed that only highly proliferating T cell clonotypes, which included SARS-CoV-2-specific cells, were maintained post-infection and shared between the CD8+ GZMB+ and GZMK+ subsets. Overall, this study describes the development of immunity against SARS-CoV-2 and identifies an effector CD8+ T cell population with memory precursor-like features.

Comparison of the Effect of Bioadhesive Polymers on Stability and Drug Release Kinetics of Biocompatible Hydrogels for Topical Application of Ibuprofen.

The study is focused on formulation of biocompatible hydrogels with a poorly soluble drug ibuprofen (5%) and comprehensive evaluation and comparison of effect of different bioadhesive polymers on their suitability for application on skin, physical stability during the accelerated and natural aging tests (by performing centrifugation test, light microscopy, differential scanning calorimetry, rheological and pH measurements), and in vitro drug release kinetics. Hydrogels, formulated with xanthan gum 1% (XIB), sodium carboxymethylcellulose 5% (CMCIB), poloxamer 407 16% (PIB), and carbomer 1% (KIB), were soft pseudoplastic semisolids with thixotropy and biocompatible pH. The type of the polymer significantly affected apparent viscosity of the hydrogels and miscibility rate with artificial sweat, their physical stability, and shape, size, and aggregation of the drug crystals and degree of crystallization. The drug release in all investigated hydrogels was diffusion-controlled in accordance with the Higuchi model and sustained for 12 h, with the drug release rate and the amount of drug released depended on the polymer. The described formulation approach enabled discrimination of the hydrogels with unsatisfactory application properties (CMCIB) and physical stability (KIB), and selection of the hydrogel with promising characteristics in terms of all investigated aspects (XIB) which could be considered for further evaluation.

Formulation of hydrogel-thickened nonionic microemulsions with enhanced percutaneous delivery of ibuprofen assessed in vivo in rats.

The study investigated usage of hydrogel of an anionic polymer xanthan gum for design of ibuprofen-loaded hydrogel-thickened microemulsions (HTMs) from the nonionic oil-in-water microemulsion (M). Xanthan gum demonstrated the performances of a thickening agent in physically stable HTMs at 5±3°C, 20±3°C, and 40±1°C during 6months. The results of physicochemical characterization (pH, conductivity, rheological behaviour, spreadability) indicated that HTMs containing 0.25-1.00% of the polymer had colloidal structure with oil nanodroplets of 14.34±0.98nm (PdI 0.220±0.075) dispersed in aqueous phase thickened with the polymer gel network which strength depended on the polymer concentration. HTMs with ibuprofen (5%) were evaluated as percutaneous drug delivery carriers. In vitro ibuprofen release from HTMs followed zero order kinetic (r>0.995) for 12h, while the referent hydrogel was described by Higuchi model. The HTM with optimized drug release rate and spreadability (HTM1) and the polymer-free microemulsion (M) were assessed and compared with the referent hydrogel in in vivo studies in rats. HTM1 and M were significantly more efficacious than reference hydrogel in producing antihyperalgesic and at lower extent antiedematous activity in prophylactic topical treatment protocol, whilst they were comparable in producing antihyperalgesic/antiedematous effects in therapeutic protocol. Topical treatments produced no obvious skin irritation.

Characterization of gelation process and drug release profile of thermosensitive liquid lecithin/poloxamer 407 based gels as carriers for percutaneous delivery of ibuprofen.

Suitability of liquid lecithin (i.e., solution of lecithin in soy bean oil with ∼ 60% w/w of phospholipids) for formation of gels, upon addition of water solution of poloxamer 407, was investigated, and formulated systems were evaluated as carriers for percutaneous delivery of ibuprofen. Formulation study of pseudo-ternary system liquid lecithin/poloxamer 407/water at constant liquid lecithin/poloxamer 407 mass ratio (2.0) revealed that minimum concentrations of liquid lecithin and poloxamer 407 required for formation of gel like systems were 15.75% w/w and 13.13% w/w, respectively, while the maximum content of water was 60.62% w/w. The systems comprising water concentrations in a range from 55 to 60.62% w/w were soft semisolids suitable for topical application, and they were selected for physicochemical and biopharmaceutical evaluation. Analysis of conductivity results and light microscopy examination revealed that investigated systems were water dilutable dispersions of spherical oligolamellar associates of phospholipids and triglyceride molecules in the copolymer water solution. Rheological behavior evaluation results indicated that the investigated gels were thermosensitive shear thinning systems. Ibuprofen (5% w/w) was incorporated by dispersing into the previously prepared carriers. Drug-loaded systems were physically stable at storage temperature from 5 ± 3°C to 40 ± 2°C, for 30 days. In vitro ibuprofen release was in accordance with the Higuchi model (rH>0.95) and sustained for 12h. The obtained results implicated that formulated LLPBGs, optimized regarding drug release and organoleptic properties, represent promising carriers for sustained percutaneous drug delivery of poorly soluble drugs.

Design of Block Copolymer Costabilized Nonionic Microemulsions and Their In Vitro and In Vivo Assessment as Carriers for Sustained Regional Delivery of Ibuprofen via Topical Administration.

Nonionic surfactants (caprylocaproyl macrogol-8 glycerides, octoxynol-12, polysorbate-20, and polyethylene glycol-40 hydrogenated castor oil) (47.03%, w/w), costabilizer (poloxamer 407) (12%-20%, w/w), oil (isopropyl myristate) (5.22%, w/w), water (q.s. ad 100%, w/w), and ibuprofen (5%, w/w) were used to develop oil-in-water microemulsions with Newtonian flow behavior, low viscosity (from 368 ± 38 to 916 ± 46 mPa s), and average droplet size from 14.79 ± 0.31 to 16.54 ± 0.75 nm. Ibuprofen in vitro release from the microemulsions was in accordance with zero-order kinetics (R0(2) > 0.99) for at least 12 h. The maximum drug release rate (3.55%h(-1) ) was from the microemulsion M3 comprising 16%, w/w of poloxamer 407. The release rate of ibuprofen from the reference hydrogel followed Higuchi kinetics (RH(2) > 0.99), and drug amount released after the 6th hour was negligible. In a rat model of inflammation, the microemulsion M3 was significantly more efficacious than the reference hydrogel in exerting antihyperalgesic effects in prophylactic topical treatment, whereas they were comparable in therapeutic treatment as well as in producing antiedematous effect in both protocols. No obvious skin irritation was observed in in vivo studies. The developed nonionic surfactants-based microemulsions containing the optimal concentration of poloxamer 407 could be promising carriers for sustained regional delivery of ibuprofen via topical administration.