Southern marsh deer (Blastocerus dichotomus) populations assessed using Amplicon Sequencing on fecal samples.

Populations in isolated and small fragments lose genetic variability very fast and are usually of conservation concern because they are at greater risk of local extinction. The largest native deer in South America, Blastocerus dichotomus (Illiger, 1815), is a Vulnerable species according to the IUCN categorization, which inhabits tropical and subtropical swampy areas. In Argentina, its presence has been restricted to four isolated fragments. Here we examine the genetic diversity and differentiation among three of them, including the three different patches that form the southernmost population, using 18 microsatellite markers genotyped by Amplicon Sequencing of DNA extracted from fecal samples. Genetic diversity was low (HE < 0.45) in all three populations studied. We found three genetic clusters compatible with the geographic location of the samples. We also found a metapopulation dynamics that involves the patches that make up the southernmost population, with evidence of a barrier to gene flow between two of them. Our results point to the creation of a corridor as a necessary and urgent management action. This is the first study, at the population level, employing microsatellite genotyping by Amplicon Sequencing with non-invasive samples in an endangered species.

Mitochondrial genome structure and composition in 70 fishes: a key resource for fisheries management in the South Atlantic.

BACKGROUND: Phylogenetic gaps of public databases of reference sequences are a major obstacle for comparative genomics and management of marine resources, particularly in the Global South, where economically important fisheries and conservation flagship species often lack closely-related references. We applied target-enrichment to obtain complete mitochondrial genomes of marine ichthyofauna from the Brazilian coast selected based on economic significance, conservation status and lack of phylogenetically-close references. These included sardines (Dorosomatidae, Alosidae), mackerels (Scombridae) croakers (Sciaenidae), groupers (Epinephelidae) and snappers (Lutjanidae). RESULTS: Custom baits were designed to enrich mitochondrial DNA across a broad phylogenetic range of fishes. Sequencing generated approximately 100k reads per sample, which were assembled in a total of 70 complete mitochondrial genomes and include fifty-two new additions to GenBank, including five species with no previous mitochondrial data. Departures from the typical gene content and order occurred in only three taxa and mostly involved tRNA gene duplications. Start-codons for all genes, except Cytochrome C Oxidase subunit I (COI), were consistently ATG, whilst a wide range of stop-codons deviated from the prevailing TAA. Phylogenetic analysis confirmed assembly accuracy and revealed signs of cryptic diversification within the Mullus genus. Lineage delimitation methods using Sardinella aurita and S. brasiliensis mitochondrial genomes support a single Operational Taxonomic Unit. CONCLUSIONS: Target enrichment was highly efficient, providing complete novel mitochondrial genomes with little sequencing effort. These sequences are deposited in public databases to enable subsequent studies in population genetics and adaptation of Latin American fish species and serve as a vital resource for conservation and management programs that rely on molecular data for species and genus-level identification.

Adeno-Associated Virus Type 5 Infection via PDGFRα Is Associated With Interstitial Lung Disease in Systemic Sclerosis and Generates Composite Peptides and Epitopes Recognized by the Agonistic Immunoglobulins Present in Patients With Systemic Sclerosis.

OBJECTIVE: The etiopathogenesis of systemic sclerosis (SSc) is unknown. Platelet-derived growth factor receptors (PDGFRs) are overexpressed in patients with SSc. Because PDGFRα is targeted by the adeno-associated virus type 5 (AAV5), we investigated whether AAV5 forms a complex with PDGFRα exposing epitopes that may induce the immune responses to the virus-PDGFRα complex. METHODS: The binding of monomeric human PDGFRα to the AAV5 capsid was analyzed by in silico molecular docking, surface plasmon resonance (SPR), and genome editing of the PDGFRα locus. AAV5 was detected in SSc lungs by in situ hybridization, immunohistochemistry, confocal microscopy, and molecular analysis of bronchoalveolar lavage (BAL) fluid. Immune responses to AAV5 and PDGFRα were evaluated by SPR using SSc monoclonal anti-PDGFRα antibodies and immunoaffinity-purified anti-PDGFRα antibodies from sera of patients with SSc. RESULTS: AAV5 was detected in the BAL fluid of 41 of 66 patients with SSc with interstitial lung disease (62.1%) and in 17 of 66 controls (25.75%) (P < 0.001). In SSc lungs, AAV5 localized in type II pneumocytes and in interstitial cells. A molecular complex formed of spatially contiguous epitopes of the AAV5 capsid and of PDGFRα was identified and characterized. In silico molecular docking analysis and binding to the agonistic anti-PDGFRα antibodies identified spatially contiguous epitopes derived from PDGFRα and AAV5 that interacted with SSc agonistic antibodies to PDGFRα. These peptides were also able to bind total IgG isolated from patients with SSc, not from healthy controls. CONCLUSION: These data link AVV5 with the immune reactivity to endogenous antigens in SSc and provide a novel element in the pathogenesis of SSc.

Low-dose interleukin-2 therapy in systemic lupus erythematosus.

In systemic lupus erythematosus (SLE), T regulatory cells (Tregs) contribute to the inhibition of autoimmune responses by suppressing self-reactive immune cells. Interleukin (IL)-2 plays an essential role in the generation, function and homeostasis of the Tregs and is reduced in SLE. Several clinical studies, including randomized trials, have shown that low-dose IL-2 therapy in SLE patients is safe and effective and can reduce disease manifestations. This review discusses the rationale for the use of low-dose IL-2 therapy in SLE, the clinical responses in patients, and the effects of this therapy on different types of T cells. Considerations are made on the current and future directions of use of low-dose IL-2 regimens in SLE.

Improved outcomes in rheumatoid arthritis with obesity after a weight loss intervention: randomized trial.

OBJECTIVE: To examine whether a weight loss intervention programme improves RA disease activity and/or musculoskeletal ultrasound synovitis measures in obese RA patients. METHODS: We conducted a proof-of-concept, 12-week, single-blind, randomized controlled trial of obese RA patients (BMI ≥ 30) with 28-joint DAS (DAS28) ≥ 3.2 and with evidence of power Doppler synovitis. Forty patients were randomized to the diet intervention (n = 20) or control group (n = 20). Diet intervention consisted of a hypocaloric diet of 1000-1500 kcal/day and high protein meal replacements. Co-primary outcomes included change in DAS28 and power Doppler ultrasound (PDUS)-34. Clinical disease activity, imaging, biomarkers, adipokines and patient-reported outcomes were monitored throughout the trial. Recruitment terminated early. All analyses were based on intent-to-treat for a significance level of 0.05. RESULTS: The diet intervention group lost an average 9.5 kg/patient, while the control group lost 0.5 kg (P < 0.001). Routine Assessment of Patient Index Data 3 (RAPID3) improved, serum leptin decreased and serum adiponectin increased significantly within the diet group and between the groups (all P < 0.03). DAS28 decreased, 5.2 to 4.2, within the diet group (P < 0.001; -0.51 [95% CI -1.01, 0.00], P = 0.056, between groups). HAQ-Disability Index (HAQ-DI) improved significantly within the diet group (P < 0.04; P = 0.065 between group). Ultrasound measures and the multi-biomarker disease activity score did not differ between groups (PDUS-34 -2.0 [95% CI -7.00, 3.1], P = 0.46 between groups). CONCLUSION: Obese RA patients on the diet intervention achieved weight loss. There were significant between group improvements for RAPID3, adiponectin and leptin levels, and positive trends for DAS28 and HAQ-DI. Longer-term, larger weight loss studies are needed to validate these findings, and will allow for further investigative work to improve the clinical management of obese RA patients. TRIAL REGISTRATION: ClinicalTrials.gov, https://clinicaltrials.gov, NCT02881307.

MIS-C: A COVID-19-as sociated condition between hypoimmunity and hyperimmunity.

Multisystem inflammatory syndrome in children (MIS-C) is a rare, severe complication of COVID-19. A better knowledge of immunological, cellular, and genetic characteristics of MIS-C could help better understand the pathogenesis of the disease and contribute to identifying specific diagnostic biomarkers and develop targeted therapies. We studied 37 MIS-C children at hospital admission and 24 healthy controls analyzing serum cytokines (IFN-α, IFN-ß, IFN-γ, IL-6, IL-10, IL-17A, IL-12p70 and TNF), lymphocyte populations by flow cytometry and 386 genes related to autoimmune diseases, autoinflammation and primary immunodeficiencies by NGS. MIS-C patients showed a significant increase of serum IFNγ (despite a significant reduction of activated Th1) and ILs, even if with a great heterogeneity among patients, revealing different pathways involved in MIS-C pathogenesis and suggesting that serum cytokines at admission may help to select the inflammatory pathways to target in each patient. Flow cytometry demonstrated a relevant reduction of T populations while the percentage of B cell was increased in agreement with an autoimmune pathogenesis of MIS-C. Genetic analysis identified variants in 34 genes and 83.3% of patients had at least one gene variant. Among these, 9 were mutated in more patients. Most genes are related to autoimmune diseases like ATM, NCF1, MCM4, FCN3, and DOCK8 or to autoinflammatory diseases associated to the release of IFNγ like PRF1, NOD2, and MEF. Thus, an incomplete clearance of the Sars-CoV2 during the acute phase may induce tissue damage and self-antigen exposure and genetic variants can predispose to hyper-reactive immune dysregulation events of MIS-C-syndrome. Type II IFN activation and cytokine responses (mainly IL-6 and IL-10) may cause a cytokine storm in some patients with a more severe acute phase of the disease, lymphopenia and multisystemic organ involvement. The timely identification of such patients with an immunocytometric panel might be critical for targeted therapeutic management.

Low STR variability in the threatened marsh deer, Blastocerus dichotomus, detected through amplicon sequencing in non-invasive samples.

Blastocerus dichotomus is the largest deer in South America. We have used 25 microsatellite markers detected and genotyped by Next Generation Sequencing to estimate the genetic variability of B. dichotomus in Argentina, where most of its populations are threatened. Primer design was based on the sequence of a shallow partial genome (15,967,456 reads; 16.66% genome coverage, mean depth 1.64) of a single individual. From the thousands of microsatellite loci found, even under high stringency selection, we chose and tested a set of 80 markers on 30 DNA samples extracted from tissue and feces from three Argentinean populations. Heterozygosity levels were low across all loci in all populations (H=0.31 to 0.40). Amplicon sequencing is a fast, easy, and affordable technique that can be very useful for the characterization of microsatellite marker sets for the conservation genetics of non-model organisms. This work is also one of the first ones to use amplicon sequencing in non-invasive samples and represents an important development for the study of threatened species.

Strategies to Use Nanoparticles to Generate CD4 and CD8 Regulatory T Cells for the Treatment of SLE and Other Autoimmune Diseases.

Autoimmune diseases are disorders of immune regulation where the mechanisms responsible for self-tolerance break down and pathologic T cells overcome the protective effects of T regulatory cells (Tregs) that normally control them. The result can be the initiation of chronic inflammatory diseases. Systemic lupus erythematosus (SLE) and other autoimmune diseases are generally treated with pharmacologic or biological agents that have broad suppressive effects. These agents can halt disease progression, yet rarely cure while carrying serious adverse side effects. Recently, nanoparticles have been engineered to correct homeostatic regulatory defects and regenerate therapeutic antigen-specific Tregs. Some approaches have used nanoparticles targeted to antigen presenting cells to switch their support from pathogenic T cells to protective Tregs. Others have used nanoparticles targeted directly to T cells for the induction and expansion of CD4+ and CD8+ Tregs. Some of these T cell targeted nanoparticles have been formulated to act as tolerogenic artificial antigen presenting cells. This article discusses the properties of these various nanoparticle formulations and the strategies to use them in the treatment of autoimmune diseases. The restoration and maintenance of Treg predominance over effector cells should promote long-term autoimmune disease remission and ultimately prevent them in susceptible individuals.

Nanoparticles Engineered as Artificial Antigen-Presenting Cells Induce Human CD4+ and CD8+ Tregs That Are Functional in Humanized Mice.

Artificial antigen-presenting cells (aAPCs) are synthetic versions of naturally occurring antigen-presenting cells (APCs) that, similar to natural APCs, promote efficient T effector cell responses in vitro. This report describes a method to produce acellular tolerogenic aAPCs made of biodegradable poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs) and encapsulating IL-2 and TGF-ß for a paracrine release to T cells. We document that these aAPCs can induce both human CD4+ and CD8+ T cells to become FoxP3+ T regulatory cells (Tregs). The aAPC NP-expanded human Tregs are functional in vitro and can modulate systemic autoimmunity in vivo in humanized NSG mice. These findings establish a proof-of-concept to use PLGA NPs as aAPCs for the induction of human Tregs in vitro and in vivo, highlighting the immunotherapeutic potential of this targeted approach to repair IL-2 and/or TGF-ß defects documented in certain autoimmune diseases such as systemic lupus erythematosus.

The pleiotropic roles of leptin in metabolism, immunity, and cancer.

The discovery of the archetypal adipocytokine leptin and how it regulates energy homeostasis have represented breakthroughs in our understanding of the endocrine function of the adipose tissue and the biological determinants of human obesity. Investigations on leptin have also been instrumental in identifying physio-pathological connections between metabolic regulation and multiple immunological functions. For example, the description of the promoting activities of leptin on inflammation and cell proliferation have recognized the detrimental effects of leptin in connecting dysmetabolic conditions with cancer and with onset and/or progression of autoimmune disease. Here we review the multiple biological functions and complex framework of operations of leptin, discussing why and how the pleiotropic activities of this adipocytokine still pose major hurdles in the development of effective leptin-based therapeutic opportunities for different clinical conditions.

Nanoparticle-mediated Delivery of IL-2 To T Follicular Helper Cells Protects BDF1 Mice from Lupus-like Disease.

We recently reported that poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs) loaded with interleukin (IL)-2 and targeted to T cells inhibited the development of lupus-like disease in BDF1 mice by inducing functional T regulatory cells (Tregs). Here we show that the protection from disease and the extended survival of BDF1 mice provided by IL-2-loaded NPs targeted to T cells is not only due to an induction of Tregs but also contributed by an inhibition of T follicular helper (TFH) cells. These results identify a dual protective activity of IL-2 in the control of lupus autoimmunity, namely the inhibition of effector TFH cells, in addition to the previously known induction of Tregs. This newly recognized activity of IL-2 delivered by NPs can help better explain the beneficial effects of low-dose IL-2 immunotherapy in systemic lupus erythematosus (SLE), and might be considered as a new strategy to slow disease progression and improve outcomes in lupus patients.

Systemic Lupus Erythematosus and Coronavirus Disease 2019.

Coronavirus disease 2019 (COVID-19) is a contagious infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It manifests with variable clinical pictures ranging from asymptomatic to mild or uncomplicated illness to severe disease with possible multi-organ involvement, with respiratory and vascular systems being the most often affected. Since COVID-19 can affect patients with autoimmune rheumatic conditions, the concomitant presence of two diseases may have clinical characteristics whose knowledge may help facilitate clinical management. This review discusses the data available in the literature on COVID-19 in systemic lupus erythematosus (SLE) patients.

DNA vaccine encoding heat shock protein 90 protects from murine lupus.

BACKGROUND: Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the presence of autoantibodies to multiple self-antigens, including heat shock proteins (HSP). Because of the increased expression of HSP90 and abnormal immune responses to it in SLE, we investigated whether an HSP90 DNA vaccine could modulate the development and clinical manifestations of SLE in lupus-prone mice. METHODS: (NZB x NZW)F1 (NZB/W) mice were vaccinated with DNA constructs encoding HSP90 or control plasmids or vehicle. The mice were then monitored for survival, circulating anti-dsDNA autoantibodies, and immune phenotypes. Renal disease was evaluated by immunohistochemistry and by the measurement of proteinuria. RESULTS: Vaccination with HSP90 DNA reduced lupus disease manifestations and prolonged the survival of NZB/W mice. The protective effects of the HSP90 DNA vaccine associated with the induction of tolerogenic dendritic cells (DCs) and an expansion of T regulatory cells (Tregs). CONCLUSIONS: The beneficial effects of DNA vaccination with HSP90 in murine SLE support the possibility of HSP90-based therapeutic modalities of intervention in SLE.

Serafino Zappacosta: An Enlightened Mentor and Educator.

With this article, the authors aim to honor the memory of Serafino Zappacosta, who had been their mentor during the early years of their career in science. The authors discuss how the combination of Serafino Zappacosta's extraordinary commitment to teaching and passion for science created a fostering educational environment that led to the creation of the "Ruggero Ceppellini Advanced School of Immunology." The review also illustrates how the research on the MHC and the inspirational scientific context in the Zappacosta's laboratory influenced the authors' early scientific interests, and subsequent professional work as immunologists.

DNA Vaccination With Hsp70 Protects Against Systemic Lupus Erythematosus in (NZB × NZW)F1 Mice.

OBJECTIVE: To address whether a targeted modulation of the abnormal expression of Hsp70 and autoantibodies against this molecule in systemic lupus erythematosus can influence disease. METHODS: Lupus-prone (NZB × NZW)F1 mice that had been DNA-vaccinated with plasmids encoding Hsp70 and controls were monitored for lupus disease parameters including anti-double stranded DNA (anti-dsDNA) autoantibodies and cytokines using enzyme-linked immunosorbent assay, and for kidney function and pathology. The phenotypic and numerical changes in relevant immune cells were evaluated by flow cytometry, and cell function was assessed. RESULTS: Mice that had been DNA-vaccinated with Hsp70 displayed marked suppression of anti-dsDNA antibody production, reduced renal disease, and antiinflammatory responses that are associated with a significantly extended survival, compared to controls. These protective effects in Hsp70-vaccinated mice were associated with an induction of tolerogenic immune responses and an expansion of functional Treg cells. CONCLUSION: DNA vaccination with Hsp70 suppresses murine lupus by inducing tolerogenic immune responses and antiinflammatory immune responses associated with reduced disease manifestations and increased mouse survival.