Evaluation of maternal-infant dyad inflammatory cytokines in pregnancies affected by maternal SARS-CoV-2 infection in early and late gestation.

Objective: SARS-CoV-2 infection induces significant inflammatory cytokine production in adults, but infant cytokine signatures in pregnancies affected by maternal SARS-CoV-2 are less well characterized. We aimed to evaluate cytokine profiles of mothers and their infants following COVID-19 in pregnancy. Study Design: Serum samples at delivery from 31 mother-infant dyads with maternal SARS-CoV-2 infection in pregnancy (COVID) were examined in comparison to 29 control dyads (Control). Samples were evaluated using a 13-plex cytokine assay. Results: In comparison with controls, interleukin (IL)-6 and interferon gamma-induced protein 10 (IP-10) were higher in COVID maternal and infant samples (p<0.05) and IL-8 uniquely elevated in COVID infant samples (p<0.05). Significant elevations in IL-6, IP-10 and IL-8 were found among both early (1st/2nd Trimester) and late (3rd Trimester) maternal SARS-CoV-2 infections. Conclusions: Maternal SARS-CoV-2 infections throughout gestation are associated with increased maternal and infant inflammatory cytokines at birth with potential to impact long-term infant health.

Synergistic Anti-Leishmanial Activities of Morphine and Imiquimod on Leishmania infantum (MCAN/ES/98/LIM-877)

Background: This study was performed to evaluate in vitro and in vivo Leishmanicidal potential of morphine (Mph), imiquimod (IQ), and their combination. Methods: Leishmania infantum promastigote and amastigote assays were performed at the presence of 0.015–150µM Mph, 0.04–416µM IQ, and their combination. The inhibition effects of these drugs on promastigotes were evaluated after 24, 48, and 72h. The cytotoxic effects of the drugs were evaluated by MTT as well as flow cytometry after 72h. We explored the therapeutic effects of Mph and IQ in BALB/c mice at the end of the treatment using parasite load de­termination and cytokine assay. One group of mice received Mph for three weeks before infection. Results: The results of promastigote and amastigote assays showed the cytotoxic effects of the drugs at low concentra­tions. The cytotoxic effects were higher on promastigotes than amastigotes (p< 0.05). There was a negative correlation between drug concentration and amastigote/promastigote viability. Imiquimod alone or combined with Mph showed remarkable cytotoxic effects at all concentrations (p< 0.05). Flow cytometry results revealed apoptosis in the parasite following exposure to the drug combinations. Accordingly, the reduction of parasite loads in the spleen and liver was observed (p< 0.05) with simultaneous increases in IFN-γ and IL-4. We believe that the in vivo leishmanicidal effect was mediated by Mph through IL-4 and by IQ through both IL-4 and IFN-γ. Conclusion: Results pointed out the promising effects of Mph and IQ at low concentrations, especially when combined.

PSX-A-8 Late-Breaking: Development of multiplex assays for equine cytokines IL-1β, IL-4, IL-6, IL-8, IL-10, and TNFα

Equine-specific assays to quantify cytokine concentrations are limited and often have a restricted range such that physiological concentrations of many cytokines are below detectable limits of the assay. We aimed to develop custom multiplex assays for equine interleukin (IL)-1β, IL-4, IL-6, IL-8, IL-10, and tumor necrosis factor (TNF) α using the Meso Scale Discovery U-PLEX platform. Equine-specific ELISA kits containing unlabeled and biotinylated polyclonal antibodies and the specific recombinant equine cytokine were purchased for each cytokine. Each biotinylated antibody was coupled to a linker specific for a unique spot within each well of the U-PLEX plates. The unlabeled antibodies were conjugated with electrochemiluminescent labels to serve as detection antibodies. Each cytokine assay was optimized individually prior to optimization in multiplex. Two preliminary experiments were performed: 1) multiplexed conjugation of equine IL-10 and TNFα to the U-PLEX plates; and 2) multiplexed conjugation of equine IL-1β, IL-4, IL-6, and IL-8 to the U-PLEX plates. Standard curves were run at concentrations ranging from 0 to 5,000 pg/mL for TNFα and IL-8, to 12,500 pg/mL for IL-10, to 25,000 pg/mL for IL-1β and IL-6, and to 50,000 pg/mL for IL-4. The minimum average concentrations measured by the standard curves were 0.065, 0.006, 0.017, 0.00013, 0.196, and 0.050 pg/mL for IL-1β, IL-4, IL-6, IL-8, IL-10, and TNFα, respectively. Test samples of equine serum (n = 5) and bronchoalveolar fluid (n = 3) before and after exercise and of ConA-stimulated equine peripheral mononuclear cell supernatants (positive control) were analyzed for each multiplexed assay. With the exception of serum from one horse, all samples ran within detectable limits of each assay. This preliminary work indicates the U-PLEX platform is a viable option to simultaneously quantify concentrations of multiple equine cytokines, allowing for expansion of research efforts focused on understanding immune responses in the horse.

High Dose IL-17A Enhances the Antiosteoarthritic Effect of Dental Pulp Mesenchymal Stem Cells by Reducing Inflammatory Response and Promoting Chondrocyte Proliferation

Objective: Mesenchymal stem cells (MSCs) have promising potential in regenerative therapy, partially through their potent immune modulation. The inflammatory cytokine IL-17A has been suggested to improve the therapeutic efficacy of MSCs. Herein, we determined whether pretreatment of dental pulp MSCs (DPMSCs) with IL-17A enhances the therapeutic potency of DPMSCs for osteoarthritis (OA).Methods: DPMSCs pretreated with IL-17A (0-75 ng/ml) were co-cultured with T lymphocytes for cytokine assay. In a rat OA model, DPMSCs pretreated with IL-17A were evaluated for antiosteoarthritic activity. Rat knee joints were harvested for color Doppler ultrasonography and histological staining, and synovial fluid was collected for cytokine assay. Additionally, chondrocytes isolated form joint cartilage of OA rats were co-cultured with IL-17A-pretreated DPMSCs for cell proliferation assay.Results: DPMSCs demonstrated a pluripotent capacity to differentiate into osteocytes, adipocytes, and chondrocytes. For in vitro co-culture, although DPMSCs pretreated with low dose of IL-17A (25 ng/ml) had inflammation-promoting effect, indicated by increased IL-2 and TNF-α secretion and decreased IL-4 release, high dose IL-17A (75 ng/ml) pretreatment enhanced the anti-inflammatory activity of DPMSCs. In vivo, high dose IL-17A-pretreated DPMSCs showed a stronger antiosteoarthritic activity than untreated DPMSCs, indicated by significantly reducing joint swelling, synovial fluid formation, the OARSI scores, and the production of inflammatory cytokines in synovial fluid. Besides, high dose IL-17A- pretreated DPMSCs enhanced the stimulatory activity of DPMSCs in chondrocyte proliferation in vitro.Conclusion: high dose IL-17A enhances the immunosuppressive property of DPMSCs, which helps alleviate osteoarthritis by inhibiting inflammatory response and promoting chondrocyte proliferation in the affected joints.

Gene signature and immune cell profiling by high-dimensional, single-cell analysis in COVID-19 patients, presenting Low T3 syndrome and coexistent hematological malignancies

Background Low T3 syndrome is frequent in patients admitted to intensive care units for critical illness and pneumonia. It has been reported also in patients with COVID-19, Hodgkin disease and chronic lymphocytic leukemia. We analyzed the clinical relevance of Low T3 syndrome in COVID-19 patients and, in particular, in those with associated hematological malignancies. Methods Sixty-two consecutive patients, hospitalized during the first wave of SARS-CoV-2 outbreak in Sant’Andrea University Hospital in Rome, were subdivided in 38 patients (Group A), showing low levels of FT3, and in 24 patients (Group B), with normal FT3 serum values. During the acute phase of the disease, we measured serum, radiologic and clinical disease severity markers and scores, in search of possible correlations with FT3 serum values. In addition, in 6 COVID-19 patients, 4 with Low T3 syndrome, including 2 with a hematological malignancy, and 2 with normal FT3 values, we performed, high-dimensional single-cell analysis by mass cytometry, multiplex cytokine assay and gene expression profiling in peripheral blood mononuclear cells (PBMC). Results Low FT3 serum values were correlated with increased Absolute Neutrophil Count, NLR and dNLR ratios and with reduced total count of CD3+, CD4+ and CD8+ T cells. Low FT3 values correlated also with increased levels of inflammation, tissue damage and coagulation serum markers as well as with SOFA, LIPI and TSS scores. The CyTOF analysis demonstrated reduction of the effector memory and terminal effector subtypes of the CD4+ T lymphocytes. Multiplex cytokine assay indicates that mainly IL-6, IP-10 and MCAF changes are associated with FT3 serum levels, particularly in patients with coexistent hematological malignancies. Gene expression analysis using Nanostring identified four genes differently expressed involved in host immune response, namely CD38, CD79B, IFIT3 and NLRP3. Conclusions Our study demonstrates that low FT3 serum levels are associated with severe COVID-19. Our multi-omics approach suggests that T3 is involved in the immune response in COVID-19 and coexistent hematological malignancy and new possible T3 target genes in these patients have been identified.

First computational design of Covid-19 coronavirus vaccine using lambda superstrings

In this work we have developed, by employing lambda superstrings, a map of candidate vaccines against SARS-CoV-2 with lengths between 9 and 200, based on estimations of the immunogenicity of the epitopes and the binding affinity of epitopes to MHC class I molecules using tools from the IEDB Analysis Resource, as well as the overall predictions obtained using the VaxiJen tool. We have synthesized one of the peptides, specifically the one of length 22, and we have carried out an immunogenicity assay and a cytokine assay, which has given positive results in both cases.

Identification of new susceptibility loci associated with rheumatoid arthritis

ObjectivesThe present study aimed to discover novel susceptibility loci associated with risk of rheumatoid arthritis (RA).MethodsWe performed a new genome-wide association study (GWAS) in Chinese subjects (1027 RA cases and 2879 controls) and further conducted an expanded meta-analysis with previous GWAS summary data and replication studies. The functional roles of the associated loci were interrogated using publicly available databases. Dual-luciferase reporter and cytokine assay were also used for exploring variant function.ResultsWe identified five new susceptibility loci (IL12RB2, BOLL-PLCL1, CCR2, TCF7 and IQGAP1; pmeta <5.00E−08) with same effect direction in each study cohort. The sensitivity analyses showed that the genetic association of at least three loci was reliable and robust. All these lead variants are expression quantitative trait loci and overlapped with epigenetic marks in immune cells. Furthermore, genes within the five loci are genetically associated with risk of other autoimmune diseases, and genes within four loci are known functional players in autoimmunity, which supports the validity of our findings. The reporter assay showed that the risk allele of rs8030390 in IQGAP1 have significantly increased reporter activity in HEK293T cells. In addition, the cytokine assay found that the risk allele of rs244672 in TCF7 was most significantly associated with increased plasma IL-17A levels in healthy controls. Finally, identified likely causal genes in these loci significantly interacted with RA drug targets.ConclusionThis study identified novel RA risk loci and highlighted that comprehensive genetic study can provide important information for RA pathogenesis and drug therapy.