Sirolimus- and cyclosporine-loaded nanostructured lipid carriers: Development, characterization, and in vitro evaluation in T-cell profiles of patients with a history of recurrent pregnancy loss.

Purpose: The authors developed nanostructured lipid carriers (NLCs) loaded with sirolimus (SRL) and cyclosporine (CsA) to improve their therapeutic efficacy in recurrent pregnancy loss (RPL) patients. Methods: Mono-delivery and co-delivery of SRL and CsA by NLCs (S-NLCs, C-NLCs, and S-C-NLCs) were developed. The MTT assay was used to study the optimum dose of formulations. PCR, Western blotting, and ELISA were also conducted. Results: Well-designed nanodrugs with a suitable size, zeta potential, desirable encapsulation efficiency drug loading, and cellular uptake confirmed optimum formulations. Based on cell viability, the amounts of SRL and CsA could be reduced greatly due to the co-delivery by NLCs. Following S-NLCs and C-NLCs interventions in T cells of patients with RPL and immune abnormality, a significant difference was observed in transcription factors and cytokine levels of Th1, Th17, and Tregs compared with healthy samples. Thus, a higher level of pro-inflammatory cytokines (IFN-γ, TNF-α, IL-17, and IL-21) and their regulators (T-bet and RORγt), as well as a lower level of an anti-inflammatory cytokine (IL-10) and its regulatory (Foxp3), were observed. However, no significant difference was found following the S-C-NLCs intervention. Conclusions: S-C-NLCs effectively balance the immune responses in peripheral T cells in RPL patients to induce maternal immune tolerance.

Expression level of immune checkpoint inhibitory factors in preeclampsia.

Preeclampsia (PE) is a severe complication in pregnancy, and its symptoms (proteinuria and hypertension) manifest after 20 weeks of gestation, affecting up to 8 % of pregnancies. The pregnant women's immune system uses different tolerance mechanisms to deal with a semi-allogeneic fetus. The T-cell subsets including CD8+, CD4+, and Treg play a critical role in maintaining pregnancies. The expression of immune checkpoint molecules in T-cells can ensure pregnancy at the feto-maternal interface by controlling immune responses. This research aims to evaluate the expression level of immune checkpoint factors, including PD-1, LAG-3, CTLA-4, and TIM-3 in normal pregnant women and PE patients. Decidual tissue was collected from 50 participants (25 PE and 25 control). For evaluating the genes expression, real-time PCR was employed. The western blot was used to assess the proteins level. The results of real-time PCR indicated significantly decreased expression level of these immune checkpoints in PE patients. In parallel to gene expression results, the protein level of PD-1, LAG-3, CTLA-4, and TIM-3 in the PE group was also reduced. We revealed that the profile of proteins and genes expression of immune checkpoints in the decidua of PE mothers are different from normal pregnancy and these results indicate aberrant expression of immune checkpoints such as PD-1, LAG-3, CTLA-4, and TIM-3 may cause maladaptation immune response which results in PE manifestation.

A comprehensive evaluation of the immune system response and type-I Interferon signaling pathway in hospitalized COVID-19 patients.

BACKGROUND: The COVID-19 pandemic has become the world's main life-threatening challenge in the third decade of the twenty-first century. Numerous studies have been conducted on SARS-CoV2 virus structure and pathogenesis to find reliable treatments and vaccines. The present study aimed to evaluate the immune-phenotype and IFN-I signaling pathways of COVID-19 patients with mild and severe conditions. MATERIAL AND METHODS: A total of 100 COVID-19 patients (50 with mild and 50 with severe conditions) were enrolled in this study. The frequency of CD4 + T, CD8 + T, Th17, Treg, and B lymphocytes beside NK cells was evaluated using flow cytometry. IFN-I downstream signaling molecules, including JAK-1, TYK-2, STAT-1, and STAT-2, and Interferon regulatory factors (IRF) 3 and 7 expressions at RNA and protein status were investigated using real-time PCR and western blotting techniques, respectively. Immune levels of cytokines (e.g., IL-1ß, IL-6, IL-17, TNF-α, IL-2R, IL-10, IFN-α, and IFN-ß) and the existence of anti-IFN-α autoantibodies were evaluated via enzyme-linked immunosorbent assay (ELISA). RESULTS: Immune-phenotyping results showed a significant decrease in the absolute count of NK cells, CD4 + T, CD8 + T, and B lymphocytes in COVID-19 patients. The frequency of Th17 and Treg cells showed a remarkable increase and decrease, respectively. All signaling molecules of the IFN-I downstream pathway and IRFs (i.e., JAK-1, TYK-2, STAT-1, STAT-2, IRF-3, and IRF-7) showed very reduced expression levels in COVID-19 patients with the severe condition compared to healthy individuals at both RNA and protein levels. Of 50 patients with severe conditions, 14 had anti-IFN-α autoantibodies in sera. Meanwhile, this result was 2 and 0 for patients with mild symptoms and healthy controls, respectively. CONCLUSION: Our results indicate a positive association of the existence of anti-IFN-α autoantibodies and immune cells dysregulation with the severity of illness in COVID-19 patients. However, comprehensive studies are necessary to find out more about this context. Video abstract.

Isolation of cfDNA from spent culture media and its association with implantation rate and maternal immunomodulation.

OBJECTIVES: This investigation aims to evaluate the association between the concentration of cell-free DNA (cfDNA) in the spent culture medium (SCM) with implantation rate and the maternal immune system in the invitro fertilization (IVF). In this study, 30 embryos were cultured and scored according to Gardner's criteria. SCM was gathered on day five from every embryo to analyze the quantity of cfDNA. The real-time PCR technique evaluated the expression level of transcription factors, including Foxp3, RORγt, GATA3, and T-bet. The percentage of Th1, Th2, Th17, Treg, NK cells, and NK cells cytotoxicity was evaluated by flow cytometry. RESULTS: The concentration of cfDNA in the ß-HCG (-), ß-HCG ( +), and ongoing pregnancy groups were 20.70 ± 9.224 ng/µL, 27.97 ± 7.990 ng/µL, and 28.91 ± 8.566 ng/µL, respectively. The ratio of Th1/Th2 and Th17/Treg reduced significantly in pregnant women, as well as the level of NK cells and NK cytotoxicity cells fell dramatically in the ongoing pregnancy group. The expression level of RORγt and T-bet declined while the expression level of Foxp3 and GATA3 increased considerably in pregnant mothers. Our investigation revealed that the concentration level of cfDNA in SCM could not be associated with implantation rate, prediction of ongoing pregnancy, and maternal immune system.

Application of cell free DNA in ART.

Various biopsy and sampling methods are used for preimplantation genetic diagnosis (PGD) of embryo. This method benefits blastomer/trophectoderm biopsy to improve the clinical outcome of in vitro fertilization (IVF). However, all of these procedures are invasive and have adverse effects on embryo development. Additionally, these procedures require expensive equipment and well-experienced technicians. Regarding these limitations, designing non-invasive methods is necessary. One of the recently proposed non-invasive and applicable methods is cell free DNA (cfDNA) molecule evaluation that have opened up exciting opportunities in the molecular diagnosis of embryo and fetus chromosomal aneuploidy. cfDNA is present in body fluids; especially blood, follicular fluid, amniotic fluid, spent embryo culture medium (SCM) and blastocoel fluid. Overall, this review highlights the cfDNA biomarker might constitute a supplemental tool for improving IVF and pregnancy outcomes, female infertility management. However, the successful application of cfDNA demands an understanding of its biological properties, kinetics, time of collection, high sensitivity and specificity cfDNA detection methods, and their limitation and challenges in the clinical settings. In this review we also describe ethical aspects of cfDNA testing.