Search Results (8)

Background: Understanding the duration and quality of immune memory following SARS-CoV-2 infection and vaccination is critical for informing public health strategies and vaccine development. While waning antibody levels have raised concerns about long-term protection, the persistence of memory B cells (MBCs) and T cells plays a vital role in sustaining immunity. Materials and Methods: We conducted a longitudinal prospective study over 12 months, enrolling 285 participants in total, either after natural infection or vaccination with BNT162b2 or mRNA-1273. Peripheral blood samples were collected at four defined time points (baseline, 1–2 months, 6–7 months, and 12–13 months after vaccination or disease onset). Immune responses were assessed through serological assays quantifying anti-RBD IgG and neutralizing antibodies, B-ELISPOT, and multiparameter flow cytometry for S1-specific memory B cells. Results: Both mRNA vaccines induced robust B cell and antibody responses, exceeding those observed after natural infection. Memory B cell frequencies peaked at 6 months and declined by 12 months, but remained above the baseline. The mRNA-1273 vaccine elicited stronger and more durable humoral and memory B-cell-mediated immunity compared to BNT162b2, likely influenced by its higher mRNA dose and longer prime-boost interval. Class-switched memory B cells and S1-specific B cells were significantly expanded in vaccine recipients. Natural infection induced more heterogeneous immune memory. Conclusions: Both mRNA vaccination and natural SARS-CoV-2 infection induce a comparable expansion of memory B cell subsets, reflecting a consistent pattern of humoral immune responses across all studied groups. These findings highlight the importance of vaccination in generating sustained immunological memory and suggest that the vaccine platform and dosage influence the magnitude and durability of immune responses against SARS-CoV-2. Full article

Background/Objectives: TDP-43 mutation-driven Amyotrophic Lateral Sclerosis (ALS) motor neuron disease is one of the most prominent forms (approximately 97%) in cases of sporadic ALS. Dysfunctional autophagy and lysosomal function are the prime mechanisms behind ALS. Mitoxantrone (Mito), a synthetic doxorubicin analog, is an inhibitor of DNA and RNA synthesis/repair via intercalating with nitrogenous bases and inhibiting topoisomerase II. The therapeutic potential of miRNAs associated with disease conditions has also been reported. This study explores the therapeutic potential of Mito along with miRNAs against mutated TDP-43 protein-induced proteinopathy in human-induced pluripotent stem cell (hiPSC)-derived human neural progenitor cells (hNPCs). Methods: HiPSCs mutated for TDP-43 were differentiated into hNPCs and used to explore the therapeutic potential of Mito at a concentration of 1 μM for 24 h (the identified non-cytotoxic dose). The therapeutic effects of Mito on miRNA expression and various cellular parameters such as mitochondrial dynamics, autophagy, and stress granules were assessed using the high-throughput Open Array technique, immunocytochemistry, flow cytometry, immunoblotting, and mitochondrial bioenergetic assay. Results: Mutated TDP-43 protein accumulation causes stress granule formation (G3BP1), mitochondrial bioenergetic dysfunction, SOD1 accumulation, hyperactivated autophagy, and ER stress in hNPCs. The mutated hNPCs also show dysregulation in six miRNAs (miR-543, miR-34a, miR-200c, miR-22, miR-29b, and miR-29c) in mutated hNPCs. A significant restoration of TDP-43 mutation-induced alterations could be witnessed upon the exposure of mutated hNPCs to Mito. Conclusions: Our study indicates that miR-543, miR-29b, miR-22, miR-200c, and miR-34a have antisense therapeutic potential alone and in combination with Mitoxantrone. Full article

Small interfering RNA (siRNA) therapy in acute myeloid leukemia (AML) is a promising strategy as the siRNA molecule can specifically target proteins involved in abnormal cell proliferation. The development of a clinically applicable method for delivering siRNA molecules is imperative due to the challenges involved in effectively delivering the siRNA into cells. We investigated the delivery of siRNA to AML MOLM-13 cells with the use of two lipid-substituted polyethyleneimines (PEIs), a commercially available reagent (Prime-Fect) and a recently reported reagent with improved lipid substitution (PEI1.2k-PHPA-Lin9). The siRNAs utilized in this study were targeting the oncogenes FLT3 and KMT2A::MLLT3. Both lipopolymers gave similar-size siRNA complexes (210–220 nm) with positive ζ-potentials (+17 to +25 mV). While the binding efficiency of both lipopolymers to siRNA were similar, PEI1.2k-PHPA-Lin9 complexes were more resistant to heparin-induced dissociation. The quantitative analysis of gene silencing performed by qPCR as well as immunostaining/flow cytometry indicated significant reduction in both FLT3 expression and FLT3 protein after specific siRNA delivery. The desired inhibition of cell growth was attained with both FLT3 and KMT2A::MLLT3 siRNAs, and the combination provided more potent effects in both cell growth and colony formation assays. Induction of apoptosis was confirmed after specific siRNA treatments using the Annexin V assay. Using Luc(+) MOLM-13 cells, the growth of the xenografted cells was shown to be retarded with Prime-Fect-delivered FLT3 siRNA, unlike the siRNA delivered with PEI1.2k-PHPA-Lin9. These results demonstrate the potential of designed lipopolymers in implementing RNAi (via delivery of siRNA) for inhibition of leukemia growth and provide evidence for the feasibility of targeting different oncogenes using siRNA-mediated therapy. Full article

CD30-positive germinal center (GC)-derived B cell lymphomas are frequently linked to Epstein–Barr Virus (EBV) infection. However, a suitable animal model for the investigation of the interplay between γ-herpesvirus and host cells in B cell pathogenesis is currently lacking. Here, we present a novel in vivo model enabling the analysis of genetically modified viruses in combination with genetically modified GC B cells. As a murine γ-herpesvirus, we used MHV-68 closely mirroring the biology of EBV. Our key finding was that Cre-mediated recombination can be successfully induced by an MHV-68 infection in GC B cells from Cγ1-Cre mice allowing for deletion or activation of loxP-flanked cellular genes. The implementation of PrimeFlow RNA assay for MHV-68 demonstrated the enrichment of MHV-68 in GC and isotype-switched B cells. As illustrations of virus and cellular modifications, we inserted the EBV gene LMP2A into the MHV-68 genome and induced constitutively active CD30-signaling in GC B cells through MHV-68 infections, respectively. While the LMP2A-expressing MHV-68 behaved similarly to wildtype MHV-68, virally induced constitutively active CD30-signaling in GC B cells led to the expansion of a pre-plasmablastic population. The findings underscore the potential of our novel tools to address crucial questions about the interaction between herpesviral infections and deregulated cellular gene-expression in future studies. Full article

The anti-inflammatory cytokine interleukin-37 (IL-37) plays a key role in inhibiting innate and adaptive immunity. Past results have shown that IL-37 is elevated in human Treg cells compared to other T cell subsets and contributes to enhancing the Treg transcription factor, forkhead box protein P3 (FOXP3). However, it is unknown if ectopic expression of IL-37 in non-Treg CD4+ T cells can lead to the development of Treg phenotype and function. In the present study, we used a PrimeFlow^® RNA assay and confirmed elevated IL37 expression in human Treg cells. We then stably transfected the non-Treg CD4+ T cell leukemia cell line, E6 Jurkat cells, with IL37 and found significant induction of the Treg phenotype. These IL-37-expressing Jurkat cells had elevated CTLA-4 and FOXP3 and produced IL-10. In conjunction with the Treg phenotype, IL-37-expressing Jurkat cells suppressed T cell activation/proliferation, comparable to human primary Treg cells. The creation of this stable human Treg-like cell line has the potential to provide further assistance for in vitro studies of human Treg cells, as it is more convenient than the use of primary human Treg cells. Furthermore, it provides insights into Treg cell biology and function. Full article

Oropouche orthobunyavirus (OROV) is an emerging arbovirus with a high potential of dissemination in America. Little is known about the role of peripheral blood mononuclear cells (PBMC) response during OROV infection in humans. Thus, to evaluate human leukocytes susceptibility, permissiveness and immune response during OROV infection, we applied RNA hybridization, qRT-PCR and cell-based assays to quantify viral antigens, genome, antigenome and gene expression in different cells. First, we observed OROV replication in human leukocytes lineages as THP-1 monocytes, Jeko-1 B cells and Jurkat T cells. Interestingly, cell viability and viral particle detection are maintained in these cells, even after successive passages. PBMCs from healthy donors were susceptible but the infection was not productive, since neither antigenome nor infectious particle was found in the supernatant of infected PBMCs. In fact, only viral antigens and small quantities of OROV genome were detected at 24 hpi in lymphocytes, monocytes and CD11c⁺ cells. Finally, activation of the Interferon (IFN) response was essential to restrict OROV replication in human PBMCs. Increased expression of type I/III IFNs, ISGs and inflammatory cytokines was detected in the first 24 hpi and viral replication was re-established after blocking IFNAR or treating cells with glucocorticoid. Thus, in short, our results show OROV is able to infect and remain in low titers in human T cells, monocytes, DCs and B cells as a consequence of an effective IFN response after infection, indicating the possibility of leukocytes serving as a trojan horse in specific microenvironments during immunosuppression. Full article

Dendritic cells (DCs) are professional antigen-presenting cells which instruct both the innate and adaptive immune systems. Once mature, they have the capacity to activate and prime naïve T cells for recognition and eradication of pathogens and tumor cells. These characteristics make them excellent candidates for vaccination strategies. Most DC vaccines have been generated from ex vivo culture of monocytes (mo). The use of mo-DCs as vaccines to induce adaptive immunity against cancer has resulted in clinical responses but, overall, treatment success is limited. The application of primary DCs or DCs generated from CD34⁺ stem cells have been suggested to improve clinical efficacy. Cord blood (CB) is a particularly rich source of CD34⁺ stem cells for the generation of DCs, but the dynamics and plasticity of the specific DC lineage development are poorly understood. Using flow sorting of DC progenitors from CB cultures and subsequent RNA sequencing, we found that CB-derived DCs (CB-DCs) exclusively originate from CD115⁺-expressing progenitors. Gene set enrichment analysis displayed an enriched conventional DC profile within the CD115-derived DCs compared with CB mo-DCs. Functional assays demonstrated that these DCs matured and migrated upon good manufacturing practice (GMP)-grade stimulation and possessed a high capacity to activate tumor-antigen-specific T cells. In this study, we developed a culture protocol to generate conventional DCs from CB-derived stem cells in sufficient numbers for vaccination strategies. The discovery of a committed DC precursor in CB-derived stem cell cultures further enables utilization of conventional DC-based vaccines to provide powerful antitumor activity and long-term memory immunity. Full article

In the ovary, the luteinizing hormone (LH) surge suppresses the proliferation and induces the luteinization of preovulatory granulosa cells (GCs), which is crucial for the survival of terminally-differentiated GCs. Krüppel-like factor 4 (Klf4) has been shown to play a role in regulating the cell cycle and apoptosis in various cell types. The rapid induction of Klf4 expressions by LH was observed in preovulatory GCs. To evaluate whether Klf4 affects GC proliferation and survival, primary rat GCs were isolated from pregnant mare serum gonadotropin-primed Sprague–Dawley rat ovaries and transfected with a Klf4 expression vector or Klf4-specific siRNA, followed by determination of the transcript levels of apoptosis-related and cell cycle-related genes. Cell proliferation, viability, and apoptosis were analyzed by BrdU incorporation, a Cell Counting Kit-8 assay, a bioluminescence caspase 3/7 assay, and flow cytometry. LH treatment increased Klf4 mRNA expression in preovulatory GCs. Transcripts of B-cell lymphoma 2 (Bcl-2) and cell cycle promoters (Cyclin D1 and Cyclin D2) decreased, whereas those of the cell cycle inhibitor, p21, increased. Altering the expression of Klf4 by overexpression or knockdown consistently affected the expression of Bcl-2 and Cyclin D1. In agreement with this, Klf4 overexpression reduced cell viability, increased the fraction of apoptotic cells, and arrested cell cycle progression in G1 phase. We conclude that Klf4 increases the susceptibility of preovulatory GCs to apoptosis by down-regulating Bcl-2, and promotes LH-induced cell cycle exit. It appears to be a key regulator induced by the LH surge that determines the fate of GCs in preovulatory follicles during the luteal transition. Full article