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Background: At the beginning of the pandemic, COVID-19 convalescent plasma (CCP) containing anti-SARS-CoV-2 antibodies was suggested as a source of therapy. In the last 3 years, many trials have demonstrated the limited usefulness of CCP therapy. This led us to the hypothesis that CCP could contain other elements, along with the desired neutralizing antibodies, which could potentially prevent it from having a therapeutic effect, among them cytokines, chemokines, growth factors, clotting factors, and autoantibodies. Methods: In total, 39 cytokines were analyzed in the plasma of 190 blood donors, and further research focused on the levels of 23 different cytokines in CCP (sCD40L, eotaxin, FGF-2, FLT-3L, ractalkine, GRO-α, IFNα2, IL-1β, IL-1RA, IL-5, IL-6, IL-8, IL-12, IL-13, IL-15, IL-17E, IP-10, MCP-1, MIP-1b, PDGF-AA, TGFα, TNFα, and TRAIL). Anti-SARS-CoV-2 antibodies and neutralizing antibodies were detected in CCP. Results: We found no significant differences between CCP taken within a maximum of 180 days from the onset of the first COVID-19 symptoms and the controls. We also made a comparison of the cytokine levels between the low neutralizing antibodies (<160) group and the high neutralizing antibodies (≥160) group and found there were no differences between the groups. Our research also showed no correlation either to levels of anti-SARS-CoV-2 IgG Ab or to the levels of neutralizing antibodies. There were also no significant changes in cytokine levels based on the period after the start of COVID-19 symptoms. Conclusions: No elements which could potentially be responsible for preventing CCP from having a therapeutic effect were found. [ABSTRACT FROM AUTHOR]

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Previously, we have shown platelet lysate (PL) can be used as a non-xenogeneic serum supplement for generation of monocyte-derived dendritic cells (DCs). Since DC-based activation protocols are extremely sensitive to microenvironmental changes such as replacement of culture medium, we wanted to examine the behavior of DCs cultured in the presence of PL under various type-1 activation conditions and assess their type 1 polarization capacity. We compared the quality of DCs cultured in 10% PL-supplemented RPMI medium (plDCs) with clinical-grade DCs obtained using commercially available serum-free medium (sfDCs), frequently used in established DC vaccine protocols. The DC maturation protocols consisted of either monophosphoryl lipid A/IFN-γ, poly I:C/TNF-α/IFN-α or poly I:C/R848. In general, plDCs were inferior to sfDCs in most aspects of their functional type 1 polarization characteristics. After maturation, the expression of co-stimulatory, HLA class II and lymph node-homing molecules was strongly up-regulated, with some noticeable differences. The expression of CD80 and CD86 was more extensive on plDCs, which was particularly evident in case of CCR7. However, after observing their functional capacity, plDCs had significantly lower allo-stimulatory capacity both in terms of CD4+ and CD8+ T cell stimulation. The high expression of CCR7 corresponded to higher CCL-19 directed DC migration of plDCs compared to sfDCs. Finally, their capacity to induce granzyme B and IFN-γ production in CD8+ T cells was significantly reduced in comparison to sfDCs. Based on these findings, the use of PL as an alternative serum supplement for generation of monocyte-derived DC anti-tumor vaccines is questionable. Abbreviations: Ag: antigen; CCL: chemokine ligand; CCR: chemokine receptor; DC: dendritic cells; DC-SIGN: dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin; FBS: fetal bovine serum; GMP: good manufacturing practice; IFN: interferon; IL: interleukin; MPLA: monophosphoryl lipid A; PGE: prostaglandin E; pI:C: polyinosinic:polycytidylic acid; pl: platelet lysate; sf: serum free; TLR: toll-like receptor; TNF: tumor necrosis factor. [ABSTRACT FROM AUTHOR]

In the past, interferon (IFN)-γ and vitamin D3 (vit D3) have both been associated with induction of tolerogenic characteristics in human dendritic cells (DCs). Although there are only a few reports on interdependency of their actions, the interplay between IFN-γ and vit D3 has been clearly demonstrated in certain aspects of immune reactivity. Since both agents have been associated with regulation of immune responses, we set out to examine their functional and mechanistic interactions in context of principal regulators of immunity, the DCs. Combined treatment with vit D3 and IFN-γ caused an extensive expression of immunoglobulin-like transcript (ILT)-3 and programmed death ligand (PDL)-1 on γ/D3DCs, significantly greater than that caused by vit D3 alone. Such γ/D3DCs retained all general DC characteristics. After CD40 ligand-induced activation, they produced increased amounts of IL-10 with almost absent production of IL-12p70. On the other hand, the co-stimulatory potential of γ/D3DCs was weak, with cells possessing the capacity to inhibit CD4+ T cell, CD8+ T cell, as well as memory T cell responses. Naive CD4+ T cells stimulated with γ/D3DCs produced increased amounts of IL-10 with concomitantly low IFN-γ production, upon T cell receptor activation. Additionally, γ/D3DCs completely inhibited granzyme B expression by CD8+ T cells. The percentage of FoxP3-positive cells in co-cultures with naive CD4+ T cells was significantly higher where γ/D3DCs were used as stimulators compared to DCs treated with vit D3 alone and it could be partially reversed by PDL-1 blockade. Interestingly, γ/D3DCs were inefficient at suppressing mDC-induced CD4+ T cell proliferation, but were twice as effective as D3DCs at suppressing mDC-induced CD8+ T cell proliferation. Blockade of indoleamine-2,3-dioxygenase did not reduce the tolerogenic phenotype induced by IFN-γ and vit D3 treatment. Examination of signaling pathways activation revealed a tendency toward increased ERK and Akt phosphorylation in γ/D3DCs. Inhibition of MEK/ERK and PI3K/mTOR pathways significantly reduced the expression of ILT-3 and PDL-1 on γ/D3DCs. In summary, we present the first evidence for existing synergy between IFN-γ and vit D3 in shaping a unique tolerogenic DC activation state. [ABSTRACT FROM AUTHOR]