Background: Epstein–Barr virus (EBV) is often kept silent and asymptomatic; however, its reactivation induces a chronic and/or recurrent infection that is associated with numerous diseases, including cancer and inflammation-related disorders. As no specific treatment is currently available, the immune factors-based micro-immunotherapy (MI) medicine 2LEBV^® could be considered a valuable therapeutic option to sustain the immune system in EBV reactivation. Methods: The present work aimed to investigate, for the first time, the effect of 2LEBV^® in several in vitro models of uninfected immune-related cells. Results: 2LEBV^® displayed phagocytosis-enhancing capabilities in granulocytes. In human peripheral blood mononuclear cells (PBMCs), it increased the intra- and extra-cellular expression of interleukin (IL)-2. Moreover, it modulated the secretion of other cytokines, increasing IL-4, IL-6, and tumor necrosis factor-α levels or lowering other cytokines levels such as IL-9. Finally, 2LEBV^® reduced the expression of human leukocyte antigen (HLA)-II in endothelial cells and macrophages. Conclusions: Although these data are still preliminary and the chosen models do not consider the underlying EBV-reactivation mechanisms, they still provide a better understanding of the mechanisms of action of 2LEBV^®, both at functional and molecular levels. Furthermore, they open perspectives regarding the potential targets of 2LEBV^® in its employment as a therapeutic intervention for EBV-associated diseases.

As one of the major cytokines implicated in the orchestration of immune responses, interleukin 6 (IL-6) can either act as a pro- or an anti-inflammatory factor, depending on the micro-environment. In micro-immunotherapy (MI) medicines, IL-6 is employed at low doses (LD) and ultra-low doses (ULD), expressed in centesimal Hahnemannian (CH), and used alone or in combination with other immune regulators to modulate patients’ immune responses. The present study focused on assessing the in vitro immune-modulatory effects of two IL-6-containing MI products: (i) the unitary IL-6 (4 CH) and (ii) the complex MI-medicine (MIM) 2LALERG^®, which includes IL-6 (17 CH) in association with other actives in its formulation. Our results showed that IL-6 (4 CH) activated granulocytes under basal conditions, and natural killer cells in the presence of an anti-CD3 signal, as assessed by their CD69 expression. In addition, IL-6 (4 CH) balanced the macrophages’ differentiation toward a M2a profile. On the other hand, the tested 2LALERG^® capsule inhibited the histamine degranulation of rats’ peritoneal mast cells and reduced the release of IL-6 itself in inflamed human macrophages. Altogether, these data provide novel pieces of evidence on the double-edged potential of the LD and ULD of IL-6 in immune responses modulation, when employed in MI.

Human papillomavirus (HPV) is the second most common infectious agent causing cancer. Persistent infection with high-risk (HR)-HPV can lead to cervical intra-epithelial neoplasia and cervical carcinomas (CC). While host immune response is necessary for viral clearance, chronic immune activation contributes to a low-grade inflammation that can ultimately lead to carcinogenesis. The micro-immunotherapy medicine (MIM) 2LPAPI^® could be a valuable tool to manage the clearance of the virus and reduce the risk of developing CC. In this in vitro study, we aimed to investigate its mode of action. We showed that actives from the MIM increased the IL-6, IFN-γ, and IP-10 secretion in human peripheral blood mononuclear cells (PBMCs) exposed to peptides derived from the HPV-16 capsid (HPV16_(L1)). This could reflect an increase in the immune activity toward HPV-16. At the same time, some active substances reduced the lympho-proliferation and the expression of T-cell activation markers. Finally, some of the MIM actives displayed antiproliferative effects in CC-derived HeLa cells under serum-starvation conditions. Altogether, this body of data highlighted for the first time the dual effect of MIM in the framework of HR-HPV infections as a potential (i) immune modulator of HPV16_(L1)-treated PBMCs and (ii) antiproliferative agent of HPV-positive CC cells.

KLEPTOSE^® CRYSMEB methylated cyclodextrin derivative displays less methylated group substitution than randomly methylated cyclodextrin. It has demonstrated an impact on atherosclerosis and neurological diseases, linked in part to cholesterol complexation and immune response, however, its impact on inflammatory cascade pathways is not clear. Thus, the impact of KLEPTOSE^® CRYSMEB on various pharmacological targets was assessed using human umbilical vein endothelial cells under physiological and inflammatory conditions, followed by screening against twelve human primary cell-based systems designed to model complex human tissue and disease biology of the vasculature, skin, lung, and inflammatory tissues using the BioMAP^® Diversity PLUS^® panel. Finally, its anti-inflammatory mechanism was investigated on peripheral blood mononuclear cells to evaluate anti-inflammatory or pro-resolving properties. The results showed that KLEPTOSE^® CRYSMEB can modulate the immune system in vitro and potentially manage vascular issues by stimulating the expression of molecules involved in the crosstalk between immune cells and other cell types. It showed anti-inflammatory effects that were driven by the inhibition of pro-inflammatory cytokine secretion and could have different impacts on different tissue types. Moreover, this cyclodextrin showed no clear impact on pro-resolving lipid mediators. Additionally, it appeared that the mechanism of action of KLEPTOSE^® CRYSMEB seems to not be shared by other well-known anti-inflammatory molecules. Finally, KLEPTOSE^® CRYSMEB may have an anti-inflammatory impact, which could be due to its effect on receptors such as TLR or direct complexation with LPS or PGE2, and conversely, this methylated cyclodextrin could stimulate a pro-inflammatory response involving lipid mediators and on proteins involved in communication with immune cells, probably via interaction with membrane cholesterol.

Tumor-associated macrophages (TAMs) play a pivotal role in cancer regulation by influencing tumor growth, metastasis, and the immune microenvironment. By providing low doses and ultra-low doses (ULD) of immune regulators to the organism, micro-immunotherapy (MI) medicines (MIM) could be seen as valuable adjuvant drugs in the context of a wide range of pathological conditions, including cancers. Thus, these MIM could target TAMs, affecting their phenotype and activities. In this study, the anti-tumor and the immune-stimulatory effects of four capsules out of the ten composing the Labo’life’s MIM 2LC1^® (2LC1-1, 2LC1-6, 2LC1-7, and 2LC1-8), as well as the specific nucleic acid (SNA^®) sequence SNA-MYC present at ULD in this medicine have been evaluated in vitro, in several cancer models, and in human monocyte-derived macrophages. Our results showed that the tested MI formulations increased the tumor cell death of spheroids from HCT-116 colon cancer cells, while reducing the spheroid volume. Moreover, the treatments impaired the clonogenic capabilities of two cancer cell lines from epithelial origin, the LNCaP prostate cancer and the MCF-7 breast cancer cells. Interestingly, ULD of the SNA-MYC shared similar anti-cancer capabilities in those models, and it led to a significant reduction in the expression of C-MYC when evaluated in a model of human M2 macrophages. In the same model, the MI formulations also increased the expression of CD86 and HLA-DR, two markers of M1 anti-tumor macrophages. In addition, the tested items modulated the secretion of a panel of chemokines related to macrophage activity and immune cell recruitment. Finally, our results showed that 2LC1-8 increased the phagocytosis capabilities of human monocyte-derived macrophages, thus possibly contributing to sustaining the immune functions of M1, which are crucial in the context of cancer. Even if more research is needed to uncover their exact mechanism of action, these results suggest that the tested capsules of 2LC1 as well as ULD of SNA-MYC display both anti-tumor and immune-enhancing effects.

KLEPTOSE® CRYSMEB is a methylated cyclodextrin derivative displaying less methylated group substitution than randomly methylated cyclodextrin. It has demonstrated an impact on atherosclerosis and neurological diseases. linked in part to cholesterol complexation and immune response, but the impact on inflammatory cascade pathways was not clear. Thus, the impact of KLEPTOSE® CRYSMEB on various pharmacological targets was assessed using human umbilical vein endothelial cells in physiological and inflammatory conditions, followed by screening against twelve human primary cell-based systems designed to model complex human tissue and disease biology of the vasculature, skin, lung, and inflammatory tissues using the BioMAP® Diversity PLUS® Panel. Finally, the anti-inflammatory mechanism was investigated on peripheral blood mononuclear cells to evaluate anti-inflammatory or pro-resolving properties. The results showed that KLEPTOSE® CRYSMEB can modulate the immune system in vitro and potentially manage vascular issues by stimulating the expression of molecules involved in the crosstalk between immune cells and other cell types. It showed anti-inflammatory effects driven by pro-inflammatory cytokine secretion inhibition and could have different impacts on different tissue types. Moreover, this cyclodextrin showed no clear impact on pro-resolving lipid mediators. Finally, it appeared that the mechanism of action of KLEPTOSE® CRYSMEB seems to not be shared by well-known anti-inflammatory molecules.