How an Immune-Factor-Based Formulation of Micro-Immunotherapy Could Interfere with the Physiological Processes Involved in the Atopic March.

Allergic diseases consist of improper inflammatory reactions to antigens and are currently an important healthcare concern, especially considering their increasing worldwide development in recent decades. The "atopic march" defines the paradigm of allergic diseases occurring in chronological order and displaying specific spatial manifestations, as they usually start as atopic dermatitis (AD) and food allergies during infancy and progressively evolve into allergic asthma (AA) and allergic rhinitis (AR) or rhino-conjunctivitis in childhood. Many immune cell subtypes and inflammatory factors are involved in these hypersensitivity reactions. In particular, the T helpers 2 (Th2) subset, through its cytokine signatures made of interleukins (ILs), such as IL-4, IL-5, IL-10, and IL-13, as well as mast cells and their related histamine pathways, contribute greatly to the perpetuation and evolution of the atopic march. By providing low doses (LD) and ultra-low doses (ULD) of ILs and immune factors to the body, micro-immunotherapy (MI) constitutes an interesting therapeutic strategy for the management of the atopic march and its symptoms. One of the aims of this review is to shed light on the current concept of the atopic march and the underlying immune reactions occurring during the IgE-mediated responses. Moreover, the different classes of traditional and innovative treatments employed in allergic diseases will also be discussed, with a special emphasis on the potential benefits of the MI medicine 2LALERG® formulation in this context.

A Sequential Micro-Immunotherapy Medicine Increases Collagen Deposition in Human Gingival Fibroblasts and in an Engineered 3D Gingival Model under Inflammatory Conditions.

Periodontal therapies use immune mediators, but their side effects can increase with dosage. Micro-immunotherapy (MI) is a promising alternative that employs immune regulators at low and ultralow doses to minimize adverse effects. In this study, the effects of 5 capsules and the entire 10-capsule sequence of the sequential MI medicine (MIM-seq) were tested in two in vitro models of periodontitis. Firstly, human gingival fibroblasts (hGFs) exposed to interleukin (IL)-1ß to induce inflammation were treated with five different capsules of MIM-seq for 3 days or with MIM-seq for 24 days. Subsequently, MIM-seq was analyzed in a 3D model of human tissue equivalent of gingiva (GTE) under the same inflammatory stimulus. Simultaneously, a non-IL-1ß-treated control and a vehicle were included. The effects of the treatments on cytotoxicity, collagen deposition, and the secreted levels of IL-1α, IL-6, prostaglandin E2 (PGE2), matrix metalloproteinase-1 (MMP-1), and tissue inhibitor of metalloproteinases-1 (TIMP-1) were evaluated. None of the tested items were cytotoxic. The complete sequence of MIM-seq decreased PGE2 release and restored collagen deposition levels induced by IL-1ß treatment in hGFs exposed to IL-1ß. MIM-seq treatment restored collagen production levels in both models. These promising preclinical findings suggest that MIM-seq should be further investigated for periodontitis treatment.

A Micro-Immunotherapy Sequential Medicine MIM-seq Displays Immunomodulatory Effects on Human Macrophages and Anti-Tumor Properties towards In Vitro 2D and 3D Models of Colon Carcinoma and in an In Vivo Subcutaneous Xenograft Colon Carcinoma Model.

In this study, the immunomodulatory effects of a sequential micro-immunotherapy medicine, referred as MIM-seq, were appraised in human primary M1 and M2 macrophages, in which the secretion of pro-inflammatory cytokines, such as interleukin (IL)-1ß, IL-6, IL-12, IL-23, and tumor necrosis factor (TNF)-alpha, was inhibited. In addition, the potential anti-proliferative effects of MIM-seq on tumor cells was assessed in three models of colorectal cancer (CRC): an in vitro two-dimensions (2D) model of HCT-116 cells, an in vitro tri-dimensional (3D) model of spheroids, and an in vivo model of subcutaneous xenografted mice. In these models, MIM-seq displayed anti-proliferative effects when compared with the vehicle. In vivo, the tumor growth was slightly reduced in MIM-seq-treated animals. Moreover, MIM-seq could slightly reduce the growth of our spheroid models, especially under serum-deprivation. When MIM-seq was combined with two well-known anti-cancerogenic agents, either resveratrol or etoposide, MIM-seq could even further reduce the spheroid's volume, pointing up the need to further assess whether MIM-seq could be beneficial for CRC patients as an adjuvant therapy. Altogether, these data suggest that MIM-seq could have anti-tumor properties against CRC and an immunomodulatory effect towards the mediators of inflammation, whose systemic dysregulation is considered to be a poor prognosis for patients.

The Unitary Micro-Immunotherapy Medicine Interferon-γ (4 CH) Displays Similar Immunostimulatory and Immunomodulatory Effects than Those of Biologically Active Human Interferon-γ on Various Cell Types.

As a cytokine, gamma-interferon (IFN-γ) is considered a key player in the fine-tuned orchestration of immune responses. The extreme cellular sensitivity to cytokines is attested by the fact that very few of these bioactive molecules per cell are enough to trigger cellular functions. These findings can, at least partially, explain how/why homeopathically-prepared cytokines, and especially micro-immunotherapy (MI) medicines, are able to drive cellular responses. We focused our fundamental research on a unitary MI preparation of IFN-γ, specifically employed at 4 CH, manufactured and impregnated onto sucrose-lactose pillules as all other MI medicines. We assessed the IFN-γ concentration in the medium after dilution of the IFN-γ (4 CH)-bearing pillules and we evaluated in vitro drug responses in a wide range of immune cells, and in endothelial cells. Our results showed that IFN-γ (4 CH) stimulated the proliferation, the activation and the phagocytic capabilities of primary immune cells, as well as modulated their cytokine-secretion and immunity-related markers' expression in a trend that is quite comparable with the well-recognized biological effects induced by IFN-γ. Altogether, these data provide novel and additional evidences on MI medicines, and specifically when active substances are prepared at 4 CH, thus suggesting the need for more investigations.

Ultra-Low Dose Cytokines in Rheumatoid Arthritis, Three Birds with One Stone as the Rationale of the 2LARTH® Micro-Immunotherapy Treatment.

Tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) are two cytokines involved in the perpetuation of the chronic inflammation state characterizing rheumatoid arthritis (RA). Significant advances in the treatment of this pathology have been made over the past ten years, partially through the development of anti-TNF and anti-IL-1 therapies. However, major side effects still persist and new alternative therapies should be considered. The formulation of the micro-immunotherapy medicine (MIM) 2LARTH® uses ultra-low doses (ULD) of TNF-α, IL-1ß, and IL-2, in association with other immune factors, to gently restore the body's homeostasis. The first part of this review aims at delineating the pivotal roles played by IL-1ß and TNF-α in RA physiopathology, leading to the development of anti-TNF and anti-IL-1 therapeutic agents. In a second part, an emphasis will be made on explaining the rationale of using multiple therapeutic targets, including both IL-1ß and TNF-α in 2LARTH® medicine. Particular attention will be paid to the ULD of those two main pro-inflammatory factors in order to counteract their overexpression through the lens of their molecular implication in RA pathogenesis.

The Micro-Immunotherapy Medicine 2LEID Exhibits an Immunostimulant Effect by Boosting Both Innate and Adaptive Immune Responses.

This study aimed at evaluating the effects of the micro-immunotherapy medicine (MIM) 2LEID, both in vitro and in vivo, on several components of the innate and adaptive immune system. MIM increased the phagocytic activity of macrophages, and it augmented the expression of the activation markers CD69 and HLA-DR in NK cells and monocytes/macrophages, respectively. The effect of MIM was evaluated in a model of respiratory infection induced by influenza A virus administration to immunocompetent mice in which it was able to improve neutrophil recruitment within the lungs (p = 0.1051) and slightly increased the circulating levels of IgM (p = 0.1655). Furthermore, MIM stimulated the proliferation of CD3-primed T lymphocytes and decreased the secretion of the immunosuppressive cytokine IL-10 in CD14+-derived macrophages. Human umbilical vein endothelial cells were finally used to explore the effect of MIM on endothelial cells, in which it slightly increased the expression of immune-related markers such as HLA-I, CD137L, GITRL, PD-L1 and ICAM-1. In conclusion, the present study suggests that MIM might be a promising nonspecific (without antigen specificity) immunostimulant drug in preventing and early treating respiratory infections, but not only exclusively, as it would gently support several facets of the immune system and host defenses.

Exploring the Potential of Micro-Immunotherapy in the Treatment of Periodontitis.

Periodontitis, characterized by the progressive destruction of dental support tissues due to altered immune responses, poses a significant concern for public health. This condition involves intricate interactions between the immune response and oral microbiome, where innate and adaptive immune responses, with their diverse cell populations and inflammatory mediators, play crucial roles in this immunopathology. Indeed, cytokines, chemokines, growth factors, and immune cells perform key functions in tissue remodeling. Focusing on periodontal therapies, our attention turns to micro-immunotherapy (MI), employing low doses (LDs) and ultra-low doses (ULDs) of immunological signaling molecules like cytokines, growth factors, and hormones. Existing studies across various fields lay the groundwork for the application of MI in periodontitis, highlighting its anti-inflammatory and regenerative potential in soft tissue models based on in vitro research. In summary, this review underscores the versatility and potential of MI in managing periodontal health, urging further investigations to solidify its clinical integration. MI supports an innovative approach by modulating immune responses at low doses to address periodontitis.

Understanding the Mode of Action of a Micro-Immunotherapy Formulation: Pre-Clinical Evidence from the Study of 2LEBV® Active Ingredients.

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.

The Micro-Immunotherapy Medicine 2LPAPI® Displays Immune-Modulatory Effects in a Model of Human Papillomavirus Type-16 L1-Protein Capsid-Treated Human Peripheral Blood Mononuclear Cells and Antiproliferative Effects in a Model of Cervical Cancer Cells.

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.

Actives from the Micro-Immunotherapy Medicine 2LMIREG® Reduce the Expression of Cytokines and Immune-Related Markers Including Interleukin-2 and HLA-II While Modulating Oxidative Stress and Mitochondrial Function.

Introduction: Micro-immunotherapy (MI) is a therapeutic option employing low doses (LD) and ultra-low doses (ULD) of cytokines and immune factors to help the organism at modulating the immune responses. In an overpowering inflammatory context, this strategy may support the restoration of the body's homeostasis, as the active ingredients of MI medicines' (MIM) could boost or slow down the physiological functions of the immune cells. The aim of the study is to evaluate for the first time the in vitro anti-inflammatory properties of some actives employed by the MIM of interest in several human immune cell models. Methods: In the first part of the study, the effects of the actives from the MIM of interest were assessed from a molecular standpoint: the expression of HLA-II, interleukin (IL)-2, and the secretion of several other cytokines were evaluated. In addition, as mitochondrial metabolism is also involved in the inflammatory processes, the second part of the study aimed at assessing the effects of these actives on the mitochondrial reactive oxygen species (ROS) production and on the mitochondrial membrane potential. Results: We showed that the tested actives decreased the expression of HLA-DR and HLA-DP in IFN-γ-stimulated endothelial cells and in LPS-treated-M1-macrophages. The tested MIM slightly reduced the intracellular expression of IL-2 in CD4+ and CD8+ T-cells isolated from PMA/Iono-stimulated human PBMCs. Additionally, while the secretion of IL-2, IL-10, and IFN-γ was diminished, the treatment increased IL-6, IL-9, and IL-17A, which may correspond to a "Th17-like" secretory pattern. Interestingly, in PMA/Iono-treated PBMCs, we reported that the treatment reduced the ROS production in B-cells. Finally, in PMA/Iono-treated human macrophages, we showed that the treatment slightly protected the cells from early cell death/apoptosis. Discussion: Overall, these results provide data about the molecular and functional anti-inflammatory effects of several actives contained in the tested MIM in immune-related cells, and their impact on two mitochondria-related processes.

In Vitro Study of Interleukin-6 when Used at Low Dose and Ultra-Low Dose in Micro-Immunotherapy.

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.

Potential Role of the Micro-Immunotherapy Medicine 2LALERG in the Treatment of Pollen-Induced Allergic Inflammation.

In this study, we evaluated the efficacy of a micro-immunotherapy medicine (MIM), 2LALERG, in a preclinical model of allergic respiratory disease sensitized with birch pollen extract (BPE). BALB/c mice were immunized with BPE, or saline solution, and were then challenged. Micro-immunotherapy medicine pillules were diluted in water, and 3 doses (0.75; 1.5; 3 mg/mouse) were tested and compared to vehicle control (3 mg/mouse). Treatments and vehicle were orally administered by gavage for 10 days. Micro-immunotherapy medicine (0.75 mg/mouse) reduced the number of total cells as well as the levels of interleukin (IL)-13 in bronchoalveolar lavage fluid (BALF) compared to vehicle control. Eosinophils in BALF tended to be lower compared to vehicle group, and the difference is close to significance. Histological analysis in the lungs confirms a moderate effect of MIM (0.75 mg/mice) on inflammatory infiltration and mucus production. Serum levels of IL-5 in MIM (0.75 mg/mouse)-treated mice were lower compared to vehicle; IL-4 levels tended to be lower too. Total immunoglobulin E (IgE) decreased in serum of MIM (1.5 and 0.75 mg/mouse) groups compared to vehicle control. Micro-immunotherapy medicine exerted the highest effect at the lowest dose tested. Micro-immunotherapy medicine resolved the local and systemic inflammation, even if partially, in a model of pollen-induced, IgE-mediated inflammation.

Pro-Inflammatory Cytokines at Ultra-Low Dose Exert Anti-Inflammatory Effect In Vitro: A Possible Mode of Action Involving Sub-Micron Particles?

Tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) are pro-inflammatory cytokines involved in acute and chronic inflammatory diseases. Indeed, immunotherapy blocking these 2 cytokines has been developed. Micro-immunotherapy (MI) also uses ultra-low doses (ULD) of pro-inflammatory cytokines, impregnated on lactose-sucrose pillules, to counteract their overexpression. The study has been conducted with 2 objectives: examine the anti-inflammatory effect in vitro and the capacity of 2 unitary medicines, TNF-α (27 CH) and IL-1ß (27 CH), to reduce the secretion of TNF-α in human primary monocytes and THP-1 cells differentiated with phorbol-12-myristate-13-acetate, after lipopolysaccharide (LPS) exposure; then, investigate the presence of particles possibly containing starting materials using tunable resistive pulse sensing technique. The results show that the unitary medicines, tested at 3 pillules concentrations (5.5, 11 and 22 mM), have reduced the secretion of TNF-α in both models by about 10-20% vs. vehicle control, depending on concentration. In this exploratory study, particles (150-1000 nm) have been detected in MI ULD-impregnated pillules and a hypothesis for MI medicines mode of action has been proposed. Conscious that more evaluations are necessary, authors are cautious in the conclusions because the findings described in the study are still limited, and future investigations may lead to different hypothesis.

Effect of the micro-immunotherapy medicine 2LPARK® on rat primary dopaminergic neurons after 6-OHDA injury: oxidative stress and survival evaluation in an in vitro model of Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disease characterized by motor impairments and resulting from progressive degenerative loss of midbrain dopaminergic (DAergic) neurons in the substantia nigra. Although the main cause of the loss of DAergic neurons is still unknown, various etiopathogenic mechanisms are distinguished, including release and accumulation of endogenous excitotoxic mediators along with the production of oxidative free radicals. Several neurotrophic and growth factors are known to increase DAergic neuronal survival and enhance antioxidant mechanisms. In this context, the micro-immunotherapy (MI) approach consists to regulate the immune system in order to protect DAergic neurons and control oxidative stress. PURPOSE: The aim of the present study was to investigate the effect of the MI medicine (MIM), 2LPARK® (Labo'Life), on oxidative stress and on the number of neurons positive for tyrosine hydroxylase (TH), in an in vitro model of PD. METHODS: Rat primary mesencephalic DAergic neurons cultures were pre-treated for 1 hr with the MIM (10 µM and 10 mM), placebo (10 µM and 10 mM) or brain-derived neurotrophic factor (BDNF; 3.3 µM) and then intoxicated with 6-hydroxydopamine (6-OHDA; 20 µM) for 48 hrs. After incubation, cells were incubated 30 mins at 37°C with CellROX green reagent and number of labeled cells were quantified. Then, cells were fixed and incubated with anti-TH antibody and the number of TH+ neurons was evaluated. RESULTS: We showed that, contrary to placebo, MIM was able to reduce oxidative stress and protect DAergic neurons from 6-OHDA-induced cell death. CONCLUSION: Our results demonstrate the in vitro efficacy of MIM on two essential mechanisms of PD and propose the MI approach as a new ally in the regulation of neuroinflammation and in the treatment of this degenerative disease.