Introduction. Previous studies have shown that probiotics have positive effects on both motor and non-motor symptoms in Parkinson’s disease (PD). Additionally, in preclinical settings, probiotics have demonstrated the ability to counteract neuronal loss and alpha-synuclein aggregation, important pathological hallmarks of PD. Notably, preliminary in vitro studies have revealed the immunomodulatory properties of probiotics. This study aims to evaluate the impact of probiotics on symptoms and peripheral cytokines levels in PD patients compared to placebo. Methods. Patients were enrolled and blindly randomized to receive either active probiotics (comprising Bifidobacterium animalis subsp. lactis BS01 LMG P-21384, Bifidobacterium longum BL03 DSM 16603, Bifidobacterium adolescentis BA02 DSM 18351, Fructo-oligosaccharides and Maltodextrin-Group A) or placebo (Maltodextrin-Group B). Clinical evaluations and plasma levels cytokines (TNF-α, IFN-γ, IL-6, and TGF-β) were also assessed at enrollment and after 12 weeks. Anti-parkinsonian therapy remained stable throughout the study. Results. Forty PD patients were recruited. After 12 weeks, Group A showed significant improvement in motor symptoms (UPDRS III: 13.89 ± 4.08 vs. 12.74 ± 4.57, p = 0.028) and non-motor symptoms (NMSS: 34.32 ± 21.41 vs. 30.11 ± 19.89, p = 0.041), with notable improvement in the gastrointestinal sub-item (3.79 ± 4.14 vs. 1.89 ± 2.54, p = 0.021). A reduction of IFN-γ levels was observed in both groups, but group A also showed a significant decrease in IL-6 and a slight increase in the anti-inflammatory cytokine TGF-β. Conclusions. Our data suggest that probiotics may modulate peripheral cytokines levels and improve clinical symptoms in PD patients. Probiotics may, therefore, represent a valuable adjunctive therapy to conventional anti-parkinsonian drugs.

Background: Probiotics are live microorganisms known for their health-promoting effects, particularly in modulating immune responses and reducing inflammation within the gastrointestinal tract. Emerging evidence suggests probiotics may also influence respiratory health, prompting investigation into their potential therapeutic application in lung inflammation. Methods: This study examined the anti-inflammatory effects of Ligilactobacillus salivarius (LS01 DSM 22775) and Bifidobacterium breve (B632 DSM 24706) on inflamed pulmonary epithelial cells. Lung carcinoma epithelial cells (A549) and normal bronchial epithelial cells (16HBE) were stimulated with IL-1β and treated with viable and heat-treated probiotics. Results: CCL-2 levels were significantly reduced by up to 40%, in A549 by viable form (10⁵–10⁷ AFU/g), instead of in 16HBE by heat-treated form (10⁷–10⁹ TFU/g). In A549 cells, TNF-α decreased by 20–80% with all formulations; instead, in 16HBE cells, IL-8 was reduced by viable strains (10⁷ AFU/g) by approximately 50%, while heat-treated strains (10⁹ TFU/g) decreased both IL-6 and IL-8 by 50%. All effective treatments completely inhibited IL-4 and eotaxin and suppressed NF-κB activation in both cell lines, with up to 80% reduction in phospho-p65 levels. In A549 cells, heat-treated strains fully blocked PGE2 production; instead, all four probiotics significantly inhibited COX-2 expression by approximately 50%. Conclusions: These findings demonstrate that both viable and heat-treated probiotics can modulate inflammatory responses in pulmonary epithelial cells, suggesting their potential application in inflammatory respiratory diseases. Heat-treated formulations may be particularly suited for local administration via inhalation, offering a promising strategy for targeting airway inflammation directly.

Prolonged exposure to ultraviolet (UV) radiations represents a significant risk factor and may lead to various skin disorders, premature aging, and an increased susceptibility to skin cancers. Recently, probiotics have emerged as promising candidates for fortifying the skin’s natural defences through their diverse mechanisms. The aim of the present work was exploring the potential of five heat-treated probiotics (Skinbac^TM, Probiotical Research S.r.l., Novara, Italy), as protective agents against UVA and UVB damages on human keratinocyte line (HaCaT) and human skin 3D model (Phenion^® Full-Thickness Skin Model, Henkel AG & Co. KGaA, Dusseldorf, Germany). The protective role toward artificially induced oxidative stress was evaluated by determining the residual viability after UV exposure and analyzing gene expression of markers involved in apoptosis (Tumor protein 53), inflammation/immunosuppression (Interleukin 6), oxidative stress (oxidative stress response enzyme heme oxygenase 1), investigated using quantitative real-time PCR. Additionally, we examined the protective effects of these strains, testing them on Normal Human Epidermal Keratinocytes (NHEK) irradiated with UVC, specifically, evaluating the expression of tight junction proteins, including claudin 1, claudin 4, and occludin, by ELISA. The tested heat-treated probiotics effectively protected from UVA, UVB, and UVC damage on all end points analyzed, revealing their capacity to enhance barrier protection in cases of damage and their potential for innovative skincare strategies centered around probiotic-based formulations for enhanced protection against UV-induced skin damage.

Background: The skin microbiome plays a crucial role in maintaining barrier function and preventing inflammaging. Heat-treated probiotics offer stability advantages for topical formulations while potentially maintaining bioactive properties. Objective: To evaluate the safety, molecular mechanisms, and clinical efficacy of heat-treated Lactiplantibacillus plantarum Skinbac™ SB01 and Bifidobacterium animalis spp. lactis Skinbac™ SB05 in reducing visible signs of skin aging. Methods: In vitro studies assessed cytotoxicity (MTT/LDH assays), Aquaporin-3 (AQP3) expression, and reactive oxygen species (ROS) production in Normal Human Epidermal Keratinocytes (NHEK). A 30-day open-label clinical study (n = 20 females, 18–70 years) evaluated three formulations (face cream, serum, and eye contour) using instrumental measurements of hydration, elasticity, density, and roughness parameters. Results: In vitro testing showed a significant increase in AQP3 expression (+22% ± 3%, p = 0.03) and a non-significant reduction in ROS levels (−33% ± 9%, p = 0.06) at 10⁷ TFU/well, with no cytotoxicity observed. Clinical evaluation demonstrated statistically significant improvements: eye contour formulation achieved +10.5% deep skin hydration (p < 0.0001) and −11% average roughness (p < 0.0001); serum showed +28.7% immediate hydration (p < 0.0001); and face cream improved gross skin elasticity by +6.3% (p < 0.01). No adverse events were reported. An independent and methodologically distinct placebo-controlled study was included for contextual support and was not directly compared with the present trial; this study evaluated a related 1% postbiotic formulation and reported statistically significant improvements over placebo in roughness, wrinkle depth, hydration, and biomechanical parameters. Conclusions: This pilot study provides preliminary evidence that heat-treated L. plantarum SB01 and B. animalis spp. lactis SB05 formulations could safely improve skin hydration and reduce roughness parameters. While in vitro results show a significant increase in AQP3 expression and an exploratory (non-significant) reduction in ROS levels, larger controlled trials are warranted to confirm clinical efficacy.

Probiotic research is constrained by three pervasive yet insufficiently challenged assumptions: the requirement for a minimum of one billion colony-forming units for efficacy, the necessity for gut colonization, and the inherent superiority of live over inactivated preparations. This study addresses these gaps through a fully factorial experimental design evaluating ten Gram-positive probiotic strains in both viable (Active Fluorescent Units, AFU) and heat-inactivated (Total Fluorescent Units, TFU) forms across three flow cytometry-verified concentrations (10 ⁵ , 10 ⁶ , 10 ⁷ cells/well per ISO 19344:2015) in primary human peripheral blood mononuclear cells (PBMC) from a single healthy male Caucasian donor (58 years), with simultaneous quantification of 17 cytokines by BioPlex suspension array. Viable preparations induced profoundly greater absolute cytokine responses than heat-inactivated preparations across 14 of 17 analytes, heat-inactivated preparations demonstrated stronger dose-response correlations (mean within-strain Spearman ρ up to 1.00) for 13 of 17 cytokines, a finding we attribute to the uncontrolled proliferation of live bacteria during 24-hour co-culture compressing the effective concentration range. Six of ten viable strains exhibited monotonically increasing profiles; two strains displayed non-monotonic bell-shaped kinetics with peak activity at 10 ⁶ AFU/well and significant attenuation at 10 ⁷ , directly falsifying the assumption that dose escalation uniformly increases immunological activity. MCP-1 was the sole cytokine showing no significant difference between viability states (p = 0.61, fold-change 1.1), providing an internal methodological control. In this single-donor model, unsupervised hierarchical clustering identified three immunological phenotype clusters, requiring multi-donor validation before these groupings can be treated as generalizable biological phenotypes, with Random Forest classification achieving 86.7% internal partition-recovery consistency (clusters derived from the same data; not an estimate of generalization to novel strains) versus 33.3% chance; In this single-donor experiment, IL-13, IL-12p70, and IFN-γ, not IL-6 or IL-1β, were the primary discriminators of strain identity; generalizability of this ranking requires multi-donor validation. Heat-inactivated preparations achieved ≥70% functional equivalence relative to viable preparations at 10 ⁷ TFU/well for the majority of responsive strains (Functional Equivalence Dose, FED ⁷⁰ ), while one strain remained immunologically inert in heat-inactivated form across all concentrations, a finding subject to the caveat that no positive control stimulus was included to formally verify PBMC functional competence on the experimental day. These findings establish a methodological framework integrating flow cytometric standardization, multiplex immunophenotyping, and machine learning for evidence-based dose characterization, postbiotic functional equivalence assessment, and data-driven strain classification in probiotic research (all p-values are descriptive within a single-donor experimental context). CONTRIBUTION TO THE FIELD: The probiotic field faces a structural paradox: products are commercialized at doses spanning three orders of magnitude without any functional evidence anchoring a specific concentration to a specific immunological outcome, and existing colony-forming unit (CFU)-based quantification methodology is inherently inapplicable to the growing category of heat-inactivated postbiotic preparations. This study addresses both gaps simultaneously and introduces a third layer of novelty — machine learning-based functional classification of probiotic strains by their cytokine-induction phenotype — for which no published precedent exists in the literature. By systematically characterizing ten Gram-positive probiotic strains in both viable and heat-inactivated forms across three concentration levels (10 ⁵ , 10 ⁶ , 10 ⁷ cells/well) using flow cytometric input standardization (Active Fluorescent Units/Total Fluorescent Units per ISO 19344:2015) and a 17-plex BioPlex cytokine readout in primary human PBMC from a single healthy donor, this work provides the first systematic multi-strain dose-response immunophenotyping dataset for Gram-positive probiotic strains. Three distinct immunological phenotype clusters are identified, including non-monotonic bell-shaped kinetics in two strains and near-complete immunological inertness in the heat-inactivated form of one strain, establishing that dose-response shape and thermal inactivation sensitivity are strain-intrinsic properties that cannot be generalized across species or formulations. Data-driven hierarchical clustering of cytokine fingerprints identified three candidate functional phenotype groups, internally re-recovered by Random Forest with 86.7% Leave-One-Out consistency — an internal partition-recovery metric that requires external validation on independent strain cohorts before the groups can be treated as generalizable biological phenotypes. Seventeen cytokines were quantified simultaneously via Bio-Plex multiplex immunoassay. Live bacteria induced significantly greater responses across 14 of 17 cytokines, with tumor necrosis factor-alpha showing a 356.9-fold increase compared to heat-inactivated preparations. However, heat-inactivated bacteria exhibited stronger dose-dependency (Spearman rho up to 0.70), suggesting a more faithful representation of true dose-response relationships unconfounded by bacterial proliferation during co-culture. Strain-specific profiling revealed three distinct immunomodulatory phenotypes emerging entirely from data-driven analysis without prior functional annotation.

Background: The skin barrier plays a fundamental role in preventing transepidermal water loss (TEWL), regulating immune responses, and protecting against pathogen colonization. Disruption of this barrier underlies xerosis, sensitive skin, and clinical cracking. Heat-treated probiotics (postbiotics) represent a stable and biologically active approach to topical formulation. Objective: To evaluate the safety, molecular mechanisms, and clinical efficacy of heat-treated Lactiplantibacillus plantarum Skinbac™ SB14 (SB14) in improving skin barrier function, hydration, and the appearance of dry and cracked skin. Methods: In vitro studies assessed cell viability (MTT assay) and cytotoxicity (LDH release assay), Aquaporin-3 (AQP3) expression, Claudin-1 expression recovery following UV-induced damage (post-damage treatment model), cytokine modulation in Normal Human Epidermal Keratinocytes (NHEK) and Peripheral Blood Mononuclear Cells (PBMCs), and antipathogen activity against Staphylococcus aureus biofilm. A 30-day open-label, placebo-controlled clinical study (n = 20 healthy volunteers, both sexes, age > 18 years) evaluated an emulsion containing 1% SB14 versus placebo using instrumental measurements of superficial hydration (Corneometer^® CM825) and TEWL (Tewameter^® TM300), and clinical scoring of skin hydration (Kligman scale 1–4) and skin cracking (ODS Overall Dry Skin Score 1–5) via C-Cube imaging. Results: In vitro testing confirmed the safety of SB14 (full cell viability by MTT assay; no cytotoxicity by LDH release assay) and demonstrated significant AQP3 upregulation (p < 0.05), partial Claudin-1 recovery in UV-damaged cells following post-damage SB14 application (p < 0.1 vs. UV damage), significant reduction in pro-inflammatory IL-8 and IL-23 in NHEK (p < 0.01 and p < 0.05), strong innate immune activation in PBMCs (TNF-α and IL-6, p < 0.001), and 21% inhibition of S. aureus biofilm at 72 h. Clinically, the SB14 formulation significantly increased superficial skin hydration by +46.1% at T14 (p = 0.0451) and +33.6% at T30 (p = 0.0144) versus baseline, while TEWL decreased by −14.5% at T14 (p = 0.0205). Clinical hydration scores improved significantly from a median of 3.0 (moderate dry skin) at baseline to 2.0 (slightly dry skin) at T30 (p = 0.0073). Cracking scores improved significantly from a median of 3.5 at baseline to 2.0 at T30 (p = 0.0037), with 80% of subjects showing improvement at T30. All parameters remained non-significant in the placebo group. No adverse events were reported. Conclusions: SB14 is safe, biologically active across multiple barrier-relevant mechanisms, and clinically effective in improving hydration and reducing visible skin cracking in subjects with dry, barrier-compromised skin.