Exploring the Effects of Probiotic Treatment on Urinary and Serum Metabolic Profiles in Healthy Individuals.

Probiotics are live microorganisms that confer health benefits when administered in adequate amounts. They are used to promote gut health and alleviate various disorders. Recently, there has been an increasing interest in the potential effects of probiotics on human physiology. In the presented study, the effects of probiotic treatment on the metabolic profiles of human urine and serum using a nuclear magnetic resonance (NMR)-based metabonomic approach were investigated. Twenty-one healthy volunteers were enrolled in the study, and they received two different dosages of probiotics for 8 weeks. During the study, urine and serum samples were collected from volunteers before and during probiotic supplementation. The results showed that probiotics had a significant impact on the urinary and serum metabolic profiles without altering their phenotypes. This study demonstrated the effects of probiotics in terms of variations of metabolite levels resulting also from the different probiotic posology. Overall, the results suggest that probiotic administration may affect both urine and serum metabolomes, although more research is needed to understand the mechanisms and clinical implications of these effects. NMR-based metabonomic analysis of biofluids is a powerful tool for monitoring host-gut microflora dynamic interaction as well as for assessing the individual response to probiotic treatment.

Evaluation of Antimicrobial, Antiadhesive and Co-Aggregation Activity of a Multi-Strain Probiotic Composition against Different Urogenital Pathogens.

The urogenital microbiota is dominated by Lactobacillus that, together with Bifidobacterium, creates a physiological barrier counteracting pathogen infections. The aim of this study was to evaluate the efficacy of a multi-strain probiotic formulation (Lactiplantibacillus plantarum PBS067, Lacticaseibacillus rhamnosus LRH020, and Bifidobacterium animalis subsp. lactis BL050) to inhibit adhesion and growth of urogenital pathogens. The antimicrobial and antiadhesive properties of the probiotic strains and their mixture were evaluated on human vaginal epithelium infected with Candida glabrata, Neisseria gonorrheae, Trichomonas vaginalis, and Escherichia coli-infected human bladder epithelium. The epithelial tissue permeability and integrity were assessed by transepithelial/transendothelial electrical resistance (TEER). Co-aggregation between probiotics and vaginal pathogens was also investigated to elucidate a possible mechanism of action. The multi-strain formulation showed a full inhibition of T. vaginalis, and a reduction in C. glabrata and N. gonorrheae growth. A relevant antimicrobial activity was observed for each single strain against E. coli. TEER results demonstrated that none of the strains have negatively impaired the integrity of the 3D tissues. All the probiotics and their mixture were able to form aggregates with the tested pathogens. The study demonstrated that the three strains and their mixture are effective to prevent urogenital infections.

Probiotics as Potential Biological Immunomodulators in the Management of Oral Lichen Planus: What's New?

Oral lichen planus (OLP) is a T cell-mediated chronic inflammatory disorder with multifactorial aetiology and malignant transformation potential. Despite the treatments so far identified, new tailored and safe specific measures are needed. Recently, human microbiota imbalance has been linked to several immune-mediated diseases, opening new therapeutic perspectives for probiotics; besides their ability to directly interact with the host microbiota, they also display a strain-specific immune-modulatory effect. Thus, this non-systematic review aims to elucidate the molecular pathways underlying probiotic activity, mainly those of Lactobacilli and Bifidobacteria and their metabolites in OLP pathogenesis and malignant transformation, focusing on the most recent in vitro and in vivo research evidence. Findings related to their activity in other immune-mediated diseases are here included, suggesting a probiotic translational use in OLP. Probiotics show immune-modulatory and microbiota-balancing activities; they protect the host from pathogens, hamper an excessive effector T cell response, reduce nuclear factor-kappa B (NF-kB) signalling and basal keratinocytes abnormal apoptosis, shifting the mucosal response towards the production of anti-inflammatory cytokines, thus preventing uncontrolled damage. Therefore, probiotics could be a highly encouraging prevention and immunotherapeutic approach for a safer and more sustainable OLP management.

In Vitro Selection of Lactobacillus and Bifidobacterium Probiotic Strains for the Management of Oral Pathobiont Infections Associated to Systemic Diseases.

The human oral pathobionts Aggregatibacter actinomycetemcomitans, Streptococcus mitis and Streptococcus mutans, in dysbiosis-promoting conditions, lead to oral infections, which also represent a threat to human systemic health. This scenario may be worsened by antibiotic misuse, which favours multi-drug resistance, making the research on pathogen containment strategies more than crucial. Therefore, we aimed to in vitro select the most promising probiotic strains against oral pathogen growth, viability, biofilm formation, and co-aggregation capacity, employing both the viable probiotics and their cell-free supernatants (CFSs). Interestingly, we also assessed probiotic efficacy against the three-pathogen co-culture, mimicking an environment similar to that in vivo. Overall, the results showed that Lactobacillus CFSs performed better than the Bifidobacterium, highlighting Limosilactobacillus reuteri LRE11, Lacticaseibacillus rhamnosus LR04, Lacticaseibacillus casei LC04, and Limosilactobacillus fermentum LF26 as the most effective strains, opening the chance to deeper investigation of their action and CFS composition. Altogether, the methodologies presented in this study can be used for probiotic efficacy screenings, in order to better focus the research on a viable probiotic, or on its postbiotics, suitable in case of infections.

Oral microbiota and vitamin D impact on oropharyngeal squamous cell carcinogenesis: a narrative literature review.

An emerging body of research is revealing the microbiota pivotal involvement in determining the health or disease state of several human niches, and that of vitamin D also in extra-skeletal regions. Nevertheless, much of the oral microbiota and vitamin D reciprocal impact in oropharyngeal squamous cell carcinogenesis (OPSCC) is still mostly unknown. On this premise, starting from an in-depth scientific bibliographic analysis, this narrative literature review aims to show a detailed view of the state of the art on their contribution in the pathogenesis of this cancer type. Significant differences in the oral microbiota species quantity and quality have been detected in OPSCC-affected patients; in particular, mainly high-risk human papillomaviruses (HR-HPVs), Fusobacterium nucleatum, Porphyromonas gingivalis, Pseudomonas aeruginosa, and Candida spp. seem to be highly represented. Vitamin D prevents and fights infections promoted by the above identified pathogens, thus confirming its homeostatic function on the microbiota balance. However, its antimicrobial and antitumoral actions, well-described for the gut, have not been fully documented for the oropharynx yet. Deeper investigations of the mechanisms that link vitamin D levels, oral microbial diversity and inflammatory processes will lead to a better definition of OPSCC risk factors for the optimization of specific prevention and treatment strategies.

Non-Melanoma Skin Cancer: news from microbiota research.

Recently, research has been deeply focusing on the role of the microbiota in numerous diseases, either affecting the skin or other organs. What it is well established is that its dysregulation promotes several cutaneous disorders (i.e. psoriasis and atopic dermatitis). To date, little is known about its composition, mediators and role in the genesis, progression and response to therapy of Non-Melanoma Skin Cancer (NMSC). Starting from a bibliographic study, we classified the selected articles into four sections: i) normal skin microbiota; ii) in vitro study models; iii) microbiota and NMSC and iv) probiotics, antibiotics and NMSC. What has emerged is how skin microflora changes, mainly represented by increases of Staphylococcus aureus, Streptococcus pyogenes and Pseudomonas aeruginosa strains, modifications in the mutual quantity of ß-Human papillomavirus genotypes, of Epstein Barr Virus and Malassezia or candidiasis, may contribute to the induction of a state of chronic self-maintaining inflammation, leading to cancer. In this context, the role of S. aureus and that of specific antimicrobial peptides look to be prominent. Moreover, although antibiotics may contribute to carcinogenesis, due to their ability to influence the microbiota balance, specific probiotics, such as Lacticaseibacillus rhamnosus GG, Lactobacillus johnsonii NCC 533 and Bifidobacteria spp., may be protective.

Human papillomavirus type 16 E6 and E7 oncoproteins interact with the nuclear p53-binding protein 1 in an in vitro reconstructed 3D epithelium: new insights for the virus-induced DNA damage response.

BACKGROUND: Despite vaccination and screening measures, anogenital cancer, mainly promoted by HPV16 oncoproteins, still represents the fourth tumor and the second cause of death among women. Cell replication fidelity is the result of the host DNA damage response (DDR). Unlike many DNA viruses that promote their life cycle through the DDR inactivation, HR-HPVs encourage cells proliferation despite the DDR turned on. Why and how it occurs has been only partially elucidated. During HPV16 infection, E6 links and degrades p53 via the binding to the E6AP LXXLL sequence; unfortunately, E6 direct role in the DDR response has not clearly identified yet. Similarly, E7 increases DDR by competing with E2F1-pRb interaction, thus leading to the inactivation of pRb, and promotion, E2F1 mediated, of DDR genes translation, by binding to the pRb-like proteins CBP/p300 and p107, that also harbour LXXLL sequence, and via the interaction and activation of several DDR proteins. METHODS: To gain information regarding E6 and E7 contribution in DDR activation, we produced an in vitro 3D HPV16-E6E7 infected epithelium, already consolidated study model for HPVs, and validated it by assessing H&E staining and BrdU, HPV16 DNA, E6E7 proteins and γH2A.X/53BP1 double-strand break (DSBs) sensors expression; then we made an immuno-colocalization of E6 and E7 with cyclin E2 and B1. Since 53BP1, like E6 and E7, also binds p53 and pRb, we supposed their possible direct binding. To explore this hypothesis, we performed a double immunofluorescence of E6 and E7 with 53BP1, a sequence analysis of 53BP1 within its BRCT2 domain and then an in situ PLA within CaSki, E6E7HPV16 NHEKs and the 3D model. RESULTS: The in vitro epithelium resembled the histology and the events typical of in vivo infected tissues. E6E7HPV16 were both expressed in basal and differentiated strata and induced H2A.X phosphorylation and 53BP1 increment into nuclear foci. After highlighting E6 and E7 co-expression with 53BP1 and a LKVLL sequence within the 53BP1 BRCT2 domain, we demonstrated the bindings via the PLA technique. CONCLUSIONS: Our results reinforce E6 and E7 role in cellular function control providing potentially new insights into the activity of this tumor virus.

Fimbrial cells exposure to catalytic iron mimics carcinogenic changes.

OBJECTIVE: Recent evidence strongly suggests that the fallopian tube is a site of origin of ovarian cancer. Although histological data show iron deposition in the fallopian tubes, its role remains unclear. To establish whether catalytic iron has a possible role in ovarian carcinogenesis, we isolated human fimbrial secretory epithelial cells (FSECs). METHODS: Fimbrial secretory epithelial cells, isolated from women undergoing isteroannessiectomy, were treated with different doses of catalytic iron (0.05-100 mM) to study cell viability; NO production; p53, Ras, ERK/MAPK, PI3K/Akt, Ki67, and c-Myc protein expressions through Western blot analysis; and immunocytochemistry or immunofluorescence. RESULTS: In FSECs treated with catalytic iron for up to 6 days, we observed an increase in cell viability, NO production, and p53, pan-Ras, ERK/MAPK, PI3K/Akt, Ki67, and c-Myc activations (P < 0.05) in a dose-dependent and time-dependent manner. These same results were also observed in FSECs maintained for respectively 2 and 4 weeks in the absence of catalytic iron after 6 days of stimulation. CONCLUSIONS: Our model aimed at studying the main nongenetic risk factor for ovarian cancer, providing an alternative interpretation for the role of menstruation in increasing risk of this pathology. This in vitro model mimics several features of the precursor lesions and opens new scenarios for further investigations regarding the correlation between damages produced by repeated retrograde menstruation carcinogenic stimuli.

Growth Conditions Influence Lactobacillus Cell-Free Supernatant Impact on Viability, Biofilm Formation, and Co-Aggregation of the Oral Periodontopathogens Fusobacterium nucleatum and Porphyromonas gingivalis.

Fusobacterium nucleatum and Porphyromonas gingivalis human periodontopathogens play a leading part in oral squamous cell carcinoma through cell proliferation, invasion, and persistent inflammation promotion and maintenance. To explore how the activity of Lactobacillus-derived cell-free supernatants (CFSs) can be influenced by growth medium components, CFSs were produced both in the standard MRS and the novel animal-derivative-free "Terreno Industriale Lattobacilli" (TIL) media, and in vitro screened for the containment of F. nucleatum and P. gingivalis both single and co-cultured and also for the interference on their co-aggregation. The viability assay demonstrated that the Limosilactobacillus reuteri LRE11 and Ligilactobacillus salivarius LS03 MRS-produced CFSs were significantly more effective against single and co-cultured pathogens. All the other CFSs significantly improved their efficacy when produced in TIL. Both MRS- and TIL-produced CFSs significantly inhibited the single and co-cultured pathogen biofilm formation. Only Levilactobacillus brevis LBR01 CFS in MRS specifically reduced F. nucleatum and P. gingivalis co-aggregation, while viable LBR01, LS03, and LRE11 in MRS significantly co-aggregated with the pathogens, but only LS03 cultivated in TIL improved this effect. This work paves the way to better consider environmental growth conditions when screening for probiotic and postbiotic efficacy as crucial to pathogen aggregation, adhesion to the host's niches, and exclusion.

Lactobacillus johnsonii LJO02 (DSM 33828) Cell-Free Supernatant and Vitamin D Improve Wound Healing and Reduce Interleukin-6 Production in Staphylococcus aureus-Infected Human Keratinocytes.

Methicillin-resistant biofilm-forming Staphylococcus spp. are found in about 25% of the overall cases of chronic wounds, which can undergo malignant degeneration and be associated with skin cancer. Although antimicrobial agents are clinically used to counteract pathogens and promote wound healing, they are increasingly ineffective against multi-drug resistant bacteria. Moreover, they can induce dysbiosis, which favors opportunistic pathogen infections and alters immune responses. Consequently, research on pathogen containment strategies is crucial. We aimed to evaluate the potential beneficial effect of Lactobacillus johnsonii LJO02 cell-free supernatant (CFS) and vitamin D, as single treatments or in combination, on cell viability, wound healing, and the pro-inflammatory interleukin-6 (IL-6) production of a Staphylococcus aureus-infected human immortalized keratinocyte cell line (HaCaT) in vitro model. The analysis showed that LJO02 CFS 20% v/v ratio and 100 nM vitamin D promoted infected cell viability and wound healing and significantly reduced IL-6 production. However, their effect was not synergic, since no significant difference between the single and combined treatments was observed. LJO02 CFS topic application and vitamin D supplementation could provide a valuable strategy for attenuating S. aureus-induced pathogenesis, promoting wound healing and opening new therapeutic strategies supporting the conventional approaches.

An animal derivative-free medium enhances Lactobacillus johnsonii LJO02 supernatant selective efficacy against the methicillin (oxacillin)-resistant Staphylococcus aureus virulence through key-metabolites.

The spread of multidrug-resistant bacteria, such as the skin commensal Staphylococcus aureus, is a worldwide health challenge; new methods to counteract opportunistic pathogen growth and virulence are urgent. We compared the activity of Lacticaseibacillus rhamnosus LR06 (DSM 21981) and Lactobacillus johnsonii LJO02 (DSM 33828) cell-free supernatants (CFSs) produced in the conventional animal derivative-based MRS medium and an innovative animal derivative-free broth (TIL) versus the MDR S. aureus (ATCC 43300). CFS influence was assessed towards the viability, metabolic activity, and ability to form biofilm of the MDR strain through optical density, alamarBlue assay, and crystal violet staining; their content in short-chain fatty acids, lactic acid, and proteins was analysed via high-resolution mass spectrometry and gas chromatography. All CFSs reduce viable and metabolically active S. aureus, being TIL more efficient compared to MRS in stimulating lactic acid bacteria metabolism and decreasing S. aureus biofilm formation. Particularly, the CFS from LJO02 grown in TIL has the best efficacy, revealing a high amount of lactic acid and 59 peculiar proteins; its effectiveness is partially maintained upon trypsin and proteinase K treatments, but not by pepsin and pH basification. Therefore, antagonistic CFSs may represent a strategic prevention approach, with bacteriotherapeutic and bio-repair potential.

Macrophages expressing TREM-1 are involved in the progression of HPV16-related oropharyngeal squamous cell carcinoma.

INTRODUCTION: Many types of research have been performed to improve the diagnosis, therapy, and prognosis of oropharyngeal carcinomas (OP-SCCs). Since they arise in lymphoid-rich areas and intense lymphocytic infiltration has been related to a better prognosis, a TREM-1 putative function in tumour progression and survival has been hypothesized. MATERIALS AND METHODS: Twenty-seven human papillomavirus (HPV) 16+ OP-SCC specimens have been analyzed to relate TREM-1 expression with histiocytic and lymphocytic markers, HPV presence and patients' outcome. RESULTS: No differences have been shown between intratumoral and stromal CD4+ cells, while intratumoral CD8+ lymphocytes are higher with respect to the tumour stroma (p = .0005). CD68+ cells are more than CD35+ and TREM-1+; their presence is related to CD35± and TREM-1± histiocytes (p = .005 and .026, respectively). Intratumoral CD4+ lymphocytes are higher in p16+ cases (11/27) than in p16- (p = .042); moreover, p16 positivity correlates to a better survival (p = .034). CD4+, CD8+ and CD35+ cells have no impact on survival, while CD68 expression heavily influences progression and bad outcome (p = .037). TREM-1 positivity also leads to worst overall survival (p = .001): peritumoral expression and death-cause relationship are always significant, particularly when the cause is OP-SCC (p = .000). CONCLUSION: While p16 shows to better stratify HPV16+ patients' outcome, TREM-1+ macrophages suggest their key importance in HPV-related OP-SCCs progression.KEY MESSAGESTREM-1 positivity correlates to the worst overall survival of HPV16-positive OPSCCs-affected patients.p16-positive HPV16 related OPSCCs patients have a better prognosis with respect to p16-negative ones.

Effects of Probiotics Administration on Human Metabolic Phenotype.

The establishment of the beneficial interactions between the host and its microbiota is essential for the correct functioning of the organism, since microflora alterations can lead to many diseases. Probiotics improve balanced microbial communities, exerting substantial health-promoting effects. Here we monitored the molecular outcomes, obtained by gut microflora modulation through probiotic treatment, on human urine and serum metabolic profiles, with a metabolomic approach. Twenty-two subjects were enrolled in the study and administered with two different probiotic types, both singularly and in combination, for 8 weeks. Urine and serum samples were collected before and during the supplementation and were analyzed by nuclear magnetic resonance (NMR) spectroscopy and statistical analyses. After eight weeks of treatment, probiotics deeply influence the urinary metabolic profiles of the volunteers, without significantly altering their single phenotypes. Anyway, bacteria supplementation tends to reduce the differences in metabolic phenotypes among individuals. Overall, the effects are recipient-dependent, and in some individuals, robust effects are already well visible after four weeks. Modifications in metabolite levels, attributable to each type of probiotic administration, were also monitored. Metabolomic analysis of biofluids turns out to be a powerful technique to monitor the dynamic interactions between the microflora and the host, and the individual response to probiotic assumption.

Implications on pathogenesis and risk of oral lichen planus neoplastic transformation: an ex-vivo retrospective immunohistochemical study.

AIMS: To evaluate OPN, MCM7, Ki-67, p53, Bcl-2 and 53BP1 presence, together with the abnormal adaptive CD4 and CD8 T-cell response markers expression in a series of oral lichen planus (OLP) affected patients and assess their combined contribution for a more objective disease classification. METHODS AND RESULTS: In this ex-vivo retrospective analysis, biopsy specimens from 28 adults with a clinical diagnosis of OLP at different progression degree (16 reticular, 2 plaque-like, 1 erosive and 9 mixed type) were collected. Sections were immunohistochemically investigated for the proinflammatory cytokine osteopontin (OPN), alpha-beta CD4 and CD8 positive T cells, DNA replication licensing factor (MCM7), proliferating cell marker (Ki-67), apoptotic and tumor antigen (p53), apoptosis modulator (Bcl-2) and cellular response regulator to double-strand breaks tumor suppressor p53-binding protein 1 expression. Statistical analysis revealed that 53BP1 is highly represented among the OLP study patients (p<0.05). Moreover, on the basis of the quantification results of the highly expressed parameters, two illness categories with different severity were evidenced. The classification hypothesis was confirmed by i) OLP lesion persistence, ii) the development of oral severe lesions in the patients belonging to high grade activity OLP group (HGA-OLPs) and iii) the ascertainment of the same evidence both in the oral squamous cell tumor controls (OSCC) and in HGA-OLP cases. CONCLUSION: This study completes the scenario with respect to early detection, thanks to a more precise histological analysis, for rationalizing the clinical and histological findings toward a sharable international disease scoring system.