The Ability of Postimmunobiotics from L. rhamnosus CRL1505 to Protect against Respiratory Syncytial Virus and Pneumococcal Super-Infection Is a Strain-Dependent Characteristic.

Previously, we demonstrated that the non-viable strain Lacticaseibacillus rhamnosus CRL1505 (NV1505) or its purified peptidoglycan (PG1505) differentially modulated the respiratory innate antiviral immune response triggered by Toll-like receptor (TLR)-3 activation in infant mice, improving the resistance to primary respiratory syncytial virus (RSV) infection and secondary pneumococcal pneumonia. In this work, we evaluated the effect of other non-viable L. rhamnosus strains and their peptidoglycans on the respiratory immune response and their impact on primary and secondary respiratory infections. In addition, the duration of the protective effect induced by NV1505 and PG1505 as well as their ability to protect against different Streptococcus pneumoniae serotypes were evaluated. Our results showed that among the five selected L. rhamnosus strains (CRL1505, CRL498, CRL576, UCO25A and IBL027), NV1505 and NVIBL027 improved the protection against viral and pneumococcal infections by modulating the respiratory immune response. Of note, only the PG1505 presented immunomodulatory activities when compared with the other purified peptidoglycans. Studies on alveolar macrophages showed that NV1505 and PG1505 differentially modulated the expression of IL-6, IFN-γ, IFN-ß, TNF-α, OAS1, RNAseL and IL-27 genes in response to RSV infection, and IL-6, IFN-γ, IL-1ß, TNF-α, CCL2, CXCL2, CXCL10 and IL-27 in response to pneumococcal challenge. Furthermore, we demonstrated that NV1505 and PG1505 treatments protected mice against secondary pneumococcal pneumonia produced by different serotypes of S. pneumoniae until 30 days after stimulation with poly(I:C). This work advances the characterization of the protective effect of NV1505 and PG1505 by demonstrating that they increase resistance against the pneumococcal serotypes 3, 6B, 14 and 19F, with an effect that lasts up to 30 days after the primary viral inflammation. The results also confirm that the immunomodulatory properties of NV1505 and PG1505 are unique and are not shared by other members of this species, and suggest the existence of a capacity to stimulate trained immunity in alveolar macrophages.

The Role of Alveolar Macrophages in the Improved Protection against Respiratory Syncytial Virus and Pneumococcal Superinfection Induced by the Peptidoglycan of Lactobacillus rhamnosus CRL1505.

The nasal priming with nonviable Lactobacillus rhamnosus CRL1505 (NV1505) or its purified peptidoglycan (PG1505) differentially modulates the respiratory innate immune response in infant mice, improving their resistance to primary respiratory syncytial virus (RSV) infection and secondary pneumococcal pneumonia. In association with the protection against RSV-pneumococcal superinfection, it was found that NV1505 or PG1505 significantly enhance the numbers of CD11c+SiglecF+ alveolar macrophages (AMs) producing interferon (IFN)-ß. In this work, we aimed to further advance in the characterization of the beneficial effects of NV1505 and PG1505 in the context of a respiratory superinfection by evaluating whether their immunomodulatory properties are dependent on AM functions. Macrophage depletion experiments and a detailed study of their production of cytokines and antiviral factors clearly demonstrated the key role of this immune cell population in the improvement of both the reduction of pathogens loads and the protection against lung tissue damage induced by the immunobiotic CRL1505 strain. Studies at basal conditions during primary RSV or S. pneumoniae infections, as well as during secondary pneumococcal pneumonia, brought the following five notable findings regarding the immunomodulatory effects of NV1505 and PG1505: (a) AMs play a key role in the beneficial modulation of the respiratory innate immune response and protection against RSV infection, (b) AMs are necessary for improved protection against primary and secondary pneumococcal pneumonia, (c) the generation of activated/trained AMs would be essential for the enhanced protection against respiratory pathogens, (d) other immune and nonimmune cell populations in the respiratory tract may contribute to the protection against bacterial and viral infections, and (e) the immunomodulatory properties of NV1505 and PG1505 are strain-specific. These findings significantly improve our knowledge about the immunological mechanisms involved in the modulation of respiratory immunity induced by beneficial microbes.

The Exopolysaccharide of Lactobacillus fermentum UCO-979C Is Partially Involved in Its Immunomodulatory Effect and Its Ability to Improve the Resistance against Helicobacter pylori Infection.

Lactobacillus fermentum UCO-979C (Lf979C) beneficially modulates the cytokine response of gastric epithelial cells and macrophages after Helicobacter pylori infection in vitro. Nevertheless, no in vivo studies were performed with this strain to confirm its beneficial immunomodulatory effects. This work evaluated whether Lf979C improves protection against H. pylori infection in mice by modulating the innate immune response. In addition, we evaluated whether its exopolysaccharide (EPS) was involved in its beneficial effects. Lf979C significantly reduced TNF-α, IL-8, and MCP-1 and augmented IFN-γ and IL-10 in the gastric mucosa of H. pylori-infected mice. The differential cytokine profile induced by Lf979C in H. pylori-infected mice correlated with an improved reduction in the pathogen gastric colonization and protection against inflammatory damage. The purified EPS of Lf979C reduced IL-8 and enhanced IL-10 levels in the gastric mucosa of infected mice, while no effect was observed for IFN-γ. This work demonstrates for the first time the in vivo ability of Lf979C to increase resistance against H. pylori infection by modulating the gastric innate immune response. In addition, we advanced knowledge of the mechanisms involved in the beneficial effects of Lf979C by demonstrating that its EPS is partially responsible for its immunomodulatory effect.

Characterization of the immunomodulatory and anti-Helicobacter pylori properties of the human gastric isolate Lactobacillus rhamnosus UCO-25A.

The ability to form biofilms and the potential immunomodulatory properties of the human gastric isolate Lactobacillus rhamnosus UCO-25A were characterized in vitro. It was demonstrated that L. rhamnosus UCO-25A is able to form biofilms on abiotic and cell surfaces, and to modulate the inflammatory response triggered by Helicobacter pylori infection in gastric epithelial cells and THP-1 macrophages. L. rhamnosus UCO-25A exhibited a substantial anti-inflammatory effect in both cell lines and improved IL-10 levels produced by challenged macrophages. Additionally, UCO-25A protected AGS cells against H. pylori infection with a higher pathogen inhibition when a biofilm was formed. Given the importance of inflammation in H. pylori-mediated diseases, the differential modulation of the inflammatory response in the gastric mucosa by an autochthonous strain is an attractive alternative for improving H. pylori eradication and reducing the severity of the diseases that arise from the resulting chronic inflammation.

Personalization of the Microbiota of Donor Human Milk with Mother's Own Milk.

The American Academy of Pediatrics recommends that extremely preterm infants receive mother's own milk (MOM) when available or pasteurized donor breast milk (DBM) when MOM is unavailable. The goal of this study was to determine whether DBM could be inoculated with MOM from mothers of preterm infants to restore the live microbiota (RM). Culture dependent and culture independent methods were used to analyze the fluctuations in the overall population and microbiome, respectively, of DBM, MOM, and RM samples over time. Using MOM at time = 0 (T0) as the target for the restoration process, this level was reached in the 10% (RM-10) and 30% (RM-30) mixtures after 4 h of incubation at 37°C, whereas, the larger dilutions of 1% (RM-1) and 5% (RM-5) after 8 h. The diversity indexes were similar between MOM and DBM samples, however, different genera were prevalent in each group. Interestingly, 40% of the bacterial families were able to expand in DBM after 4 h of incubation indicating that a large percentage of the bacterial load present in MOM can grow when transferred to DBM, however, no core microbiome was identified. In summary, the microbiome analyses indicated that each mother has a unique microbiota and that live microbial reestablishment of DBM may provide these microbes to individual mothers' infants. The agreement between the results obtained from the viable bacterial counts and the microbiome analyses indicate that DBM incubated with 10-30% v/v of the MOM for 4 h is a reasonable restoration strategy.

Lactobacillus johnsonii N6.2 Modulates the Host Immune Responses: A Double-Blind, Randomized Trial in Healthy Adults.

Lactobacillus johnsonii N6.2 mitigates the onset of type 1 diabetes (T1D) in biobreeding diabetes-prone rats, in part, through changes in kynurenine:tryptophan (K:T) ratios. The goal of this pilot study was to determine the safety, tolerance, and general immunological response of L. johnsonii N6.2 in healthy subjects. A double-blind, randomized clinical trial in 42 healthy individuals with no known risk factors for T1D was undertaken to evaluate subject responses to the consumption of L. johnsonii N6.2. Participants received 1 capsule/day containing 108 colony-forming units of L. johnsonii N6.2 or placebo for 8 weeks. Comprehensive metabolic panel (CMP), leukocyte subpopulations by complete blood count (CBC) and flow cytometry, serum cytokines, and relevant metabolites in the indoleamine-2,3-dioxygenase pathway were assessed. L. johnsonii N6.2 survival and intestinal microbiota was analyzed. Daily and weekly questionnaires were assessed for potential effects of probiotic treatment on general wellness. The administration of L. johnsonii N6.2 did not modify the CMP or CBC of participants suggesting general safety. In fact, L. johnsonii N6.2 administration significantly decreased the occurrence of abdominal pain, indigestion, and cephalic syndromes. As predicted, increased serum tryptophan levels increased resulting in a decreased K:T ratio was observed in the L. johnsonii N6.2 group. Interestingly, immunophenotyping assays revealed that monocytes and natural killer cell numbers were increased significantly after washout (12 weeks). Moreover, an increase of circulating effector Th1 cells (CD45RO+CD183+CD196-) and cytotoxic CD8+ T cells subset was observed in the L. johnsonii N6.2 group. Consumption of L. johnsonii N6.2 is well tolerated in adult control subjects, demonstrates systemic impacts on innate and adaptive immune populations, and results in a decreased K:T ratio. These data provide support for the safety and feasibility of using L. johnsonii N6.2 in prevention trials in subjects at risk for T1D. TRIAL REGISTRATION: This trial was registered at http://clinicaltrials.gov as NCT02349360.

Influence of oregano essential oil on traditional Argentinean cheese elaboration: Effect on lactic starter cultures.

The aim of this work is to study the oregano essential oil (OEO) composition from Northwestern Argentinean regions and to evaluate its effect on the lactic starter cultures. The oregano used, Origanum vulgare var hirtum, was obtained from Andalgalá, Catamarca. The essential oil presented high amounts of α-terpinene (10%), γ-terpinene (15.1%), terpinen-4-ol (15.5%) and thymol (13.0%) as the main components. No negative effect on growth or metabolic activity of lactic acid bacteria Streptococcus thermophilus CRL 728 and CRL 813, Lactobacillus delbrueckii subsp. bulgaricus CRL 656 and CRL 468, and Lactococcus lactis subsp. lactis CRL 597 up to the maximum concentration (200µg/g) assayed was observed. No differences in the organoleptic characteristics of semi-hard cheeses flavored with oregano essential oil (200µg/g) and homemade cheeses flavored with oregano leaves were found. With respect to the microbiological quality of the products, neither enterobacteria nor mold and yeast were detected during ripening in essential-oil flavored cheese compared to control cheese (enterobacteria 2×103UFC/g) and cheese flavored with oregano leaves (mold/yeast 4×104CFU/g). Our results showed that the use of oregano essential oil and lactic starter culture considerably improved cheese quality.

Influencia del aceite esencial de orégano en la elaboración tradicional de quesos: efecto sobre el fermento láctico / Influence of oregano essential oil on traditional Argentinean cheese elaboration: Effect on lactic starter cultures

The aim of this work is to study the oregano essential oil (OEO) composition from Northwestern Argentinean regions and to evaluate its effect on the lactic starter cultures. The oregano used, Origanum vulgare var hirtum, was obtained from Andalgalá, Catamarca. The essential oil presented high amounts of α-terpinene (10%), γ-terpinene (15.1%), terpinen-4-ol (15.5%) and thymol (13.0%) as the main components. No negative effect on growth or metabolic activity of lactic acid bacteria Streptococcus thermophilus CRL 728 and CRL 813, Lactobacillus delbrueckii subsp. bulgaricus CRL 656 and CRL 468, and Lactococcus lactis subsp. lactis CRL 597 up to the maximum concentration (200 μg/g) assayed was observed. No differences in the organoleptic characteristics of semi-hard cheeses flavored with oregano essential oil (200 μg/g) and homemade cheeses flavored with oregano leaves were found. With respect to the microbiological quality of the products, neither enterobacteria nor mold and yeast were detected during ripening in essential-oil flavored cheese compared to control cheese (enterobacteria 2 × 10³ UFC/g) and cheese flavored with oregano leaves (mold/yeast 4 × 10(4) CFU/g). Our results showed that the use of oregano essential oil and lactic starter culture considerably improved cheese quality.

El objetivo de este trabajo fue estudiar la composición del aceite esencial de orégano recolectado en el noroeste argentino y evaluar su efecto sobre algunos fermentos lácticos. El orégano recolectado correspondió a la especie Origanum vulgare var. hirtum proveniente de Andalgalá, Catamarca. En su aceite esencial (obtenido por arrastre con vapor de agua) se detectó principalmente α-terpineno (10%), γ-terpineno (15,1%), terpinen-4-ol (15,5%) y timol (13,0%). El aceite esencial no tuvo efecto inhibitorio (máxima concentración ensayada 200 μg/g) sobre el crecimiento ni sobre la actividad metabólica de Streptococcus thermophilus CRL 728 y CRL 813, de Lactobacillus delbrueckii subsp. bulgaricus CRL 656 y CRL 468, y de Lactococcus lactis subsp. lactis CRL 597. No se observaron diferencias en las características organolépticas de quesos semiduros aromatizados con el aceite esencial (200 μg/g) comparados con quesos artesanales aromatizados con hojas de orégano. Respecto de la calidad microbiológica de los productos, no se detectaron enterobacterias ni hongos o levaduras durante la maduración en los quesos aromatizados con el aceite esencial de orégano comparados con los quesos control, que presentaron desarrollo de enterobacterias (2 × 10³ UFC/g), y con los quesos elaborados con hojas de orégano, en los que hubo desarrollo de hongos/levaduras (4 × 10(4) CFU/g). Los resultados obtenidos demostraron que el uso del aceite esencial de orégano y del fermento láctico incrementó la calidad general de los quesos artesanales.

Intraspecific variation in essential oil composition of the medicinal plant Lippia integrifolia (Verbenaceae). Evidence for five chemotypes.

The aerial parts of Lippia integrifolia (incayuyo) are widely used in northwestern and central Argentina for their medicinal and aromatic properties. The essential oil composition of thirty-one wild populations of L. integrifolia covering most of its natural range was analyzed by GC and GC-MS. A total of one hundred and fifty two terpenoids were identified in the essential oils. Sesquiterpenoids were the dominant components in all but one of the collections analyzed, the only exception being a sample collected in San Juan province where monoterpenoids amounted to 51%. Five clearly defined chemotypes were observed. One possessed an exquisite and delicate sweet aroma with trans-davanone as dominant component (usually above 80%). Another with an exotic floral odour was rich in oxygenated sesquiterpenoids based on the rare lippifoliane and africanane skeletons. The trans-davanone chemotype is the first report of an essential oil containing that sesquiterpene ketone as the main constituent. The absolute configuration of trans-davanone from L. integrifolia was established as 6S, 7S, 10S, the enantiomer of trans-davanone from 'davana oil' (Artemisia pallens). Wild plants belonging to trans-davanone and lippifolienone chemotypes were propagated and cultivated in the same parcel of land in Santa Maria, Catamarca. The essential oil compositions of the cultivated plants were essentially identical to the original plants in the wild, indicating that the essential oil composition is largely under genetic control. Specimens collected near the Bolivian border that initially were identified as L. boliviana Rusby yielded an essential oil practically identical to the trans-davanone chemotype of L. integrifolia supporting the recent view that L. integrifolia (Gris.) Hieron. and L. boliviana Rusby are synonymous.

Gastroprotection as an example: antiadhesion against Helicobacter pylori, anti-inflammatory and antioxidant activities of aqueous extracts from the aerial parts of Lippia integrifolia Hieron.

ETHNOPHARMACOLOGICAL RELEVANCE: The aerial parts of Lippia integrifolia (Gris.) Hieronymus (Verbenaceae), known as incayuyo, are used by the peasant population of Argentina for treatment of diseases related to a gastrointestinal system, mainly for inflammation of the stomach and have also been included into the Argentina Food Code. This study aimed to investigate the phytochemical profile of hydrophilic extracts from the herbal material by LC-MS and to evaluate potential pharmacological mechanisms rationalizing the traditional use of incayuyo aqueous extracts potential anti-inflammatory treatment of gastrointestinal disorders. MATERIALS AND METHODS: Phytochemical profiling: LC-MS of an aqueous decoction. Antiadhesive effects against Helicobacter pylori: in vitro FACS assay using FITC-labeled bacteria and AGS human stomach cells. Influence of extracts on stomach cells and RAW 264.7 macrophages: MTT viability assay and BrdU proliferation ELISA. Influence of extracts on IL-6 and IL-8 secretion from stomach cells was quantitated by ELISA after infection of the cells with Helicobacter pylori. Influence of test extracts on macrophages: phagocytosis of FITC-labeled Zymosan particles and NO production. Antioxidative capacity: DPPH assay and O2-induced caroten oxidation. RESULTS: LC-MS profiling indicated the presence of compounds 1-20 with flavonoid hexosides, phenylethanoides (acteoside, isoacteoside) and sesquiterpenes [(epi)lippidulcine, peroxylippidulcine] in the decoction extract and subfraction PhF. The extract exhibits strong in vitro antioxidative capacity and inhibited adhesion of Helicobacter pylori to stomach cells up to 40%, while an EtOH-soluble fraction showed inhibition rates of up to 60%. Decoction increased the cellular viability of AGS cells significantly at >10 µg/mL, while proliferation rate was not influenced. Helicobacter pylori induced IL-8 secretion was significantly reduced by coincubation of AGS cells with the extracts. Aqueous extracts stimulated phagocytosis rate of macrophages and inhibited the LPS-induced NO-secretion. CONCLUSIONS: The traditional use of aqueous extracts from Lippia integrifolia for gastric inflammation seems to be rationalized: besides anti-inflammatory effects on stomach cells antiadhesive properties of the extracts against the main bacterial inductor of gastritis Helicobacter pylori were identified. Additional effects for stimulation of innate immunity and potential radical scavenging effects may additionally contribute to the activity of the extracts.

Effects of polysaccharide isolated from Streptococcus thermophilus CRL1190 on human gastric epithelial cells.

EPS1190 was isolated from skim milk fermented with Stretococcus thermophilus CRL1190. The polysaccharide consisted of 33% glucose and 66% galactose with 1,4- and 1,4,6-galactose residues as main building blocks beside a high amount of 1,4-linked glucose. The polymer was characterized additionally concerning viscosity and zeta potential. EPS1190 stimulated cellular vitality and proliferation of human stomach AGS cells and human buccal KB cells significantly. EPS1190 stimulated phagocytosis rate of murine macrophages RAW264.7 significantly. NO-release or anti-inflammatory effects by inhibition of LPS-induced NO release were not observed. Confocal laser scanning microscopy revealed that EPS1190 is partially internalized into AGS cells via endosomes. The bioadhesive absorption of FITC-labeled EPS1190 into the mucus layer on the apical side of the epithelium using histological tissue sections from human stomach was observed. Specific interaction of EPS1190 with mucin can be excluded as shown by microviscosimetry studies. EPS1190 increased the adhesion of H. pylori to AGS cells, which resulted in increased secretion of proinflammatory cytokines TNFa, IL-6 and IL-8. Summarizing, EPS1190 seems to stimulate epithelial cell regeneration and immunological innate defense mechanisms, which again can rationalized the use of this polysaccharide as cytoprotective compound in probiotioc preparations.

Chemical constituents, anti-inflammatory and antioxidant activities of bark extracts from Prunus tucumanensis Lillo.

The anti-inflammatory, antioxidant, antimicrobial and cytotoxic activities of the hexane (HE), chloroform (CE) and methanol (ME) extracts obtained from the bark of Prunus tucumanensis Lillo were investigated. Both ME and CE extracts displayed a significant in vitro anti-inflammatory activity similar to dexamethasone and to a commercial formulation (Pygeum) used for the treatment of benign prostatic hyperplasia (BPH). ME exhibited powerful antioxidant (67.6% relative to BHT) and free radical scavenging (RC50 = 5 ppm) activities, antimicrobial activities against Staphylococcus aureus and Mycobacterium smegmatis and did not show any cytotoxic effect on human-derived macrophage cells. Chemical analyses showed that (2 R,3 R)-3,5,7,3',5'-pentahydroxyflavan, ß-sitosterol and ß-sitosterol-3-O-ß-D-glucopyranoside (daucosterol) are relevant components of ME.