The effect of probiotic Bifidobacterium lactis Bl-04 on innate antiviral responses invitro.

Consumption of certain probiotic strains may be beneficial for reducing the risk of acute upper respiratory tract infections (URTIs), however, underlying immunological mechanisms are elusive. Bifidobacterium lactis Bl-04™ has been reported in humans to significantly reduce the risk of URTIs, affect the innate immunity in the nasal mucosa, and reduce nasal lavage virus titer after a rhinovirus (RV) challenge. To study the immunological mechanisms, we investigated the effect of Bl-04 on cytokine production and transcriptomes of human monocyte-derived macrophages (Mfs) and dendritic cells (DCs), and further on RV replication and cytokine production in MRC-5 fibroblasts. The results showed that Bl-04 modulates antiviral immune responses and potentiates cytokine production during viral challenge mimic in immune cells. However, effect of Bl-04 on RV replication and cytokine production in fibroblasts was negligible. Overall, the findings suggest that Bl-04 mildly stimulates antiviral immunity in Mfs and DCs, and potentially influences viral replication in fibroblasts that however warrants further investigations.

Enhancing Bioaccessibility of Plant Protein Using Probiotics: An In Vitro Study.

As plant-based diets become more popular, there is an interest in developing innovations to improve the bioaccessibility of plant protein. In this study, seven probiotic strains (Bifidobacterium animalis subsp. lactis B420, B. lactis Bl-04, Lactobacillus acidophilus NCFM, Lacticaseibacillus rhamnosus HN001, Lacticaseibacillus paracasei subsp. paracasei Lpc-37, Lactiplantibacillus plantarum Lp-115, and Lactococcus lactis subsp. lactis Ll-23) were evaluated for their capacity to hydrolyze soy and pea protein ingredients in an in vitro digestion model of the upper gastrointestinal tract (UGIT). Compared to the control digestion of protein without a probiotic, all the studied strains were able to increase the digestion of soy or pea protein, as evidenced by an increase in free α-amino nitrogen (FAN) and/or free amino acid concentration. The increase in FAN varied between 13 and 33% depending on the protein substrate and probiotic strain. The survival of probiotic bacteria after exposure to digestive fluids was strain-dependent and may have affected the strain's capacity to function and aid in protein digestion in the gastrointestinal environment. Overall, our results from the standardized in vitro digestion model provide an approach to explore probiotics for improved plant protein digestion and bioaccessibility of amino acids; however, human clinical research is needed to evaluate the efficacy of probiotics on amino acid absorption and bioavailability in vivo.

Effects of Bacterial Lysates and Metabolites on Collagen Homeostasis in TNF-α-Challenged Human Dermal Fibroblasts.

During skin aging, the production of extracellular matrix (ECM) proteins, such as type I collagen, decreases and the synthesis of ECM-degrading matrix metalloproteinases (MMPs) rises, leading to an imbalance in homeostasis and to wrinkle formation. In this study, we examined the effects of bacterial lysates and metabolites from three bifidobacteria and five lactobacilli on collagen homeostasis in human dermal fibroblasts during challenge with tumor necrosis factor alpha (TNF-α), modeling an inflammatory condition that damages the skin's structure. Antiaging properties were measured, based on fibroblast cell viability and confluence, amount of type I pro-collagen, ratio of MMP-1 to type I pro-collagen, cytokines, and growth factors. The TNF-α challenge increased the MMP-1/type I pro-collagen ratio and levels of proinflammatory cytokines, as expected. With the probiotics, differences were clearly dependent on bacterial species, strain, and form. In general, the lysates elicited less pronounced responses in the biomarkers. Of all strains, the Bifidobacterium animalis ssp. lactis strains Bl-04 and B420 best maintained type I pro-collagen production and the MMP-1/collagen type I ratio under no-challenge and challenge conditions. Metabolites that were produced by bifidobacteria, but not their lysates, reduced several proinflammatory cytokines (IL-6, IL-8, and TNF-α) during the challenge, whereas those from lactobacilli did not. These results indicate that B. animalis ssp. lactis-produced metabolites, especially those of strains Bl-04 and B420, could support collagen homeostasis in the skin.

The Effect of Human Milk Oligosaccharides and Bifidobacterium longum subspecies infantis Bi-26 on Simulated Infant Gut Microbiome and Metabolites.

Human milk oligosaccharides (HMOs) shape the developing infant gut microbiota. In this study, a semi-continuous colon simulator was used to evaluate the effect of 2 HMOs-2'-fucosyllactose (2'-FL) and 3-fucosyllactose (3-FL)-on the composition of infant faecal microbiota and microbial metabolites. The simulations were performed with and without a probiotic Bifidobacterium longum subspecies infantis Bi-26 (Bi-26) and compared with a control that lacked an additional carbon source. The treatments with HMOs decreased α-diversity and increased Bifidobacterium species versus the control, but the Bifidobacterium species differed between simulations. The levels of acetic acid and the sum of all short-chain fatty acids (SCFAs) trended toward an increase with 2'-FL, as did lactic acid with 2'-FL and 3-FL, compared with control. A clear correlation was seen between the consumption of HMOs and the increase in SCFAs (-0.72) and SCFAs + lactic acid (-0.77), whereas the correlation between HMO consumption and higher total bifidobacterial numbers was moderate (-0.46). Bi-26 decreased propionic acid levels with 2'-FL. In conclusion, whereas infant faecal microbiota varied between infant donors, the addition of 2'-FL and 3-FL, alone or in combination, increased the relative abundance and numbers Bifidobacterium species in the semi-continuous colon simulation model, correlating with the production of microbial metabolites. These findings may suggest that HMOs and probiotics benefit the developing infant gut microbiota.

In Vitro Screen of Lactobacilli Strains for Gastrointestinal and Vaginal Benefits.

Traditional probiotics comprise mainly lactic acid bacteria that are safe for human use, tolerate acid and bile, and adhere to the epithelial lining and mucosal surfaces. In this study, one hundred commercial and non-commercial strains that were isolated from human feces or vaginal samples were tested with regards to overall growth in culture media, tolerance to acid and bile, hydrogen peroxide (H2O2) production, and adhesion to vaginal epithelial cells (VECs) and to blood group antigens. As a result, various of the tested lactobacilli strains were determined to be suitable for gastrointestinal or vaginal applications. Commercial strains grew better than the newly isolated strains, but tolerance to acid was a common property among all tested strains. Tolerance to bile varied considerably between the strains. Resistance to bile and acid correlated well, as did VEC adhesion and H2O2 production, but H2O2 production was not associated with resistance to bile or acid. Except for L. iners strains, vaginal isolates had better overall VEC adhesion and higher H2O2 production. Species- and strain-specific differences were evident for all parameters. Rank-ordered clustering with nine clusters was used to identify strains that were suitable for gastrointestinal or vaginal health, demonstrating that the categorization of strains for targeted health indications is possible based on the parameters that were measured in this study.

Selective Utilization of the Human Milk Oligosaccharides 2'-Fucosyllactose, 3-Fucosyllactose, and Difucosyllactose by Various Probiotic and Pathogenic Bacteria.

Prebiotic human milk oligosaccharides (HMOs) are found in human milk, which are not digested by infants but are metabolized by beneficial gut bacteria. We determined the ability of 57 bacterial strains within the Family Lactobacillaceae and genera Bifidobacterium and Bacteroides and potentially pathogenic bacteria to ferment the HMOs 2'-fucosyllactose, 3-fucosyllactose, and difucosyllactose. In addition, prebiotic galacto-oligosaccharides (GOS), lactose, fucose, and glucose were evaluated as carbon sources for these bacterial strains. Bacterial growth was monitored using the automatic Bioscreen C system. Only certain bifidobacteria, such as Bifidobacterium longum subsp. infantis and Bifidobacterium bifidum, as well as Bacteroides fragilis, Bacteroides vulgatus, and Bacteroides thetaiotaomicron utilized the studied HMOs as their sole carbon source, whereas almost all studied bacterial strains were able to utilize GOS, lactose, and glucose. The selectivity in utilization of HMOs by only certain bacteria can be advantageous by promoting beneficial microbes but not supporting the harmful pathogens in contrast to other less selective prebiotics.

Effect of Phytase on in Vitro Hydrolysis of Phytate and the Formation of myo-Inositol Phosphate Esters in Various Feed Materials.

Phytase is commonly used as a feed enzyme in monogastric animals to increase the bioavailability of phytate phosphorus and other nutrients. The accumulation of myo-inositol phosphate intermediates during phytate degradation in various segments of the gastrointestinal tract (GIT) is poorly understood. The aim of this study was to determine the efficacy of Buttiauxella spp. phytase in degrading the phytate in corn, soybean meal, and complete corn-soybean meal diet to myo-inositol phosphate esters (IP1-IP5) and completely dephosphorylated myo-inositol rings using an in vitro model of the poultry upper GIT. Our results show that the phytase hydrolyzes phytate efficiently to small IP esters, whereas the myo-inositol level remains constant between control and phytase treatments. Although the in vitro digestion model does not incorporate all factors that govern phytate hydrolysis, it is a valuable tool for evaluating phytase efficacy at various enzyme doses and with different feed ingredients.

The effect of 2'-fucosyllactose on simulated infant gut microbiome and metabolites; a pilot study in comparison to GOS and lactose.

Human milk oligosaccharides (HMOs) shape gut microbiota during infancy by acting as fermentable energy source. Using a semi-continuous colon simulator, effect of an HMO, 2'-fucosyllactose (2'-FL), on composition of the infant microbiota and microbial metabolites was evaluated in comparison to galacto-oligosaccharide (GOS) and lactose and control without additional carbon source. Data was analysed according to faecal sample donor feeding type: breast-fed (BF) or formula-fed (FF), and to rate of 2'-FL fermentation: fast or slow. Variation was found between the simulations in the ability to utilise 2'-FL. The predominant phyla regulated by 2'-FL, GOS and lactose were significant increase in Firmicutes, numerical in Actinobacteria, and numerical decrease in Proteobacteria compared to control. Verrucomicrobia increased in FF accounted for Akkermansia, whereas in fast-fermenting simulations Actinobacteria increased with trend for higher Bifidobacterium, and Proteobacteria decrease accounted for Enterobacteriaceae. Short-chain fatty acids and lactic acid with 2'-FL were produced in intermediate levels being between ones generated by the control and GOS or lactose. In 2'-FL fast-fermenting group, acetic acid specifically increased with 2'-FL, whereas lactose and GOS also increased lactic acid. The results highlight specificity of 2'-FL as energy source for only certain microbes over GOS and lactose in the simulated gut model.

High-Fat Diet, Betaine, and Polydextrose Induce Changes in Adipose Tissue Inflammation and Metabolism in C57BL/6J Mice.

SCOPE: High-fat diets are a likely cause of low-grade inflammation and obesity-related pathologies. This study measures the effects of a high-fat diet, in combination with two dietary supplements-betaine and polydextrose-on metabolism and inflammation in the adipose tissue of diet-induced obese mice. METHODS AND RESULTS: Forty male C57BL/6J mice are fed a high-fat diet for 8 weeks and compared with low-fat-diet-fed control animals (n = 10). For the last 4 weeks, the high-fat-diet-fed animals are supplemented with 1% betaine, 3.33% polydextrose, their combination, or plain water. Fat depots from subcutaneous and visceral adipose tissue are analyzed for inflammatory markers and nontargeted metabolomics by quantitative PCR and LC-QTOF-MS. The high-fat diet significantly increases adipose tissue inflammation in both fat depots. By metabolic profiling, clear differences are noted between low-fat-diet and high-fat-diet groups with regard to the levels of several metabolite species-primarily carnitines, lipids, and amino acids. Dietary betaine mitigates the high-fat-diet-induced IL-6 expression and significantly increases betaine and butyrobetaine levels in adipose tissue. CONCLUSIONS: The high-fat diet induces patent changes in carnitine and lipid metabolism in adipose tissue. Betaine supplementation elevates the levels of betaine and its derivatives and certain carnitine species, as reported in muscle and liver, and moderately reduces inflammation.

Increased HSF1 expression predicts shorter disease-specific survival of prostate cancer patients following radical prostatectomy.

Prostate cancer is a highly heterogeneous disease and the clinical outcome is varying. While current prognostic tools are regarded insufficient, there is a critical need for markers that would aid prognostication and patient risk-stratification. Heat shock transcription factor 1 (HSF1) is crucial for cellular homeostasis, but also a driver of oncogenesis. The clinical relevance of HSF1 in prostate cancer is, however, unknown. Here, we identified HSF1 as a potential biomarker in mRNA expression datasets on prostate cancer. Clinical validation was performed on tissue microarrays from independent cohorts: one constructed from radical prostatectomies from 478 patients with long term follow-up, and another comprising of regionally advanced to distant metastatic samples. Associations with clinical variables and disease outcomes were investigated. Increased nuclear HSF1 expression correlated with disease advancement and aggressiveness and was, independently from established clinicopathological variables, predictive of both early initiation of secondary therapy and poor disease-specific survival. In a joint model with the clinical Cancer of the Prostate Risk Assessment post-Surgical (CAPRA-S) score, nuclear HSF1 remained a predictive factor of shortened disease-specific survival. The results suggest that nuclear HSF1 expression could serve as a novel prognostic marker for patient risk-stratification on disease progression and survival after radical prostatectomy.

A high-content image analysis approach for quantitative measurements of chemosensitivity in patient-derived tumor microtissues.

Organotypic, three-dimensional (3D) cancer models have enabled investigations of complex microtissues in increasingly realistic conditions. However, a drawback of these advanced models remains the poor biological relevance of cancer cell lines, while higher clinical significance would be obtainable with patient-derived cell cultures. Here, we describe the generation and data analysis of 3D microtissue models from patient-derived xenografts (PDX) of non-small cell lung carcinoma (NSCLC). Standard of care anti-cancer drugs were applied and the altered multicellular morphologies were captured by confocal microscopy, followed by automated image analyses to quantitatively measure phenotypic features for high-content chemosensitivity tests. The obtained image data were thresholded using a local entropy filter after which the image foreground was split into local regions, for a supervised classification into tumor or fibroblast cell types. Robust statistical methods were applied to evaluate treatment effects on growth and morphology. Both novel and existing computational approaches were compared at each step, while prioritizing high experimental throughput. Docetaxel was found to be the most effective drug that blocked both tumor growth and invasion. These effects were also validated in PDX tumors in vivo. Our research opens new avenues for high-content drug screening based on patient-derived cell cultures, and for personalized chemosensitivity testing.

Global tests for novelty.

Outlier detection covers the wide range of methods aiming at identifying observations that are considered unusual. Novelty detection, on the other hand, seeks observations among newly generated test data that are exceptional compared with previously observed training data. In many applications, the general existence of novelty is of more interest than identifying the individual novel observations. For instance, in high-throughput cancer treatment screening experiments, it is meaningful to test whether any new treatment effects are seen compared with existing compounds. Here, we present hypothesis tests for such global level novelty. The problem is approached through a set of very general assumptions, making it innovative in relation to the current literature. We introduce test statistics capable of detecting novelty. They operate on local neighborhoods and their null distribution is obtained by the permutation principle. We show that they are valid and able to find different types of novelty, e.g. location and scale alternatives. The performance of the methods is assessed with simulations and with applications to real data sets.

Optimization of Invasion-Specific Effects of Betulin Derivatives on Prostate Cancer Cells through Lead Development.

The anti-invasive and anti-proliferative effects of betulins and abietane derivatives was systematically tested using an organotypic model system of advanced, castration-resistant prostate cancers. A preliminary screen of the initial set of 93 compounds was performed in two-dimensional (2D) growth conditions using non-transformed prostate epithelial cells (EP156T), an androgen-sensitive prostate cancer cell line (LNCaP), and the castration-resistant, highly invasive cell line PC-3. The 25 most promising compounds were all betulin derivatives. These were selected for a focused secondary screen in three-dimensional (3D) growth conditions, with the goal to identify the most effective and specific anti-invasive compounds. Additional sensitivity and cytotoxicity tests were then performed using an extended cell line panel. The effects of these compounds on cell cycle progression, mitosis, proliferation and unspecific cytotoxicity, versus their ability to specifically interfere with cell motility and tumor cell invasion was addressed. To identify potential mechanisms of action and likely compound targets, multiplex profiling of compound effects on a panel of 43 human protein kinases was performed. These target de-convolution studies, combined with the phenotypic analyses of multicellular organoids in 3D models, revealed specific inhibition of AKT signaling linked to effects on the organization of the actin cytoskeleton as the most likely driver of altered cell morphology and motility.

Quantification of dynamic morphological drug responses in 3D organotypic cell cultures by automated image analysis.

Glandular epithelial cells differentiate into complex multicellular or acinar structures, when embedded in three-dimensional (3D) extracellular matrix. The spectrum of different multicellular morphologies formed in 3D is a sensitive indicator for the differentiation potential of normal, non-transformed cells compared to different stages of malignant progression. In addition, single cells or cell aggregates may actively invade the matrix, utilizing epithelial, mesenchymal or mixed modes of motility. Dynamic phenotypic changes involved in 3D tumor cell invasion are sensitive to specific small-molecule inhibitors that target the actin cytoskeleton. We have used a panel of inhibitors to demonstrate the power of automated image analysis as a phenotypic or morphometric readout in cell-based assays. We introduce a streamlined stand-alone software solution that supports large-scale high-content screens, based on complex and organotypic cultures. AMIDA (Automated Morphometric Image Data Analysis) allows quantitative measurements of large numbers of images and structures, with a multitude of different spheroid shapes, sizes, and textures. AMIDA supports an automated workflow, and can be combined with quality control and statistical tools for data interpretation and visualization. We have used a representative panel of 12 prostate and breast cancer lines that display a broad spectrum of different spheroid morphologies and modes of invasion, challenged by a library of 19 direct or indirect modulators of the actin cytoskeleton which induce systematic changes in spheroid morphology and differentiation versus invasion. These results were independently validated by 2D proliferation, apoptosis and cell motility assays. We identified three drugs that primarily attenuated the invasion and formation of invasive processes in 3D, without affecting proliferation or apoptosis. Two of these compounds block Rac signalling, one affects cellular cAMP/cGMP accumulation. Our approach supports the growing needs for user-friendly, straightforward solutions that facilitate large-scale, cell-based 3D assays in basic research, drug discovery, and target validation.

Low cdc27 and high securin expression predict short survival for breast cancer patients.

Cell cycle regulators cdc27 and securin participate in control of the mitotic checkpoint and survey the mitotic spindle to maintain chromosomal integrity. This is achieved by their functions in metaphase-anaphase transition, DNA damage repair, enhancement of mitotic arrest and apoptosis. We report on the roles of cdc27 and securin in aneuploidy and prognosis of breast cancer. The study comprises 429 breast cancer patients with up to 22 years of follow-up. DNA content was determined by image cytometry, and immunopositivity for cdc27 and securin was based on tissue microarrays. An inverse association between cdc27 and securin expression was observed in both image cytometric and immunohistochemical analyses. Low cdc27 and high securin expression identified patients with significant difference in disease outcome. Cdc27 and securin immunoexpression identified patients at risk of early cancer death within five years from diagnosis. In multivariate analysis, the combination of cdc27 and securin immunohistochemistry was the strongest predictor of cancer death after lymph node status. We demonstrate, for the first time in human breast cancer, the prognostic value of cdc27 and securin immunohistochemistry. Cdc27 and securin appear promising biomarkers for applications in predicting disease progression, prognostication of individual patients and potential in anti-mitotic drug development.