Microbiome, probiotics and neurodegenerative diseases: deciphering the gut brain axis.

The gut microbiota is essential to health and has recently become a target for live bacterial cell biotherapies for various chronic diseases including metabolic syndrome, diabetes, obesity and neurodegenerative disease. Probiotic biotherapies are known to create a healthy gut environment by balancing bacterial populations and promoting their favorable metabolic action. The microbiota and its respective metabolites communicate to the host through a series of biochemical and functional links thereby affecting host homeostasis and health. In particular, the gastrointestinal tract communicates with the central nervous system through the gut-brain axis to support neuronal development and maintenance while gut dysbiosis manifests in neurological disease. There are three basic mechanisms that mediate the communication between the gut and the brain: direct neuronal communication, endocrine signaling mediators and the immune system. Together, these systems create a highly integrated molecular communication network that link systemic imbalances with the development of neurodegeneration including insulin regulation, fat metabolism, oxidative markers and immune signaling. Age is a common factor in the development of neurodegenerative disease and probiotics prevent many harmful effects of aging such as decreased neurotransmitter levels, chronic inflammation, oxidative stress and apoptosis-all factors that are proven aggravators of neurodegenerative disease. Indeed patients with Parkinson's and Alzheimer's diseases have a high rate of gastrointestinal comorbidities and it has be proposed by some the management of the gut microbiota may prevent or alleviate the symptoms of these chronic diseases.

Design and validation of an orally administrated active L. fermentum-L. acidophilus probiotic formulation using colorectal cancer Apc Min/+ mouse model.

Probiotics have been shown to have beneficial properties in attenuating the risk of colorectal cancer (CRC) development. However, functional evidence to support such effects for some probiotic bacteria are relatively unknown. Here, we document a significant antioxidant, anti-proliferative and pro-apoptotic activities of Lactobacillus acidophilus ATCC 314 and Lactobacillus fermentum NCIMB 5221 on CRC cells, particularly when used in combination (La-Lf). Furthermore, a superior synergistic activity on the inhibition of tumor growth and modulation of cell proliferation and epithelial markers in the Apc Min/+ CRC mouse model was explored, based on the expression levels of Ki-67, E-cadherin, ß-catenin, and cleaved caspase-3 (CC3) proteins. The anti-cancer activity of La-Lf co-culture was significantly enhanced in vitro with significant reduced proliferation (38.8 ± 6.9 %, P = 0.009) and increased apoptosis (413 RUL, P < 0.001) towards cancer cells, as well as significant protection of normal colon cell growth from toxic treatment (18.6 ± 9.8 %, P = 0.001). La-Lf formulation (1010cfu/animal/day) altered aspects of intestinal tumorigenesis by significantly reducing intestinal tumor multiplicity (1.7-fold, P = 0.016) and downregulating cellular proliferation markers, including ß-catenin (P = 0.041) and Ki-67 (P = 0.008). In conclusion, La-Lf showed greater protection against intestinal tumorigenesis supporting a potential use as a biotherapeutic for the prevention of CRC.

Use of artificial cell microcapsule containing thalidomide for treating TNBS-induced Crohn's disease in mice.

In this study, we examined the in-vivo characteristics of a novel microencapsulated thalidomide formulation in a murine model of experimental Crohn's disease. Crohn's disease was induced with a single intra-colonic injection of 120 mg/kg of bodyweight of 2,5,6-trinitrobenzene sulfonic acid (TNBS) dissolved in 30% ethanol in Balb/c mice. Level of tumor necrosis factor alpha (TNF-α), interleukin one beta (IL-1ß), interleukin 6 (IL-6) and nitric oxide (NO) were measured in tissue homogenate. Moreover, myeloperoxidase (MPO) activity was determined to assess the extent of neutrophil infiltration. Dose response study showed that treating the mice with microencapsulated thalidomide (100 mg/kg of bodyweight) for two weeks significantly decreased the degree of intestinal inflammation related to Crohn's disease. Higher and lower doses (0, 25, 50 and 200 mg/kg of bodyweight) did not exhibit comparable effects. The present study validates the success of alginate-poly-L-lysine-alginate (APA) microcapsules containing thalidomide in reducing colonic inflammation, and proposes a potential remedy for Crohn's disease.

Enrichment of Bifidobacterium longum subsp. infantis ATCC 15697 within the human gut microbiota using alginate-poly-L-lysine-alginate microencapsulation oral delivery system: an in vitro analysis using a computer-controlled dynamic human gastrointestinal model.

This study evaluates alginate-poly-L-lysine-alginate Bifidobacterium longum subsp. infantis ATCC 15697-loaded microcapsules to enrich the human gut microbiota. The cell survival of alginate-poly-L-lysine-alginate microencapsulated B. infantis ATCC 15697 in gastric acid, bile, and through human gastrointestinal transit was investigated, as well as the formulation's effect on the gut microbiota. Results show that microencapsulation increases B. infantis ATCC 15697 cell survival at pH1.0 (33.54 ± 2.80% versus <1.00 ± 0.00%), pH1.5 (41.15 ± 2.06% versus <1.00 ± 0.00%), pH2.0 (60.88 ± 1.73% versus 36.01 ± 2.63%), pH3.0 (75.43 ± 1.23% versus 46.30 ± 1.43%), pH4.0 (71.40 ± 2.02% versus 47.75 ± 3.12%) and pH5.0 (73.88 ± 3.79% versus 58.93 ± 2.26%) (p < 0.05). In addition, microencapsulation increases cell survival at 0.5% (76.85 ± 0.80% versus 70.77 ± 0.64%), 1.0% (59.99 ± 0.97% versus 53.47 ± 0.58%) and 2.0% (53.10 ± 1.87% versus 44.59 ± 1.52%) (p < 0.05) (w/v) bile. Finally, daily administration of alginate-poly-L-lysine-alginate microencapsulated B. infantis ATCC 15697 in a human gastrointestinal model induces a significant enrichment of B. infantis within the ascending (184.51 ± 17.30% versus 53.83 ± 17.82%; p < 0.05), transverse (174.79 ± 25.32% versus 73.17 ± 15.30%; p < 0.05) and descending (94.90 ± 25.22% versus 46.37 ± 18.93%; p > 0.05) colonic microbiota.

Effect of orally administered L. fermentum NCIMB 5221 on markers of metabolic syndrome: an in vivo analysis using ZDF rats.

Metabolic syndrome, encompassing type 2 diabetes mellitus and cardiovascular disease, is a growing health concern of industrialized countries. Ferulic acid (FA) is a phenolic acid found in foods normally consumed by humans that has demonstrated antioxidant activity, cholesterol-lowering capabilities, and anti-tumorigenic properties. Select probiotic bacteria, including Lactobacillus fermentum NCIMB 5221, produce FA due to intrinsic ferulic acid esterase activity. The aim of the present research was to investigate a FA-producing probiotic, L. fermentum NCIMB 5221, as a biotherapeutic for metabolic syndrome. The probiotic formulation was administered daily for 8 weeks to Zucker diabetic fatty (ZDF) rats, a model of hyperlipidemia and hyperglycemia. Results show that the probiotic formulation reduced fasting insulin levels and insulin resistance, significantly reduced serum triglycerides (p = 0.016), lowered serum low-density lipoprotein cholesterol levels (p = 0.008), and significantly reduced the atherogenic (p = 0.016) and atherosclerosis (p = 0.012) index as compared to the control animals. In addition, the probiotic formulation significantly increased high-density lipoprotein cholesterol levels (p = 0.041) as compared to the control animals. This research indicates that administration of the FA-producing L. fermentum NCIMB 5221 has the potential to reduce insulin resistance, hyperinsulinemia, hypercholesterolemia, and other markers involved in the pathogenesis of metabolic syndrome. Further studies are required to investigate the human clinical potential of the probiotic formulation in affecting the markers and pathogenesis of metabolic syndrome.

Probiotics for the prevention and treatment of allergies, with an emphasis on mode of delivery and mechanism of action.

Allergy, also termed type I hypersensitivity, is defined as a "disease following a response by the immune system to an otherwise innocuous antigen". The prevalence of allergies is high and escalating, with almost half the populations of North America and Europe having allergies to one or more common environmental antigens. Although rarely life-threatening allergies cause much distress and pose an important economic burden. Recent studies demonstrate the importance of the commensal bacteria of the gastrointestinal tract, termed the microbiota, in stimulating and modulating the immune system. This goes hand-in-hand with the hygiene hypothesis, proposed by Strachan in 1989. With this in mind, the use of pre- and probiotics has gained interest to prevent and treat allergies through modulation of the gut microbiota and the immune system. Probiotics, namely Lactobacilli and Bifidobacteria, are live microorganisms that can be incorporated in the diet in the form of functional foods or dietary supplements to beneficially influence the host. In recent studies, probiotic formulations demonstrated the capability to successfully modulate allergic rhinitis, atopic disorders and food-related allergies. A number of probiotic mechanisms of action are involved in controlling hypersensitivity responses, many of which are still not yet understood. Microencapsulation has gained importance as a device for the oral delivery of probiotic cells and may play an important role in the development of a successful probiotic formulation to treat and prevent allergies. Despite the promising research on probiotic biotherapeutics, further investigations are required to develop a successful therapeutic to treat and prevent allergies.

Development and characterization of chitosan-PEG-TAT nanoparticles for the intracellular delivery of siRNA.

Recently, cell-penetrating peptides have been proposed to translocate antibodies, proteins, and other molecules in targeted drug delivery. The proposed study presents the synthesis and characterization of a peptide-based chitosan nanoparticle for small interfering RNA (siRNA) delivery, in-vitro. Specifically, the synthesis included polyethylene glycol (PEG), a hydrophilic polymer, and trans-activated transcription (TAT) peptide, which were chemically conjugated on the chitosan polymer. The conjugation was achieved using N-Hydroxysuccinimide-PEG-maleimide (heterobifunctional PEG) as a cross-linker, with the bifunctional PEG facilitating the amidation reaction through its N-Hydroxysuccinimide group and reacting with the amines on chitosan. At the other end of PEG, the maleimide group was chemically conjugated with the cysteine-modified TAT peptide. The degree of substitution on chitosan with PEG and on PEG with TAT was confirmed using colorimetric assays. The resultant polymer was used to form nanoparticles complexing siRNA, which were then characterized for particle size, morphology, cellular uptake, and cytotoxicity. The nanoparticles were tested in-vitro on mouse neuroblastoma cells (Neuro2a). Particle size and surface charge were characterized and an optimal pH condition and PEG molecular weight were determined to form sterically stable nanoparticles. Results indicate 7.5% of the amines in chitosan polymer were conjugated to the PEG and complete conjugation of TAT peptide was observed on the synthesized PEGylated chitosan polymer. Compared with unmodified chitosan nanoparticles, the nanoparticles formed at pH 6 were monodispersed and of <100 nm in size, exhibiting maximum cell transfection ability and very low cytotoxicity. Thus, this research may be of significance in translocating biotherapeutic molecules for intracellular delivery applications.

Probiotics in colorectal cancer (CRC) with emphasis on mechanisms of action and current perspectives.

Colorectal cancer (CRC) is the third most common form of cancer. Diverse therapies such as chemotherapy, immunotherapy and radiation have shown beneficial effects, but are limited because of their safety and toxicity. Probiotic formulations have shown great promise in CRC as preventive and early stage therapeutics. This review highlights the importance of a balanced intestinal microbiota and summarizes the recent developments in probiotics for treating CRC. Specifically, this report describes evidence of the role of probiotics in modulating the microbiota, in improving the physico-chemical conditions of the gut and in reducing oxidative stress. It also discusses the mechanisms of probiotics in inhibiting tumour progression, in producing anticancer compounds and in modulating the host immune response. Even though some of these effects were observed in several clinical trials, when probiotic formulations were used as a supplement to CRC therapies, the application of probiotics as biotherapeutics against CRC still needs further investigation.

Effect of probiotics Lactobacillus and Bifidobacterium on gut-derived lipopolysaccharides and inflammatory cytokines: an in vitro study using a human colonic microbiota model.

Gut-derived lipopolysaccharides (LPS) are critical to the development and progression of chronic low-grade inflammation and metabolic diseases. In this study, the effects of probiotics Lactobacillus and Bifidobacterium on gut-derived lipopolysaccharide and inflammatory cytokine concentrations were evaluated using a human colonic microbiota model. Lactobacillus reuteri, L. rhamnosus, L. plantarum, Bifidobacterium animalis, B. bifidum, B. longum, and B. longum subsp. infantis were identified from the literature for their anti-inflammatory potential. Each bacterial culture was administered daily to a human colonic microbiota model during 14 days. Colonic lipopolysaccharides, and Gram-positive and negative bacteria were quantified. RAW 264.7 macrophage cells were stimulated with supernatant from the human colonic microbiota model. Concentrations of TNF-alpha, IL-1beta, and IL-4 cytokines were measured. Lipopolysaccharide concentrations were significantly reduced with the administration of B. bifidum (-46.45 +/- 5.65%), L. rhamnosus (-30.40 +/- 5.08%), B. longum (-42.50 +/- 1.28%), and B. longum subsp. infantis (-68.85 +/- 5.32%) (p < 0.05). Cell counts of Gram-negative and positive bacteria were distinctly affected by the probiotic administered. There was a probiotic strain-specific effect on immunomodulatory responses of RAW 264.7 macrophage cells. B. longum subsp. infantis demonstrated higher capacities to reduce TNF-alpha concentrations (-69.41 +/- 2.78%; p < 0.05) and to increase IL-4 concentrations (+16.50 +/- 0.59%; p < 0.05). Colonic lipopolysaccharides were significantly correlated with TNF-alpha and IL-1beta concentrations (p < 0.05). These findings suggest that specific probiotic bacteria, such as B. longum subsp. infantis, might decrease colonic lipopolysaccharide concentrations, which might reduce the proinflammatory tone. This study has noteworthy applications in the field of biotherapeutics for the prevention and/or treatment of inflammatory and metabolic diseases.

Microencapsulation for the Therapeutic Delivery of Drugs, Live Mammalian and Bacterial Cells, and Other Biopharmaceutics: Current Status and Future Directions.

Microencapsulation is a technology that has shown significant promise in biotherapeutics, and other applications. It has been proven useful in the immobilization of drugs, live mammalian and bacterial cells and other cells, and other biopharmaceutics molecules, as it can provide material structuration, protection of the enclosed product, and controlled release of the encapsulated contents, all of which can ensure efficient and safe therapeutic effects. This paper is a comprehensive review of microencapsulation and its latest developments in the field. It provides a comprehensive overview of the technology and primary goals of microencapsulation and discusses various processes and techniques involved in microencapsulation including physical, chemical, physicochemical, and other methods involved. It also summarizes the state-of-the-art successes of microencapsulation, specifically with regard to the encapsulation of microorganisms, mammalian cells, drugs, and other biopharmaceutics in various diseases. The limitations and future directions of microencapsulation technologies are also discussed.

Probiotic Ferulic Acid Esterase Active Lactobacillus fermentum NCIMB 5221 APA Microcapsules for Oral Delivery: Preparation and in Vitro Characterization.

Probiotics possess potential therapeutic and preventative effects for various diseases and metabolic disorders. One important limitation for the oral delivery of probiotics is the harsh conditions of the upper gastrointestinal tract (GIT) which challenge bacterial viability and activity. One proposed method to surpass this obstacle is the use of microencapsulation to improve the delivery of bacterial cells to the lower GIT. The aim of this study is to use alginate-poly-L-lysine-alginate (APA) microcapsules to encapsulate Lactobacillus fermentum NCIMB 5221 and characterize its enzymatic activity and viability through a simulated GIT. This specific strain, in previous research, was characterized for its inherent ferulic acid esterase (FAE) activity which could prove beneficial in the development of a therapeutic for the treatment and prevention of cancers and metabolic disorders. Our findings demonstrate that the APA microcapsule does not slow the mass transfer of substrate into and that of the FA product out of the microcapsule, while also not impairing bacterial cell viability. The use of simulated gastrointestinal conditions led to a significant 2.5 log difference in viability between the free (1.10 × 104 ± 1.00 × 103 cfu/mL) and the microencapsulated (5.50 × 106 ± 1.00 × 105 cfu/mL) L. fermentum NCIMB 5221 following exposure. The work presented here suggests that APA microencapsulation can be used as an effective oral delivery method for L. fermentum NCIMB 5221, a FAE-active probiotic strain.