Docket 92037-A-PCT/JPW/PT/AWG POLYMERIC CARRIER FOR PROBIOTICS [0001] This application claims the benefit of U.S. Provisional Application No. 63/381,741, filed October 31, 2022, the content of which is hereby incorporated by reference. [0002] Throughout this application, various publications are referenced, including referenced in parenthesis. The disclosures of all publications mentioned in this application in their entireties are hereby incorporated by reference into this application in order to provide additional description of the art to which this invention pertains and of the features in the art which can be employed with this invention. TECHNICAL FIELDS [0003] This application relates to novel probiotic delivery devices for reducing respiratory inflammation, infection, and exacerbations in COPD patients. The natural activity of probiotics against specific respiratory pathogens is put to work in a device in which these probiotics and their active compounds are released in a controlled way. BACKGROUND OF THE INVENTION [0004] Chronic Obstructive Pulmonary Disease (COPD) is a prevalent, progressive, often fatal disease associated with advanced chronic inflammation of the respiratory tract that results in airflow limitation in the lungs and concomitant small airway obstruction. With 3.2 million fatalities reported worldwide in 2015 ¹ , COPD is currently the third leading cause of death in the U.S., behind heart disease and cancer ² . The standard of care, including long-acting bronchodilators and inhaled corticosteroids, relieves symptoms through smooth muscle relaxation mechanisms, yet fails to reverse underlying chronic tissue inflammation and fibrosis ³ , and also fails to mitigate and treat exacerbations related to respiratory infections. In fact, inhaled steroids have been found to increase risk for pneumonia ^4-5 , which is a concern in the COPD population. Given this backdrop, COPD treatments targeting both chronic inflammation and infection are needed to improve the outcomes and quality of life for COPD patients. BRIEF SUMMARY [0005] In an embodiment, there is provided a respiratory tract insertable device comprising a mesh that carries a probiotic composition. [0006] There is also provided a method of treating or preventing chronic obstructive pulmonary disease (COPD) or a respiratory infection, the method comprising having a subject suffering from, or at risk of developing, COPD use the device. [0007] In another embodiment, there is provided a method of treating or preventing COPD or a respiratory infection, the method comprising controlled intra-oral release of a probiotic composition, or molecules released from the probiotic composition, to the respiratory tract in an effective amount to treat or prevent the COPD or respiratory infection in a human subject. BRIEF DESCRIPTION OF THE DRAWINGS [0008] Fig. 1: Research approach diagram - Inhibition of pathogen establishment and regulation of microbiome composition in the lung using novel probiotic scaffold system applied directly into the oral cavity. [0009] Figs.2A-2B: Dose dependent zone clearing of Haemophilus influenzae with Bacillus coagulans. Haemophilus influenzae was streaked onto chocolate agar, membranes loaded with increasing concentration of probiotics were applied to the streaked plates and incubated overnight at 37°C. (n=3, 50µl *: p<0.05) (Fig. 2A). Quantification of the zones of inhibition are shown in Fig.2B. [0010] Figs. 3A-3C: Mechanism of Probiotic Action via Biofilm Disruption. Fig. 3A - Biofilm formation/liftoff. Fig. 3B - P. aeruginosa biofilms grown in 96-well microtiter plate in quadruplicate pretreated with increasing concentrations of probiotics (top to bottom). Bottom arrow: biofilm, top arrows: dispersed cells. Fig. 3C - Pseudomonas aeruginosa reduction due to increasing concentrations of pretreatment with probiotics shown on the. O.D. readings of microtiter-well contents at 540 nm (n=3, *: p<0.05). [0011] Figs.4A-4C: Probiotic Growth on Nano-Mesh. Fig.4A - Mesh readily conformed to 3D-printed human tooth model (mesh diameter = 559±258 nm, n=40, SEM). Probiotics (Lactobacillus acidophilus & Bifidobacterium lactis) grown on the mesh remained viable over time (Live/Dead at 24 and 48 hrs, green=live, red=dead). Also shown is agar control. Fig.4B - Probiotic viability (n=5, *:p<0.05) was quantified by detecting the fluorescence at wavelength 485–535nm (SYTO9/Live Dye) and 485–635nm (propidium iodide/Dead Dye. For control, mesh is placed atop the agar but without probiotic pre-seeding. Fig. 4C - The ratio of the Live/Dead ratio was maintained on the mesh (n=5), reflecting microbe life cycle. [0012] Fig. 5: Illustration of transport mechanisms and steady state measurement of mass transfer coefficient in flow. An example equation which can be used to determine the transport of peptides and cells released from the scaffold is provided below: [0013] Figs.6A-6B: Flow cell used for the evaluation of release of metabolites and life cycle of the probiotic scaffolds. The flow cell is sealed with a glass slide and tested with dye tracers under laminar flow at 2 mL/min for characterization purposes (Fig. 6A). Blue shows a bolus release of blue dextran while yellow shows a controlled steady state release of tartrazine formulated in microcrystalline cellulose, as measured visually and by UV-VIS spectroscopy (Fig.6B). [0014] Figs.7A-7D: Dose dependent zone clearing of Haemophilus influenzae (at increasing concentrations from left to right: 25 uL, 50 uL, 100 uL) with membranes containing Bacillus coagulans on both TSA (top) and chocolate agar (bottom) plates (Fig. 7A). A duplicate experimental set shown is shown in Fig. 7B. Results of probiotics (increasing concentration from left to right) on growth of microorganism on chocolate agar (Fig. 7C). The different colored columns correspond to the various concentrations of H. influenzae treated with probiotic. Results of probiotics (increasing concentration from left to right) on growth of microorganism on TSA plates (Fig. 7D). The different colored columns correspond to the various concentrations of H. influenzae treated with probiotic. [0015] Fig. 8: Process development diagram for characterization of bacteriocins from Bacillus coagulans for applications in COPD. [0016] Fig.9: OD600 of Bacillus coagulans culture over time. Visible cell number increases over time. [0017] Fig.10: pH of Bacillus coagulans over time. pH value decreases over time [0018] Fig.11: Standard Nisin LC-MS results. [0019] Fig.12: Bacillus coagulans sample cation-exchange results. [0020] Fig.13: Bacillus coagulans sample Columbia University LC-MS results. [0021] Fig.14: Bacillus coagulans sample Touro School LC-MS results. [0022] Fig. 15: Bacillus coagulans sample Columbia University LC-MS results zoom in at 2.31 min. [0023] Fig.16: Bacillus coagulans sample Touro School LC-MS results zoom in at 2.31 min. DETAILED DESCRIPTION [0024] In order to facilitate an understanding of the subject matter disclosed herein, each of the following terms, as used herein, shall have the meaning set forth below, except as expressly provided otherwise herein. [0025] As used herein, “respiratory tract” includes all the elements of the upper respiratory tract and lower respiratory tract, including the nose, nasal cavity, pharynx, larynx, trachea, bronchi, and lungs. [0026] As used herein, “respiratory tract insertable device” shall mean a device that is able to be inserted and maintained in the respiratory tract of a subject on the order of days to hours. For example, a respiratory tract insertable device includes, but is not limited to, a device that is able to be maintained in the oral cavity, nasal cavity, or trachea of a subject. An example of respiratory tract insertable device is an orally wearable device, such as a mouthguard. [0027] As used herein, all numerical ranges provided are intended to expressly include at least the endpoints and all numbers that fall between the endpoints of ranges. [0028] The use of probiotics for the treatment of medical conditions including CODP, respiratory infections, and also for the maintenance of both GI and oral health, is driven on the one hand by an interest in understanding and leveraging the role of microbiome regulation in human health, and on the other, by documented clinical benefits. Probiotics are active against respiratory pathogens and inflammation, regulate microbiome composition in vitro and in vivo, and may be formulated and delivered intraorally via controlled release devices. [0029] This application discloses a device designed to deliver probiotics and their signaling molecules to the respiratory tract of a subject in order to reduce inflammation in the lung tissue and to prevent respiratory infections and exacerbations related to COPD. In an embodiment, the device is an intraoral device and delivers probiotics intraorally. The delivery of probiotics intraorally stands in direct contrast to ingestion into the stomach and gut and opens up a completely different method of delivery as well as mechanisms of action. In a preferred embodiment, the selected probiotics are based on their activity against common respiratory pathogens. [0030] The premise of one of the novel treatment approaches to COPD is the connection between the oral, nasal, and lung microbiomes, and epidemiological studies that collectively suggest a strong association between oral health and COPD conditions ^6-7 . The upper airways comprise a continuous surface that terminates in the lungs through which air continuously passes in and out via the oral and nasal cavities; this air flow combined with both microaspiration and gravity flow of mucus and saliva downwards is the reason the lower airway is seeded with microbes from the oral and nasal cavities ^8-9 . As the described devices are intended to be worn on the order of hours or days, some level of direct exposure to the lungs will be possible, for both the probiotics as well as their metabolites and signaling molecules. [0031] Notably, clinical success has been observed based on probiotic use, for example, in human fecal microbiota transplantation ¹⁰ , the complete elimination of S. aureus by Bacillus in humans via quorum sensing ¹¹ , and burn wound healing in rats using probiotics in combination with a collagen hydrogel scaffold ¹² . There are also instances of probiotics delivered intranasally offering prophylaxis against respiratory infection in mice ¹³ , whilst delivery to the gut in humans has been associated with more systemic reduction of inflammation, presumably by modulating inflammation response through immune cell release of anti- and pro-inflammatory cytokines ^14-16 . [0032] This technology demonstrates that Lactobacillus acidophilus & Bifidobacterium lactis probiotic strains isolated from ingestible probiotic tablets, can be grown onto mesh of electrospun PCL via a seeding technique and were successfully migrated from agar to the mesh. Of note is that the mesh pore size is such that the microbes can become established inside the mesh as well as on the exterior. Confocal fluorescence microscopy shows that these colonies establish themselves in various morphologies. The PCL is attractive because it has robust mechanical properties, long shelf life, and has the flexibility and character of a fabric that can adopt various shapes and configurations. These matrices are flexible in that nutrients, adjuvants, and also other drugs and agents may be introduced to facilitate the growth of the probiotics. The use of probiotics delivered to the oral cavity to treat respiratory disease has not been well studied, and this disclosure describes developing a scaffold and delivery technology, as well as the selection of appropriate microorganisms, for this purpose. [0033] The following embodiments and examples (including details thereof) are set forth to aid in an understanding of the subject matter of this disclosure but are not intended to, and should not be construed to, limit in any way the invention that is claimed. [0034] In this disclosure, a respiratory tract insertable device comprising a scaffold that carries a probiotic composition is described. In some embodiments, the scaffold comprises a mesh, film, and/or a gel. [0035] In this disclosure, a respiratory tract insertable device comprising a mesh that carries a probiotic composition is described. [0036] In some embodiments, the mesh comprises polylactide-co-glycolide (PLGA) and/or poly-e-caprolactone (PCL). [0037] In some embodiments, the mesh comprises electrospun polylactide-co-glycolide (PLGA) and/or poly-e-caprolactone (PCL). [0038] In some embodiments, the mesh comprises 5:1 PLGA / PCL. [0039] In some embodiments, the mesh comprises fibers, wherein each fiber having a diameter between 200-900 nm, more preferably between 300-800 nm, more preferably between 500-600 nm. [0040] In some embodiments, the mesh comprises sodium alginate and or collagen. [0041] In some embodiments, the probiotic composition comprises a probiotic microbe culture. [0042] In some embodiments, the probiotic microbe culture comprises any one of Lactobacillus acidophilus, Bifidobacterium lactis, Bacillus coagulans and Saccharomyces boulardii, or a mixture of probiotics selected from the group consisting of Lactobacillus acidophilus, Bifidobacterium lactis, Bacillus coagulans and Saccharomyces boulardii. [0043] In some embodiments, the probiotic composition further comprises a food supply for the probiotic microbe culture. [0044] In some embodiments, the probiotic composition further comprises a nutrient, adjuvant, drug, and/or agent which facilitates growth of the probiotic microbe culture. [0045] In some embodiments, the probiotic microbe culture has a live/dead ratio greater than 2, more preferably greater than 3, more preferably greater than 3.5, more preferably greater than 4. [0046] In some embodiments, the probiotic microbe culture has a live/dead ratio of about 4. [0047] In some embodiments, the probiotic composition further comprises lactic acid. [0048] In some embodiments, the probiotic microbe culture is grown or seeded onto the mesh. [0049] In some embodiments, the device is a an orally wearable device, a mouthguard, or a nasally wearable device. In some embodiments, the device may be inserted into the trachea. [0050] In some embodiments, the mesh is conformed to a 3D-printed human tooth model. [0051] In this disclosure, a method is described for treating or preventing chronic obstructive pulmonary disease (COPD), the method comprising having a subject suffering from or at risk of developing COPD use any one of the respiratory tract insertable devices described herein. [0052] In this disclosure, a method is described for treating or preventing a respiratory infection, the method comprising having a subject suffering from or at risk of developing a respiratory infection use any one of the respiratory tract insertable devices described herein. [0053] In some embodiments, the respiratory infection is caused by any one of Pseudomonas aeruginosa, Moraxella catarrhalis, Haemophilus influenzae, and Streptococcus pneumoniae. [0054] There is also provided any one of the respiratory tract insertable devices described herein for use in treating or preventing chronic obstructive pulmonary disease (COPD), or for use in treating or preventing a respiratory infection. The respiratory tract infection may be caused by, for example, any one of Pseudomonas aeruginosa, Moraxella catarrhalis, Haemophilus influenzae, and Streptococcus pneumoniae. [0055] In some embodiments, treatment or prevention of COPD or a respiratory infection utilizing any one of the respiratory tract insertable devices described herein results in a decrease of inflammatory cytokines present in the respiratory tract. [0056] In some embodiments, the respiratory microbiome composition of a subject is normalized after utilizing any one of the respiratory tract insertable devices described herein. [0057] In some embodiments, any one of the respiratory tract insertable devices described herein is used for a period of about 2, about 4, about 6, about 8, about 10, about 12, about 18, about 24, about 36, or about 48 hours. [0058] In some embodiments, the period of using the device is repeated daily, weekly, or monthly. [0059] There is also provided a method of treating or preventing COPD or a respiratory infection, the method comprising controlled intra-oral release of a probiotic composition, or molecules released from the probiotic composition, to the respiratory tract in an effective amount to treat or prevent the COPD or the respiratory infection in a human subject. [0060] In some embodiments, the probiotic composition comprises one or more of the microbes selected from the group consisting of Lactobacillus acidophilus, Bifidobacterium lactis, Bacillus coagulans, and Saccharomyces boulardii. [0061] In some embodiments, the respiratory infection is caused by any one of Pseudomonas aeruginosa, Moraxella catarrhalis, Haemophilus influenzae, and Streptococcus pneumoniae. [0062] In summary, this technology identifies the use of probiotic delivered through a wearable scaffold mesh to treat or prevent infection from respiratory pathogens. Probiotics, such as Lactobacillus acidophilus and Bifidobacterium lactis, can be isolated from tablets and colonies can be then established on the scaffold mesh. The flexible matrices have robust mechanical properties, a long shelf life, and can allow nutrients, adjuvants and other drugs to facilitate probiotic growth. Characterization of the probiotics and scaffold suggest an optimal 24-hour culture period of the probiotics on the scaffold. Probiotics may prevent infection and reduce inflammation and therefore, probiotic-carrying scaffolds may be useful wearable delivery devices for COPD treatment and prevention. [0063] Various other inventive aspects can be integrated or employed, such as probiotic delivery system to treat other GI tract related diseases in addition to COPD or to improve microbiome composition in a subject. Standard Methods [0064] Standard methods in molecular biology are described Sambrook, Fritsch and Maniatis (1982 & 19892nd Edition, 20013rd Edition) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3 ^rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA, Vol.217, Academic Press, San Diego, CA). Standard methods also appear in Ausbel, et al. (2001) Current Protocols in Molecular Biology, Vols.1-4, John Wiley and Sons, Inc. New York, NY, which describes cloning in bacterial cells and DNA mutagenesis (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugates and protein expression (Vol.3), and bioinformatics (Vol.4). [0065] Methods for protein purification including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization are described (Coligan, et al. (2000) Current Protocols in Protein Science, Vol.1, John Wiley and Sons, Inc., New York). Chemical analysis, chemical modification, post-translational modification, production of fusion proteins, glycosylation of proteins are described (see, e.g., Coligan, et al. (2000) Current Protocols in Protein Science, Vol.2, John Wiley and Sons, Inc., New York; Ausubel, et al. (2001) Current Protocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, NY, pp. 16.0.5- 16.22.17; Sigma-Aldrich, Co. (2001) Products for Life Science Research, St. Louis, MO; pp. 45-89; Amersham Pharmacia Biotech (2001) BioDirectory, Piscataway, N.J., pp. 384-391). Production, purification, and fragmentation of polyclonal and monoclonal antibodies are described (Coligan, et al. (2001) Current Protcols in Immunology, Vol.1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, supra). Standard techniques for characterizing ligand/receptor interactions are available (see, e.g., Coligan, et al. (2001) Current Protocols in Immunology, Vol.4, John Wiley, Inc., New York). Equivalents and Incorporation by Reference [0066] All references cited herein are incorporated by reference to the same extent as if each individual publication, database entry (e.g. Genbank sequences or GeneID entries), patent application, or patent, was specifically and individually indicated to be incorporated by reference in its entirety, for all purposes. This statement of incorporation by reference is intended by Applicants, pursuant to 37 C.F.R. §1.57(b)(1), to relate to each and every individual publication, database entry (e.g. Genbank sequences or GeneID entries), patent application, or patent, each of which is clearly identified in compliance with 37 C.F.R. §1.57(b)(2), even if such citation is not immediately adjacent to a dedicated statement of incorporation by reference. The inclusion of dedicated statements of incorporation by reference, if any, within the specification does not in any way weaken this general statement of incorporation by reference. Citation of the references herein is not intended as an admission that the reference is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. [0067] While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention. EXAMPLES Example 1: Specific Aims and Research Strategy [0068] The first aim of the research plan is to develop intra-oral devices to deliver probiotics and their signaling molecules, including bacteriocins, to the oral cavity and respiratory tract in order to reduce inflammation in the lung tissue and to prevent respiratory infections and flare- ups in COPD patients. The objective is to create and characterize a prototype system comprised of probiotic organisms, a probiotic carrier, and a formulation, and to test the hypothesis that in the oral cavity, this approach may suppresses respiratory pathogens, regulate respiratory microbiome composition ^{9, 17} vs. triggers ¹⁸ , and ultimately, reduce tissue inflammation for COPD in vitro. [0069] The hypothesis is that probiotics are active against respiratory pathogens and inflammation, regulate microbiome composition in vitro and in vivo, and may be formulated and delivered intra-orally via controlled release devices. Fig. 1 shows a graphical representation of this hypothesis with the scaffold as part of a wearable mouthguard. [0070] The aims of the research plan are: ^ Aim 1A - Screening of probiotic strains: Lactobacillus acidophilus, Bifidobacterium lactis, Bacillus coagulans and Saccharomyces boulardii will be screened against the respiratory pathogens Pseudomonas aeruginosa, Moraxella catarrhalis, Haemophilus influenzae, Streptococcus pneumoniae to evaluate the potential of anti-microbial activity and also with inflammatory models to determine anti-inflammatory activity. The selection criteria are 1) significant zone of clearing as measured by a microbial disc assay, and 2) significant reduction in gene expression levels for pro-inflammatory cytokines as measured by microarrays. ^ Aim 1B - Structural chemical characterization: For probiotic candidates that show sufficient activity, structural chemical characterization of their metabolites and signaling molecules will be characterized in the presence and absence of pathogens, using high powered chemical characterization techniques (e.g. LC-MS/ NMR) to create a molecular level understanding of the pathways and metabolite releases. ^ Aim 2A - Scaffold design: Design of controlled-release device elements, e.g. films, gels, or meshes that contain the probiotics, nutrient matrix, and delivery capability enabling these microbes to grow, reproduce, and be established in saliva in a controlled manner, during a time frame on the order of 8 hours. Design criteria include the ability to control the time frame of release, the dose, as well as underlying control of the nutrients and scaffolding on which the probiotics grow and detach. ^ Aim 2B - Transport properties: Characterize pharmaceutically relevant transport properties of the active metabolites and the lifecycle of the probiotics identified in Aim 1B in a representative formulation developed in Aim 2A, in order to determine the release rate and appropriate dose regimen. [0071] The long term impact of this approach is anticipated to be reduction of chronic inflammation in the lungs, protection against respiratory microbes that may enter the lungs via the oral cavity and nose, and finally restoration of the oral microbiome to a healthy condition in those COPD patients who may have also lost some of their teeth and suffer from poor oral microbiome condition and periodontal disease ^6-7 . Significance [0072] The use of probiotics for the treatment of medical conditions including burns ¹² , asthma ¹⁹ , respiratory infections ²⁰ , psoriasis ²¹ , and also for the maintenance of both GI ^22-23 and oral health ²⁴ , is driven on the one hand by an interest in understanding and leveraging the role of microbiome regulation in human health, and on the other, by documented clinical benefits. However, mechanisms of action are often not well understood, nor are treatments controlled or regulated in a traditional pharmaceutical sense. A sensible direction is to develop more controlled and nuanced ways of using probiotics, with an aim of increasing understanding, as well as advancing technology for medical applications. This proposal is significant in that it aims to develop the materials and methods to grow probiotic microorganisms onto scaffolds that may be applied intraorally for the prevention of infection and the reduction of inflammation in COPD patients. Innovation [0073] Cell scaffold technology has advanced such that many cell types can be grown on a diverse set of scaffolds in a predictable manner. Here, a similar approach is attempted for the purpose of growing and delivering probiotic cells into the oral cavity. The use of probiotics delivered to the oral cavity to treat respiratory disease has not been well studied, and this proposal aims to take a first step in this direction by developing the scaffold and delivery technology, as well as the selection of appropriate microorganisms; both of which are needed to progress into animal studies to test the overall hypothesis. This proposal seeks to create tailored probiotic scaffolds for intra-oral delivery, as well to test the hypothesis that this technology can be applied in COPD. Success of this proposal will lead to better ways to utilize probiotics in medicine generally, and also specifically for COPD. Aim 1A – Screening of Probiotic Strains [0074] Our work suggests that probiotics are active against respiratory pathogens and can regulate the microbiome composition in vitro and in vivo, as well as the associated inflammation. Our initial data supporting the reduction of growth of respiratory pathogens was demonstrated with Haemophilus influenzae, a key pathogen implicated in COPD exacerbations ²⁵ . Using a membrane disc assay, a zone of inhibition with the probiotic Bacillus coagulans is evident (Figs. 2A-2B), and is dose responsive. No such response was seen with other probiotics such as Lactobacillus acidophilus, Bifidobacterium lactis or Saccharomyces bouldarii. [0075] Preliminary results evaluating the activity of probiotics on the biofilm life cycle of respiratory pathogens was shown with Pseudomonas aeruginosa, and the data is presented in Figs. 3A-3C. In the presence of previously incubated probiotics Lactobacillus and Bifidobacterium, P. aeruginosa grown in wells pretreated with the probiotics exhibited a significant cell reduction relative to control. As the concentration of probiotics used to pretreat the wells increases, there is clear reduction in not only total biofilm, but particularly in dispersed cells (upper ring in Fig.3B). Experimental Design [0076] The probiotic strains Lactobacillus acidophilus, Bifidobacterium lactis, Bacillus coagulans, and Saccharomyces boulardii will be screened against the respiratory pathogens Pseudomonas aeruginosa, Moraxella catarrhalis, Haemophilus influenzae, and Streptococcus pneumoniae using traditional techniques of microbiology, and also in a tissue inflammation model. The selection criteria are: 1) significant zone of clearing as measured by a microbial disc assay, 2) reduction in dispersed biofilm cells, and 3) significant reduction in gene expression levels for pro-inflammatory cytokines as measured by microarrays. Strains that show the best activity will be selected for further development in the device designs. [0077] Microbial Growth: Probiotic strains will be screened to determine whether probiotics impede the growth of respiratory pathogens. The respiratory pathogens to be screened include those implicated in COPD exacerbation. Membrane discs will be prepared by immersion of discs into probiotic solutions of varying concentrations for 30 minutes at room temperature. After 30 minutes, the discs are air-dried in a sterile petri plate. The discs are then placed on cultures of respiratory pathogens that have been streaked onto the surface of selective media agar plates followed by incubation overnight at 37°C. Effect of probiotic is determined by measuring the clearance distance surrounding the membrane disc. [0078] Biofilm Life Cycle: To observe the effect of probiotics on growth and survival of a biofilm the method described by O’Toole ²⁶ will be used. An overnight culture of a respiratory pathogenic organism will be grown in the presence of varying concentrations of probiotics in a 96-well microtiter plate. Each concentration of probiotic will be evaluated in quadruplicates. The 96-well plate will be incubated at 37°C for a 24-hour period. After 24 hours the contents of the plates will be removed and wells will be rinsed and stained as described ²⁶ . [0079] Anti-inflammatory Mediators: To evaluate probiotics as anti-inflammatory mediators, the pulmonary adenocarcinoma derived cell line A549 will be used. A549 cells will be purchased from Sigma-Aldrich (catalog number 86012804) and cultured in Dulbecco’s modified eagle’s medium with 10% fetal calf serum (FCS), 100 units/mL penicillin and 100 mG/mL streptomycin, under a humidified atmosphere 5% CO2 plus 95% air at 37ºC. [0080] Agonists: To model COPD, cigarette smoke extract (CSE) and lipopolysaccharide (LPS) will be added as described by Nachmias et. al. ²⁷ . Research cigarettes (1RGF) will be obtained from the Kentucky Tobacco Research Center. LPS will be purchased from Sigma- Aldrich Co. St. Louis, MO. Treatments will include 2%, 4% and 10% CSE. LPS will be added to the cultures as described by Victoni et al. to stimulate COPD exacerbation conditions. [0081] Antagonists: We have extensive experience in assaying inflammatory gene expression levels using Affymetrix microarrays. To evaluate the effect of probiotics on inflammation, gene expression profiles will be performed as described Offenbacher et al. ²⁸ . [0082] The in vitro studies will test the potential anti-inflammatory activity of probiotics. Comparison will be made to cells not treated with probiotics. After treatment with or without antagonists, cells will be harvested for mRNA extraction (Qiagen Inc. Germantown, MD). RNA quality will be assessed with a Bioanalyzer 2100. Gene expression profiles will be performed using Affymetrix recommended procedures (Affymetrix Inc. Santa Clara, CA). Gene chip targets will be synthesized from the RNA using Affymetrix target synthesis procedures. Targets will be hybridized to gene chips and scanned using photoluminescence. Affymetrix GeneChip Microarray software will be used for scanning and initial analysis. Aim 1B - Chemical Structural Characterization [0083] This aim is to characterize by structural and chemical analysis the probiotic metabolites and signaling molecules that demonstrated clearing in the membrane disc assay. High powered characterization (e.g. LC-MS/ NMR) as well as requisite purification (electrophoresis/ chromatography) will be used to create a molecular level understanding of the signaling pathways and metabolites released. The objective of this work is to elucidate mechanism of behavior when possible. An example of prior work is the showing that fengycin peptides released by Bacillus are responsible for inhibition of quorum sensing in S. aureus ¹¹ . In this case, RP-HPLC was conducted to fractionate the cyclic peptides before further analysis and quantitation on HPLC-MS and HPLC-MS-MS. Expected Outcomes, Interpretation and Alternative Strategies: [0084] Based upon our preliminary results, identification of a probiotic that confirms our hypothesis that commensal microbes (i.e. probiotics) can impede the growth of respiratory microorganisms implicated in COPD exacerbation, alter the biofilm life cycle of the respiratory microbes, and contribute to the anti-inflammatory response, is expected. We have extensive experience with the oral and respiratory microbiomes. All methods of analysis used in this Aim are well established and routinely used by the Loewy lab and therefore we do not foresee major setbacks in experimental methodologies. Although not described in this proposal, as Aim 1A studies progress, exploring different combinations of probiotics to determine any additive or synergistic effects in pathogen control is possible. [0085] For Aim 1B, lactic acid is expected as a major secondary metabolite from most of the probiotic bacterial strains; however, it is expected that minor, more complex metabolites secreted will be of more interest. Possible pitfalls of this chemical characterization include assay variability, instrument sensitivity and resolution, and reproducibility. These will be overcome by the judicious use of control experiments as well as establishing limits of detection with known signal peptides for both LC-MS and NMR measurements. In addition, scale up of the probiotic cultures will be conducted to generate larger quantities for purification and further experimentation. Aim 2A - Probiotic Carrier Design [0086] Sodium alginate, collagen, and electrospun meshes of polylactide-co-glycolide (PLGA) / poly-e-caprolactone (PCL) have been explored extensively for mammalian cell culture by us and others ^{12, 29-30, 35} , and these may be adapted for use in bacteria culture ^36,37 . Moreover, nutrients, adjuvants, and also other drugs and agents may be introduced to facilitate the growth of the probiotics. The strategy is to select lead probiotic strains (ideally both a yeast and a bacteria) identified in Aim 1A, and then grown them on mesh-based carriers (see Figs. 4A-4C) in a saliva model, compare their response to those grown in alginate hydrogel and answer the question which of these carriers are best suited to which strains, and under what conditions. The viability and growth of probiotics, as well as their ability to kill COPD-related pathogens (Aim 1A) will be measured and compared. [0087] Our results demonstrate that Bacillus coagulans can be grown onto mesh of electrospun PCL via our customized seeding technique and the probiotic can be successfully migrated from agar to the mesh. Briefly, probiotics were cultured in Lennox (LB) agar and broth (9mL, Invitrogen, CA) and enumerated for 24 hours. Prior to probiotic culture, the PLGA (5:1) PCL fibrous meshes (n=5) were immersed in LB broth for 1 hour and pressed onto a 100 mm petri-dish using forceps. After which 20 μL of probiotic containing LB broth was inoculated mesh onto each polymeric mesh and cultured until the time of analysis under aerobic conditions (37 ^o C, 5% CO2). To assess cell viability (n=2), LIVE/DEAD Baclight (Invitrogen, CA) staining was used following the manufacturer’s specification. First the cultured meshes were rinsed in phosphate buffered saline and imaged using confocal microscopy (Olympus Fluoview FV100, Center Valley, PA) at excitation wavelengths of 488 nm and 568 nm (Fig. 4A). Fig. 4A demonstrates the presence of live probiotics on the polymeric meshes in comparison with agar control after 24 hours and 48 hours under culture. To compare the ratio of live and dead probiotics (n=5), fluorescence was detected at excitation and emission wavelengths of 485 and 535 nm for detection of live bacteria. Excitation and emission wavelengths of 485 and 635 nm were used for detection of dead bacteria with a Tecan microplate reader. For measurements, polymeric meshes were immersed in the LIVE/DEAD solution for 15 minutes and 100 μL of the mesh containing solution was added to a 96 well plate (Fig. 4B and Fig. 4C). Fig. 4B shows that there is fluorescence for live probiotics is significantly higher than dead probiotics after 24 and 48 hours under culture, further providing evidence that the substrate supports cell viability. In addition, Fig. 4C shows that the ratio of live versus dead probiotics peaked after 24 hours in culture in comparison to broth control and 48 hours of culturing. Thus suggesting an optimal culture period of 24 hours. [0088] These preliminary data demonstrate that probiotics can be cultured on the electrospun meshes, and most importantly, the probiotics remain viable over time with a constant live/dead ratio on the mesh, attesting to the feasibility of the approach in Aim 2. The PCL mesh is attractive because it has robust mechanical properties, long shelf life, and has the flexibility and character of a fabric that can adopt various shapes and configurations, to be readily applied in the oral cavity. Expected Results, Interpretation, and Alternative Strategies [0089] It is expected that the probiotic organisms will be able to be grown reproducibly on the mesh and in the hydrogel in vitro over an 8-48 hr period, and this will be monitored with cell imaging techniques as well as quantified via fluorometer readings. It is also expected that construction of a fit for purpose wearable device containing the scaffold and probiotics will be straightforward, for the purposes of demonstration in vivo; the development of the device specifics are part of this proposal. One potential pitfall is proteolysis of active peptides in the saliva and this will be assessed using salivary enzymes. Looking ahead, an animal study would logically follow, but this proposal is focused primarily on the design components and physical chemical behavior of the scaffolds and their active microbes in presence and absence of pathogens in vitro. Aim 2B - Transport Properties: [0090] The release characteristics, life cycle of the probiotic cells, and transport properties of the secreted molecules elucidated in Aim 1B will be characterized in representative formulations developed in Aim 2A, as illustrated schematically in Fig. 5. Flow cells such as those shown in Figs. 6A-6B will be used in which the probiotic mesh can be placed, and observed microscopically and evaluated in flow. Key characteristics including release rate, cell lifecycle, mass transfer coefficients, diffusion coefficients, as well as susceptibility to proteolytic salivary enzymes can all be estimated and quantified in a simple flow configuration. These studies will help us to better understand the mechanism of action of probiotics on COPD biofilm formation and elimination, and ultimately help us to optimize the probiotic carrier design. [0091] Statistical Analysis: For quantitative outcomes in Aims 1 and 2, analysis of variance (ANOVAs) will be used followed by Tukey’s post hoc tests (SAS Institute) to determine differences between groups. Sample sizes for the planned studies are chosen based on published studies and our preliminary data. [0092] Project Time Line and Looking Ahead: Once a suitable scaffold material, probiotic strains, and a device design have been thoroughly evaluated in vitro, these findings would be readily adapted to a murine model and tested in vivo for both safety in healthy animals but also for prophylaxis against infection in a COPD model as well as for inflammatory modulation in a COPD model. Such an animal test would be a logical next step of this work. Several murine models of COPD exist ^31-32 as well as methods to introduce animal dentures ^33-34 . Example 2: Effect of Probiotics on Respiratory Pathogens Objectives: [0093] To study if probiotics can inhibit the growth of respiratory pathogens and thereby prevent COPD exacerbations. [0094] To determine whether the effects of probiotics at inhibiting the growth of pathogenic microorganisms are dose dependent. [0095] To find a material that will allow growth of probiotics and have the capacity to release the probiotics in a controlled release manner. Introduction [0096] Patients with COPD are more prone to respiratory infections by organisms like Haemophilus influenzae- the most common pathogenic microorganism found when sputum of patients who were experiencing COPD exacerbations were cultured. ³⁸ The goal of treatment of exacerbations and inflammation of the lungs is to reduce the negative symptoms associated with the current exacerbation and to prevent future exacerbations.¹ A novel approach is warranted for treatment due to the shortcomings of current treatment options, such as antibiotic resistance. ³⁹ Natural attempts to eradicate the pathogen would be useful to treat these exacerbations and thereby improve patient outcomes. Experimental Procedure Overview [0097] Grow Media: ^ Prepare chocolate agar and TSA plates ^ Dispense the following concentrations of H. influenzae onto the plates: 25 µl, 50 µl, or 100 µl. [0098] Dissolve Probiotics: ^ Dissolve Bacillus coagulans in sterile water at various concentrations: o (Control) 6 mL sterile water o (A) 4 billion cells BC / 6 mL o (B) 8 billion cells BC / 6 mL o (C) 12 billion cells BC / 6 mL [0099] Incubate: ^ Place membranes in each probiotic solution prepared in previous step for 30 minutes then air dry for 30 minutes ^ After incubation, place membranes on corresponding sections of the plates ^ Incubate plates at 37ºC overnight Experimental Procedures [00100] Effect of Probiotic on Growth of H. influenzae Cells: Through many studies, probiotics have demonstrated the ability to diminish the growth and effects of certain bacteria. The Kirby-Bauer disc diffusion was utilized in this experiment to observe probiotic susceptibility against the pathogen H. influenzae which is thought to contribute to exacerbations in patients with respiratory infections. Discs containing various concentrations of B. coagulans were applied on both TSA and chocolate agar plates. They were incubated overnight. Susceptibility of the cells to the probiotic was determined by the presence of inhibition zones around the bacteria. The expectation is to see larger zones of clearance from discs that were submerged in solutions of probiotics of higher concentrations. While we expect to see similar trends in the inhibition of growth of the pathogenic bacteria between the TSA and chocolate agar plates, the results from the chocolate agar plates are expected to be more accurate because chocolate agar is better suited for the growth of fastidious organisms. [00101] Growth of Probiotic Culture on Fiber Mesh: Effects of probiotics are optimized when released in a controlled manner. The goal of this procedure was to grow probiotic in an unconventional method, so that the element could be incorporated into a new drug delivery system. In order to design a probiotic scaffold that would have the ability to release the probiotic metabolites and signaling molecules in a controlled manner the seeding technique was utilized. The probiotic of choice, B. coagulans, was grown onto mesh of electrospun PCL. PCL has attractive features such as robust chemical properties and a long shelf life. Discussion [00102] B. coagulans is successful in inhibiting the growth of the respiratory pathogen, H. influenzae, a key pathogen implicated in COPD exacerbations. [00103] Based on the measured zones of clearing the effects of probiotic on inhibiting pathogenic microorganisms are dose dependent. [00104] Confocal fluorescence microscopy showed that colonies of probiotics establish themselves in various morphologies on fibrous mesh. [00105] B. coagulans can successfully be migrated from agar to electro spun PCL mesh. Conclusion [00106] B. coagulans has demonstrated antimicrobial activity and therefore has clinical applications in terms of reducing inflammation in the lung tissue and preventing exacerbations in patients with chronic respiratory disease. Probiotics are effective due to their ability to alter the biofilm lifecycle and thereby impede the growth of respiratory pathogenic microorganisms. The eradication of the growth of the biofilms is dose-dependent and therefore a minimum dosage of probiotic treatment is required to demonstrate beneficial outcomes in COPD patients. B. coagulans can be grown onto mesh of electrospun polycaprolactone (PCL); this scaffold design serves as a site where probiotics can grow, reproduce and detach in a controlled manner. These studies are directed toward the development of a novel drug delivery device that will deliver probiotics and their signaling molecules to the respiratory tract to treat and prevent exacerbations in COPD patients. Example 3: Characterization of Bacteriocins from Bacillus Coagulans for Applications in COPD Motivation [00107] Chronic Obstructive Pulmonary Disease (COPD) is a common and preventable disease, while current treatments for COPD are insufficient, and more research is needed to address treatment options. [00108] This proposal is to characterize and purify active peptides derived from Bacillus coagulans, a probiotic bacteria, that inhibits pathogens associated with COPD flare ups, specifically Haemophilus influenzae. [00109] Preliminary unpublished data indicates the activity of Bacillus coagulans against Haemophilus influenzae (Fig. 2A), and the activity is believed to be related to small cyclic peptides and lipopeptides ⁴⁰ . Objectives [00110] The goal of this summer project is to purify, isolate and characterize the bacteriocins from Bacillus coagulans, including their structures and activity against Haemophilus influenzae. [00111] With this basic technique, cation exchange is the main purification method, followed by other refinement ⁴¹ . Conclusion [00112] The concentration of bacteriocins in the broth is 0.06 mg/ml, calculated from the Cation Exchange Experiment. [00113] The strains from Columbia University cultivations are very similar to those from Touro School; by comparing Fig.13 and Fig.14, a highly similar eluting time of 2.31 min was discovered; additionally, the "TOF MS ES+" data in Fig. 15 and Fig. 16 have a similar range of roughly 720 to 782 M/Z. These comparison shows possible similarity of peptides between the Columbia University sample and the Touro School sample. 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