An intravenous pancreatic cancer therapeutic: Characterization of CRISPR/Cas9n-modified Clostridium novyi-Non Toxic.

Clostridium novyi has demonstrated selective efficacy against solid tumors largely due to the microenvironment contained within dense tumor cores. The core of a solid tumor is typically hypoxic, acidic, and necrotic-impeding the penetration of current therapeutics. C. novyi is attracted to the tumor microenvironment and once there, can both lyse and proliferate while simultaneously re-activating the suppressed immune system. C. novyi systemic toxicity is easily mitigated by knocking out the phage DNA plasmid encoded alpha toxin resulting in C. novyi-NT; but, after intravenous injection spores are quickly cleared by phagocytosis before accomplishing significant tumor localization. C. novyi-NT could be designed to accomplish intravenous delivery with the potential to target all solid tumors and their metastases in a single dose. This study characterizes CRISPR/Cas9 modified C. novyi-NT to insert the gene for RGD, a tumor targeting peptide, expressed within the promoter region of a spore coat protein. Expression of the RGD peptide on the outer spore coat of C. novyi-NT indicates an increased capacity for tumor localization of C. novyi upon intravenous introduction based on the natural binding of RGD with the αvß3 integrin commonly overexpressed on the epithelial tissue surrounding a tumor, and lead to immune stimulation.

Hacking the Immune Response to Solid Tumors: Harnessing the Anti-Cancer Capacities of Oncolytic Bacteria.

Oncolytic bacteria are a classification of bacteria with a natural ability to specifically target solid tumors and, in the process, stimulate a potent immune response. Currently, these include species of Klebsiella, Listeria, Mycobacteria, Streptococcus/Serratia (Coley's Toxin), Proteus, Salmonella, and Clostridium. Advancements in techniques and methodology, including genetic engineering, create opportunities to "hijack" typical host-pathogen interactions and subsequently harness oncolytic capacities. Engineering, sometimes termed "domestication", of oncolytic bacterial species is especially beneficial when solid tumors are inaccessible or metastasize early in development. This review examines reported oncolytic bacteria-host immune interactions and details the known mechanisms of these interactions to the protein level. A synopsis of the presented membrane surface molecules that elicit particularly promising oncolytic capacities is paired with the stimulated localized and systemic immunogenic effects. In addition, oncolytic bacterial progression toward clinical translation through engineering efforts are discussed, with thorough attention given to strains that have accomplished Phase III clinical trial initiation. In addition to therapeutic mitigation after the tumor has formed, some bacterial species, referred to as "prophylactic", may even be able to prevent or "derail" tumor formation through anti-inflammatory capabilities. These promising species and their particularly favorable characteristics are summarized as well. A complete understanding of the bacteria-host interaction will likely be necessary to assess anti-cancer capacities and unlock the full cancer therapeutic potential of oncolytic bacteria.

The Correlation of Sleep Disturbance and Location of Glioma Tumors: A Narrative Review.

Sleep disturbance can occur when sleep centers of the brain, regions that are responsible for coordinating and generating healthy amounts of sleep, are disrupted by glioma growth or surgical resection. Several disorders cause disruptions to the average duration, quality, or patterns of sleep, resulting in sleep disturbance. It is unknown whether specific sleep disorders can be reliably correlated with glioma growth, but there are sufficient numbers of case reports to suggest that a connection is possible. In this manuscript, these case reports and retrospective chart reviews are considered in the context of the current primary literature on sleep disturbance and glioma diagnosis to identify a new and useful connection which warrants further systematic and scientific examination in preclinical animal models. Confirmation of the relationship between disruption of the sleep centers in the brain and glioma location could have significant implications for diagnostics, treatment, monitoring of metastasis/recurrence, and end-of-life considerations.

Revisiting the COVID-19 fatality rate and altitude association through a comprehensive analysis.

The emergence of COVID-19 virus has led to a pandemic with staggering morbidity and mortality. There is evidence showing that pre-existing conditions and environmental factors are associated with worse COVID-19 outcomes. Among these conditions, altitude is of particular interest. Altitude has been shown to influence the morbidity and mortality of multiple chronic pathologies such as cardiovascular disease, chronic obstructive pulmonary disease and lung cancer. COVID-19 fatality rate has been associated with as altitude as well, but findings are disputed. Therefore, we revisit this assessment with a comprehensive analysis of the relationship between COVID-19 fatality rates and altitude for the Mountain region of the United States while considering the effect of additional comorbidities and sociodemographic factors. A Generalized Additive Model (GAM) approach using one year of county data adjusted by population density was performed to evaluate associations within states and for the whole region. Our analysis revealed a consistent effect where COVID-19 case-fatality rate is decreased with higher altitude, even when controlling for pre-existing conditions and certain demographic variables. In summary, the work presented provides evidence that suggests that the protective effects of high altitude are likely to be influenced by physiologic factors but demographic trends that are associated with life at high altitude must also be considered.

Pathophysiological Link between Insulin Resistance and Adrenal Incidentalomas.

Adrenal incidentalomas are incidentally discovered adrenal masses greater than one centimeter in diameter. An association between insulin resistance and adrenal incidentalomas has been established. However, the pathophysiological link between these two conditions remains incompletely characterized. This review examines the literature on the interrelationship between insulin resistance and adrenal masses, their subtypes, and related pathophysiology. Some studies show that functional and non-functional adrenal masses elicit systemic insulin resistance, whereas others conclude the inverse. Insulin resistance, hyperinsulinemia, and the anabolic effects on adrenal gland tissue, which have insulin and insulin-like growth factor-1 receptors, offer possible pathophysiological links. Conversely, autonomous adrenal cortisol secretion generates visceral fat accumulation and insulin resistance. Further investigation into the mechanisms and timing of these two pathologies as they relate to one another is needed and could be valuable in the prevention, detection, and treatment of both conditions.

Potential Therapeutic Targets for Combination Antibody Therapy against Pseudomonas aeruginosa Infections.

Despite advances in antimicrobial therapy and even the advent of some effective vaccines, Pseudomonas aeruginosa (P. aeruginosa) remains a significant cause of infectious disease, primarily due to antibiotic resistance. Although P. aeruginosa is commonly treatable with readily available therapeutics, these therapies are not always efficacious, particularly for certain classes of patients (e.g., cystic fibrosis (CF)) and for drug-resistant strains. Multi-drug resistant P. aeruginosa infections are listed on both the CDC's and WHO's list of serious worldwide threats. This increasing emergence of drug resistance and prevalence of P. aeruginosa highlights the need to identify new therapeutic strategies. Combinations of monoclonal antibodies against different targets and epitopes have demonstrated synergistic efficacy with each other as well as in combination with antimicrobial agents typically used to treat these infections. Such a strategy has reduced the ability of infectious agents to develop resistance. This manuscript details the development of potential therapeutic targets for polyclonal antibody therapies to combat the emergence of multidrug-resistant P. aeruginosa infections. In particular, potential drug targets for combinational immunotherapy against P. aeruginosa are identified to combat current and future drug resistance.

The Evolution and Future of Targeted Cancer Therapy: From Nanoparticles, Oncolytic Viruses, and Oncolytic Bacteria to the Treatment of Solid Tumors.

While many classes of chemotherapeutic agents exist to treat solid tumors, few can generate a lasting response without substantial off-target toxicity despite significant scientific advancements and investments. In this review, the paths of development for nanoparticles, oncolytic viruses, and oncolytic bacteria over the last 20 years of research towards clinical translation and acceptance as novel cancer therapeutics are compared. Novel nanoparticle, oncolytic virus, and oncolytic bacteria therapies all start with a common goal of accomplishing therapeutic drug activity or delivery to a specific site while avoiding off-target effects, with overlapping methodology between all three modalities. Indeed, the degree of overlap is substantial enough that breakthroughs in one therapeutic could have considerable implications on the progression of the other two. Each oncotherapeutic modality has accomplished clinical translation, successfully overcoming the potential pitfalls promising therapeutics face. However, once studies enter clinical trials, the data all but disappears, leaving pre-clinical researchers largely in the dark. Overall, the creativity, flexibility, and innovation of these modalities for solid tumor treatments are greatly encouraging, and usher in a new age of pharmaceutical development.

Methods and Techniques to Facilitate the Development of Clostridium novyi NT as an Effective, Therapeutic Oncolytic Bacteria.

The tumor microenvironment is characterized by anomalous vascularization, hypoxia, and acidity at the core of solid tumors that culminates in concentrated necrosis and immune system dysregulation among other effects. While this environment presents several challenges for the development of oncotherapeutics that deliver their activity via the enhanced permeability and retention (EPR) effect of the leaky blood vessels around a tumor, oncolytic bacteria, or a class of bacteria with a noted capacity to lyse solid tumors, are attracted to the very environment found at the center of solid tumors that confounds other therapeutics. It is this capacity that allows for a potent, active penetration from the tumor margins into the core, and subsequent colonization to facilitate lysis and immune reactivation. Clostridium novyi in particular has recently shown great promise in preclinical and clinical trials when administered directly to the tumor. These studies indicate that C. novyi is uniquely poised to effectively accomplish the long sought after "holy grail" of oncotherapeutics: selective tumor localization via intravenous delivery. This study reports the development of efficient methods that facilitate experimental work and therapeutic translation of C. novyi including the ability to work with this obligate micro-anaerobe on the benchtop. Additionally, this study seeks to utilize this newfound experimental flexibility to address several gaps in the current knowledge regarding the efficacy of CRIPSR/Cas9-mediated gene insertion in this species to further develop this oncolytic bacteria and the genetic customization of bacteria in general.

The next frontier of oncotherapy: accomplishing clinical translation of oncolytic bacteria through genetic engineering.

The development of a 'smart' drug capable of distinguishing tumor from host cells has been sought for centuries, but the microenvironment of solid tumors continues to confound therapeutics. Solid tumors present several challenges for current oncotherapeutics, including aberrant vascularization, hypoxia, necrosis, abnormally high pH and local immune suppression. While traditional chemotherapeutics are limited by such an environment, oncolytic microbes are drawn to it - having an innate ability to selectively infect, colonize and eradicate solid tumors. Development of an oncolytic species would represent a shift in the cancer therapeutic paradigm, with ramifications reaching from the medical into the socio-economic. Modern genetic engineering techniques could be implemented to customize 'Frankenstein' bacteria with advantageous characteristics from several species.

Lay abstract Side effects of chemotherapeutics are thought to often be a reflection of our inability to target these toxic substances to only cancer cells; hence, scientists have spent centuries searching for alternative treatments that would confine their actions to tumor cells, sparing healthy tissue. Unfortunately, the dense nature of tumor tissue along with altered blood vessels, that lead to diminished tumor tissue oxygenation, altered tissue pH and cellular metabolic inactivity or even cell death have proven challenging. Importantly, these barriers have contributed to local and even sometimes systemic suppression of the patient's immune system that can allow the tumor to grow and progress unchecked. While most non-cancer cells are inhibited by the local tumor environment, certain microbes, including some bacteria and viruses, are drawn to it, possessing a natural ability to selectively infect, colonize and eradicate solid tumors. These microbes may also restore the patient's immune balance. However, use of these microbes is not without its own problems; nevertheless, modern genetic engineering techniques could be implemented to develop customized, safe, effective bacteria with advantageous characteristics. The development and clinical translation of cancer-fighting bacteria would represent a shift in cancer therapeutics and would have ramifications that reach beyond medical efficacy into the realm of socioeconomics. This review seeks to marry the current field of oncolytic bacteria with the expanding field of modern bacterial genetic engineering techniques in prospect of such a therapeutic.

Overcoming Hurdles in Nanoparticle Clinical Translation: The Influence of Experimental Design and Surface Modification.

Nanoparticles are becoming an increasingly popular tool for biomedical imaging and drug delivery. While the prevalence of nanoparticle drug-delivery systems reported in the literature increases yearly, relatively little translation from the bench to the bedside has occurred. It is crucial for the scientific community to recognize this shortcoming and re-evaluate standard practices in the field, to increase clinical translatability. Currently, nanoparticle drug-delivery systems are designed to increase circulation, target disease states, enhance retention in diseased tissues, and provide targeted payload release. To manage these demands, the surface of the particle is often modified with a variety of chemical and biological moieties, including PEG, tumor targeting peptides, and environmentally responsive linkers. Regardless of the surface modifications, the nano-bio interface, which is mediated by opsonization and the protein corona, often remains problematic. While fabrication and assessment techniques for nanoparticles have seen continued advances, a thorough evaluation of the particle's interaction with the immune system has lagged behind, seemingly taking a backseat to particle characterization. This review explores current limitations in the evaluation of surface-modified nanoparticle biocompatibility and in vivo model selection, suggesting a promising standardized pathway to clinical translation.

The Role of the Gut Microbiota in Sustained Weight Loss Following Roux-en-Y Gastric Bypass Surgery.

BACKGROUND: The aim of the study was to investigate the role of the gut microbiota in weight regain or suboptimal weight loss following Roux-en-Y gastric bypass (RYGB). MATERIALS AND METHODS: The gut microbiota composition in post-RYGB patients who experienced successful weight loss (SWL, n = 6), post-RYGB patients who experienced poor weight loss (PWL, n = 6), and non-surgical controls (NSC, n = 6) who were age- and BMI-matched to the SWL group (NSC, n = 6) were characterized through 16S rRNA gene sequencing. To further investigate the impact of the gut microbiota on weight profile, human fecal samples were transplanted into antibiotic-treated mice. RESULTS: Orders of Micrococcales and Lactobacillales were enriched in SWL and PWL groups compared to the NSC group. No significant difference was observed in the gut microbiota composition between PWL and SWL patients. However, transfer of the gut microbiota from human patients into antibiotic-treated mice resulted in significantly greater weight gain in PWL recipient mice compared to SWL recipient mice. A few genera that were effectively transferred from humans to mice were associated with weight gain in mice. Among them, Barnesiella was significantly higher in PWL recipient mice compared to SWL and NSC recipient mice. CONCLUSION: These results indicate that the gut microbiota are at least functionally, if not compositionally, different between PWL and SWL patients. Some taxa may contribute to weight gain after surgery. Future studies will need to determine the molecular mechanisms behind the effects of the gut bacteria on weight regain after RYGB.

Energy-triggered drug release from polymer nanoparticles for orthopedic applications.

Sequestra, present in many cancers and orthopedic infections, provide a safe harbor for the development of drug resistance. In the face of burgeoning drug resistance, the importance of nanoscale, microenvironment-triggered drug delivery cannot be overestimated. Such strategies may preserve pharmaceutical efficacy and significantly alter the etiology of many orthopedic diseases. Although temperature-, pH- and redox-responsive nanoparticle-based systems have been extensively studied, local drug delivery from polymeric nanoparticles can be triggered by a variety of energy forms. This review offers an overview of the state of the field as well as a perspective on the safety and efficacy of ultrasound, hyperthermia and radio frequency-triggered internal delivery systems in a variety of applications.

Prostate-Specific Membrane Antigen Targeted Polymersomes for Delivering Mocetinostat and Docetaxel to Prostate Cancer Cell Spheroids.

Prostate cancer cells overexpress the prostate-specific membrane antigen (PSMA) receptors on the surface. Targeting the PSMA receptor creates a unique opportunity for drug delivery. Docetaxel is a Food and Drug Administration-approved drug for treating metastatic and androgen-independent prostate cancer, and mocetinostat is a potent inhibitor of class I histone deacetylases. In this study, we prepared reduction-sensitive polymersomes presenting folic acid on the surface and encapsulating either docetaxel or mocetinostat. The presence of folic acid allowed efficient targeting of the PSMA receptor and subsequent internalization of the polymeric vesicles in cultured LNCaP prostate cancer cell spheroids. The intracellular reducing agents efficiently released docetaxel and mocetinostat from the polymersomes. The combination of the two drug-encapsulated polymersome formulations significantly (p < 0.05) decreased the viability of the LNCaP cells (compared to free drugs or control) in three-dimensional spheroid cultures. The calculated combination index value indicated a synergistic effect for the combination of mocetinostat and docetaxel. Thus, our PSMA-targeted drug-encapsulated polymersomes has the potential to lead to a new direction in prostate cancer therapy that decreases the toxicity and increases the efficacy of the drug delivery systems.

A resorbable antibiotic eluting bone void filler for periprosthetic joint infection prevention.

Periprosthetic joint infection (PJI) following total knee arthroplasty is a globally increasing procedural complication. These infections are difficult to treat and typically require revision surgery. Antibiotic-loaded bone cement is frequently utilized to deliver antibiotics to the site of infection; however, bone cement is a nondegrading foreign body and known to leach its antibiotic load, after an initial burst release, at subtherapeutic concentrations for months. This work characterized a resorbable, antibiotic-eluting bone void filler designed to restore bone volume and prevent PJI. Three device formulations were fabricated, consisting of different combinations of synthetic inorganic bone graft material, degradable polymer matrices, salt porogens, and antibiotic tobramycin. These formulations were examined to determine the antibiotic's elution kinetics and bactericidal potential, the device's degradation in vitro, as well as osteoconductivity and device resorption in vivo using a pilot rabbit bone implant model. Kirby-Bauer antibiotic susceptibility tests assessed bactericidal activity. Liquid chromatography with tandem mass spectrometry measured antibiotic elution kinetics, and scanning electron microscopy was used to qualitatively assess degradation. Results indicated sustained antibiotic release from all three formulations above the Staphylococcus aureus minimum inhibitory concentration for a period of 5 to 8 weeks. Extensive degradation was observed with the Group 3 formulation after 90 days in phosphate-buffered saline, with a lesser degree of degradation observed in the other two formulations. Results from the pilot rabbit study showed the Group 3 device to be biocompatible, with minimal inflammatory response and no fibrous encapsulation in bone. The device was also highly osteoconductive-exhibiting an accelerated mineral apposition rate. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1632-1642, 2016.

Optimal tube length for the submerged printing of ovarian cancer cells.

Ovarian cancer is the fifth most common cancer affecting US women, killing more women each year than all other gynecologic cancers combined. Treatment of ovarian cancer is challenging with an overall 5-year survival rates of only 28-46% based on the metastatic state of the disease. While overall survival has improved with modern chemotherapy, poor outcomes have persisted. One of the greatest challenges in cancer therapeutic research remains that late-stage drug development trials for drug candidates have high attrition rates, up to 70% in Phase II and 59% in Phase III trials. The development of in vitro, high-throughput, cell based assays could provide a tool to overcome the challenges associated with high attrition rates by allowing for controlled cell deposition with a defined, controlled phenotype. Submerged, three-dimensional (3D) microfluidic printing technology is uniquely capable of controlling cell deposition without sacrificing the viability of cells for cell-based assays. Here, we investigate the phenotypic effects of tube length during printing on the cells. We observe that the length of the tube has minimal effects on the viability and density of A2780 ovarian cancer cells different cell lines. This study details foundational information for developing a high-throughput cell-based assays (CBA) for screening effective cancer drug candidates.

Comparison of submerged and unsubmerged printing of ovarian cancer cells.

A high-throughput cell based assay would greatly aid in the development and screening of ovarian cancer drug candidates. Previously, a three-dimensional microfluidic printer that is not only capable of controlling the location of cell deposition, but also of maintaining a liquid, nutrient rich environment to preserve cellular phenotype has been developed (Wasatch Microfluidics). In this study, we investigated the impact (i.e., viability, density, and phenotype) of depositing cells on a surface submerged in cell culture media. It was determined that submersion of the microfluidic print head in cell media did not alter the cell density, viability, or phenotype.. This article describes an in depth study detailing the impact of one of the fundamental components of a 3D microfluidic cell printer designed to mimic the in vivo cell environment. Development of such a tool holds promise as a high-throughput drug-screening platform for new cancer therapeutics.

Molded polymer-coated composite bone void filler improves tobramycin controlled release kinetics.

Infection remains a significant problem associated with biomedical implants and orthopedic surgeries, especially in revision total joint replacements. Recent advances in antibiotic-releasing bone void fillers (BVF) provide new opportunities to address these types of device-related orthopedic infections that often lead to substantial economic burdens and reduced quality of life. We report improvements made in fabrication and scalability of an antibiotic-releasing polycaprolactone-calcium carbonate/phosphate ceramic composite BVF using a new solvent-free, molten-cast fabrication process. This strategy provides the ability to tailor drug release kinetics from the BVF composite based on modifications of the inorganic substrate and/or the polymeric component, allowing extended tobramycin release at bactericidal concentrations. The mechanical properties of the new BVF composite are comparable to many reported BVFs and validate the relative homogeneity of fabrication. Most importantly, fabrication quality controls are correlated with favorable drug release kinetics, providing bactericidal activity to 10 weeks in vitro when the polycaprolactone component exceeds 98% w/w of the total polymer fraction. Furthermore, in a time kill study, tobramycin-releasing composite fragments inhibited S. aureus growth over 48 h at inoculums as high as 10(9) CFU/mL. This customizable antibiotic-releasing BVF polymer-inorganic biomaterial should provide osseointegrative and osteoconductive properties while contributing antimicrobial protection to orthopedic sites requiring the use of bone void fillers.

Polymer-controlled release of tobramycin from bone graft void filler.

Despite clinical, material, and pharmaceutical advances, infection remains a major obstacle in total joint revision surgery. Successful solutions must extend beyond bulk biomaterial and device modifications, integrating locally delivered pharmaceuticals and physiological cues at the implant site, or within large bone defects with prominent avascular spaces. One approach involves coating clinically familiar allograft bone with an antibiotic-releasing rate-controlling polymer membrane for use as a matrix for local drug release in bone. The kinetics of drug release from this system can be tailored via alterations in the substrate or the polymeric coating. Drug-loaded polycaprolactone coating releases bioactive tobramycin from both cadaveric-sourced cancellous allograft fragments and synthetic hybrid coralline ceramic bone graft fragments with similar kinetics over a clinically relevant 6-week timeframe. However, micron-sized allograft particulate provides extended bioactive tobramycin release. Addition of porogen polyethylene glycol to the polymer coating formulation changes tobramycin release kinetics without significant impact on released antibiotic bioactivity. Incorporation of oil-microencapsulated tobramycin into the polymer coating did not significantly modify tobramycin release kinetics. In addition to releasing inhibitory concentrations of tobramycin, antibiotic-loaded allograft bone provides recognized beneficial osteoconductive potential, attractive for decreasing orthopedic surgical infections with improved filling of dead space and new bone formation.

Cultured primary macrophage activation by lipopolysaccharide depends on adsorbed protein composition and substrate surface chemistry.

Recent efforts show that significantly reducing implant-adsorbed proteins does not avoid the foreign body response. Fluorinated surfaces are commonly used to passivate cell-mediated inflammatory responses to implanted materials but adsorb host proteins and facilitate the attachment and proliferation of macrophages. This study considers in vitro macrophage activation to fluorinated TeflonAF(®) compared to tissue-culture polystyrene using pre-adsorbed proteins (fibrinogen, BSA, collagen and elastin). Primary macrophage cultures adhere on all pre-adsorbed protein surfaces in a protein concentration-dependent manner and activate to the same extent after 72 h, regardless of surface chemistry. However, macrophages alter their cultured adherent morphology depending on which protein is pre-adsorbed to these surfaces. Macrophages cultured on TeflonAF(®) on all pre-adsorbed proteins produced overall higher levels of the pro-inflammatory cytokines - TNF-α, IL-6, IL-1ß or MCP-1 - than those cultured on tissue-culture polystyrene and those cultured in serum-free media. However, at 72 h, macrophages adherent on BSA or fibrinogen pre-adsorbed surfaces failed to exhibit increased amounts of TNF-a, IL-6 or IL-1/S on either TeflonAF(®) or TCPS, as well as MCP-1 on TCPS, in the presence of activating lipopolysaccharide. Different cell responses to pre-adsorbed proteins reflect substrate-specific regulation of macrophage cytokine secretion, indicative of LPS tolerance distinct from secondary macrophage cultures, and also distinct from macrophages adherent to surfaces in the absence of proteins. This result has bearing on connecting macrophage adhesion via adsorbed proteins on (fluorinated) biomaterials, and their resulting chronic activation that yields the FBR and possibly reduces effective macrophage clearance of microbes around implanted materials.