Loratadine is an anti-inflammatory agent against C. difficile toxin B.

BACKGROUND: Clostridium difficile infection (CDI) is a debilitating nosocomial infection. C. difficile produces toxins A and B, which cause inflammation. Existing therapies have issues with recurrence, cost, and safety. We aim to discover a safe, effective, and economical non-microbiological therapeutic approach against CDI. METHODS: We included human primary peripheral blood mononuclear cells (PBMCs), fresh human colonic explants, and humanized HuCD34-NCG mice. Toxin A+B+ VPI10463 and A-B+ ribotype 017 C. difficile strains were used. We used single-cell RNA profiling and high-throughput screening to find actionable toxin B-dependent pathways in PBMCs. RESULTS: Histamine 1 receptor-related drugs were found among the hit compounds that reversed toxin-mediated macrophage inflammatory protein one alpha (MIP-1α) expression in PBMCs. We identified Loratadine as the safest representative antihistamine for therapeutic development. Loratadine inhibited toxin B-induced MIP-1α secretion in fresh human colonic tissues. Oral Loratadine (10 mg/kg/day) maintained survival, inhibited intestinal Ccl3 mRNA expression, and prevented vancomycin-associated recurrence in the VPI10463-infected mice and ribotype 017-infected hamsters. Splenocytes from Loratadine-treated mice conferred anti-inflammatory effects to the VPI10463-infected T/B cell-deficient Rag-/- mice. Oral Loratadine suppressed human MIP-1α expression in monocytes/macrophages in toxin B-expressing ribotype 017-infected humanized HuCD34-NCG mice. CONCLUSIONS: Loratadine may be repurposed to optimize existing therapies against CDI.

Loratadine is an FDA-approved antihistamine that inhibits toxin B-mediated pro-inflammatory macrophage inflammatory protein one alpha secretion from immune cells. The anti-inflammatory effect of Loratadine ameliorates intestinal inflammation in C. difficile-infected animals. Loratadine may be repurposed to optimize existing therapies.

A fine-tuned yeast surface-display/secretion platform enables the rapid discovery of neutralizing antibodies against Clostridioides difficile toxins.

BACKGROUND: Neutralizing antibody plays a key role in protecting hosts from invasive pathogens and their virulent components. Current high-throughput assays for antibody screening are based on binding activities. However, those antibodies with high affinity may not have neutralizing activities. Subsequent functionality assays are necessary to identify neutralizing antibodies from binders with high affinity to their target antigens, which is laborious and time-consuming. Therefore, a versatile platform that can rapidly identify antibodies with both high binding affinity and neutralizing activity is desired to curb future pandemics like COVID-19. RESULTS: In this proof-of-concept study, we adapted Saccharomyces cerevisiae to either display human antibodies on the yeast surface or secrete soluble antibodies into the cultivation supernatant under a controllable 'switch' through different carbon source induced promoters. Initially, an engineered chimeric-bispecific Fab antibody, derived from humanized nanobodies against both Clostridioides difficile toxin A and B (TcdA and TcdB), was successfully expressed either on the yeast cell surface or in the culture medium with intact bioactivity, suggesting the applicability of our system in antibody display and secretion. Next, a combinatorial Fab library was constructed from B cells isolated from a convalescent patient with a high serological neutralizing titer against TcdB. Following three rounds of magnetic bead enrichment and one round of flow cytometry sorting, antibodies against TcdB were enriched efficiently. We then sorted out single binders with high binding affinity and induced them to express soluble antibodies in culture medium. The neutralizing activity of culture supernatant was analyzed using cell-based assay immediately. This way, we rapidly identified two unique neutralizers (out of seven binders) that can neutralize the cytotoxicity of TcdB. CONCLUSION: The antibody screening platform described here simplifies the neutralizing antibody discovery procedure and will be an attractive alternative for screening functional antibodies against infectious diseases.

Delivery of a therapeutic antibody to the lower gastrointestinal tract for the treatment of Clostridium difficile infection (CDI).

The colonic delivery system of toxin neutralizing antibody is a promising method for treating Clostridium difficile infection (CDI) and has some advantages over the parental administration of a neutralizing antibody. However, colonic delivery of biologics presents several challenges, including instability of biologics during encapsulation into the delivery system and harsh conditions in the upper GI tract. In this work, we described a multi-particulate delivery system encapsulating a tetra-valent antibody ABAB-IgG1 with the potential to treat CDI. This work first approved that the cecum injection of ABAB-IgG1 into the lower GI tract of mice could relieve the symptoms, enhance the clinical score, and improve the survival rate of mice during CDI. Then, the antibody was spray layered onto mannitol beads and then enteric coated with pH-sensitive polymers to achieve colon-targeting release. The in vitro release of antibody from the multi-particulate system and the pH-sensitive release of antibody was monitored. The in vivo efficacy of this system was further examined and confirmed in mice and hamsters. In summary, the findings of this study should provide practical information and potential treatment options for CDI through colonic delivery of antibody therapeutics to the lower GI tract using a multi-particulate delivery system.

Genistein Inhibits Clostridioides difficile Infection via Estrogen Receptors and Lysine-Deficient Protein Kinase 1.

BACKGROUND: Clostridioides difficile infection (CDI) is a debilitating nosocomial disease. Postmenopausal women may have an increased risk of CDI, suggesting estrogen influence. Soybean products contain a representative estrogenic isoflavone, genistein. METHODS: The anti-inflammatory and antiapoptotic effects of genistein were determined using primary human cells and fresh colonic tissues. The effects of oral genistein therapy among mice and hamsters were evaluated. RESULTS: Within 10 days of CDI, female c57BL/6J mice in a standard environment (regular diet) had a 50% survival rate, while those with estrogen depletion and in an isoflavone-free environment (soy-free diet) had a 25% survival rate. Oral genistein improved their 10-day survival rate to 100% on a regular diet and 75% in an isoflavone-free environment. Genistein reduced macrophage inflammatory protein-1α (MIP-1α) secretion in fresh human colonic tissues exposed to toxins. Genistein inhibited MIP-1α secretion in primary human peripheral blood mononuclear cells, abolished apoptosis and BCL-2-associated X (BAX) expression in human colonic epithelial cells, and activated lysine-deficient protein kinase 1 (WNK1) phosphorylation in both cell types. The anti-inflammatory and antiapoptotic effects of genistein were abolished by inhibiting estrogen receptors and WNK1. CONCLUSIONS: Genistein reduces CDI disease activity by inhibiting proinflammatory cytokine expression and apoptosis via the estrogen receptor/G-protein estrogen receptor/WNK1 pathways.

Establishment of a gnotobiotic pig model of Clostridioides difficile infection and disease.

Clostridioides difficile (C. difficile) is a gram-positive, spore-forming, anaerobic bacterium known to be the most common cause of hospital-acquired and antibiotic-associated diarrhea. C. difficile infection rates are on the rise worldwide and treatment options are limited, indicating a clear need for novel therapeutics. Gnotobiotic piglets are an excellent model to reproduce the acute pseudomembranous colitis (PMC) caused by C. difficile due to their physiological similarities to humans and high susceptibility to infection. Here, we established a gnotobiotic pig model of C. difficile infection and disease using a hypervirulent strain. C. difficile-infected pigs displayed classic signs of C. difficile infection, including severe diarrhea and weight loss. Inoculated pigs had severe gross and microscopic intestinal lesions. C. difficile infection caused an increase in pro-inflammatory cytokines in samples of serum, large intestinal contents, and pleural effusion. C. difficile spores and toxins were detected in the feces of inoculated animals as tested by anaerobic culture and cytotoxicity assays. Successful establishment of this model is key for future work as therapeutics can be evaluated in an environment that accurately mimics what happens in humans. The model is especially suitable for evaluating potential prophylactics and therapeutics, including vaccines and passive immune strategies.

The development of live biotherapeutics against Clostridioides difficile infection towards reconstituting gut microbiota.

Clostridioides difficile is the most prevalent pathogen of nosocomial diarrhea. In the United States, over 450,000 cases of C. difficile infection (CDI), responsible for more than 29,000 deaths, are reported annually in recent years. Because of the emergence of hypervirulent strains and strains less susceptible to vancomycin and fidaxomicin, new therapeutics other than antibiotics are urgently needed. The gut microbiome serves as one of the first-line defenses against C. difficile colonization. The use of antibiotics causes gut microbiota dysbiosis and shifts the status from colonization resistance to infection. Hence, novel CDI biotherapeutics capable of reconstituting normal gut microbiota have become a focus of drug development in this field.

ADS024, a Bacillus velezensis strain, protects human colonic epithelial cells against C. difficile toxin-mediated apoptosis.

Clostridioides difficile infection (CDI) causes intestinal injury. Toxin A and toxin B cause intestinal injury by inducing colonic epithelial cell apoptosis. ADS024 is a Bacillus velezensis strain in development as a single-strain live biotherapeutic product (SS-LBP) to prevent the recurrence of CDI following the completion of standard antibiotic treatment. We evaluated the protective effects of the sterile filtrate and ethyl acetate extract of conditioned media from ADS024 and DSM7 (control strain) against mucosal epithelial injury in toxin-treated human colonic tissues and apoptosis in toxin-treated human colonic epithelial cells. Ethyl acetate extracts were generated from conditioned culture media from DSM7 and ADS024. Toxin A and toxin B exposure caused epithelial injury in fresh human colonic explants. The sterile filtrate of ADS024, but not DSM7, prevented toxin B-mediated epithelial injury in fresh human colonic explants. Both sterile filtrate and ethyl acetate extract of ADS024 prevented toxin-mediated apoptosis in human colonic epithelial cells. The anti-apoptotic effects of ADS024 filtrate and ethyl acetate extract were dependent on the inhibition of caspase 3 cleavage. The sterile filtrate, but not ethyl acetate extract, of ADS024 partially degraded toxin B. ADS024 inhibits toxin B-mediated apoptosis in human colonic epithelial cells and colonic explants.

Spray layering of human immunoglobulin G: Optimization of formulation and process parameters.

Spray layering is a technique used to apply drug or functional polymers onto carrier beads; in addition, it can be used as an alternative method for protein drying and to layer protein on a multiparticulate delivery system. In this study, the effects of formulation variables and process parameters on human immunoglobulin G (IgG) properties during spray layering were studied. Excipients including polyvinylpyrrolidone (PVP), trehalose, sucrose, L-arginine monohydrochloride were studied for their effects on improving IgG stability during spray layering. Process parameters including protein solution feed rate, inlet air temperature, inlet air flow rate, and atomization pressure of spray solution were studied using 24 full factorial design with three replicated center points. Adding PVP into the formulation significantly decreased the turbidity of the reconstitution solution and increased the IgG recovery. Adding trehalose, sucrose, or arginine further improved protein recovery after reconstitution and decreased the percentage of IgG aggregation. The Design of Experiments (DOE) results showed no significant effects from the four process factors on the process yield and IgG protein recovery in the range of parameters studied. All main factors except atomization pressure had significant effects on monomer percentage, among which air flow represented the most significant influence. In addition, the inlet air temperature had significant effects on the in vitro binding activity of IgG after spray layering. By optimizing the formulation, we were able to recover the most spray layered IgG and reduce the IgG aggregation during the process. The DOE studies gave insight into how process variables affect the spray layered products.

A probiotic yeast-based immunotherapy against Clostridioides difficile infection.

Antibiotic-resistant Clostridioides difficile is an anaerobic Gram-positive bacterium that colonizes the colon and is responsible for more than 29,000 deaths in the United States each year. Hence, C. difficile infection (CDI) poses an urgent threat to public health. Antibody-mediated neutralization of TcdA and TcdB toxins, the major virulence factors of CDI, represents an effective strategy to combat the disease without invoking antibiotic resistance. However, current antitoxin approaches are mostly based on parenteral infusion of monoclonal antibodies that are costly, narrow spectrum, and not optimized against the intestinal disease. Here, we engineered probiotic Saccharomyces boulardii to constitutively secrete a single tetra-specific antibody that potently and broadly neutralized both toxins and demonstrated protection against primary and recurrent CDI in both prophylactic and therapeutic mouse models of disease. This yeast immunotherapy is orally administered, can be used concurrently with antibiotics, and may have potential as a prophylactic against CDI risk and as a therapeutic for patients with CDI.

Therapeutic Mechanism of Macrophage Inflammatory Protein 1 α Neutralizing Antibody (CCL3) in Clostridium difficile Infection in Mice.

BACKGROUND: Clostridium difficile infection (CDI) causes diarrhea and colitis. We aimed to find a common pathogenic pathway in CDI among humans and mice by comparing toxin-mediated effects in human and mouse colonic tissues. METHOD: Using multiplex enzyme-linked immunosorbent assay, we determined the cytokine secretion of toxin A- and B-treated human and mouse colonic explants. RESULTS: Toxin A and toxin B exposure to fresh human and mouse colonic explants caused different patterns of cytokine secretion. Toxin A induced macrophage inflammatory protein (MIP) 1α secretion in both human and mouse explants. Toxin A reduced the expression of chloride anion exchanger SLC26A3 expression in mouse colonic explants and human colonic epithelial cells. Patients with CDI had increased colonic MIP-1 α expression and reduced colonic SLC26A3 (solute carrier family 26, member 3) compared with controls. Anti-MIP-1 α neutralizing antibody prevented death, ameliorated colonic injury, reduced colonic interleukin 1ß (IL-1ß) messenger RNA expression, and restored colonic SLC26a3 expression in C. difficile-infected mice. The anti-MIP-1 α neutralizing antibody prevented CDI recurrence. SLC26a3 inhibition augmented colonic IL-1 ß messenger RNA expression and abolished the protective effect of anti-MIP-1 α neutralizing antibody in mice with CDI. CONCLUSION: MIP-1 α is a common toxin A-dependent chemokine in human and mouse colon. MIP-1 α mediates detrimental effects by reducing SLC26a3 and enhancing IL-1 ß expression in the colon.

Designed Ankyrin Repeat Protein (DARPin) Neutralizers of TcdB from Clostridium difficile Ribotype 027.

Clostridium difficile infection (CDI) is a leading cause of hospital-acquired diarrhea. In recent decades, the emergence of the "hypervirulent" BI/NAP1/027 strains of C. difficile significantly increased the morbidity and mortality of CDI. The pathogenesis of CDI is primarily mediated by the action of two toxins, TcdA and TcdB, with TcdB being the major virulent factor in humans. In this report, we describe the engineering of a panel of designed ankyrin repeat proteins (DARPins) that potently neutralize TcdB from the BI/NAP1/027 strains (e.g., TcdBUK1). The most effective DARPin, D16, inhibits TcdBUK1 with a 50% effective concentration (EC50) of 0.5 nM, which is >66-fold lower than that of the FDA-approved anti-TcdB antibody bezlotoxumab (EC50, ∼33 nM). Competitive enzyme-linked immunosorbent assays (ELISAs) showed that D16 blocks interactions between TcdB and its receptor, chondroitin sulfate proteoglycan 4 (CSPG4). The dimeric DARPin U3D16, which pairs D16 with DARPin U3, a disrupter of the interaction of TcdB with Frizzled 1/2/7 receptor, exhibits 10-fold-to-20-fold-enhanced neutralization potency against TcdB from C. difficile strains VPI 10463 (laboratory strain) and M68 (CF/NAP9/017) but identical activity against TcdBUK1 relative to D16. Subsequent ELISAs revealed that TcdBUK1 did not significantly interact with Frizzled 1/2/7. Computation modeling revealed 4 key differences at the Frizzled 1/2/7 binding interface which are likely responsible for the significantly reduced binding affinity.IMPORTANCE We report the engineering and characterization of designed ankyrin proteins as potent neutralizers of TcdB toxin secreted by a hypervirulent ribotype 027 strain of Clostridium difficile We further show that although TcdB toxins from both ribotype 027 and VPI 10461 interact efficiently with TcdB receptors CSPG4 and Pvrl3, TcdB027 lacks significant ability to bind the only known physiologically relevant TcdB receptor, Frizzled 1/2/7.

Correction: Selection and characterization of ultrahigh potency designed ankyrin repeat protein inhibitors of C. difficile toxin B.

[This corrects the article DOI: 10.1371/journal.pbio.3000311.].

Structure of the full-length Clostridium difficile toxin B.

Clostridium difficile is an opportunistic pathogen that establishes in the colon when the gut microbiota are disrupted by antibiotics or disease. C. difficile infection (CDI) is largely caused by two virulence factors, TcdA and TcdB. Here, we report a 3.87-Å-resolution crystal structure of TcdB holotoxin that captures a unique conformation of TcdB at endosomal pH. Complementary biophysical studies suggest that the C-terminal combined repetitive oligopeptides (CROPs) domain of TcdB is dynamic and can sample open and closed conformations that may facilitate modulation of TcdB activity in response to environmental and cellular cues during intoxication. Furthermore, we report three crystal structures of TcdB-antibody complexes that reveal how antibodies could specifically inhibit the activities of individual TcdB domains. Our studies provide novel insight into the structure and function of TcdB holotoxin and identify intrinsic vulnerabilities that could be exploited to develop new therapeutics and vaccines for the treatment of CDI.

Selection and characterization of ultrahigh potency designed ankyrin repeat protein inhibitors of C. difficile toxin B.

Clostridium difficile infection (CDI) is a major nosocomial disease associated with significant morbidity and mortality. The pathology of CDI stems primarily from the 2 C. difficile-secreted exotoxins-toxin A (TcdA) and toxin B (TcdB)-that disrupt the tight junctions between epithelial cells leading to the loss of colonic epithelial barrier function. Here, we report the engineering of a series of monomeric and dimeric designed ankyrin repeat proteins (DARPins) for the neutralization of TcdB. The best dimeric DARPin, DLD-4, inhibited TcdB with a half maximal effective concentration (EC50) of 4 pM in vitro, representing an approximately 330-fold higher potency than the Food and Drug Administration (FDA)-approved anti-TcdB monoclonal antibody bezlotoxumab in the same assay. DLD-4 also protected mice from a toxin challenge in vivo. Cryo-electron microscopy (cryo-EM) studies revealed that the 2 constituent DARPins of DLD-4-1.4E and U3-bind the central and C-terminal regions of the delivery domain of TcdB. Competitive enzyme-linked immunosorbent assay (ELISA) studies showed that the DARPins 1.4E and U3 interfere with the interaction between TcdB and its receptors chondroitin sulfate proteoglycan 4 (CSPG4) and frizzled class receptor 2 (FZD2), respectively. Our cryo-EM studies revealed a new conformation of TcdB (both apo- and DARPin-bound at pH 7.4) in which the combined repetitive oligopeptides (CROPS) domain points away from the delivery domain. This conformation of the CROPS domain is in stark contrast to that seen in the negative-stain electron microscopy (EM) structure of TcdA and TcdB at the same pH, in which the CROPS domain bends toward and "kisses" the delivery domain. The ultrapotent anti-TcdB molecules from this study serve as candidate starting points for CDI drug development and provide new biological tools for studying the pathogenicity of C. difficile. The structural insights regarding both the "native" conformation of TcdB and the putative sites of TcdB interaction with the FZD2 receptor, in particular, should help accelerate the development of next-generation anti-C. difficile toxin therapeutics.

Host-targeted niclosamide inhibits C. difficile virulence and prevents disease in mice without disrupting the gut microbiota.

Clostridium difficile is the leading cause of nosocomial diarrhea and colitis in the industrialized world. Disruption of the protective gut microbiota by antibiotics enables colonization by multidrug-resistant C. difficile, which secrete up to three different protein toxins that are responsible for the gastrointestinal sequelae. Oral agents that inhibit the damage induced by toxins, without altering the gut microbiota, are urgently needed to prevent primary disease and break the cycle of antibiotic-induced disease recurrence. Here, we show that the anthelmintic drug, niclosamide, inhibits the pathogenesis of all three toxins by targeting a host process required for entry into colonocytes by each toxin. In mice infected with an epidemic strain of C. difficile, expressing all three toxins, niclosamide reduced both primary disease and recurrence, without disrupting the diversity or composition of the gut microbiota. Given its excellent safety profile, niclosamide may address an important unmet need in preventing C. difficile primary and recurrent diseases.

Cysteine Protease-Mediated Autocleavage of Clostridium difficile Toxins Regulates Their Proinflammatory Activity.

BACKGROUND & AIMS: Clostridium difficile toxin A (TcdA) and C difficile toxin toxin B (TcdB), the major virulence factors of the bacterium, cause intestinal tissue damage and inflammation. Although the 2 toxins are homologous and share a similar domain structure, TcdA is generally more inflammatory whereas TcdB is more cytotoxic. The functional domain of the toxins that govern the proinflammatory activities of the 2 toxins is unknown. METHODS: Here, we investigated toxin domain functions that regulate the proinflammatory activity of C difficile toxins. By using a mouse ilea loop model, human tissues, and immune cells, we examined the inflammatory responses to a series of chimeric toxins or toxin mutants deficient in specific domain functions. RESULTS: Blocking autoprocessing of TcdB by mutagenesis or chemical inhibition, while reducing cytotoxicity of the toxin, significantly enhanced its proinflammatory activities in the animal model. Furthermore, a noncleavable mutant TcdB was significantly more potent than the wild-type toxin in the induction of proinflammatory cytokines in human colonic tissues and immune cells. CONCLUSIONS: In this study, we identified a novel mechanism of regulating the biological activities of C difficile toxins in that cysteine protease-mediated autoprocessing regulates toxins' proinflammatory activities. Our findings provide new insight into the pathogenesis of C difficile infection and the design of therapeutics against the disease.

TPL2 Is a Key Regulator of Intestinal Inflammation in Clostridium difficile Infection.

Tumor progression locus 2 (TPL2), a serine/threonine protein kinase, is a major inflammatory mediator in immune cells. The predominant inflammatory actions of TPL2 depend on the activation of mitogen-activated protein kinases (MAPK) and the upregulated production of the cytokines tumor necrosis factor alpha (TNF-α) and interleukin 1ß (IL-1ß) in macrophages and dendritic cells in response to lipopolysaccharide (LPS). Significant increases in TNF-α, IL-6, IL-ß, and IL-8 levels in patients with Clostridium difficile infection (CDI) have been reported. Both TNF-α and IL-6 have been postulated to play key roles in the systemic inflammatory response in CDI, and IL-8 is essential for the development of local intestinal inflammatory responses in CDI. The objective of this study was to elucidate the role of TPL2 in the pathogenesis of CDI. We found that TPL2 was significantly activated in human and mouse intestinal tissues upon C. difficile toxin exposure or CDI. We further demonstrated that TPL2 knockout (TPL2-KO) mice were significantly more resistant to CDI than wild-type mice, with significantly reduced production of TNF-α, IL-6, IL-1ß, KC (a mouse homologue of IL-8), and myeloperoxidase (MPO) in the ceca and colons of TPL2-KO mice. Finally, we found that TPL2 inhibition by a specific inhibitor or TPL2 gene ablation significantly reduced TcdB-induced production of TNF-α, IL-6, IL-ß, and KC by inhibiting the activation of p38, extracellular signal-regulated kinase (ERK), and c-Jun NH2-terminal kinase (JNK). Taken together, our data suggest that TPL2 represents a potential therapeutic target for CDI treatment.

Mice with Inflammatory Bowel Disease are Susceptible to Clostridium difficile Infection With Severe Disease Outcomes.

Background: Over the past several decades, there has been a significant increase in the incidence of Clostridium difficile infection (CDI) in patients suffering from inflammatory bowel disease (IBD). However, a wild-type animal model is not available to study these comorbid diseases. Methods: We evaluated the susceptibility to CDI of mice with dextran sulfate sodium salt (DSS)-induced colitis (IBD mice) with or without antibiotic exposure; we examined the histopathology and cytokine response in the concomitant diseases after the model was created. Results: No CDI occurs in healthy control mice, wherease the incidence of CDI in IBD mice is 40%; however, in IBD mice that received antibiotics, the incidence of CDI is 100% and the disease is accompanied by high levels of toxins in the mouse feces and sera. Compared to IBD and CDI alone, those IBD mice infected with C. difficile have more severe symptoms, toxemia, histopathological damage, and higher mortality. Moreover, several proinflammatory cytokines and chemokines are significantly elevated in the colon tissues from IBD mice infected with C. difficile. Conclusions: We, for the first time, demonstrate in an animal model that mice with dextran sulfate sodium induced-inflammatory bowel disease are significantly more susceptible to C. difficile infection, and that the bacterial infection led to more severe disease and death. These findings are consistent with clinical observations, thus, the animal model will permit us to study the pathogenesis of these concurrent diseases and to develop therapeutic strategies against the comorbidity of IBD and CDI.

The role of purified Clostridium difficile glucosylating toxins in disease pathogenesis utilizing a murine cecum injection model.

Most pathogenic Clostridium difficile produce two major exotoxins TcdA and TcdB, in the absence of which the bacterium is non-pathogenic. While it is important to investigate the role of each toxin in the pathogenesis of C. difficile infection (CDI) using isogenic strains, it is impossible to precisely control the expression levels of individual toxins and exclude bacterial factors that may contribute to the toxins' effects during infection. In this study, we utilized an acute intestinal disease model by injecting purified toxins directly into mouse cecum after a midline laparotomy. We evaluated the physical condition of mice by clinical score and survival, and the intestinal tissue damage and inflammation by histology. Depending on the dose of the toxins, mice developed mild to severe colitis, experienced diarrhea or rapidly died. We found that both purified TcdA and TcdB were able to induce clinical disease, intestinal inflammation, and tissue damage that resembled CDI. TcdA was significantly faster in inducing intestinal inflammation and tissue damage, and was approximately five times more potent than TcdB in terms of inducing severe gut disease and death outcomes in mice. Moreover, we found that the two toxins had significant synergistic effects on disease induction. Comparison of the in vivo toxicity of TcdB from clinical strains revealed that TcdB from an epidemic RT 027 strain was more toxic than the others. Our study thus demonstrates that both TcdA and TcdB, independent of other factors from C. difficile bacterium, are able to cause disease that resembles CDI and highlights the importance of targeting both toxins for vaccines and therapeutics against the disease.

A Multiparticulate Delivery System for Potential Colonic Targeting Using Bovine Serum Albumin as a Model Protein : Theme: Formulation and Manufacturing of Solid Dosage Forms Guest Editors: Tony Zhou and Tonglei Li.

PURPOSE: There are many important diseases whose treatment could be improved by delivering a therapeutic protein to the colon, for example, Clostridium difficile infection, ulcerative colitis and Crohn's Disease. The goal of this project was to investigate the feasibility of colonic delivery of proteins using multiparticulate beads. METHODS: In this work, bovine serum albumin (BSA) was adopted as a model protein. BSA was spray layered onto beads, followed by coating of an enteric polymer EUDRAGIT® FS 30 D to develop a colonic delivery system. The secondary and tertiary structure change and aggregation of BSA during spray layering process was examined. The BSA layered beads were then challenged in an accelerated stability study using International Council for Harmonization (ICH) conditions. The in vitro release of BSA from enteric coated beads was examined using United States Pharmacopeia (USP) dissolution apparatus 1. RESULTS: No significant changes in the secondary and tertiary structure or aggregation profile of BSA were observed after the spray layering process. Degradation of BSA to different extents was detected after storing at 25°C and 40°C for 38 days. Enteric coated BSA beads were intact in acidic media while released BSA in pH 7.4 phosphate buffer. CONCLUSION: We showed the feasibility of delivering proteins to colon in vitro using multiparticulate system.