Spinal cord injury causes sustained disruption of the blood-testis barrier in the rat.

There is a high incidence of infertility in males following traumatic spinal cord injury (SCI). Quality of semen is frequently poor in these patients, but the pathophysiological mechanism(s) causing this are not known. Blood-testis barrier (BTB) integrity following SCI has not previously been examined. The objective of this study was to characterize the effects of spinal contusion injury on the BTB in the rat. 63 adult, male Sprague Dawley rats received SCI (n = 28), laminectomy only (n = 7) or served as uninjured, age-matched controls (n = 28). Using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), BTB permeability to the vascular contrast agent gadopentate dimeglumine (Gd) was assessed at either 72 hours-, or 10 months post-SCI. DCE-MRI data revealed that BTB permeability to Gd was greater than controls at both 72 h and 10 mo post-SCI. Histological evaluation of testis tissue showed increased BTB permeability to immunoglobulin G at both 72 hours- and 10 months post-SCI, compared to age-matched sham-operated and uninjured controls. Tight junctional integrity within the seminiferous epithelium was assessed; at 72 hours post-SCI, decreased expression of the tight junction protein occludin was observed. Presence of inflammation in the testes was also examined. High expression of the proinflammatory cytokine interleukin-1 beta was detected in testis tissue. CD68(+) immune cell infiltrate and mast cells were also detected within the seminiferous epithelium of both acute and chronic SCI groups but not in controls. In addition, extensive germ cell apoptosis was observed at 72 h post-SCI. Based on these results, we conclude that SCI is followed by compromised BTB integrity by as early as 72 hours post-injury in rats and is accompanied by a substantial immune response within the testis. Furthermore, our results indicate that the BTB remains compromised and testis immune cell infiltration persists for months after the initial injury.

THE ROLE OF TAK1 IN PANCREATIC CANCER DEVELOPMENT

Pancreatic cancer is the fourth leading cause of cancer-related mortality in the United States and the fifth leading cause of cancer-related mortality worldwide. Pancreatic cancer is a big challenge in large due to the lack of early symptoms. In addition, drug resistance is a major obstacle to the success of chemotherapy in pancreatic cancer. The underlying mechanism of drug resistance in human pancreatic cancers is not well understood. Better understanding of the mechanism of molecular pathways in human pancreatic cancers can help to identify the novel therapeutic target candidates, and develop the new preventive and clinic strategies to improve patient survival. We discovered that TAK1 is overexpressed in pancreatic cancer cell lines and patient tumor tissues. We demonstrated that the elevated activity of TAK1 is caused by its binding partner TAB1. Knocking down of TAK1 in pancreatic cancer cells with RNAi technique resulted in cell apoptosis and significantly reduces the size of tumors in mice and made a chemotherapy drug more potent. Targeting the kinase activity of TAK1 with the selective inhibitor LY2610956 strongly synergized in vitro with the antitumor activity of gemcitabine, oxaliplatin, or irinotecan on pancreatic cancer cells. These findings highlighted that TAK1 could be a potential therapeutic target for pancreatic cancer. We also demonstrated that TAK activity is regulated by its binding protein TAB1. We defined a minimum TAB1 sequence which is required and sufficient for TAK1 kinase activity. We created a recombinant TAK1-TAB1 C68 fusion form which has highly kinase activity. This active form could is used for screening TAK1 inhibitors. In addition, several posttranslational modifications were identified in our study. The acetylation of lysine 158 on TAK1 is required for kinase activity. This site is conserved throughout all of kinase. Our findings may reveal a new mechanism by which kinase activity is regulated.

A comparative assessment of endothelium from pseudophakic and phakic donor corneas stored in organ culture

AIMS To evaluate the endothelial quality of corneas obtained from pseudophakic donors and to compare the data with matched phakic controls. METHODS Corneas from eyes with posterior chamber intraocular lenses (PCIOLs) and corneas from phakic eyes (controls) were stored for 1-2 weeks in organ culture and then examined after staining with Alizarin red S. The corneas were divided into two groups according to the duration of storage. Endothelial cell density, the percentage of hexagonal cells, and the coefficient of variation (CV) were determined. RESULTS There was no statistically significant difference between the 14 PCIOL corneas and the 13 controls stored in organ culture for 7 days for any of the three parameters studied. The mean cell density was 2155 (SD 529) cells/mm(2) in the PCIOL corneas and 2118 (453) cells/mm(2) in the controls (p=0.85). The mean percentage of hexagonal cells was 52% (8%) and 58% (7%), respectively (p=0.06). The mean CV was 0.32 (0.18) in the pseudophakic corneas and 0.39 (0.18) in the controls (p=0.33). Moreover, there was no significant difference between the PCIOL corneas and the controls stored for up to 2 weeks. CONCLUSIONS The corneal endothelium from eyes with PCIOLs appears to be similar to that of phakic eyes after 1-2 weeks in organ culture. This finding suggests that corneas from pseudophakic eyes should not routinely be disqualified for transplantation. The use of at least some pseudophakic corneas may substantially increase the potential donor pool.

Memo has a novel role in S1P signaling and crucial for vascular development

Memo is a conserved protein that was identified as an essential mediator of tumor cell motility induced by receptor tyrosine kinase activation. Here we show that Memo null mouse embryonic fibroblasts (MEFs) are impaired in PDGF-induced migration and this is due to a defect in sphingosine-1-phosphate (S1P) signaling. S1P is a bioactive phospholipid produced in response to multiple stimuli, which regulates many cellular processes. S1P is secreted to the extracellular milieu where it exerts its function by binding a family of G-protein coupled receptors (S1PRs), causing their activation in an autocrine or paracrine manner. The process, termed cell-autonomous S1PR signaling, plays a role in survival and migration. Indeed, PDGF uses cell-autonomous S1PR signaling to promote cell migration; we show here that this S1P pathway requires Memo. Using vascular endothelial cells (HUVECs) with Memo knock-down we show that their survival in conditions of serum-starvation is impaired. Furthermore, Memo loss in HUVECs causes a reduction of junctional VE-cadherin and an increase in sprout formation. Each of these phenotypes is rescued by S1P or S1P agonist addition, showing that Memo also plays an important role in cell-autonomous S1PR signaling in endothelial cells. We also produced conventional and endothelial cell-specific conditional Memo knock-out mouse strains and show that Memo is essential for embryonic development. Starting at E13.5 embryos of both strains display bleeding and other vascular problems, some of the phenotypes that have been described in mouse strains lacking S1PRs. The essential role of Memo in embryonic vascular development may be due in part to alterations in S1P signaling. Taken together our results show that Memo has a novel role in the S1P pathway and that Memo is needed to promote cell-autonomous S1PR activation.

Covalent immobilisation of VEGF on plasma-coated electrospun scaffolds for tissue engineering applications.

Recent findings in the field of biomaterials and tissue engineering provide evidence that surface immobilised growth factors display enhanced stability and induce prolonged function. Cell response can be regulated by material properties and at the site of interest. To this end, we developed scaffolds with covalently bound vascular endothelial growth factor (VEGF) and evaluated their mitogenic effect on endothelial cells in vitro. Nano- (254±133 nm) or micro-fibrous (4.0±0.4 μm) poly(ɛ-caprolactone) (PCL) non-wovens were produced by electrospinning and coated in a radio frequency (RF) plasma process to induce an oxygen functional hydrocarbon layer. Implemented carboxylic acid groups were converted into amine-reactive esters and covalently coupled to VEGF by forming stable amide bonds (standard EDC/NHS chemistry). Substrates were analysed by X-ray photoelectron spectroscopy (XPS), enzyme-linked immuno-assays (ELISA) and immunohistochemistry (anti-VEGF antibody and VEGF-R2 binding). Depending on the reaction conditions, immobilised VEGF was present at 127±47 ng to 941±199 ng per substrate (6mm diameter; concentrations of 4.5 ng mm(-2) or 33.3 ng mm(-2), respectively). Immunohistochemistry provided evidence for biological integrity of immobilised VEGF. Endothelial cell number of primary endothelial cells or immortalised endothelial cells were significantly enhanced on VEGF-functionalised scaffolds compared to native PCL scaffolds. This indicates a sustained activity of immobilised VEGF over a culture period of nine days. We present a versatile method for the fabrication of growth factor-loaded scaffolds at specific concentrations.

Renal microvascular assembly and repair: power and promise of molecular definition.

Developmental assembly of the renal microcirculation is a precise and coordinated process now accessible to experimental scrutiny. Although definition of the cellular and molecular determinants is incomplete, recent findings have reframed concepts and questions about the origins of vascular cells in the glomerulus and the molecules that direct cell recruitment, specialization and morphogenesis. New findings illustrate principles that may be applied to defining critical steps in microvascular repair following glomerular injury. Developmental assembly of endothelial, mesangial and epithelial cells into glomerular capillaries requires that a coordinated, temporally defined series of steps occur in an anatomically ordered sequence. Recent evidence shows that both vasculogenic and angiogenic processes participate. Local signals direct cell migration, proliferation, differentiation, cell-cell recognition, formation of intercellular connections, and morphogenesis. Growth factor receptor tyrosine kinases on vascular cells are important mediators of many of these events. Cultured cell systems have suggested that basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF) promote endothelial cell proliferation, migration or morphogenesis, while genetic deletion experiments have defined an important role for PDGF beta receptors and platelet-derived growth factor (PDGF) B in glomerular development. Receptor tyrosine kinases that convey non-proliferative signals also contribute in kidney and other sites. The EphB1 receptor, one of a diverse class of Eph receptors implicated in neural cell targeting, directs renal endothelial migration, cell-cell recognition and assembly, and is expressed with its ligand in developing glomeruli. Endothelial TIE2 receptors bind angiopoietins (1 and 2), the products of adjacent supportive cells, to signals direct capillary maturation in a sequence that defines cooperative roles for cells of different lineages. Ultimately, definition of the cellular steps and molecular sequence that direct microvascular cell assembly promises to identify therapeutic targets for repair and adaptive remodeling of injured glomeruli.

Thrombotic thrombocytopenic purpura during pregnancy versus imitator of preeclampsia.

BACKGROUND Thrombotic thrombocytopenic purpura (TTP) is a severe disorder affecting the microcirculation of multiple organs due to a systemic endothelial cell injury secondary to a deficiency in ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 motif, member 13) activity. TTP is a rare complication of pregnancy with a poor prognosis and high fetal mortality, especially when it occurs during the first trimester. Recent data have supported that effective treatment of TTP is plasma therapy. Unfortunately a major problem remains in the delay in diagnosis due to confounding factors between other "imitators of preeclampsia." Rapid and readily available laboratory testing to quickly diagnose TTP is desperately needed to improve care and to save mother and future child life. CASE REPORT We describe a rare case of successful pregnancy after TTP manifestations occurring in the first trimester; most importantly, our experience represents the first case of atypical manifestation due to neurologic and kidney manifestations preceding laboratory assay alterations. RESULTS We treated a patient with plasma replacement of 30 mL/kg/day and daily plasmapheresis in combination with continuous infusion of fresh-frozen plasma 10 mL/kg/day. The response of clinical manifestation immediately improved. At 30 weeks, the patient had multiple episodes of high blood pressure and concomitant decrease of hemoglobin and platelet count, so a cesarean section was immediately performed. She delivered a healthy female baby. CONCLUSION Early diagnosis by ADAMTS13 activity, occasionally occurring before clinical manifestations, aided us in promptly administering commended and life-saving treatments.

Engineering an in vitro air-blood barrier by 3D bioprinting

Intensive efforts in recent years to develop and commercialize in vitro alternatives in the field of risk assessment have yielded new promising two- and three dimensional (3D) cell culture models. Nevertheless, a realistic 3D in vitro alveolar model is not available yet. Here we report on the biofabrication of the human air-blood tissue barrier analogue composed of an endothelial cell, basement membrane and epithelial cell layer by using a bioprinting technology. In contrary to the manual method, we demonstrate that this technique enables automatized and reproducible creation of thinner and more homogeneous cell layers, which is required for an optimal air-blood tissue barrier. This bioprinting platform will offer an excellent tool to engineer an advanced 3D lung model for high-throughput screening for safety assessment and drug efficacy testing.

Hypersensitive granulomatous response to mycobacterial glycolipid trehalose 6,6'-dimycolate

Protection against Mycobacterium tuberculosis infection requires an effective cell mediated immune response leading to granuloma formation and organism containment. Trehalose 6,6'-dimycolate (TDM), a glycolipid present on the mycobacterial cell wall, has been implicated as a key component in establishment of the granulomatous response. TDM has potent immunoregulatory and inflammatory properties; the acute response to TDM produces pathology resembling early Mycobacterium tuberculosis infection. We have further developed this model to study TDM-specific cell mediated immune responses that may play a role in the later stages of infection and pathology. Lungs from mice immunized with TDM in the form of a water-oil-water (w/o/w) emulsion demonstrate heightened histological damage, inflammation, lymphocytic infiltration, and vascular endothelial cell damage upon subsequent challenge with TDM. This exacerbated response can be adoptively transferred to naïve mice via transfer of non-adherent lymphocytes from TDM immunized mice. To identify the cell phenotype(s) regulating this response, purified non-adherent cell populations (CD4+ and CD8+ T cells; CD19 + B cells) were isolated from TDM immunized mice, adoptively transferred into naive mice, and subsequently challenged with TDM. Lung histopathology and cytokine production identified CD4+ cells as the critical cell phenotype regulating the TDM-specific hypersensitive response. The role of CD1d in presentation of TDM was examined. CD1d, a molecule known to present lipids to T cells, was identified as critical in development of the hypersensitive response. CD4+ cells were isolated from TDM-immunized CD1d -/- mice and adoptively transferred into naive wild type mice, followed by TDM challenge. These mice were deficient in development of the hypersensitive granulomatous response, signifying the importance of CD1d in the generation of TDM-specific CD4+ cells. The experiments presented in this dissertation provide further evidence for involvement of TDM-specific cell mediated immune response in elicitation of pathological damage during Mycobacterium tuberculosis infection. ^

Insights into the biology of lymphatic metastasis

Recent data suggest that the generation of new lymphatic vessels (i.e. lymphangiogenesis) may be a rate-limiting step in the dissemination of tumor cells to regional lymph nodes. However, efforts to study the cellular and molecular interactions that take place between tumor cells and lymphatic endothelial cells have been limited due to a lack of lymphatic endothelial cell lines available for study. ^ I have used a microsurgical approach to establish conditionally immortalized lymphatic endothelial cell lines from the afferent mesenteric lymphatic vessels of mice. Characterization of lymphatic endothelial cells, and tumor-associated lymphatic vessels revealed high expression levels of VCAM-1, which is known to facilitate adhesion of some tumor cells to vascular endothelial cells. Further investigation revealed that murine melanoma cells selected for high expression of α4, a counter-receptor for VCAM-1, demonstrated enhanced adhesion to lymphatic endothelial cells in vitro, and increased tumorigenicity and lymphatic metastasis in vivo, despite similar lymphatic vessel numbers. ^ Next, I examined the effects of growth factors that regulate lymphangiogenesis, and report that several growth factors are capable of activating survival and proliferation pathways of lymphatic endothelial cells. The dual protein tyrosine kinase inhibitor AEE788 (EGFR and VEGFR-2) inhibited the activation of Akt and MAPK in lymphatic endothelial cells responding to multiple growth factors. Moreover, oral treatment of mice with AEE788 decreased lymphatic vessel density and production of lymphatic metastasis by human colon cancer cells growing in the cecum of nude mice. ^ In the last set of experiments, I investigated the surgical management of lymphatic metastasis using a novel model of sentinel lymphadenectomy in live mice bearing subcutaneous B16-BL6 melanoma. The data demonstrate that this procedure when combined with wide excision of the primary melanoma, significantly enhanced survival of syngeneic C57BL/6 mice. ^ Collectively, these results indicate that the production of lymphatic metastasis depends on lymphangiogenesis, tumor cell adhesion to lymphatic endothelial cells, and proliferation of tumor cells in lymph nodes. Thus, lymphatic metastasis is a multi-step, complex, and active process that depends upon multiple interactions between tumor cells and tumor associated lymphatic endothelial cells. ^

Caenorhabditis elegans germinal center kinase (Gck-1) is a p-body component that inhibits precocious ectopic MAP kinase dependent meiotic maturation

The Caenorhabditis elegans germline is an excellent model system for studying meiosis, as the gonad contains germ cells in all stages of meiosis I prophase in a linear temporal and spatial pattern. To form healthy gametes, many events must be coordinated. Failure of any step in the process can reduce fertility. Here, we describe a C. elegans Germinal Center Kinase, GCK-1, that is essential for the accurate progression of germ cells through meiosis I prophase. In the absence of GCK-1, germ cells undergo precocious maturation due to the activation of a specific MAP kinase isoform. Furthermore, GCK-1 localizes to P-bodies, RNP particles that have been implicated in RNA degradation and translational control. Like two other components of C. elegans germline P-bodies, GCK-1 functions to limit physiological germ cell apoptosis. This is the first study to identify a role for a GCK-III kinase in metazoan germ cell development and to link P-body function with MAP kinase activation and germ cell maturation. ^

Downstream targets of the Rhox5 homeobox gene

The X-linked mouse Rhox gene cluster contains over 30 homeobox genes that are candidates to regulate multiple steps in male and female gametogenesis. The founding member of the Rhox gene cluster, Rhox5, is an androgen-dependent gene expressed in Sertoli cells that promotes the survival and differentiation of the adjacent male germ cells. To decipher downstream signaling pathways of Rhox5, I used in vivo and in vitro microarray profiling to identify and characterize downstream targets of Rhox5 in the testis. This led to the identification of many Rhox5 -regulated genes, two of which I focused on in more detail. One of them, Unc5c, encodes a pro-apoptotic receptor with tumor suppressor activity that I found is negatively regulated by Rhox5 through a Rhox5-response element in the Unc5c 5' untranslated region (5' UTR). Examination of other mouse Rhox family members revealed that Rhox2 and Rhox3 also have the ability to downregulate Unc5c expression. The human RHOX protein RHOXF2 also had this ability, indicating that Unc5c repression is a conserved Rhox-dependent response. The repression of Unc5c expression by Rhox5 may, in part, mediate Rhox5's pro-survival function in the testis, as I found that Unc5c mutant mice have decreased germ cell apoptosis in the testis. This along with my other data leads me to propose a model in which Rhox5 is a negative regulator upstream of Unc5c in a Sertoli-cell pathway that promotes germ-cell survival. The other Rhox5-regulated gene that I studied in detail is insulin II (Ins2). Several lines of evidence, including electrophoretic mobility shift anaylsis, promoter mutagenesis, and chromatin immuoprecipitation analysis indicated that Ins2 is a direct target of Rhox5. Structure-function analysis identified homeodomain residues and the RHOX5 amino-terminal domain crucial for conferring Ins2 inducibility. Rhox5 regulates not only the Ins2 gene but also genes encoding other secreted proteins regulating metabolism (adiponectin and resistin), the rate-liming enzyme for monosaturated fatty acid biosynthesis (SCD-1), and transcription factors crucial for regulating metabolism (the nuclear hormone receptor PPARγ). I propose that the regulation of some or all of these molecules in Sertoli cells is responsible for the Rhox5-dependent survival of the adjacent germ cells. ^

Characterization of the interactions between fibronectin and the Borrelia burgdorferi lipoprotein, BBK32

The findings presented in this dissertation detail the complex interaction between BBK32 and fibronectin and describe novel consequences of the interaction. BBK32 is a fibronectin-binding protein on Borrelia burgdorferi, the causative agent of Lyme disease. We found that BBK32 contains multiple fibronectin-binding motifs, recognizing the fibronectin N-terminal domain (NTD) and the gelatin binding domain (GBD) in an anti-parallel order, where corresponding sites in BBK32 and fibronectin are aligned so that there is a one-to-one interaction between the proteins. While characterizing this interaction, we discovered that binding of BBK32 to the GBD inhibits the migration stimulating factor's (MSF) motogenic activity. In the presence of BBK32, endothelial cells do not migrate in response to increasing concentrations of MSF or the GBD. MSF is found under wound healing conditions, and inhibition of its activity may allow the tick-transmitted spirochetes to delay wound healing and to establish an infection. ^ Biophysical structural studies, designed to identify a mechanism of interaction, revealed that BBK32 binding to the NTD leads to the unfolding of plasma fibronectin, which exposes α5β1 integrin recognition motifs. Binding assays demonstrate that the BBK32-NTD interaction enhances the plasma fibronectin-α5β1 integrin interaction, which may allow B. burgdorferi to invade host cells, and thereby evade the host immune system. ^ We also determined that BBK32 binds fibronectin F3 modules, which leads to plasma fibronectin aggregation and induction of superfibronectin. The resulting superfibronectin is conformationally distinct from plasma and cellular fibronectin, and can inhibit endothelial cell proliferation. BBK32's active superfibronectin-forming motif has been located to a region between residues 160 and 175, which contains two sequence motifs that are also found in anastellin, the only other known superfibronectin-inducing protein. ^ A potential consequence of BBK32-induced superfibronectin formation was identified. BBK32-induced superfibronectin formation results in the exposure of α4β1 integrin recognition sequences in fibronectin. The α4β1 integrin is required for leukocyte transendothelial cell migration. BBK32-induced superfibronectin inhibits this activity. The inhibition of leukocyte recruitment to the infection site may slow the activity of the host immune system, and permit the spirochetes to establish an infection. ^

IDENTIFICATION AND CHARACTERIZATION OF DISTINCT POPULATIONS OF CLONOGENIC BONE MARROW STROMAL CELLS CAPABLE OF TRANSFERRING THE HEMATOPOIETIC MICROENVIRONMENT IN VIVO AND SUPPORTING LT-HSCs IN VITRO

Bone marrow (BM) stromal cells are ascribed two key functions, 1) stem cells for non-hematopoietic tissues (MSC) and 2) as components of the hematopoietic stem cell niche. Current approaches studying the stromal cell system in the mouse are complicated by the low yield of clonogenic progenitors (CFU-F). Given the perivascular location of MSC in BM, we developed an alternative methodology to isolate MSC from mBM. An intact ‘plug’ of bone marrow is expelled from bones and enzymatically disaggregated to yield a single cell suspension. The recovery of CFU-F (1917.95+199) reproducibly exceeds that obtained using the standard BM flushing technique (14.32+1.9) by at least 2 orders of magnitude (P<0.001; N = 8) with an accompanying 196-fold enrichment of CFU-F frequency. Purified BM stromal and vascular endothelial cell populations are readily obtained by FACS. A detailed immunophenotypic analysis of lineage depleted BM identified PDGFRαβPOS stromal cell subpopulations distinguished by their expression of CD105. Both subpopulations retained their original phenotype of CD105 expression in culture and demonstrate MSC properties of multi-lineage differentiation and the ability to transfer the hematopoietic microenvironment in vivo. To determine the capacity of either subpopulation to support long-term multi-lineage reconstituting HSCs, we fractionated BM stromal cells into either the LinNEGPDGFRαβPOSCD105POS and LINNEGPDGFRαβPOSCD105LOW/- populations and tested their capacity to support LT-HSC by co-culturing each population with either 1 or 10 HSCs for 10 days. Following the 10 day co-culture period, both populations supported transplantable HSCs from 10 cells/well co-cultures demonstrating high levels of donor repopulation with an average of 65+23.6% chimerism from CD105POS co-cultures and 49.3+19.5% chimerism from the CD105NEG co-cultures. However, we observed a significant difference when mice were transplanted with the progeny of a single co-cultured HSC. In these experiments, CD105POS co-cultures (100%) demonstrated long-term multi- lineage reconstitution, while only 4 of 8 mice (50%) from CD105NEG -single HSC co-cultures demonstrated long-term reconstitution, suggesting a more limited expansion of functional stem cells. Taken together, these results demonstrate that the PDGFRαβCD105POS stromal cell subpopulation is distinguished by a unique capacity to support the expansion of long-term reconstituting HSCs in vitro.

REGULATION OF TOXIN SYNTHESIS BY CLOSTRIDIUM DIFFICILE

Clostridium difficile is the leading definable cause of nosocomial diarrhea worldwide due to its virulence, multi-drug resistance, spore-forming ability, and environmental persistence. The incidence of C. difficile infection (CDI) has been increasing exponentially in the last decade. Virulent strains of C. difficile produce either toxin A and/or toxin B, which are essential for the pathogenesis of this bacterium. Current methods for diagnosing CDI are mostly qualitative tests that detect the bacterium, the toxins, or the toxin genes. These methods do not differentiate virulent C. difficile strains that produce active toxins from non-virulent strains that do not produce toxins or produce inactive toxins. Based on the knowledge that C. difficile toxins A and B cleave a substrate that is stereochemically similar to the native substrate of the toxins, uridine diphosphoglucose, a quantitative, cost-efficient assay, the Cdifftox activity assay, was developed to measure C. difficile toxin activity. The concept behind the activity assay was modified to develop a novel, rapid, sensitive, and specific assay for C. difficile toxins in the form of a selective and differential agar plate culture medium, the Cdifftox Plate assay (CDPA). This assay combines in a single step the specific identification of C. difficile strains and the detection of active toxin(s). The CDPA was determined to be extremely accurate (99.8% effective) at detecting toxin-producing strains based on the analysis of 528 C. difficile isolates selected from 50 tissue culture cytotoxicity assay-positive clinical stool samples. This new assay advances and improves the culture methodology in that only C. difficile strains will grow on this medium and virulent strains producing active toxins can be differentiated from non-virulent strains. This new method reduces the time and effort required to isolate and confirm toxin-producing C. difficile strains and provides a clinical isolate for antibiotic susceptibility testing and strain typing. The Cdifftox activity assay was used to screen for inhibitors of toxin activity. Physiological levels of the common human conjugated bile salt, taurocholate, was found to inhibit toxin A and B in vitro activities. When co-incubated ex vivo with purified toxin B, taurocholate protected Caco-2 colonic epithelial cells from the damaging effects of the toxin. Furthermore, using a caspase-3 detection assay, taurocholate reduced the extent of toxin B-induced Caco-2 cell apoptosis. These results suggest that bile salts can be effective in protecting the gut epithelium from C. difficile toxin damage, thus, the delivery of physiologic amounts of taurocholate to the colon, where it is normally in low concentration, could be useful in CDI treatment. These findings may help to explain why bile rich small intestine is spared damage in CDI, while the bile salt poor colon is vulnerable in CDI. Toxin synthesis in C. difficile occurs during the stationary phase, but little is known about the regulation of these toxins. It was hypothesized that C. difficile toxin synthesis is regulated by a quorum sensing mechanism. Two lines of evidence supported this hypothesis. First, a small (KDa), diffusible, heat-stable toxin-inducing activity accumulates in the medium of high-density C. difficile cells. This conditioned medium when incubated with low-density log-phase cells causes them to produce toxin early (2-4 hrs instead of 12-16 hrs) and at elevated levels when compared with cells grown in fresh medium. These data suggested that C. difficile cells extracellularly release an inducing molecule during growth that is able to activate toxin synthesis prematurely and demonstrates for the first time that toxin synthesis in C. difficile is regulated by quorum signaling. Second, this toxin-inducing activity was partially purified from high-density stationary-phase culture supernatant fluid by HPLC and confirmed to induce early toxin synthesis, even in C. difficile virulent strains that over-produce the toxins. Mass spectrometry analysis of the purified toxin-inducing fraction from HPLC revealed a cyclic compound with a mass of 655.8 Da. It is anticipated that identification of this toxin-inducing compound will advance our understanding of the mechanism involved in the quorum-dependent regulation of C. difficile toxin synthesis. This finding should lead to the development of even more sensitive tests to diagnose CDI and may lead to the discovery of promising novel therapeutic targets that could be harnessed for the treatment C. difficile infections.