Pharmacodynamic measures within tumors expose differential activity of PD(L)-1 antibody therapeutics.

Macromolecules such as monoclonal antibodies (mAbs) are likely to experience poor tumor penetration because of their large size, and thus low drug exposure of target cells within a tumor could contribute to suboptimal responses. Given the challenge of inadequate quantitative tools to assess mAb activity within tumors, we hypothesized that measurement of accessible target levels in tumors could elucidate the pharmacologic activity of a mAb and could be used to compare the activity of different mAbs. Using positron emission tomography (PET), we measured the pharmacodynamics of immune checkpoint protein programmed-death ligand 1 (PD-L1) to evaluate pharmacologic effects of mAbs targeting PD-L1 and its receptor programmed cell death protein 1 (PD-1). For PD-L1 quantification, we first developed a small peptide-based fluorine-18-labeled PET imaging agent, [18F]DK222, which provided high-contrast images in preclinical models. We then quantified accessible PD-L1 levels in the tumor bed during treatment with anti-PD-1 and anti-PD-L1 mAbs. Applying mixed-effects models to these data, we found subtle differences in the pharmacodynamic effects of two anti-PD-1 mAbs (nivolumab and pembrolizumab). In contrast, we observed starkly divergent target engagement with anti-PD-L1 mAbs (atezolizumab, avelumab, and durvalumab) that were administered at equivalent doses, correlating with differential effects on tumor growth. Thus, we show that measuring PD-L1 pharmacodynamics informs mechanistic understanding of therapeutic mAbs targeting PD-L1 and PD-1. These findings demonstrate the value of quantifying target pharmacodynamics to elucidate the pharmacologic activity of mAbs, independent of mAb biophysical properties and inclusive of all physiological variables, which are highly heterogeneous within and across tumors and patients.

Evaluating Shigella flexneri Pathogenesis in the Human Enteroid Model.

The enteric pathogen Shigella is one of the leading causes of moderate-to-severe diarrhea and death in young children in developing countries. Transformed cell lines and animal models have been widely used to study Shigella pathogenesis. In addition to altered physiology, transformed cell lines are composed of a single cell type that does not sufficiently represent the complex multicellular environment of the human colon. Most available animal models do not accurately mimic human disease. The human intestinal enteroid model, derived from LGR5+ stem cell-containing intestinal crypts from healthy subjects, represents a technological leap in human gastrointestinal system modeling and provides a more physiologically relevant system that includes multiple cell types and features of the human intestine. We established the utility of this model for studying basic aspects of Shigella pathogenesis and host responses. In this study, we show that Shigellaflexneri is capable of infecting and replicating intracellularly in human enteroids derived from different segments of the intestine. Apical invasion by S. flexneri is very limited but increases ∼10-fold when enteroids are differentiated to include M cells. Invasion via the basolateral surface was at least 2-log10 units more efficient than apical infection. Increased secretion of interleukin-8 and higher expression levels of the mucin glycoprotein Muc2 were observed in the enteroids following S. flexneri infection. The human enteroid model promises to bridge some of the gaps between traditional cell culture, animal models, and human infection.

CITED2 modulates estrogen receptor transcriptional activity in breast cancer cells.

Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (CITED2) is a member of the CITED family of non-DNA binding transcriptional co-activators of the p300/CBP-mediated transcription complex. Previously, we identified CITED2 as being overexpressed in human breast tumors relative to normal mammary epithelium. Upon further investigation within the estrogen receptor (ER)-positive subset of these breast tumor samples, we found that CITED2 mRNA expression was elevated in those associated with poor survival. In light of this observation, we investigated the effect of elevated CITED2 levels on ER function. While ectopic overexpression of CITED2 in three ER-positive breast cancer cell lines (MCF-7, T47D, and CAMA-1) did not alter cell proliferation in complete media, growth was markedly enhanced in the absence of exogenous estrogen. Correspondingly, cells overexpressing CITED2 demonstrated reduced sensitivity to the growth inhibitory effects of the selective estrogen receptor modulator, 4-hydroxytamoxifen. Subsequent studies revealed that basal ER transcriptional activity was elevated in CITED2-overexpressing cells and was further increased upon the addition of estrogen. Similarly, basal and estrogen-induced expression of the ER-regulated genes trefoil factor 1 (TFF1) and progesterone receptor (PGR) was higher in cells overexpressing CITED2. Concordant with this observation, ChIP analysis revealed higher basal levels of CITED2 localized to the TFF-1 and PGR promoters in cells with ectopic overexpression of CITED2, and these levels were elevated further in response to estrogen stimulation. Taken together, these data indicate that CITED2 functions as a transcriptional co-activator of ER in breast cancer cells and that its increased expression in tumors may result in estrogen-independent ER activation, thereby reducing estrogen dependence and response to anti-estrogen therapy.

Diagnostic imaging learning resources evaluated by students and recent graduates.

Many learning resources can help students develop the problem-solving abilities and clinical skills required for diagnostic imaging. This study explored veterinary students' perceptions of the usefulness of a variety of learning resources. Perceived resource usefulness was measured for different levels of students and for academic versus clinical preparation. Third-year (n=139) and final (fifth) year (n=105) students and recent graduates (n=56) completed questionnaires on perceived usefulness of each resource. Resources were grouped for comparison: abstract/low complexity (e.g., notes, multimedia presentations), abstract/high complexity (e.g., Web-based and film case repositories), concrete/low complexity (e.g., large-group "clicker" workshops), and concrete/high complexity (e.g., small-group interpretation workshops). Lower-level students considered abstract/low-complexity resources more useful for academic preparation and concrete resources more useful for clinical preparation. Higher-level students/recent graduates also considered abstract/low-complexity resources more useful for academic preparation. For all levels, lecture notes were considered highly useful. Multimedia slideshows were an interactive complement to notes. The usefulness of a Web-based case repository was limited by accessibility problems and difficulty. Traditional abstract/low-complexity resources were considered useful for more levels and contexts than expected. Concrete/high-complexity resources need to better represent clinical practice to be considered more useful for clinical preparation.

Epithelial and Neutrophil Interactions and Coordinated Response to Shigella in a Human Intestinal Enteroid-Neutrophil Coculture Model.

Polymorphonuclear neutrophils (PMN) are recruited to the gastrointestinal mucosa in response to inflammation, injury, and infection. Here, we report the development and the characterization of an ex vivo tissue coculture model consisting of human primary intestinal enteroid monolayers and PMN, and a mechanistic interrogation of PMN-epithelial cell interaction and response to Shigella, a primary cause of childhood dysentery. Cellular adaptation and tissue integration, barrier function, PMN phenotypic and functional attributes, and innate immune responses were examined. PMN within the enteroid monolayers acquired a distinct activated/migratory phenotype that was influenced by direct epithelial cell contact as well as by molecular signals. Seeded on the basal side of the intestinal monolayer, PMN were intercalated within the epithelial cells and moved paracellularly toward the apical side. Cocultured PMN also increased basal secretion of interleukin 8 (IL-8). Shigella added to the apical surface of the monolayers evoked additional PMN phenotypic adaptations, including increased expression of cell surface markers associated with chemotaxis and cell degranulation (CD47, CD66b, and CD88). Apical Shigella infection triggered rapid transmigration of PMN to the luminal side, neutrophil extracellular trap (NET) formation, and bacterial phagocytosis and killing. Shigella infection modulated cytokine production in the coculture; apical monocyte chemoattractant protein (MCP-1), tumor necrosis factor alpha (TNF-α), and basolateral IL-8 production were downregulated, while basolateral IL-6 secretion was increased. We demonstrated, for the first time, PMN phenotypic adaptation and mobilization and coordinated epithelial cell-PMN innate response upon Shigella infection in the human intestinal environment. The enteroid monolayer-PMN coculture represents a technical innovation for mechanistic interrogation of gastrointestinal physiology, host-microbe interaction, innate immunity, and evaluation of preventive/therapeutic tools. IMPORTANCE Studies of mucosal immunity and microbial host cell interaction have traditionally relied on animal models and in vitro tissue culture using immortalized cancer cell lines, which yield nonphysiological and often unreliable results. Herein, we report the development and characterization of an ex vivo enteroid-PMN coculture consisting of normal human intestinal epithelium and a mechanistic interrogation of PMN and epithelial cell interaction and function in the context of Shigella infection. We demonstrated tissue-driven phenotypic and functional adaptation of PMN and a coordinated epithelial cell and PMN response to Shigella, a primary cause of dysentery in young children in the developing world.

Occurrence of Ulcerative Lesions in Sea Stars (Asteroidea) of theNorthern Gulf of California, USA.

To assess the variation of sea star (Asteroidea) populations in the Northern Gulf of California and look for evidence of sea star ulcerative lesions, counts of sea star species were recorded in 2019 using a standard belt-transect technique of 30 m2. During surveys, scuba divers also measured sea stars' diameter and examined them for ulcerative lesions. Ulcerative lesions were noted on Phataria unifascialis only. To verify changes in abundance and size of the two most abundant species, P. unifascialis and Pharia pyramidata, historical data from 2009, 2010, and 2016 were used as comparison and using the same methodology. To evaluate differences in abundance or diameter in sea star species over time, analysis of variance tests were used. We found a significant reduction in diameter for the species P. unifascialis and P. pyramidata over time (<0.0001), but only P. unifascialis also showed a significant decrease in abundance (P=0.018). The decrease in diameter recorded for these two species, along with the signs of ulcerative lesions found on one of them in 2019, suggest that a potential mortality event occurred and, as a result, new younger (i.e., smaller) recruits could be recovering the population. These results highlight the importance of population monitoring to understand complex reef community dynamics.

Depletion of the apical endosome in response to viruses and bacterial toxins provides cell-autonomous host defense at mucosal surfaces.

Polarized epithelial cells form an essential barrier against infection at mucosal surfaces. Many pathogens breach this barrier to cause disease, often by co-opting cellular endocytosis mechanisms to enter the cell through the lumenal (apical) cell surface. We recently discovered that the loss of the cell polarity gene PARD6B selectively diminishes apical endosome function. Here, we find that in response to the entry of certain viruses and bacterial toxins into the epithelial cells via the apical membrane, PARD6B and aPKC, two components of the PARD6B-aPKC-Cdc42 apical polarity complex, undergo rapid proteasome-dependent degradation. The perturbation of apical membrane glycosphingolipids by toxin- or virus-binding initiates degradation of PARD6B. The loss of PARD6B causes the depletion of apical endosome function and renders the cell resistant to further infection from the lumenal cell surface, thus enabling a form of cell-autonomous host defense.

Improved Radium-223 Therapy with Combination Epithelial Sodium Channel Blockade.

Background: Radium-223 dichloride ([223Ra]RaCl2) is the first approved alpha particle-emitting therapy and is indicated for treatment of bone metastatic castrate resistant prostate cancer. Approximately half of the dose is absorbed into the gastrointestinal (GI) tract within minutes of administration, limiting disease-site uptake and contributing to toxicity. Here, we investigate the role of enteric ion channels and their modulation for improved therapeutic efficacy and reduced side effects. Methods: Utilizing primary human duodenal organoids (enteroids) as in vitro models of the functional GI epithelium, we found that Amiloride (ENaC blocker) and NS-1619 (K+ channel activator) presented significant effects in 223Ra membranal transport. The radioactive drug distribution was evaluated for lead combinations in vivo, and in osteosarcoma and prostate cancer models. Results: Amiloride shifted 223Ra uptake in vivo from the gut, to nearly double the uptake at sites of bone remodeling. Bone tumor growth inhibition with the combination as measured by bioluminescent and X-ray imaging was significantly greater than single agents alone, and the combination resulted in no weight loss. Conclusion: This combination of approved agents may be readily implemented as a clinical approach to improve outcomes of bone metastatic cancer patients with the benefit of ameliorated tolerability.

Identification of prospective factors promoting osteotropism in breast cancer: a potential role for CITED2.

Breast cancer metastases develop in the bone more frequently than any other site and are a common cause of morbidity in the form of bone pain, pathological fractures, nerve compression and life-threatening hypercalcemia. Despite ongoing research efforts, the molecular and cellular mechanisms that regulate breast cancer cell homing to and colonization of the bone as well as resultant pathological bone alteration remain poorly understood. To identify key mediators promoting breast cancer metastasis to bone, we utilized an immunocompetent, syngeneic murine model of breast cancer metastasis employing the mammary tumor cell line NT2.5. Following intracardiac injection of NT2.5 cells in neu-N mice, metastases developed in the bone, liver and lung, closely mimicking the anatomical distribution of metastases in patients with breast cancer. Using an in vivo selection process, we established NT2.5 sublines demonstrating an enhanced ability to colonize the bone and liver. Genome-wide cDNA microarray analysis comparing gene expression between parental NT2.5 cells and established sublines revealed both known and novel mediators of bone metastasis and osteolysis, including the transcriptional co-activator CITED2. In further studies, we found that expression of CITED2 was elevated in human primary breast tumors and bone metastasis compared to normal mammary epithelium and was highest in breast cancer cell lines that cause osteolytic bone metastasis in animal models. In addition, reducing CITED2 expression in NT2.5 cells inhibited the establishment of bone metastasis and osteolysis in vivo, suggesting a potential role for CITED2 in promoting breast cancer bone metastasis.

Expression of cyclooxygenase isoforms in ulcerated tissues of the nonglandular portion of the stomach in horses.

OBJECTIVE: To characterize the expression of the cyclooxygenase (COX)-1 and COX-2 isoforms in naturally occurring ulcers of the nonglandular portion of the stomach in horses. SPECIMEN POPULATION: 38 specimens from ulcerated stomachs and 10 specimens from healthy stomachs. PROCEDURES: Specimens were collected at an abbatoir; for each specimen of squamous gastric mucosa, 1 portion was fixed in neutral-buffered 10% formalin for immunohistochemical analysis and another was frozen at -70 degrees C for immunoblotting analysis. Immunoreactivity to 2 antibodies, MF241 (selective for COX-1) and MF243 (selective for COX-2), was evaluated by a veterinary pathologist using a scoring system. Expression of COX-1 and COX-2 was confirmed by use of immunoblotting analyses. RESULTS: All specimens from healthy stomachs strongly expressed COX-1, whereas only 2 of 10 expressed COX-2. The expression of both isoforms varied greatly in the ulcerated mucosal specimens. Expression of COX-1 was significantly lower and expression of COX-2 was significantly higher in ulcerated versus healthy specimens. CONCLUSIONS AND CLINICAL RELEVANCE: Increased expression of COX-2 in gastric ulcers of the squamous portion of the stomach in horses suggested a role for this enzyme in gastric ulcer healing.

Use of alumni and employer surveys for internal quality assurance of the DVM program at the University of Montreal.

Annual alumni and employer surveys, initially designed as outcomes assessment tools, were integrated into a new internal quality assurance strategy to improve the doctor of veterinary medicine program at the University of Montreal's Faculté de Médecine Vétérinaire. Data collected annually from the classes of 2004-2007 indicated that alumni and their employers were generally satisfied with their level of preparation after one year of professional activity. Specific weaknesses were found in non-technical skills such as communication and resource management. These data were used in support of other forms of feedback to guide curricular reform.

Epithelial WNT2B and Desert Hedgehog Are Necessary for Human Colonoid Regeneration after Bacterial Cytotoxin Injury.

Intestinal regeneration and crypt hyperplasia after radiation or pathogen injury relies on Wnt signaling to stimulate stem cell proliferation. Mesenchymal Wnts are essential for homeostasis and regeneration in mice, but the role of epithelial Wnts remains largely uncharacterized. Using the enterohemorrhagic E. coli-secreted cytotoxin EspP to induce injury to human colonoids, we evaluated a simplified, epithelial regeneration model that lacks mesenchymal Wnts. Here, we demonstrate that epithelial-produced WNT2B is upregulated following injury and essential for regeneration. Hedgehog signaling, specifically activation via the ligand Desert Hedgehog (DHH), but not Indian or Sonic Hedgehog, is another driver of regeneration and modulates WNT2B expression. These findings highlight the importance of epithelial WNT2B and DHH in regulating human colonic regeneration after injury.

Effect of using an audience response system on learning environment, motivation and long-term retention, during case-discussions in a large group of undergraduate veterinary clinical pharmacology students.

BACKGROUND: Teaching methods that provide an opportunity for individual engagement and focussed feedback are required to create an active learning environment for case-based teaching in large groups. AIMS: A prospective observational controlled study was conducted to evaluate whether the use of an audience response system (ARS) would promote an active learning environment during case-based discussions in large groups, have an impact on student motivation and improve long-term retention. METHODS: Group A (N = 83) participated in large group case discussions where student participation was voluntary, while for group B (N = 86) an ARS was used. Data collection methods included student and teacher surveys, student focus group interviews, independent observations and 1-year post-course testing. RESULTS: Results indicated that the use of an ARS provided an active learning environment during case-based discussions in large groups by favouring engagement, observation and critical reflection and by increasing student and teacher motivation. Although final exam results were significantly improved in group B, long-term retention was not significantly different between groups. CONCLUSIONS: It was concluded that ARS use significantly improved the learning experience associated with case-based discussions in a large group of undergraduate students.

The importance of knowledge, skills, and attitude attributes for veterinarians in clinical and non-clinical fields of practice: a survey of licensed veterinarians in Quebec, Canada.

To improve content validity and the pertinence of outcome assessment tools used for the undergraduate Doctor of Veterinary Medicine program at the University of Montreal's Faculté de médecine vétérinaire, a survey of members of the Quebec veterinary association was conducted. This survey aimed to determine the importance of a list of 71 attributes-categorized as knowledge, general skills, specific skills, and attitudes-for clinical and non-clinical types of professional activities. The results indicated that all basic knowledge components, general skills, and attitudes were equally important for all types of veterinary professional activities, while the importance of specific skills was significantly different for clinical practice and non-clinical fields. It was therefore proposed that outcomes assessment surveys of stakeholders, such as alumni and employers, be analyzed separately for each type of career option.

Clostridium perfringens enterotoxin as a novel-targeted therapeutic for brain metastasis.

Brain metastasis is the most commonly occurring intracranial tumor whose incidence seems to be increasing. With standard therapy, the average survival time of patients is approximately 8 months, and treatment often leads to neurologic dysfunction in long-term survivors, emphasizing the need for novel therapeutics. Clostridium perfringens enterotoxin (CPE) has recently been shown to rapidly and specifically destroy cancer cells expressing CPE receptors claudin-3 and claudin-4. Unfortunately, the utility of CPE is precluded by systemic toxicity because its receptors are expressed in numerous organs. Here, we provide the first preclinical evidence that CPE may be uniquely suited to the local treatment of brain metastasis. By immunohistochemical analysis, claudin-3 and claudin-4 were expressed frequently in metastases from breast (15 of 18), lung (15 of 20), and colon (12 of 14) carcinoma, and infrequently in metastases from renal cell carcinoma (2 of 16) and melanoma (2 of 16). In contrast, expression of claudin-3 and claudin-4 was absent in adjacent normal brain tissue. Further examination of the central nervous system (CNS) revealed low or undetectable levels of claudin-3 and claudin-4 in all regions tested by Western and immunohistochemical analysis. Treatment of breast cancer cell lines (MCF-7, MDA-MB-468, NT2.5-luc) and normal human astrocytes with CPE in vitro resulted in rapid and dose-dependent cytolysis exclusively in breast cancer cells, correlating with claudin-3 and claudin-4 expression. Moreover, intracranial CPE treatment significantly inhibited tumor growth and increased survival in two murine models of breast cancer brain metastasis, without any apparent local or systemic toxicity. These data suggest that CPE therapy may have efficacy against a wide variety of brain metastases without CNS toxicity.

Comparison of efficacy and safety of paste formulations of firocoxib and phenylbutazone in horses with naturally occurring osteoarthritis.

OBJECTIVE: To compare efficacy and safety of paste formulations of firocoxib and phenylbutazone in horses with naturally occurring osteoarthritis. DESIGN: Randomized controlled clinical trial. ANIMALS: 253 client-owned horses with naturally occurring osteoarthritis. PROCEDURES: Horses were treated with firocoxib (0.1 mg/kg [0.045 mg/lb], PO, q 24 h) or phenylbutazone (4.4 mg/kg [2 mg/lb], PO, q 24 h) for 14 days. Physical examinations and lameness evaluations were performed prior to treatment and after 7 and 14 days. Clinical improvement was defined as a reduction of at least 1 lameness grade or a combined reduction of at least 3 points in scores for pain during manipulation or palpation, joint swelling, joint circumference, and range of motion. RESULTS: Proportion of horses clinically improved on day 14 for the firocoxib group (104/123 [84.6%]) was not significantly different from the proportion for the phenylbutazone group (103/119 [86.6%]). Proportion of horses that were improved on day 14 was significantly greater for horses treated with firocoxib than for horses treated with phenylbutazone with regard to score for pain on manipulation or palpation (P = 0.028), joint circumference score (P = 0.026), and range of motion score (P = 0.012), but not for overall lameness score or joint swelling score. No direct treatment-related adverse effects were detected during the study. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that overall clinical efficacy of a paste formulation of firocoxib in horses with naturally occurring osteoarthritis was comparable to efficacy of a paste formulation of phenylbutazone.

CITED2 attenuates macrophage recruitment concordant with the downregulation of CCL20 in breast cancer cells.

The transcriptional co-regulator Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain-2 (CITED2) may promote breast tumor growth; however, the mechanisms by which its effects are mediated remain to be fully elucidated. Tumor-associated macrophages serve an important function in tumor development and progression and are recruited by chemotactic factors produced by cells within the tumor microenvironment. The present study assessed the effects of CITED2 silencing on macrophage recruitment in two xenograft mouse models of human breast cancer, one in which tumor growth was sensitive to CITED2 silencing (MDA-MB-231) and one in which it was insensitive (MDA-MB-468). The present study identified that silencing CITED2 significantly attenuated macrophage infiltration in MDA-MB-231 but not MDA-MB-468 orthotopic tumors, concordant with its effect on tumor growth. Correspondingly, conditioned media obtained from CITED2-silenced MDA-MB-231 cells exhibited a significantly decreased ability to induce macrophage recruitment by Transwell migration assay, whereas the chemotactic effect of MDA-MB-468 conditioned media was unaffected. Examining the expression of macrophage chemoattractants within orthotopic tumors and tumor cell-conditioned media revealed a significant decrease in C-C motif chemokine ligand (CCL)20 mRNA and protein expression following CITED2-silencing in MDA-MB-231 cells, compared with that in cells transfected with scramble shRNA. However, mRNA and protein expression was unaffected by CITED2-silencing in MDA-MB-468 cells. Furthermore, chromatin immunoprecipitation analysis revealed that CITED2 was localized to the CCL20 promoter in MDA-MB-231 cells, suggesting that it serves a direct function in its regulation, which is consistent with the effect of CITED2 silencing on CCL20 expression. Lastly, neutralizing CCL20 in the conditioned media of MDA-MB-231 cells significantly inhibited macrophage recruitment. Collectively, these results suggest that CITED2 is involved in modulating macrophage recruitment, representing a novel mechanism through which it may influence tumor growth. This may be partly mediated by regulating tumor cell production of the chemokine CCL20.

Label-free Raman spectroscopy provides early determination and precise localization of breast cancer-colonized bone alterations.

Breast neoplasms frequently colonize bone and induce development of osteolytic bone lesions by disrupting the homeostasis of the bone microenvironment. This degenerative process can lead to bone pain and pathological bone fracture, a major cause of cancer morbidity and diminished quality of life, which is exacerbated by our limited ability to monitor early metastatic disease in bone and assess fracture risk. Spurred by its label-free, real-time nature and its exquisite molecular specificity, we employed spontaneous Raman spectroscopy to assess and quantify early metastasis driven biochemical alterations to bone composition. As early as two weeks after intracardiac inoculations of MDA-MB-435 breast cancer cells in NOD-SCID mice, Raman spectroscopic measurements in the femur and spine revealed consistent changes in carbonate substitution, overall mineralization as well as crystallinity increase in tumor-bearing bones when compared with their normal counterparts. Our observations reveal the possibility of early stage detection of biochemical changes in the tumor-bearing bones - significantly before morphological variations are captured through radiographic diagnosis. This study paves the way for a better molecular understanding of altered bone remodeling in such metastatic niches, and for further clinical studies with the goal of establishing a non-invasive tool for early metastasis detection and prediction of pathological fracture risk in breast cancer.

Enterohemorrhagic E. coli (EHEC)-Secreted Serine Protease EspP Stimulates Electrogenic Ion Transport in Human Colonoid Monolayers.

One of the characteristic manifestations of Shiga-toxin-producing Escherichia coli (E. coli) infection in humans, including EHEC and Enteroaggregative E. coli O104:H4, is watery diarrhea. However, neither Shiga toxin nor numerous components of the type-3 secretion system have been found to independently elicit fluid secretion. We used the adult stem-cell-derived human colonoid monolayers (HCM) to test whether EHEC-secreted extracellular serine protease P (EspP), a member of the serine protease family broadly expressed by diarrheagenic E. coli can act as an enterotoxin. We applied the Ussing chamber/voltage clamp technique to determine whether EspP stimulates electrogenic ion transport indicated by a change in short-circuit current (Isc). EspP stimulates Isc in HCM. The EspP-stimulated Isc does not require protease activity, is not cystic fibrosis transmembrane conductance regulator (CFTR)-mediated, but is partially Ca2+-dependent. EspP neutralization with a specific antibody reduces its potency in stimulating Isc. Serine Protease A, secreted by Enteroaggregative E. coli, also stimulates Isc in HCM, but this current is CFTR-dependent. In conclusion, EspP stimulates colonic CFTR-independent active ion transport and may be involved in the pathophysiology of EHEC diarrhea. Serine protease toxins from E. coli pathogens appear to serve as enterotoxins, potentially significantly contributing to watery diarrhea.

TGF-beta promotes the establishment of renal cell carcinoma bone metastasis.

UNLABELLED: Bone metastases develop in approximately 30% of patients with RCC, and the mechanisms responsible for this phenomenon are unknown. We found that TGF-beta1 stimulation of RCC bone metastasis cells promotes tumor growth and bone destruction possibly by stimulating paracrine interactions between tumor cells and the bone. INTRODUCTION: Bone metastasis is a frequent complication and causes marked morbidity in patients with renal cell carcinoma (RCC). Surprisingly, the specific mechanisms of RCC interaction with bone have been scarcely studied despite the inability to prevent or effectively treat bone metastasis. Bone is a reservoir for various growth factors including the pleiotropic cytokine TGF-beta1. TGF-beta1 has been shown to have tumor-supportive effects on advanced cancers and evidence suggests its involvement in promoting the development of breast cancer bone metastasis. Here, we studied the potential role of TGF-beta1 in the growth of RCC bone metastasis (RBM). MATERIALS AND METHODS: To inhibit TGF-beta1 signaling, RBM cells stably expressing a dominant-negative (DN) TGF-betaRII cDNA were generated. The in vivo effect of TGF-beta1 on RBM tumor growth and osteolysis was determined by histological and radiographic analysis, respectively, of athymic nude mice after intratibial injection of parental, empty vector, or DN RBM cells. The in vitro effect of TGF-beta1 on RBM cell growth was determined after TGF-beta1 treatment by MTT assay. RESULTS: TGF-beta1 and the TGF-beta receptors I and II (TGF-betaRI/II) were consistently expressed in both RBM tissues and cell lines. Inhibition of TGF-beta1 signaling in RBM cells significantly reduced tumor establishment and osteolysis observed in vivo after injection into the murine tibia, although no effect on tumor establishment was observed after injection of RBM cells subcutaneously or into the renal subcapsule. Treatment of five RBM cell lines with TGF-beta1 in vitro either had no effect (2/5) or resulted in a significant inhibition (3/5) of cell growth, suggesting that TGF-beta1 may promote RBM tumor growth indirectly in vivo. CONCLUSIONS: TGF-beta1 stimulation of RBM cells plays a role in promoting tumor growth and subsequent osteolysis in vivo, likely through the initiation of tumor-promoting paracrine interactions between tumor cells and the bone microenvironment. These data suggest that inhibition of TGF-beta1 signaling may be useful in the treatment of RBM.