Clostridium Perfringens Enterotoxin Research Articles

Developing a COVID‐19 Yeast Oral Vaccine for Low Income Countries

Despite the scientific ingenuity of their design, traditional SARS‐CoV‐2 mRNA vaccines are expensive and require ultracold freezers for storage that are not available in many parts of the world. This makes them inaccessible to third world countries, and no shelf stable, low‐cost alternative currently exists. In response, we have constructed a novel COVID‐19 vaccine delivery system using the probiotic yeast Saccharomyces boulardii. We engineered a plasmid to express the receptor binding domain of the SARS CoV‐2 spike protein. The yeast mating type pheromone secretion signal was fused to the N‐terminus of the immunogen and the Claudin‐4 targeting sequence of the Clostridium perfringens enterotoxin was fused to the C‐terminus. Yeast cells secreting this construct are currently being tested in an animal model to determine if they can elicit an immune response.

Disruption of Claudin-Made Tight Junction Barriers by Clostridium perfringens Enterotoxin: Insights from Structural Biology.

Claudins are a family of integral membrane proteins that enable epithelial cell/cell interactions by localizing to and driving the formation of tight junctions. Via claudin self-assembly within the membranes of adjoining cells, their extracellular domains interact, forming barriers to the paracellular transport of small molecules and ions. The bacterium Clostridium perfringens causes prevalent gastrointestinal disorders in mammals by employing an enterotoxin (CpE) that targets claudins. CpE binds to claudins at or near tight junctions in the gut and disrupts their barrier function, potentially by disabling their assembly or via cell signaling means—the mechanism(s) remain unclear. CpE ultimately destroys claudin-expressing cells through the formation of a cytotoxic membrane-penetrating β-barrel pore. Structures obtained by X-ray crystallography of CpE, claudins, and claudins in complex with CpE fragments have provided the structural bases of claudin and CpE functions, revealing potential mechanisms for the CpE-mediated disruption of claudin-made tight junctions. This review highlights current progress in this space—what has been discovered and what remains unknown—toward efforts to elucidate the molecular mechanism of CpE disruption of tight junction barriers. It further underscores the key insights obtained through structure that are being applied to develop CpE-based therapeutics that combat claudin-overexpressing cancers or modulate tight junction barriers.

Autophagic degradation of claudin‐5 mediated by its binding to a Clostridium perfringens enterotoxin fragment modulates endothelial barrier permeability

The blood-brain barrier (BBB) protects the central nervous system (CNS) from harmful elements, while it also restricts efficient drug delivery into the CNS. Previously, we generated a mutated fragment of Clostridium perfringens enterotoxin (cCPEYWSH ) which specifically binds to the endothelial tight junction protein claudin-5. Here, we explore the mechanisms regulating the dynamics of membranous claudin-5 and BBB permeability. Following cCPEYWSH binding to claudin-5, caveolin-1 mediates the redistribution of claudin-5 to the cytosol. This abnormal cytosolic aggregation triggers the autophagic degradation of claudin-5, leading to an increase in BBB permeability. Enhancement or inhibition of autophagy accelerates or inhibits the degradation of cytosolic claudin-5, respectively. Our findings may pave the way for improving BBB permeability for drug delivery.

Claudin-3 regulates luminal fluid accumulation in the developing chick lung

Claudins are a family of tight junction proteins expressed in epithelial tissues during development and in postnatal life. We hypothesized that claudins are required for branching morphogenesis in the developing chick lung. To test this hypothesis, we exposed cultured chick lung explants at embryonic day 5 to a truncated non-toxic form of the Clostridium perfringens enterotoxin known as C-CPE that removes C-CPE-sensitive claudins from tight junctions. Using in situ hybridization and immunofluorescence studies, we established that only one C-CPE-sensitive claudin, Claudin-3, was expressed in the chick lung at this stage. C-CPE treated lung explants did not exhibit any defect in lung branching compared to controls. However, they did exhibit a significantly smaller lumen area, suggesting that paracellular permeability was perturbed. The decrease in lumen area was associated with a loss of Claudin-3 expression within tight junctions of the respiratory epithelium and an increase in permeability of the respiratory epithelium. When C-CPE-treated lung explants were treated with forskolin, lumen area was restored. In summary, removal of a sealing claudin, Claudin-3, from tight junctions in embryonic lung epithelium results in a decrease in lumen area and in hydrostatic pressure needed for lung development.

In silico prediction of amino acids involved in cCPE290-319 interaction with claudin 4.

Among the 26 human claudin proteins, the food-poisoning bacterium Clostridium perfringens produces an enterotoxin (~ 35.00 kDa) that specifically targets human claudin 4, causing diarrhea by fluid accumulation in the intestinal cavity. The Clostridium perfringens enterotoxin (CPE) C-terminal domain (cCPE ~ 15.00 kDa) tightly binds to claudin 4 and disrupts the tight junction barriers in the intestines. In this study, we aimed to determine the contribution and type of amino acid interactions involved in association between claudin 4 and the C-terminal CPE. First, the three-dimensional format of claudin 4 was downloaded from RCSB. Then, during 60.00 nanoseconds (nsec), molecular dynamics simulation was conducted using the GROMACS package on CPE of crystallographic structure. The results indicated that the simulations performed well during the simulation times and there were no noticeable problems or artifacts. We found that Coulombic (glycine 317, proline 311 and serine 313) and Lennard-Jones (tyrosine 310, leucine 315, serine 313 and glycine 317) interactions played a significant role in complex stability. This information localized the C-terminal of CPE as a linear sequence sufficient for recognition and binding to the eukaryotic CPE receptor. A detailed description of the dissociation process brings valuable insight into the interaction of the claudin 4-cCPE290-319 complexes, which could help in the future to design more potent drugs.

Claudin-Targeted Suicide Gene Therapy for Claudin-Overexpressing Tumor Cells by Using Modified Clostridium perfringens Enterotoxin (CPE).

Bacterial toxins gain growing attention as potential cancer treatment due to their potent cytotoxic effects. Among the very different toxins with diverse modes of action, the Clostridium perfringens enterotoxin (CPE) is in focus to treat solid cancers. This toxin targets the tight junction proteins claudin-3 and -4 (Cldn-3/4), which are frequently overexpressed in solid cancers. Binding to these claudins induces pore formation in the host cell plasma membrane leading to rapid oncoleaking cell death of tumor cells. Based on this, extending the targeting of CPE beyond Cldn-3/4 is of interest, since other claudins, such as claudin-1 or -5 are often overexpressed in various cancer entities such as non-small-cell lung cancer (NSCLC) or papillary thyroid carcinoma. In this chapter we describe the modification of a CPE-encoding vector by structure-directed mutagenesis to either preferentially target Cldn-1 and -5 or to expand targeting to Cldn1-9 for improved broadened cytotoxic targeting of claudin-overexpressing tumors such as but not limited to lung cancer via CPE gene transfer.

High claudin-4 antigen expression in triple-negative breast cancer by the immunohistochemistry method.

Background:Triple-negative breast cancer is a heterogeneous subtype of breast cancer. Claudin is an epithelial tight junctional protein, and also it is a receptor for clostridium perfringens enterotoxin and shows impairment of expression in several cancers. The chief purpose of this study is to assess the claudin-4 expression in triple-negative breast cancer (TNBC) Iranian patients and evaluate its correlation with some clinicopathological factors.Materials and Methods:In this study, 81 TNBC patients were evaluated for the claudin-4 expression by immunohistochemistry. The slides’ staining intensity was examined and scored from 0 to 3. Then, slides were reviewed to assess the percentage of cells with membrane and cytoplasmic staining; the obtaining scores were 1–4. Finally, added the resulting two numbers from two stages, and the final number was a maximum of 7. Final scores of 0–3 were considered the low expression, and 4–7 were considered the high expression. Finally, the collected data were analyzed using the Chi-square test.Results:Eighty-one women with breast cancer and a mean age of 49 ± 12 years participated in the study. In 80% of the patients, there was a high expression of claudin-4 marker, and 20% had low expression. The expression level of the marker was not significantly correlated with age, tumor size, lymph node involvement, tumor grade, disease stage, Ki-67, and metastasis.Conclusion:The present study confirmed the high frequency of claudin-4 antigen expression in TNBC patients, and no significant correlation was observed between the expression of antigen and demographic or clinicopathological factors.

NanI Sialidase Enhances the Action of Clostridium perfringens Enterotoxin in the Presence of Mucus.

ABSTRACTClostridium perfringens enterotoxin (CPE) is the main virulence factor for C. perfringens type F strains to cause human gastrointestinal diseases, which can involve lethal enterotoxemia. During type F disease, CPE encounters an adherent mucus layer overlying the intestines, so the current study evaluated if NanI potentiates CPE activity in the presence of adherent mucus. CPE alone caused more cytotoxicity transepithelial electrical resistance (TEER) and permeability to fluorescent dextran (FD) for minimal mucus-producing HT29 cells versus that in their derivative HT29-MTX-E12 cells, which produce abundant adherent mucus. However, for HT29-MTX-E12 cells, the presence of NanI significantly increased CPE binding and pore formation, which enhanced their sensitivity to CPE effects on cytotoxicity, TEER, and FD permeability. When the ability of NanI to potentiate CPE-induced enterotoxemia was then tested in a mouse small intestinal loop enterotoxemia model, a pathophysiologically relevant 50 μg/mL dose of CPE did not kill mice. However, the copresence of purified NanI resulted in significant CPE-induced lethality. More CPE was detected in the sera of mice challenged with 50 μg/mL of CPE when NanI was copresent during challenge. The copresence of NanI and CPE during challenge also significantly increased intestinal histologic damage compared to that after challenge with CPE alone, suggesting that NanI enhancement of CPE-induced intestinal damage may increase CPE absorption into blood. Overall, these results indicate that (i) mucus inhibits CPE action and (ii) NanI can potentiate CPE action in the presence of mucus, which may help explain why type F strains that produce relatively low levels of CPE are still pathogenic.IMPORTANCE NanI is a sialidase produced by some Clostridium perfringens type F strains. Here, we found that NanI can significantly increase the action of C. perfringens enterotoxin (CPE), which is the main toxin responsible for severe human enteric disease caused by type F strains. This effect likely helps to explain why even some type F strains that produce small amounts of CPE are pathogenic.

Ablation of Red Stable Transfected Claudin Expressing Canine Prostate Adenocarcinoma and Transitional Cell Carcinoma Cell Lines by C-CPE Gold-Nanoparticle-Mediated Laser Intervention.

Claudin (CLDN) proteins are commonly expressed in cancers and targeted in novel therapeutic approaches. The C-terminal of Clostridium perfringens enterotoxin (C-CPE) efficiently binds several claudins. In this study, recombinant C-CPE conjugated to gold nanoparticles (AuNPs) has been used for prostate adenocarcinoma (PAC) and transitional cell carcinoma (TCC) cell killing in vitro using gold-nanoparticle-mediated laser perforation (GNOME-LP). A PAC and TCC cell lines, as well as red fluorescence variants, allowing deep tissue imaging, were used. CLDN-3, -4, and -7 expression was confirmed by qPCR and immunofluorescences. The binding of C-CPE-AuNPs complexes on the cell surface was examined by scanning electron microscopy (SEM). Further, transcriptome analysis was carried out to evaluate the effect of C-CPE binder on the biological response of treated cells. Directed C-CPE-AuNP binding verified the capability to target CLDN receptors. Transcriptome analysis showed that C-CPE binding may activate immune and inflammatory responses but does not directly affect cell survival. Cancer cells ablation was demonstrated using a combination of GNOME-LP and C-CPE-AuNPs treatment reducing tumor cell viability to less than 10% depending on cell line. The fluorescent cell lines and the verified proof of concept in vitro provide the basis for perspective xenograft studies in an animal model.

Targeting prostate cancer with Clostridium perfringens enterotoxin functionalized nanoparticles co-encapsulating imaging cargo enhances magnetic resonance imaging specificity

Magnetic resonance is a key imaging tool for the detection of prostate cancer; however, better tools focusing on cancer specificity are required to distinguish benign from cancerous regions. We found higher expression of claudin-3 (CLDN-3) and -4 (CLDN-4) in higher grade than lower-grade human prostate cancer biopsies (n = 174), leading to the design of functionalized nanoparticles (NPs) with a non-toxic truncated version of the natural ligand Clostridium perfringens enterotoxin (C-CPE) that has a strong binding affinity to Cldn-3 and Cldn-4 receptors. We developed a first-of-its-type, C-CPE-NP-based MRI detection tool in a prostate tumor-bearing mouse model. NPs with an average diameter of 152.9 ± 15.7 nm (RS1) had a 2-fold enhancement of tumor specificity compared to larger (421.2 ± 33.8 nm) NPs (RS4). There was a 1.8-fold (P < 0.01) and 1.6-fold (P < 0.01) upregulation of the tumor-to-liver signal intensities of C-RS1 and C-RS4 (functionalized NPs) compared to controls, respectively. Also, tumor specificity was 3.1-fold higher (P < 0.001) when comparing C-RS1 to C-RS4. This detection tool improved tumor localization of contrast-enhanced MRI, supporting potential clinical applicability.

Effective Oncoleaking Treatment of Pancreatic Cancer by Claudin-Targeted Suicide Gene Therapy with Clostridium perfringens Enterotoxin (CPE).

Simple SummaryCurrent therapies for pancreas carcinoma (PC) are of limited efficacy due to tumor aggressiveness and therapy resistance. Bacterial toxins with pore-forming (oncoleaking) potential are promising tools in cancer therapy. We have developed a novel, suicide gene therapy treatment, based on Clostridium perfringens enterotoxin (CPE)-mediated oncoleaking. This is achieved by CPE suicide gene therapy to treat PC, which overexpresses the claudin-3 and -4 (Cldn3/4) tight junction proteins, which are targets of CPE action. This targeted gene therapy causes rapid eradication of Cldn3/4 overexpressing PC cells via oncoleaking and initiation of apoptotic/necrotic signaling. We demonstrate efficacy of this approach in vitro and after nonviral in vivo gene transfer in cell lines and in patient derived xenograft PC models. This therapy approach has translational potential for treatment of pancreas carcinomas and could also be translated into new combination settings with conventional chemotherapy.Pancreatic cancer (PC) is one of the most lethal cancers worldwide, associated with poor prognosis and restricted therapeutic options. Clostridium perfringens enterotoxin (CPE), is a pore-forming (oncoleaking) toxin, which binds to claudin-3 and -4 (Cldn3/4) causing selective cytotoxicity. Cldn3/4 are highly upregulated in PC and represent an effective target for oncoleaking therapy. We utilized a translation-optimized CPE vector (optCPE) for new suicide approach of PC in vitro and in cell lines (CDX) and patient-derived pancreatic cancer xenografts (PDX) in vivo. The study demonstrates selective toxicity in Cldn3/4 overexpressing PC cells by optCPE gene transfer, mediated by pore formation, activation of apoptotic/necrotic signaling in vitro, induction of necrosis and of bystander tumor cell killing in vivo. The optCPE non-viral intratumoral in vivo jet-injection gene therapy shows targeted antitumoral efficacy in different CDX and PDX PC models, leading to reduced tumor viability and induction of tumor necrosis, which is further enhanced if combined with chemotherapy. This selective oncoleaking suicide gene therapy improves therapeutic efficacy in pancreas carcinoma and will be of value for better local control, particularly of unresectable or therapy refractory PC.

Structural basis for Clostridium perfringens enterotoxin targeting of claudins at tight junctions in mammalian gut

The bacterium Clostridium perfringens causes severe, sometimes lethal gastrointestinal disorders in humans, including enteritis and enterotoxemia. Type F strains produce an enterotoxin (CpE) that causes the third most common foodborne illness in the United States. CpE induces gut breakdown by disrupting barriers at cell-cell contacts called tight junctions (TJs), which are formed and maintained by claudins. Targeted binding of CpE to specific claudins, encoded by its C-terminal domain (cCpE), loosens TJ barriers to trigger molecular leaks between cells. Cytotoxicity results from claudin-bound CpE complexes forming pores in cell membranes. In mammalian tissues, ∼24 claudins govern TJ barriers-but the basis for CpE's selective targeting of claudins in the gut was undetermined. We report the structure of human claudin-4 in complex with cCpE, which reveals that enterotoxin targets a motif conserved in receptive claudins and how the motif imparts high-affinity CpE binding to these but not other subtypes. The structural basis of CpE targeting is supported by binding affinities, kinetics, and half-lives of claudin-enterotoxin complexes and by the cytotoxic effects of CpE on claudin-expressing cells. By correlating the binding residence times of claudin-CpE complexes we determined to claudin expression patterns in the gut, we uncover that the primary CpE receptors differ in mice and humans due to sequence changes in the target motif. These findings provide the molecular and structural element CpE employs for subtype-specific targeting of claudins during pathogenicity of C. perfringens in the gut and a framework for new strategies to treat CpE-based illnesses in domesticated mammals and humans.

CITED4 enhances the metastatic potential of lung adenocarcinoma.

BackgroundCITED4 belongs to the CBP/p300‐interacting transactivator with glutamic acid and aspartic acid‐rich tail (CITED) family which is induced by various cytokines and participates in cytokine‐induced proliferation and differentiation. CITED4 is induced by HB‐EGF in lung cancer cells. However, it is unclear whether and how CITED4 contributes to the invasion and metastasis of lung adenocarcinoma (ADC).MethodsCITED4 expression in lung adenocarcinoma and its association with disease‐free survival (DFS) and overall survival were analyzed based on a cohort of 261 patients. The roles of CITED4 were validated via loss‐of‐function and gain‐of‐function experiments. The relationship between CITED4 and CLDN3 was validated by immunohistochemistry, Western blotting, and luciferase reporter assays. The function of the CITED4‐CTNNB1‐CLDN3 complex was fully validated and described.ResultsCITED4 expression was significantly upregulated in ADC tissues and cells and a predictor for DFS. Downregulation of CITED4 attenuated the proliferation and invasion, whereas CITED4 overexpression enhanced these effects. Overexpression and knockdown of CITED4 resulted in the upregulation and downregulation of CLDN3, respectively. Moreover, CITED4 downregulation suppressed CLDN3‐mediated ADC cell metastasis in vivo. CITED4 was highly expressed and positively correlated with CLDN3. Mechanistically, CITED4 interacted with CTNNB1 and functioned synergistically to enhance CLDN3 transcription. Importantly, CITED4 induced ADC invasion via a CLDN3‐dependent pathway. CITED4 determined the level of CLDN3, which in turn affected the sensitivity of tumors to Clostridium perfringens enterotoxin treatment.ConclusionsThe CITED4‐CTNNB1‐CLDN3 axis plays a key role in the invasion and metastasis of ADC and provides a novel therapeutic target for lung cancer treatment.

Current notions about etiopathogenic and genetics specific features of &lt;i&gt;Сlostridium perfringens&lt;/i&gt; toxins

The review presents modern data on the genetics and etiopathogenetic features of Clostridium perfringens toxins, including the role of Clostridium perfringens enterotoxin, in the development of food poisoning and a number of intestinal diseases in humans, animals and birds.

Targeting of claudin-4 by Clostridium perfringens enterotoxin-conjugated polysialic acid nanoparticles for pancreatic cancer therapy

Despite recent advances in chemotherapy, pancreatic cancer remains a leading cause of cancer-related deaths. Moreover, although targeted therapy has shown promising therapeutic efficacy in many types of cancers, no such effective targeting strategy for treatment of pancreatic cancer, which is in desperate need for new treatment approaches. Here, we developed claudin-4–targeting Clostridium perfringens enterotoxin (CPE) peptide-conjugated polysialic acid nanoparticles (C-SNPs) for pancreatic cancer-targeted therapy. Doxorubicin-loaded C-SNPs (DOX-C-SNPs) higly accumulated in the targeted pancreatic cancer via enhanced peameability and retention (EPR) effect, targeting claudin-4 in pancreatic cancer that becomes superficially exposed owing to the disruption of tight junctions. Notably, DOX-C-SNP accumulation in the non-targeted, normal pancreas was significantly reduced because of hindered access to claudin-4 in tight junctions. As a result, DOX-C-SNPs substantially suppressed tumor growth in an orthotopic pancreatic cancer model while exerting minimal toxicity against non-targeted, normal tissues. Collectively, these findings indicate that claudin-4–targeting DOX-C-SNPs may have promise in treating pancreatic cancers through targeting of exposed claudin-4.

COMPARISON OF FECAL CYTOLOGY AND PRESENCE OF CLOSTRIDIUM PERFRINGENS ENTEROTOXIN IN CAPTIVE BLACK-FOOTED FERRETS (MUSTELA NIGRIPES) BASED ON DIET AND FECAL QUALITY.

The black-footed ferret (Mustela nigripes) is an endangered mustelid native to North America. Gastroenteritis is a documented cause of morbidity and mortality in managed individuals, particularly by infectious agents. Fecal cytology is an inexpensive and rapid test that can help guide clinical management strategies for animals with enteritis; however, normal parameters have not been established in black-footed ferrets. The objective of this study was to characterize fecal cytological findings of 50 fecal samples from 18 black-footed ferrets that received two different diet types (ground meat versus whole prey) and that were visibly judged to be normal or abnormal. This study also tested for the presence of Clostridium perfringens enterotoxin by enzyme-linked immunosorbent assay in all abnormal and a subset of normal fecal samples. Significantly higher spore-forming bacteria and yeast prevalence were present in normal feces from individuals following the meat-based compared with the whole-prey diet. Samples from individuals with abnormal feces had significantly more spore-forming bacteria than normal feces, regardless of diet. Normal feces had higher diplococci and spore-forming bacteria compared with domestic canine and feline standards. A single abnormal fecal sample was positive for enterotoxin and originated from the only animal requiring treatment. Results indicate that low numbers of spore-forming bacteria can be found in fecal samples from clinically normal black-footed ferrets. Fecal cytology shows significantly increased spore-formers in clinically abnormal ferrets and in clinically normal ferrets following a ground meat-based diet.

Identifying the Virulent Factors of Clostridium perfringens Locally Isolated from Different Species

Clostridium perfringens incriminated in many diseases among different species of animals due to its ability to produce many virulence factors. In the current study, 135 intestinal samples were collected from different animal species of different localities in Egypt. Samples were subjected to isolation and identification (morphologically and biochemically) for obtaining Clostridium perfringens isolates (n=26, 19.25%). The PCR was carried out to elucidate the virulence factors. It was indicated that all the 26 Clostridium perfringens isolates had CPA gene and Clostridium perfringens enterotoxin (CPE gene), whereas 23% of isolates of chicken and cattle intestinal samples contained CPA, Net B, and CPE genes as virulence factors. Consequently, those isolates are highly recommended to be used in the preparation of enterotoxemia and necrotic enteritis vaccines as they are more virulent strains.

Probing Ligand-Receptor Interaction in Living Cells Using Force Measurements With Optical Tweezers.

This work probes the binding kinetics of COOH-terminus of Clostridium perfringens enterotoxin (c-CPE) and claudin expressing MCF-7 cells using force spectroscopy with optical tweezers. c-CPE is of high biomedical interest due to its ability to specifically bind to claudin with high affinity as well as reversibly disrupt tight junctions whilst maintaining cell viability. We observed single-step rupture events between silica particles functionalized with c-CPE and MCF-7 cells. Extensive calibration of the optical tweezers’ trap stiffness and displacement of the particle from trap center extracted a probable bond rupture force of ≈ 18 pN. The probability of rupture events with c-CPE functionalized silica particles increased by 50% compared to unfunctionalized particles. Additionally, rupture events were not observed when probing cells not expressing claudin with c-CPE coated particles. Overall, this work demonstrates that optical tweezers are invaluable tools to probe ligand-receptor interactions and their potential to study dynamic molecular events in drug-binding scenarios.

Anti-tumor effect of a recombinant Bifidobacterium strain secreting a claudin-targeting molecule in a mouse breast cancer model

Bifidobacterium is a nonpathogenic strain of anaerobic bacteria that selectively localizes and proliferates in tumors. It has emerged as a specific carrier of anticancer proteins against malignant tumors. Claudins are tetraspanin transmembrane proteins that form tight junctions. Claudin-4 is overexpressed in certain epithelial malignant cancers. The C-terminal fragment of the Clostridium perfringens enterotoxin (C-CPE), an exotoxin without the cytotoxic domain, strongly binds to claudin-4. The C-CPE fusion toxin (C-CPE-PE23), which targets claudin-4, strongly suppresses tumor growth; however, C-CPE fusion toxins exhibit hepatic toxicity. In this study, we successfully generated a strain of Bifidobacterium longum that secreted C-CPE-PE23 (B. longum-C-CPE-PE23) and was specific to and cross reactive with human and mouse claudin-4. We evaluated the therapeutic potential of this strain against triple-negative breast cancer using a mouse model. C-CPE-PE23 decreased cell viability in a dose-dependent manner in human and mouse breast cancer cell lines. After intravenous injection, Bifidobacterium was specifically distributed in the tumors of mice bearing breast cancer tumors. Moreover, B. longum-C-CPE-PE23 significantly suppressed tumor growth in mice with breast cancer without serious side effects, such as weight loss or hepatic and renal damage. We suggest that B. longum-C-CPE-PE23 is a good candidate for breast cancer treatment. Bifidobacterium could also be used as a drug delivery system for hepatotoxic agents.

Role of Claudins in Renal Branching Morphogenesis.

Claudins are a family of tight junction proteins that are expressed during mouse kidney development. They regulate paracellular transport of solutes along the nephron and contribute to the final composition of the urinary filtrate. To understand their roles during development, we used a protein reagent, a truncated version of the Clostridium perfringens enterotoxin (C‐CPE), to specifically remove a subset of claudin family members from mouse embryonic kidney explants at embryonic day 12. We observed that treatment with C‐CPE decreased the number and the complexity of ureteric bud tips that formed: there were more single and less bifid ureteric bud tips when compared to control‐treated explants. In addition, C‐CPE‐treated explants exhibited ureteric bud tips with larger lumens when compared to control explants (p < .05). Immunofluorescent analysis revealed decreased expression and localization of Claudin‐3, −4, −6, and −8 to tight junctions of ureteric bud tips following treatment with C‐CPE. Interestingly, Claudin‐7 showed higher expression in the basolateral membrane of the ureteric bud lineage and poor localization to the tight junctions of the ureteric bud lineage both in controls and in C‐CPE‐treated explants. Taken together, it appears that claudin proteins may play a role in ureteric bud branching morphogenesis through changes in lumen formation that may affect the efficiency by which ureteric buds emerge and branch.