A Novel Peptide Prevents Enterotoxin- and Inflammation-Induced Intestinal Fluid Secretion by Stimulating Sodium-Hydrogen Exchanger 3 Activity.

BACKGROUND & AIMS: Acute diarrheal diseases are the second most common cause of infant mortality in developing countries. This is contributed to by lack of effective drug therapy that shortens the duration or lessens the volume of diarrhea. The epithelial brush border sodium (Na+)/hydrogen (H+) exchanger 3 (NHE3) accounts for a major component of intestinal Na+ absorption and is inhibited in most diarrheas. Because increased intestinal Na+ absorption can rehydrate patients with diarrhea, NHE3 has been suggested as a potential druggable target for drug therapy for diarrhea. METHODS: A peptide (sodium-hydrogen exchanger 3 stimulatory peptide [N3SP]) was synthesized to mimic the part of the NHE3 C-terminus that forms a multiprotein complex that inhibits NHE3 activity. The effect of N3SP on NHE3 activity was evaluated in NHE3-transfected fibroblasts null for other plasma membrane NHEs, a human colon cancer cell line that models intestinal absorptive enterocytes (Caco-2/BBe), human enteroids, and mouse intestine in vitro and in vivo. N3SP was delivered into cells via a hydrophobic fluorescent maleimide or nanoparticles. RESULTS: N3SP uptake stimulated NHE3 activity at nmol/L concentrations under basal conditions and partially reversed the reduced NHE3 activity caused by elevated adenosine 3',5'-cyclic monophosphate, guanosine 3',5'-cyclic monophosphate, and Ca2+ in cell lines and in in vitro mouse intestine. N3SP also stimulated intestinal fluid absorption in the mouse small intestine in vivo and prevented cholera toxin-, Escherichia coli heat-stable enterotoxin-, and cluster of differentiation 3 inflammation-induced fluid secretion in a live mouse intestinal loop model. CONCLUSIONS: These findings suggest pharmacologic stimulation of NHE3 activity as an efficacious approach for the treatment of moderate/severe diarrheal diseases.

mOrange2, a Genetically Encoded, pH Sensitive Fluorescent Protein, is an Alternative to BCECF-AM to Measure Intracellular pH to Determine NHE3 and DRA Activity.

BACKGROUND/AIMS: NHE3 (Na+/H+ exchanger3) and SLC26A3 (Cl-/HCO3- exchanger, DRA) are the major components of the intestinal neutral NaCl absorptive process and based on the intestinal segment, contribute to HCO3- absorption and HCO3- secretion. NHE3 and DRA are highly regulated by changes in second messengers, cAMP, cGMP and Ca2+. Precise and convenient measurement of exchanger activity is necessary to allow rapid study of physiologic and pharmacologic functions. Some epithelial cells are difficult to load with AM ester dyes and loading may not be uniform. METHODS: The use of a genetically modified fluorescent protein, mOrange2 was explored as an intracellular pH sensor protein to measure exchange activity of NHE3 and DRA. The model used was FRT cells stably expressing NHE3 or DRA with intracellular pH measured by changes of mOrange2 fluorescence intensity. Intracellular pH was monitored using a) Isolated single clones of FRT/mOrange2/HA-NHE3 cells studied in a confocal microscope with time-lapse live cell imaging under basal conditions and when NHE3 was inhibited by exposure to forskolin and stimulated by dexamethasone, b) coverslip grown FRT/mOrange2 cells expressing NHE3 or DRA using a computerized fluorometer with a perfused cuvette with standardization of the mOrange2 absorption and emission signal using K+/Nigericin as an internal standard in each experiment. RESULTS: A similar rate of intracellular alkalization by Na+ addition in cells expressing NHE3 and by Cl- removal in cells expressing DRA was found in mOrange2 expressing cells compared to the same cells loaded with BCECF-AM,both using the same pH calibration with K+/Nigericin. Using mOrange2 as the pH sensor, NHE3 basal activity was quantitated and shown to be inhibited by forskolin and stimulated by dexamethasone, and DRA was oppositely shown to be stimulated by forskolin, responses similar to results found using BCECF-AM. CONCLUSION: This study demonstrates that mOrange2 protein can be an effective alternate to BCECF-AM in measuring intracellular pH (preferred setting Ex520nm, Em 563nm) as affected by NHE3 and DRA activity, with the advantage, compared to AM ester dyes, that genetic expression can provide uniform expression of the pH sensor.

New Technologies to Image Tumors.

The premise of this book is the importance of the tumor microenvironment (TME). Until recently, most research on and clinical attention to cancer biology, diagnosis, and prognosis were focused on the malignant (or premalignant) cellular compartment that could be readily appreciated using standard morphology-based imaging.

NHERF3 is necessary for Escherichia coli heat-stable enterotoxin-induced inhibition of NHE3: differences in signaling in mouse small intestine and Caco-2 cells.

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of childhood death from diarrhea and the leading cause of Traveler's diarrhea. E. coli heat-stable enterotoxin (ST) is a major virulence factor of ETEC and inhibits the brush border Na/H exchanger NHE3 in producing diarrhea. NHE3 regulation involves multiprotein signaling complexes that form on its COOH terminus. In this study, the hypothesis was tested that ST signals via members of the Na/H exchanger regulatory factor (NHERF) family of scaffolding proteins, NHERF2, which had been previously shown to have a role, and now with concentration on a role for NHERF3. Two models were used: mouse small intestine and Caco-2/BBe cells. In both models, ST rapidly increased intracellular cGMP, inhibited NHE3 activity, and caused a quantitatively similar decrease in apical expression of NHE3. The transport effects were NHERF3 and NHERF2 dependent. Also, mutation of the COOH-terminal amino acids of NHERF3 supported that NHERF3-NHERF2 heterodimerization was likely to account for this dual dependence. The ST increase in cGMP in both models was partially dependent on NHERF3. The intracellular signaling pathways by which ST-cGMP inhibits NHE3 were different in mouse jejunum (activation of cGMP kinase II, cGKII) and Caco-2 cells, which do not express cGKII (elevation of intracellular Ca2+ concentration [Ca2+]i). The ST elevation of [Ca2+]i was from intracellular stores and was dependent on NHERF3-NHERF2. This study shows that intracellular signaling in the same diarrheal model in multiple cell types may be different; this has implications for therapeutic strategies, which often assume that models have similar signaling mechanisms.

HGAL localization to cell membrane regulates B-cell receptor signaling.

Human germinal center-associated lymphoma (HGAL) is specifically expressed only in germinal center (GC) B lymphocytes and GC-derived lymphomas. HGAL protein decreases lymphocyte motility by inhibiting the ability of myosin to translocate actin via direct interaction with F-actin and myosin II and by activating RhoA signaling via direct interactions with RhoA-specific guanine nucleotide exchange factors. HGAL protein also regulates B-cell receptor (BCR) signaling by directly binding to and enhancing Syk kinase activity and activation of its downstream effectors. Herein we demonstrate that HGAL protein can be myristoylated and palmitoylated and that these modifications localize HGAL to cellular membrane raft microdomains with distinct consequences for BCR signaling and chemoattractant-induced cell mobility. In BCR signaling, raft localization of HGAL facilitates interaction with Syk and modulation of the BCR activation and signaling, which induces HGAL phosphorylation and redistribution from lipid raft to bulk membrane and cytoplasm, followed by degradation. In contrast, HGAL myristoylation and palmitoylation avert its inhibitory effects on chemoattractant-induced cell motility. These findings further elucidate the growing and complex role of HGAL in B-cell biology and suggest that membrane-bound and cytoplasmic HGAL protein differently regulates distinct biological processes.

HGAL, a germinal center specific protein, decreases lymphoma cell motility by modulation of the RhoA signaling pathway.

HGAL is a germinal center (GC)-specific gene that negatively regulates lymphocyte motility and whose expression predicts improved survival of patients with diffuse large B-cell lymphoma (DLBCL) and classical Hodgkin lymphoma (cHL). We demonstrate that HGAL serves as a regulator of the RhoA signaling pathway. HGAL enhances activation of RhoA and its down-stream effectors by a novel mechanism - direct binding to the catalytic DH-domain of the RhoA-specific guanine nucleotide exchange factors (RhoGEFs) PDZ-RhoGEF and LARG that stimulate the GDP-GTP exchange rate of RhoA. We delineate the structural domain of HGAL that mediates its interaction with the PDZ-RhoGEF protein. These observations reveal a novel molecular mechanism underlying the inhibitory effects of GC-specific HGAL protein on the motility of GC-derived lymphoma cells. This mechanism may underlie the limited dissemination and better outcome of patients with HGAL-expressing DLBCL and cHL.

Hepatitis C virus inhibits DNA damage repair through reactive oxygen and nitrogen species and by interfering with the ATM-NBS1/Mre11/Rad50 DNA repair pathway in monocytes and hepatocytes.

Hepatitis C virus (HCV) infection is associated with the development of hepatocellular carcinoma and putatively also non-Hodgkin's B cell lymphoma. In this study, we demonstrated that PBMCs obtained from HCV-infected patients showed frequent chromosomal aberrations and that HCV infection of B cells in vitro induced enhanced chromosomal breaks and sister chromatid exchanges. HCV infection hypersensitized cells to ionizing radiation and bleomycin and inhibited nonhomologous end-joining repair. The viral core and nonstructural protein 3 proteins were shown to be responsible for the inhibition of DNA repair, mediated by NO and reactive oxygen species. Stable expression of core protein induced frequent chromosome translocations in cultured cells and in transgenic mice. HCV core protein binds to the NBS1 protein and inhibits the formation of the Mre11/NBS1/Rad50 complex, thereby affecting ATM activation and inhibiting DNA binding of repair enzymes. Taken together, these data indicate that HCV infection inhibits multiple DNA repair processes to potentiate chromosome instability in both monocytes and hepatocytes. These effects may explain the oncogenicity and immunological perturbation of HCV infection.

Identification of Intestinal NaCl Absorptive-Anion Secretory Cells: Potential Functional Significance.

Use of human enteroids studied in the undifferentiated and differentiated state that mimic the intestinal crypt and villus, respectively, has allowed studies of multiple enterocyte populations, including a large population of enterocytes that are transitioning from the crypt to the villus. This population expresses NHE3, DRA, and CFTR, representing a combination of Na absorptive and anion secretory functions. In this cell population, these three transporters physically interact, which affects their baseline and regulated activities. A study of this cell population and differentiated Caco-2 cells transduced with NHE3 and endogenously expressing DRA and CFTR has allowed an understanding of previous studies in which cAMP seemed to stimulate and inhibit DRA at the same time. Understanding the contributions of these cells to overall intestinal transport function as part of the fasting and post-prandial state and their contribution to the pathophysiology of diarrheal diseases and some conditions with constipation will allow new approaches to drug development.

Apoptosis, mastocytosis, and diminished adipocytokine gene expression accompany reduced epididymal fat mass in long-standing diet-induced obese mice.

BACKGROUND: Obesity is characterized by increased cell death and inflammatory reactions in the adipose tissue. Here, we explored pathophysiological alterations taking place in the adipose tissue in long-standing obesity. In the epididymal fat of C57BL/6 mice fed a high-fat diet for 20 weeks, the prevalence and distribution of dead adipocytes (crown-like structures), mast cells (toluidine blue, mMCP6), macrophages (F4/80), and apoptotic cells (cleaved caspase-3) were measured. Moreover, gene and/or protein expression of several adipocytokines (leptin, adiponectin, TNF-α, IL-10, IL-6, MCP-1), F4/80, mMCP6, cleaved caspase-3 were determined. RESULTS: We observed that the epididymal fat mass was lower in obese than in lean mice. In obese mice, the epididymal fat mass correlated inversely with body weight and liver mass. Dead adipocytes, mast cells, macrophages, and apoptotic cells were abundant in the epididymal fat of obese mice, especially in the rostral vs. caudal zone. Accordingly, mMCP6, F4/80, and cleaved caspase-3 gene and/or protein expression was increased. Conversely, adiponectin, leptin, IL-6, and MCP-1 gene expression levels were lower in the epididymal fat of obese than lean mice. Although TNF-α and IL-10 gene expression was higher in the epididymal fat of obese mice, their expression relative to F4/80 and mMCP6 expression were lower in the heavily infiltrated rostral than caudal zone. CONCLUSIONS: This study demonstrates that in mice with long-standing obesity diminished gene expression of several adipocytokines accompany apoptosis and reduced mass of the epididymal fat. Our findings suggest that this is due to both increased prevalence of dead adipocytes and altered immune cell activity. Differential distribution of metabolically challenged adipocytes is indicative of the presence of biologically diverse zones within the epididymal fat.

Conditional overexpression of connective tissue growth factor disrupts postnatal lung development.

Connective tissue growth factor (CTGF) is a member of an emerging family of immediate-early gene products that coordinates complex biological processes during development, differentiation, and tissue repair. Overexpression of CTGF is associated with mechanical ventilation with high tidal volume and oxygen exposure in newborn lungs. However, the role of CTGF in postnatal lung development and remodeling is not well understood. In the present study, a double-transgenic mouse model was generated with doxycycline-inducible overexpression of CTGF in respiratory epithelial cells. Overexpression of CTGF from Postnatal Days 1-14 resulted in thicker alveolar septa and decreased secondary septal formation. This is correlated with increased myofibroblast differentiation and disorganized elastic fiber deposition in alveolar septa. Overexpression of CTGF also decreased alveolar capillary network formation. There were increased alpha-smooth muscle actin expression and collagen deposition, and dramatic thickening in the peribronchial/peribronchiolar and perivascular regions in the double-transgenic lungs. Furthermore, overexpression of CTGF increased integrin-linked kinase expression, activated its downstream signaling target, Akt, as well as increased mRNA expression of fibronectin. These data demonstrate that overexpression of CTGF disrupts alveologenesis and capillary formation, and induces fibrosis during the critical period of alveolar development. These histologic changes are similar to those observed in lungs of infants with bronchopulmonary dysplasia.

Hepatitis C virus causes uncoupling of mitotic checkpoint and chromosomal polyploidy through the Rb pathway.

Hepatitis C virus (HCV) infection is associated with the development of hepatocellular carcinoma and probably also non-Hodgkin's B-cell lymphoma. The molecular mechanisms of HCV-associated carcinogenesis are unknown. Here we demonstrated that peripheral blood mononuclear cells obtained from hepatitis C patients and hepatocytes infected with HCV in vitro showed frequent chromosomal polyploidy. HCV infection or the expression of viral core protein alone in hepatocyte culture or transgenic mice inhibited mitotic spindle checkpoint function because of reduced Rb transcription and enhanced E2F-1 and Mad2 expression. The silencing of E2F-1 by RNA interference technology restored the function of mitotic checkpoint in core-expressing cells. Taken together, these data suggest that HCV infection may inhibit the mitotic checkpoint to induce polyploidy, which likely contributes to neoplastic transformation.

Very rapid cloning, expression and identifying specificity of T-cell receptors for T-cell engineering.

Neoantigens can be predicted and in some cases identified using the data obtained from the whole exome sequencing and transcriptome sequencing of tumor cells. These sequencing data can be coupled with single-cell RNA sequencing for the direct interrogation of the transcriptome, surfaceome, and pairing of αß T-cell receptors (TCRαß) from hundreds of single T cells. Using these 2 large datasets, we established a platform for identifying antigens recognized by TCRαßs obtained from single T cells. Our approach is based on the rapid expression of cloned TCRαß genes as Sleeping Beauty transposons and the determination of the introduced TCRαßs' antigen specificity and avidity using a reporter cell line. The platform enables the very rapid identification of tumor-reactive TCRs for the bioengineering of T cells with redirected specificity.

Antitumorigenic effect of brain proline rich polypeptide-1 in human chondrosarcoma.

Proline rich polypeptide (PRP-1) produced by neurosecretory cells of the hypothalamus is one of the fragments of neurophysin-vasopressin-associated glycoprotein. The primary structure of the neuropeptide PRP-1 isolated from neurosecretory granules of bovine neurohypophysis. We investigated PRP-1 action on chondrosarcoma, the second most common malignancy in bone, which primarily affects the cartilage cells. This deadly disease does not have any effective treatment. Earlier we demonstrated MYC oncogene inactivating effect by 1 lg/ml concentration brain PRP-1 In the present study we observed reduced viable sarcoma JJ012 cell numbers in comparison with control (89% growth inhibition) when treated with low concentrations of PRP-1 (0.5­1 lg/ml). Higher concentrations did not exhibit inhibitory effect. We assume that PRP-1 in low concentration impedes cell cycle progression. The fact that low concentrations of PRP-1 abolished Myc activity prompts to think that the antitumorigenic effect of PRP-1 in low concentrations is mediated through oncogene inactivation.

Combining adoptive cellular and immunocytokine therapies to improve treatment of B-lineage malignancy.

Currently, the lineage-specific cell-surface molecules CD19 and CD20 present on many B-cell malignancies are targets for both antibody- and cell-based therapies. Coupling these two treatment modalities is predicted to improve the antitumor effect, particularly for tumors resistant to single-agent biotherapies. This can be shown using an immunocytokine, composed of a CD20-specific monoclonal antibody fused to biologically active interleukin 2 (IL-2), combined with ex vivo expanded human umbilical cord blood-derived CD8(+) T cells, that have been genetically modified to be CD19 specific, for adoptive transfer after allogeneic hematopoietic stem-cell transplantation. We show that a benefit of targeted delivery of recombinant IL-2 by the immunocytokine to the CD19(+)CD20(+) tumor microenvironment is improved in vivo persistence of the CD19-specific T cells, and this results in an augmented cell-mediated antitumor effect. Phase I trials are under way using anti-CD20-IL-2 immunocytokine and CD19-specific T cells as monotherapies, and our results warrant clinical trials using combination of these two immunotherapies.

Modeling Wnt signaling by CRISPR-Cas9 genome editing recapitulates neoplasia in human Barrett epithelial organoids.

Primary organoid cultures generated from patient biopsies comprise a novel improved platform for disease modeling, being genetically stable and closely recapitulating in vivo scenarios. Barrett esophagus (BE) is the major risk factor for esophageal adenocarcinoma. There has been a dearth of long-term in vitro expansion models of BE neoplastic transformation. We generated a long-term virus-free organoid expansion model of BE neoplasia from patient biopsies. Both wild-type and paired APC-knockout (APCKO) BE organoids genome-edited by CRISPR-Cas9 showed characteristic goblet cell differentiation. Autonomous Wnt activation was confirmed in APCKO organoids by overexpression of Wnt target genes and nuclear-translocated ß-catenin expression after withdrawal of Wnt-3A and R-spondin-1. Wnt-activated organoids demonstrated histologic atypia, higher proliferative and replicative activity, reduced apoptosis, and prolonged culturability. Wnt-activated organoids also showed sustained protrusive migration ability accompanied by disrupted basement membrane reorganization and integrity. This CRISPR-Cas9 editing human-derived organoid model recapitulates the critical role of aberrant Wnt/ß-catenin signaling activation in BE neoplastic transformation. This system can be used to study other 'driver' pathway alterations in BE-associated neoplasia, avoiding signaling noise present in immortalized or cancer-derived cell lines.

Most early disseminated cancer cells detected in bone marrow of breast cancer patients have a putative breast cancer stem cell phenotype.

PURPOSE: The presence of disseminated tumor cells (DTC) in the bone marrow of breast cancer patients is an acknowledged independent prognostic factor. The biological metastatic potential of these cells has not yet been shown. The presence of putative breast cancer stem cells is shown both in primary tumors and distant metastases. These cells with a CD44(+)CD24(-/low) phenotype represent a minor population in primary breast cancer and are associated with self-renewal and tumorigenic potential. Recognizing the potential effect of prevalence of putative stem cells among DTC, we evaluated the bone marrow DTC. EXPERIMENTAL DESIGN: We employed the double/triple-staining immunohistochemistry protocol and modified the established bone marrow cytokeratin (CK) staining protocol by adding steps for additional antigens, CD44 and/or CD24. We evaluated 50 bone marrow specimens, previously categorized as CK(+) from early breast cancer patients. CK(+) cells were examined for CD44 and CD24 expression by light microscopy, fluorescence microscopy, and spectral imaging. RESULTS: We detected the putative stem cell-like phenotype in all CK(+) specimens. The mean prevalence of putative stem/progenitor cells was 72% and median prevalence was 65% (range, 33-100%) among the overall DTC per patient, compared with primary tumors where this phenotype is reported in <10% of cells. CONCLUSIONS: This is the first evidence of the existence of the putative stem-like phenotype within the DTC in bone marrow in early breast cancer patients. All patients had a putative stem cell phenotype among the DTC and most individual DTC showed such phenotype. Future molecular characterization of these cells is warranted.

Microscopy and Image Analysis.

This unit provides an overview of light microscopy, including objectives, light sources, filters, film, and color photography for fluorescence microscopy and fluorescence in situ hybridization (FISH). We believe there are excellent opportunities for cytogeneticists, pathologists, and other biomedical readers, to take advantage of specimen optical clearing techniques and expansion microscopy-we briefly point to these new opportunities. © 2017 by John Wiley & Sons, Inc.

Fluorescent immunohistochemistry and in situ hybridization analysis of mouse pancreas using low-power antigen-retrieval technique.

To facilitate the immunological reaction of antibodies with antigens in fixed tissues, it is necessary to unmask or retrieve the antigens through pretreatment of the specimens. However, adjustment of heating-induced antigen retrieval is always required for different tissues and antigens. In this study, by using a low-power antigen-retrieval technique with appropriate dilution of antibodies, we successfully immunostained key antigens in pancreas such as insulin, PDX-1, glucagon, cytokeratin, and CD31, which have previously presented a particular challenge for investigators because of the rapid autodigestion and high nonspecific antibody binding in this tissue. Satisfactory results were obtained when immunohistochemistry and fluorescence in situ hybridization analysis were combined in the same slides.

A Practical Approach to Quantitative Processing and Analysis of Small Biological Structures by Fluorescent Imaging.

Standards in quantitative fluorescent imaging are vaguely recognized and receive insufficient discussion. A common best practice is to acquire images at Nyquist rate, where highest signal frequency is assumed to be the highest obtainable resolution of the imaging system. However, this particular standard is set to insure that all obtainable information is being collected. The objective of the current study was to demonstrate that for quantification purposes, these correctly set acquisition rates can be redundant; instead, linear size of the objects of interest can be used to calculate sufficient information density in the image. We describe optimized image acquisition parameters and unbiased methods for processing and quantification of medium-size cellular structures. Sections of rabbit aortas were immunohistochemically stained to identify and quantify sympathetic varicosities, >2 µm in diameter. Images were processed to reduce background noise and segment objects using free, open-access software. Calculations of the optimal sampling rate for the experiment were based on the size of the objects of interest. The effect of differing sampling rates and processing techniques on object quantification was demonstrated. Oversampling led to a substantial increase in file size, whereas undersampling hindered reliable quantification. Quantification of raw and incorrectly processed images generated false structures, misrepresenting the underlying data. The current study emphasizes the importance of defining image-acquisition parameters based on the structure(s) of interest. The proposed postacquisition processing steps effectively removed background and noise, allowed for reliable quantification, and eliminated user bias. This customizable, reliable method for background subtraction and structure quantification provides a reproducible tool for researchers across biologic disciplines.