Long term in vitro expansion of epithelial stem cells enabled by pharmacological inhibition of PAK1-ROCK-Myosin II and TGF-β signaling

Summary: Despite substantial self-renewal capability in vivo, epithelial stem and progenitor cells located in various tissues expand for a few passages in vitro in feeder-free condition before they succumb to growth arrest. Here, we describe the EpiX method, which utilizes small molecules that inhibit PAK1-ROCK-Myosin II and TGF-β signaling to achieve over one trillion-fold expansion of human epithelial stem and progenitor cells from skin, airway, mammary, and prostate glands in the absence of feeder cells. Transcriptomic and epigenomic studies show that this condition helps epithelial cells to overcome stresses for continuous proliferation. EpiX-expanded basal epithelial cells differentiate into mature epithelial cells consistent with their tissue origins. Whole-genome sequencing reveals that the cells retain remarkable genome integrity after extensive in vitro expansion without acquiring tumorigenicity. EpiX technology provides a solution to exploit the potential of tissue-resident epithelial stem and progenitor cells for regenerative medicine. : Zhang et al. screen a small-molecule collection and find that pharmacologic inhibition of TGF-β and PAK1-ROCK-Myosin II, in low calcium conditions, supports extended expansion of epithelial stem cells in 2D format. This approach enhances the potential of tissue-resident epithelial stem cells for cell therapy. Keywords: epithelial stem and progenitor cells, cell culture method, TGF-β, PAK1/ROCK/Myosin II, feeder-free, regenerative medicine, cell therap

Defective antigen presentation by monocytes in ESRD patients not responding to hepatitis B vaccination: impaired HBsAg internalization and expression of ICAM-1 and HLA-DR/Ia molecules

This study was undertaken to evaluate the monocyte function of uraemic non-responders to hepatitis B vaccination. Therefore, some parameters concerning antigen processing by monocytes (Mo) as antigen presenting cells (APC) were analysed. It was found that in uraemic non-responders, (1) the internalization of HBsAg by monocytes was significantly decreasjed—HBsAg complexed with specific IgG or as immune complex isolated from patients is better internalized compared with free HBsAg; (2) during antigen presentation the expression of adhesion (ICAM-1) and accessory (HLA-DR/Ia) molecules was significantly decreased in uraemic patients, especially in non-responders; and (3) impaired internalization of HBsAg as well as a decrease in ICAM-1 and HLA-DR/Ia expression, correlated well with the blunted proliferation of CD4+ T cells stimulated by autologous monocytes induced by HBsAg

Comparison of small intestinal contrast ultrasound with magnetic resonance enterography in pediatric Crohn's disease

Aim To compare the diagnostic yield of small intestinal contrast ultrasonography (SICUS) with magnetic resonance enterography (MRE) in routine clinical practice in a cohort of pediatric patients investigated for Crohn's disease (CD) attending a UK tertiary center. Methods and Results Patients with suspected or established CD who underwent SICUS were identified retrospectively. SICUS was compared to conventional transabdominal ultrasound (TUS), ileocolonoscopy (IC), and MRE. The accuracy and agreement of SICUS in detecting small bowel lesions and CD‐related complications were assessed using kappa (κ) coefficient statistics. A total of 93 patients (median age 15 years, range 2–17, 49 male) underwent SICUS; 58 had suspected and 35 had established CD. In suspected CD, sensitivity and specificity of SICUS in detecting CD small bowel lesions were 81.8 and 100% and for TUS 85.7 and 87.5%, respectively. In established CD, sensitivity and specificity of SICUS were 98.7 and 100% and TUS 80 and 100%, respectively. Agreement between SICUS and IC was substantial for the presence of lesions (κ = 0.73) but fair in TUS (κ = 0.31). Agreement between SICUS and IC was almost perfect for detecting strictures (κ = 0.84), with a sensitivity of 100% and specificity of 97.6%. When comparing SICUS and TUS with MRE, agreement for the presence of lesions was substantial (κ = 0.63) and moderate (κ = 0.53), respectively. Agreement between SICUS and MRE was substantial for detecting strictures (κ = 0.77) and dilatation (κ = 0.68). Conclusions SICUS offers a radiation‐free alternative for assessing pediatric small bowel CD, with diagnostic accuracy that is comparable to MRE and IC, supporting its wider use in routine practice

Depression in individuals who subsequently develop inflammatory bowel disease: a population-based nested case-control study.

OBJECTIVE: Depression is a potential risk factor for developing IBD. This association may be related to GI symptoms occurring before diagnosis. We aimed to determine whether depression, adjusted for pre-existing GI symptoms, is associated with subsequent IBD. DESIGN: We conducted a nested case-control study using the Clinical Practice Research Datalink identifying incident cases of UC and Crohn's disease (CD) from 1998 to 2016. Controls without IBD were matched for age and sex. We measured exposure to prevalent depression 4.5-5.5 years before IBD diagnosis. We created two sub-groups with prevalent depression based on whether individuals had reported GI symptoms before the onset of depression. We used conditional logistic regression to derive ORs for the risk of IBD depending on depression status. RESULTS: We identified 10 829 UC cases, 4531 CD cases and 15 360 controls. There was an excess of prevalent depression 5 years before IBD diagnosis relative to controls (UC: 3.7% vs 2.7%, CD 3.7% vs 2.9%). Individuals with GI symptoms prior to the diagnosis of depression had increased adjusted risks of developing UC and CD compared with those without depression (UC: OR 1.47, 95% CI 1.21 to 1.79; CD: OR 1.41, 95% CI 1.04 to 1.92). Individuals with depression alone had similar risks of UC and CD to those without depression (UC: OR 1.13, 95% CI 0.99 to 1.29; CD: OR 1.12, 95% CI 0.91 to 1.38). CONCLUSIONS: Depression, in the absence of prior GI symptoms, is not associated with subsequent development of IBD. However, depression with GI symptoms should prompt investigation for IBD

Role of 4-1BB Ligand in Costimulation of T Lymphocyte Growth and its Upregulation on M12 B Lymphomas by cAMP

K46J B lymphomas express a T cell costimulatory activity that is not inhibited by CTLA-4Ig, anti-B7-1, anti-B7-2, anti-intercellular adhesion molecule 1 or antibodies to heat stable antigen. In this paper we report that this costimulatory activity is mediated at least in part by 4-1BB ligand, a member of the tumor necrosis factor (TNF) gene family that binds to 4-1BB, a T cell activation antigen with homology to the TNF/nerve growth factor receptor family. A fusion protein between 4-1BB and alkaline phosphatase (4-1BB-AP) blocks T cell activation by K46J lymphomas in both an antigen-specific system and with polyclonally (anti-CD3) activated T cells. 4-1BB-AP also blocks antigen presentation by normal spleen cells. When the antigen-presenting cells express B7 molecules as well as 4-1BB ligand, we find that B7 molecules and 4-1BB-AP both contribute to T cell activation. These data suggest that 4-1BB ligand plays an important role in costimulation of IL-2 production and proliferation by T cells. The B lymphoma M12 expresses low levels of 4-1BB-L but can be induced to express higher levels by treatment of the B ceils with cAMP, which also induces B7-1 and B7-2 in these cells. Thus cAMP appears to coordinately induce several costimulatory molecules on B cells

Design, synthesis, and evaluation of curcumin-derived arylheptanoids for glioblastoma and neuroblastoma cytotoxicity

Using an innovative approach toward multiple carbon-carbon bond-formations that relies on the multifaceted catalytic properties of titanocene complexes we constructed a series of C1-C7 analogs of curcumin for evaluation as brain and peripheral nervous system anti-cancer agents. C2-Arylated analogs proved efficacious against neuroblastoma (SK-N-SH & SK-N-FI) and glioblastoma multiforme (U87MG) cell lines. Similar inhibitory activity was also evident in p53 knockdown U87MG GBM cells. Furthermore, lead compounds showed limited growth inhibition in vitro against normal primary human CD34+hematopoietic progenitor cells. Taken together, the present findings indicate that these curcumin analogs are viable lead compounds for the development of new central and peripheral nervous system cancer chemotherapeutics with the potential for little effects on normal hematopoietic progenitor cells

Astabiotics: Antimicrobial Signal Transduction Activators

poster abstractThe increasing prevalence of multi-drug resistant Gram-negative bacteria is a major public health concern. All available antibiotics are inhibitors of targets essential for virulence or growth. CpxRA is a highly conserved bacterial signal transduction system that responds to extracytoplasmic membrane stress. CpxA is a sensor with kinase and phosphatase activity; upon activation, CpxA donates a phosphate group to CpxR, activating a transcriptional response. Activation of CpxRA reduces the flow of protein traffic through the cytoplasmic membrane, dramatically reducing the expression of virulence determinants. Activation of CpxRA abolishes the virulence of Salmonella Typhimurium in mice. We found that activation of CpxRA crippled the ability of Haemophilus ducreyi to cause disease in experimentally infected human volunteers. Using an Escherichia coli reporter strain, we developed a high throughput screen to detect compounds that activate CpxRA. In a pilot screen of 36,000 compounds, we identified 1 class of compounds that shifts the equilibrium of CpxA to kinase activity, activating CpxR. Based on its potency, the calculated effective dose of the lead compound (a nitroindole) was 10 mg/kg. Female mice tolerated 100 mg/kg of the nitroindole given twice a day for 3 days. A CpxRA activating mutant constructed in uropathogenic E. coli (UPEC) was severely impaired in a murine urinary tract infection model; thus, activation of CpxRA is a valid treatment strategy for UPEC. However, when female mice were challenged with UPEC and treated with 100 mg/kg of the nitroindole using the schedule above, there were no differences in the recovered CFU in the urine, bladder, and kidney of sham and compound-treated mice. Future studies will include medicinal optimization of the nitroindole and identification and optimization other leads that activate CpxRA. Although our pilot test of efficacy was negative, astabiotics have great potential as broad-spectrum, adjunctive therapies to existing antimicrobials for treatment of Gram-negative infections

High-resolution intravital microscopy

Cellular communication constitutes a fundamental mechanism of life, for instance by permitting transfer of information through synapses in the nervous system and by leading to activation of cells during the course of immune responses. Monitoring cell-cell interactions within living adult organisms is crucial in order to draw conclusions on their behavior with respect to the fate of cells, tissues and organs. Until now, there is no technology available that enables dynamic imaging deep within the tissue of living adult organisms at sub-cellular resolution, i.e. detection at the level of few protein molecules. Here we present a novel approach called multi-beam striped-illumination which applies for the first time the principle and advantages of structured-illumination, spatial modulation of the excitation pattern, to laser-scanning-microscopy. We use this approach in two-photon-microscopy - the most adequate optical deep-tissue imaging-technique. As compared to standard two-photon-microscopy, it achieves significant contrast enhancement and up to 3-fold improved axial resolution (optical sectioning) while photobleaching, photodamage and acquisition speed are similar. Its imaging depth is comparable to multifocal two-photon-microscopy and only slightly less than in standard single-beam two-photon-microscopy. Precisely, our studies within mouse lymph nodes demonstrated 216% improved axial and 23% improved lateral resolutions at a depth of 80 µm below the surface. Thus, we are for the first time able to visualize the dynamic interactions between B cells and immune complex deposits on follicular dendritic cells within germinal centers (GCs) of live mice. These interactions play a decisive role in the process of clonal selection, leading to affinity maturation of the humoral immune response. This novel high-resolution intravital microscopy method has a huge potential for numerous applications in neurosciences, immunology, cancer research and developmental biology. Moreover, our striped-illumination approach is able to improve the resolution of any laser-scanning-microscope, including confocal microscopes, by simply choosing an appropriate detector

Hypoxia-Inducible Factor-1α Regulates CD55 in Airway Epithelium

Airway epithelial CD55 down-regulation occurs in several hypoxia-associated pulmonary diseases, but the mechanism is unknown. Using in vivo and in vitro assays of pharmacologic inhibition and gene silencing, the current study investigated the role of hypoxia-inducible factor (HIF)-1α in regulating airway epithelial CD55 expression. Hypoxia down-regulated CD55 expression on small-airway epithelial cells in vitro, and in murine lungs in vivo; the latter was associated with local complement activation. Treatment with pharmacologic inhibition or silencing of HIF-1α during hypoxia-recovered CD55 expression in small-airway epithelial cells. HIF-1α overexpression or blockade, in vitro or in vivo, down-regulated CD55 expression. Collectively, these data show a key role for HIF-1α in regulating the expression of CD55 on airway epithelium