Immature Epithelial Cells Research Articles

Secreted Metabolites of Bifidobacterium infantis and Lactobacillus acidophilus Protect Immature Human Enterocytes from IL-1β-Induced Inflammation: A Transcription Profiling Analysis.

Combination regimens of Bifidobacterium infantis and Lactobacillus acidophilus have been demonstrated to prevent necrotizing enterocolitis (NEC) in clinical trials. However, the molecular mechanisms responsible for this protective effect are not well understood. Additionally, conditioned media from individual cultures of these two probiotics show strain specific modulation of inflammation using in vitro human intestinal NEC models. Here we report a transcription profiling analysis of gene expression in immature human fetal intestinal epithelial cells (H4 cells) pretreated with conditioned media from B. infantis (BCM) or L. acidophilus (LCM) prior to IL-1β stimulation. Compared with control media, the two probiotic-conditioned media (PCM) treatments altered the expression of hundreds of genes involved in the immune response, apoptosis and cell survival, cell adhesion, the cell cycle, development and angiogenesis. In IL-1β-stimulated cells, PCM treatment decreased the upregulation of genes in the NF-κB activation pathway and downregulated genes associated with extracellular matrix (ECM) remodeling. Compared with LCM, BCM showed more significant modulatory effects on ECM remodeling, reflected by a lower p value. IL-6 and IL-8 production was significantly reduced in IL-1β-stimulated cells pretreated with PCM (p<0.05), which was consistent with their altered gene expression. Western blot analysis showed that compared with IL-1β stimulation alone, PCM treatment attenuated the decrease of cytoplasmic IκBα and NF-κB p65 levels as well as the increase of nuclear NF-κB p65 levels in the stimulated cells (p<0.05). In conclusion, PCM treatment exerted anti-inflammatory effects in immature human fetal enterocytes primarily by modulating genes in the NF-κB signaling and ECM remodeling pathways. Additionally, some components of these signaling pathways, particularly the ECM remodeling pathway, were more profoundly affected by BCM than LCM.

Protective effects of transforming growth factor β2 in intestinal epithelial cells by regulation of proteins associated with stress and endotoxin responses.

Transforming growth factor (TGF)-β2 is an important anti-inflammatory protein in milk and colostrum. TGF-β2 supplementation appears to reduce gut inflammatory diseases in early life, such as necrotizing enterocolitis (NEC) in young mice. However, the molecular mechanisms by which TGF-β2 protects immature intestinal epithelial cells (IECs) remain to be more clearly elucidated before interventions in infants can be considered. Porcine IECs PsIc1 were treated with TGF-β2 and/or lipopolysaccharide (LPS), and changes in the cellular proteome were subsequently analyzed using two-dimensional gel electrophoresis-MS and LC-MS-based proteomics. TGF-β2 alone induced the differential expression of 13 proteins and the majority of the identified proteins were associated with stress responses, TGF-β and Toll-like receptor 4 signaling cascades. In particular, a series of heat shock proteins had similar differential trends as previously shown in the intestine of NEC-resistant preterm pigs and young mice. Furthermore, LC-MS-based proteomics and Western blot analyses revealed 20 differentially expressed proteins following treatment with TGF-β2 in LPS-challenged IECs. Thirteen of these proteins were associated with stress response pathways, among which five proteins were altered by LPS and restored by TGF-β2, whereas six were differentially expressed only by TGF-β2 in LPS-challenged IECs. Based on previously reported biological functions, these patterns indicate the anti-stress and anti-inflammatory effects of TGF-β2 in IECs. We conclude that TGF-β2 of dietary or endogenous origin may regulate the IEC responses against LPS stimuli, thereby supporting cellular homeostasis and innate immunity in response to bacterial colonization, and the first enteral feeding in early life.

Disequilibrium of BMP2 Levels in the Breast Stem Cell Niche Launches Epithelial Transformation by Overamplifying BMPR1B Cell Response

SummaryUnderstanding the mechanisms of cancer initiation will help to prevent and manage the disease. At present, the role of the breast microenvironment in transformation remains unknown. As BMP2 and BMP4 are important regulators of stem cells and their niches in many tissues, we investigated their function in early phases of breast cancer. BMP2 production by tumor microenvironment appeared to be specifically upregulated in luminal tumors. Chronic exposure of immature human mammary epithelial cells to high BMP2 levels initiated transformation toward a luminal tumor-like phenotype, mediated by the receptor BMPR1B. Under physiological conditions, BMP2 controlled the maintenance and differentiation of early luminal progenitors, while BMP4 acted on stem cells/myoepithelial progenitors. Our data also suggest that microenvironment-induced overexpression of BMP2 may result from carcinogenic exposure. We reveal a role for BMP2 and the breast microenvironment in the initiation of stem cell transformation, thus providing insight into the etiology of luminal breast cancer.

Cytokeratin localization and basal cell differentiation in the epididymal epithelium during postnatal development of the mouse.

The epididymis is a male genital organ that has plays various functions, including sperm concentration, maturation, and storage. The epididymal epithelium consists of principal cells, clear cells, and basal cells. To comprehensively understand the occurrence and morphological differentiation of basal cells, we examined the expression and localization of cytokeratins (CKs) in the epididymal epithelium during postnatal development of the mouse. Immunohistochemical staining showed that, in adult mice, CK5 and CK14 were exclusively expressed in the cytoplasm of basal cells. During postnatal development, basal cells that stained positive for CK5 and CK14 first appeared in immature columnar epithelial cells in mice aged 1 week. The immunoreactivity became progressively stronger in mice aged 2-3 weeks. In mice aged 3 weeks, the immunoreactivity was strong in regions IV and V. In mice aged ≥ 4 weeks, strong immunoreactivity was observed in all epididymal regions. CK5 and CK14 could be useful markers of differentiation in epididymal basal cells. These basal cells originate from immature columnar epithelial cells and are of two types—dome-shaped and flask-shaped—. The flask-shaped cells are mainly located in the initial segment of the mouse epididymis.

Autophagy regulator BECN1 suppresses mammary tumorigenesis driven by WNT1 activation and following parity

Earlier studies reported allelic deletion of the essential autophagy regulator BECN1 in breast cancers implicating BECN1 loss, and likely defective autophagy, in tumorigenesis. Recent studies have questioned the tumor suppressive role of autophagy, as autophagy-related gene (Atg) defects generally suppress tumorigenesis in well-characterized mouse tumor models. We now report that, while it delays or does not alter mammary tumorigenesis driven by Palb2 loss or ERBB2 and PyMT overexpression, monoallelic Becn1 loss promotes mammary tumor development in 2 specific contexts, namely following parity and in association with wingless-type MMTV integration site family, member 1 (WNT1) activation. Our studies demonstrate that Becn1 heterozygosity, which results in immature mammary epithelial cell expansion and aberrant TNFRSF11A/TNR11/RANK (tumor necrosis factor receptor superfamily, member 11a, NFKB activator) signaling, promotes mammary tumorigenesis in multiparous FVB/N mice and in cooperation with the progenitor cell-transforming WNT1 oncogene. Similar to our Becn1+/−;MMTV-Wnt1 mouse model, low BECN1 expression and an activated WNT pathway gene signature correlate with the triple-negative subtype, TNFRSF11A axis activation and poor prognosis in human breast cancers. Our results suggest that BECN1 may have nonautophagy-related roles in mammary development, provide insight in the seemingly paradoxical roles of BECN1 in tumorigenesis, and constitute the basis for further studies on the pathophysiology and treatment of clinically aggressive triple negative breast cancers (TNBCs).

Abstract LB-138: Autophagy regulator BECN1 acts as a context-dependent tumor suppressor opposing parity-associated and WNT1-driven mammary tumorigenesis

Abstract Earlier studies reported allelic deletion of the essential autophagy regulator BECN1 in breast cancers implicating BECN1 loss and defective autophagy in tumorigenesis. Recent studies questioned the tumor suppressive role of autophagy, as Atg defects, including Becn1 deletion, suppress tumorigenesis in mouse tumor models. For the first time, we report that monoallelic Becn1 loss has a context-specific tumor-promoting function in parity-associated and WNT-driven mammary tumorigenesis. Our studies demonstrate that Becn1 heterozygocity promotes immature mammary epithelial cell expansion, Receptor Activator of NFκB (RANK) signaling and mammary tumorigenesis in cooperation with the progenitor cell-transforming WNT pathway. Similar to our mouse model, low BECN1 expression and WNT pathway activation in human breast tumors correlate with triple negative breast cancers (TNBCs), RANK axis activation and poor prognosis. Our results provide novel insight in the seemingly paradoxical role of BECN1 in tumorigenesis and the basis for further studies on pathophysiology and treatment of clinically aggressive TNBCs. Citation Format: Michelle Cicchini, Rumela Chakrabarti, Sameera Kongara, Sandy Price, Ritu Nahar, Fred Lozy, Hua Zhong, Alexei Vazquez, Yibin Kang, Vassiliki Karantza. Autophagy regulator BECN1 acts as a context-dependent tumor suppressor opposing parity-associated and WNT1-driven mammary tumorigenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-138. doi:10.1158/1538-7445.AM2014-LB-138

Feline Coccygeal Teratoma: Immunohistochemical Characterization of Immature Tissue Components

Congenital teratomas arising in the coccygeal region have not been reported in domestic animals. This report describes a congenital coccygeal teratoma in a male Burmese cat. A round to oval expansile mass with solid and cystic areas was found ventral to the 5th to 7th caudal vertebrae. Microscopically, the tumour was composed of derivatives of all three primordial germ layers with neuroectodermal predominance. Immunohistochemical double labelling identified immature tissue components in combination with Sox2, a regulator of stem cell proliferation and differentiation. Few Sox2-positive cells co-expressed the neural crest stem cell markers vimentin and neurotrophin receptor p75(NTR). Not all Sox2- and p75(NTR)-positive cells expressed vimentin. An overlapping expression of vimentin-negative and Sox2-, p75(NTR)-positive cells and GFAP- and p75(NTR)-positive cells may indicate a transition stage from immature to mature non-myelinating Schwann cells. Periaxin-positive myelinating Schwann cells surrounding neurofilament-positive axons were observed. Sox2 was additionally expressed in immature odontogenic epithelial cells and in immature cells of endodermal origin. Sox2 was not observed in mature cells, with the exception of satellite glial cells and mucous glands. Despite the presence of immature tissue components, no recurrence was observed 1 year after surgical removal.

Pathogenesis Implication for Necrotizing Enterocolitis Prevention in Preterm Very‐Low‐Birth‐Weight Infants

Recent reports show that the incidence of and deaths caused by necrotizing enterocolitis (NEC) in preterm very-low-birth-weight (PVLBW) infants are on the rise. Unfortunately, NEC often rapidly progresses from early signs of intestinal inflammation to extensive necrosis within a matter of hours, making treatment and secondary prevention extremely difficult to achieve. Primary prevention should thus be the priority. Recent studies provide information that enhances our understanding of the pathophysiology and provides more practical options for the prevention of NEC. The most accepted hypothesis at present is that enteral feeding (providing substrate) in the presence of abnormal intestinal colonization by pathogens provokes an inappropriately heightened inflammatory response in immature intestinal epithelial cells of PVLBW infants. Seventy-four relevant articles were reviewed. Our focus was on the present understanding of the pathophysiology of NEC in the context of developing optimal strategies to prevent NEC in PVLBW infants. Strategies such as antenatal glucocorticoids, postnatal breast milk feeding, and cautious approach to enteral feeding failed to eliminate NEC in PVLBW infants because these strategies did not address the complexity of the pathogenesis. Probiotics seem to be the most significant advance in NEC prevention at present because of the significant range of beneficial effects at various levels of gut function and defense mechanism and the present evidence based on 19 randomized controlled trials.

Oral Administration of Transforming Growth Factor-β1 (TGF-β1) Protects the Immature Gut from Injury via Smad Protein-dependent Suppression of Epithelial Nuclear Factor κB (NF-κB) Signaling and Proinflammatory Cytokine Production

Inflammatory immune responses play an important role in mucosal homeostasis and gut diseases. Nuclear factor κB (NF-κB), central to the proinflammatory cascade, is activated in necrotizing enterocolitis (NEC), a devastating condition of intestinal injury with extensive inflammation in premature infants. TGF-β is a strong immune suppressor and a factor in breast milk, which has been shown to be protective against NEC. In an NEC animal model, oral administration of the isoform TGF-β1 activated the downstream effector Smad2 in intestine and significantly reduced NEC incidence. In addition, TGF-β1 suppressed NF-κB activation, maintained levels of the NF-κB inhibitor IκBα in the intestinal epithelium, and systemically decreased serum levels of IL-6 and IFN-γ. The immature human fetal intestinal epithelial cell line H4 was used as a reductionistic model of the immature enterocyte to investigate mechanism. TGF-β1 pretreatment inhibited the TNF-α-induced IκBα phosphorylation that targets the IκBα protein for degradation and inhibited NF-κB activation. Chromatin immunoprecipitation (ChIP) assays demonstrated decreased NF-κB binding to the promoters of IL-6, IL-8, and IκBα in response to TNF-α with TGF-β1 pretreatment. These TGF-β1 effects appear to be mediated through the canonical Smad pathway as silencing of the TGF-β central mediator Smad4 resulted in loss of the TGF-β1 effects. Thus, TGF-β1 is capable of eliciting anti-inflammatory effects by inhibiting NF-κB specifically in the intestinal epithelium as well as by decreasing systemic IL-6 and IFN-γ levels. Oral administration of TGF-β1 therefore can potentially be used to protect against gastrointestinal diseases.

A Genome-Wide Analysis of Open Chromatin in Human Epididymis Epithelial Cells Reveals Candidate Regulatory Elements for Genes Coordinating Epididymal Function1

The epithelium lining the epididymis has a pivotal role in ensuring a luminal environment that can support normal sperm maturation. Many of the individual genes that encode proteins involved in establishing the epididymal luminal fluid are well characterized. They include ion channels, ion exchangers, transporters, and solute carriers. However, the molecular mechanisms that coordinate expression of these genes and modulate their activities in response to biological stimuli are less well understood. To identify cis-regulatory elements for genes expressed in human epididymis epithelial cells, we generated genome-wide maps of open chromatin by DNase-seq. This analysis identified 33,542 epididymis-selective DNase I hypersensitive sites (DHS), which were not evident in five cell types of different lineages. Identification of genes with epididymis-selective DHS at their promoters revealed gene pathways that are active in immature epididymis epithelial cells. These include processes correlating with epithelial function and also others with specific roles in the epididymis, including retinol metabolism and ascorbate and aldarate metabolism. Peaks of epididymis-selective chromatin were seen in the androgen receptor gene and the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which has a critical role in regulating ion transport across the epididymis epithelium. In silico prediction of transcription factor binding sites that were overrepresented in epididymis-selective DHS identified epithelial transcription factors, including ELF5 and ELF3, the androgen receptor, Pax2, and Sox9, as components of epididymis transcriptional networks. Active genes, which are targets of each transcription factor, reveal important biological processes in the epididymis epithelium.

Abstract 1974: Functional characterization of Basonuclin 1 (BNC1): a novel tumor suppressor gene commonly downregulated in human prostate cancer.

Abstract Prostate cancer (PCa) harbors a myriad of aberrant genomic and epigenetic alterations. Epigenetic inactivation of genes in PCa is largely based on transcriptional silencing by aberrant CpG methylation of CpG rich promoter regions. To better understand the role of aberrant methylation changes and to identify biological pathways likely to be affected in PCa, we used a genome-wide methylation array from Illumina (Infunium) to identify novel methylated genes associated to PCa. We identified several candidate genes including BNC1 as novel genes which are frequently methylated and whose methylation closely related to inactivation of gene expression in prostate cancer cell lines. Basonuclin1 is a zinc finger transcription factor that interacts with a subset of promoters of genes transcribed by both RNA polymerase-I and -II and has roles in maintaining ribosomal biogenesis and the proliferative potential of immature epithelial cells. We found that the expression of BNC1 negatively correlated with the degree of methylation at the CpG island in prostate tumor samples compared to benign prostate tissues. The forced-expression of BNC1 significantly decreased prostate cancer cell proliferation whereas the siRNA mediated BNC1 knock-down increased prostate cancer cell proliferation. Overall, our data suggest a potential tumor suppressor role for BNC1 that includes regulation of cell proliferation and cell-cell interaction in prostate carcinogenesis. Citation Format: Bernard Kwabi-Addo, Songping Wang, Paulett Furbert-Harris, Srinivasan Yegnasubramanian, Joseph Devaney. Functional characterization of Basonuclin 1 (BNC1): a novel tumor suppressor gene commonly downregulated in human prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1974. doi:10.1158/1538-7445.AM2013-1974

Cadmium Transport in a Model of Neonatal Intestinal Cells Correlates to MRP1 and Not DMT1 or FPN1

Newborns have a higher gastrointestinal uptake of cadmium than adults. In adults, the iron transporters DMT1 and FPN1 are involved in the intestinal absorption of cadmium, while in neonates, the mechanisms for cadmium absorption are unknown. We have investigated possible cadmium transporters in the neonatal intestine by applying a model of immature human intestinal epithelial Caco-2 cells. To mimic the continuous cadmium exposure via diet in neonates, cells were allowed to differentiate for 7 days in medium containing 1 μM CdCl2. A dramatic upregulation of the MT1 gene expression followed cadmium pretreatment, indicating a high sensitivity of the immature cells to cadmium. Cadmium pretreatment increased the basolateral efflux of 109Cd, without causing any effects on the passive diffusion of mannitol or the transepithelial electrical resistance. The augmented transport of cadmium was correlated to an upregulation of MRP1 gene expression and increased activity of the efflux protein MRP1. No effects were observed on gene expression of the efflux proteins MRP2 and P-gp or the iron transporters DMT1, DMT1-IRE and FPN1. In conclusion, our data indicate that continuous cadmium exposure increases the absorption of the metal in immature intestinal cells and that MRP1 is involved in the intestinal cadmium absorption in newborns.

Expression of defensins in the lungs in acute respiratory distress syndrome associated with influenza pneumonia

Summary. We investigated paraffin slices of the lungs from 35 people died during the 2009 epidemic of influenza A / H1N1. The exudative stage of acute respiratory distress syndrome (ARDS) was diagnosed in 10 cases, the proliferative stage in 16 and the fibrotic stage in 9 cases. Expression of α–defensins (HNP 1–3) in lung tissue was determined by the immunohistochemistry. The highest HNP 1–3 expression level was found during the exudative stage in neutrophils; it was similar in macrophages and alveolocytes type 2. α–defensins were also expressed by the epithelium of terminal bronchioles. During the proliferative stage, the HNP 1–3 expression in neutrophils remained at the high level, it was increased in alveolocytes type 2 and was unchanged in macrophages. During this stage, HNP 1–3 additionally expressed by immature alveolocytes and squamous metaplasia epithelial cells in the terminal bronchioles. During the fibrotic stage, the HNP 1–3 expression in neutrophils was reduced but remained the highest in comparison with that in the other cells; it did not changed in the alveolocytes type 2 and the macrophages. In this stage, HNP 1–3 also expressed in proliferating fibroblasts.

Pulmonary blastoma: A clinical study of three cases.

e18109 Background: Pulmonary blastoma (PB) is a rare biphasic neoplasm composed of both immature mesenchymal and epithelial cells representing 0.2-0.5% of pulmonary malignancies. Initial presentation ranges from asymptomatic to classical symptoms of advanced pulmonary tumors. Surgical resection is the preferred treatment with no consensus on the role of other adjuvant therapies. Methods: We report the clinical features of PB in 3 patients (pts). Retrospective review of medical records of pts diagnosed with pulmonary blastoma at Jackson Memorial Hospital, Miami, FL since 2009. Staging was done using the 2009 AJCC staging system for NSCLC. All cases required to have confirmed pathological diagnosis before inclusion. Variables analyzed were demographics, clinical presentation, treatments and outcomes. Results: Mean age at diagnosis was 53 years old; two were male; 2 were Hispanic and one African American. Two of them were heavy smokers (80 and 45 packs year, respectively). At diagnosis, all cases where symptomatic; one had mild hypercalcemia of 12.9 mg/dL and two where stage IV with confirmed metastatic disease (bone and thyroid gland). Two of the cases where treated with surgical resection and both achieved complete remission (CR); one pt still remains on CR after 8 months post-surgery and the other one relapsed after 22 months post-surgery. This pt was placed on palliative chemotherapy (3 cycles of cisplatin/docetaxel) with no response. Further palliation was radiotherapy (37 Gy) with no response. The other case was treated with chemotherapy alone (6 cycles of cisplatin/docetaxel) and attained partial remission. All 3 patients remain alive. Conclusions: In our case series, all pts were symptomatic at diagnosis and two presented with metastatic disease. Surgery was the most beneficial intervention in regard to CR and disease-free interval as in previous reports. Chemotherapy has shown mixed responses, thus, larger studies are needed to define the role of chemotherapy in the adjuvant and palliative settings.

TGF‐β2 Induces Maturation of Immature Human Intestinal Epithelial Cells and Inhibits Inflammatory Cytokine Responses Induced Via the NF‐κB Pathway

Breast milk transforming growth factor (TGF)-β2 is associated with healthy immune maturation and reduced risk of immune-mediated disease in infants. We sought to investigate whether conditioning with TGF-β2 may result in a more mature immune responder phenotype in immature human intestinal epithelial cells (IECs). Primary human fetal IECs (hFIECs) and the human fetal small intestinal epithelial cell line (H4 cells) were conditioned with breast milk levels of TGF-β2, and an inflammatory response was subsequently induced. Inflammatory cytokine secretion and mRNA expression were measured by enzyme-linked immunosorbent assay and quantitative real-time polymerase chain reaction, respectively. Alterations in activation of inflammatory signaling pathways were detected from IECs by immunoblotting and immunofluorescence. The effects of TGF-β2 conditioning on gene expression patterns in hFIECs were assessed by cDNA microarray analysis and quantitative PCR. Conditioning with TGF-β2 significantly attenuated subsequent interleukin (IL)-1β-, TNF-α-, and poly I:C-induced IL-8 and IL-6 responses in immature human IECs. Conditioning with TGF-β2 inhibited IL-1β-induced IκB-α degradation and NF-κB p65 nuclear translocation, which may partially result from TGF-β2-induced changes in the expression of genes in the NF-κB signaling pathway detected by cDNA microarray and qPCR. Conditioning with TGF-β2 attenuates the subsequent inflammatory cytokine response in immature human IECs by inhibiting signaling in the NF-κB pathway. The immunomodulatory potential of breast milk may in part be mediated by TGF-β2, which may provide a novel means of supporting intestinal immune maturation in neonates.

LPS‐binding Protein Enables Intestinal Epithelial Restitution Despite LPS Exposure

Intestinal epithelial restitution is the first part in the process of mucosal repair after injury in the intestine. Integrity of the intestinal mucosal barrier is important as a first line of defense against bacteria and endotoxin. Necrotizing enterocolitis (NEC) is a major cause of morbidity and mortality in extremely-low-birth-weight infants, but its mechanisms are not well defined. Abnormal bacterial colonization, immature barrier function, innate immunity activation, and inflammation likely play a role. Lipopolysaccharide (LPS)-binding protein (LBP) is secreted by enterocytes in response to inflammatory stimuli and has concentration-dependent effects. At basal concentrations, LBP stimulates the inflammatory response by presenting LPS to its receptor; however, at high concentrations, LBP is able to neutralize LPS and prevent an exaggerated inflammatory response. We sought to determine how LBP would affect wound healing in an in vitro model of intestinal cell restitution and protect against intestinal injury in a rodent model of NEC. Immature intestinal epithelial cells (IEC-6) were seeded in poly-L-lysine-coated 8-chamber slides and grown to confluence. A 500-μm wound was created using a cell scraper mounted on the microscope to achieve uniform wounding. Media was replaced with media containing LPS ± LBP. Slide wells were imaged after 0, 8, and 24 hours and then fixed. Cellular restitution was evaluated via digital images captured on an inverted microscope and wound closure was determined by automated analysis. Toll-like receptor 4 (TLR4) was determined by reverse transcriptase-polymerase chain reaction after RNA isolation from wounded cells 24 hours after treatment. LPS alone attenuated wound healing in immature intestinal epithelium. This attenuation is reversed by 24 hours with increasing concentrations of LBP so that wound healing is equivalent to control (P < 0.001). TLR4 was increased with LPS alone but levels returned to that of control after addition of LBP in the higher concentrations. LBP had no effect on the development of intestinal injury when given during our rodent model of NEC. Abnormal bacterial colonization and activation of innate immunity by LPS are likely involved in the pathogenesis of NEC.The attenuation of wound healing was reversed when LBP was added to LPS but only in the higher concentrations. At these same concentrations of LBP, TLR4 was decreased to that of control. These results indicate that LBP may be a novel therapeutic strategy to facilitate wound healing after the acute phase of NEC and other forms of intestinal injury.

Elucidating Prognosis and Biology of Breast Cancer Arising in Young Women Using Gene Expression Profiling

Breast cancer in young women is associated with poor prognosis. We aimed to define the role of gene expression signatures in predicting prognosis in young women and to understand biological differences according to age. Patients were assigned to molecular subtypes [estrogen receptor (ER)(+)/HER2(-); HER2(+), ER(-)/HER2(-))] using a three-gene classifier. We evaluated whether previously published proliferation, stroma, and immune-related gene signatures added prognostic information to Adjuvant! online and tested their interaction with age in a Cox model for relapse-free survival (RFS). Furthermore, we evaluated the association between candidate age-related genes or gene sets with age in an adjusted linear regression model. A total of 3,522 patients (20 data sets) were eligible. Patients aged 40 years or less had a higher proportion of ER(-)/HER2(-) tumors (P < 0.0001) and were associated with poorer RFS after adjustment for breast cancer subtype, tumor size, nodal status, and histologic grade and stratification for data set and treatment modality (HR = 1.34, 95% CI = 1.10-1.63, P = 0.004). The proliferation gene signatures showed no significant interaction with age in ER(+)/HER2(-) tumors after adjustment for Adjuvant! online. Further analyses suggested that breast cancer in the young is enriched with processes related to immature mammary epithelial cells (luminal progenitors, mammary stem, c-kit, RANKL) and growth factor signaling in two independent cohorts (n = 1,188 and 2,334). Proliferation-related prognostic gene signatures can aid treatment decision-making for young women. However, breast cancer arising at a young age seems to be biologically distinct beyond subtype distribution. Separate therapeutic approaches such as targeting RANKL or mammary stem cells could therefore be needed.

Immunohistochemical Expression of Survivin and γ-H2AX in Vulvar Intraepithelial Neoplasia and Low-Stage Squamous Cell Carcinoma

Survivin inhibits apoptosis and is involved in the regulation of cell cycle progression and in the mitotic spindle formation. It is overexpressed in many cancers. The histone γ-H2AX is a marker of activated DNA damage and is overexpressed in different cancers and their precursor lesions. It also forms early during apoptosis. Eighty-seven formalin-fixed, paraffin-embedded archival vulvar tissues originating from 55 preoperatively untreated patients were immunostained with antibodies to survivin and γ-H2AX to determine their expression in normal squamous vulvar epithelia (NE, n=25), lichen sclerosus (n=10), high-grade classic vulvar intraepithelial neoplasia (n=16), differentiated vulvar intraepithelial neoplasia (n=16), and vulvar invasive keratinizing squamous cell carcinoma (ISCC, n=20; FIGO Ib). Immunostaining for both factors was scored for moderate and strong intensities with regard to quantity. Statistical analysis was performed by the χ test and Fisher exact test. Nuclear surviving expression increased from NE and lichen scleros to high-grade classic vulvar intraepithelial neoplasia, differentiated vulvar intraepithelial neoplasia, and ISCC significantly (P=0.0001) and followed the distribution of immature squamous epithelial cells. Positive scores for γ-H2AX were found in nuclei of cells in all diagnostic cohorts, in any epithelial level with some accentuation in the upper layers, was seen in pycnotic nuclei in horn pearls of ISCC and apoptotic bodies, without relevant statistical distributions. Immunoscores did not differ between grade 1 and grades 2/3. Expression patterns were different for both factors, suggesting their involvement in different biologic mechanisms as an early event leading to resistance to apoptosis in vulvar carcinogenesis.

P63 transcriptionally regulates BNC1, a Pol I and Pol II transcription factor that regulates ribosomal biogenesis and epithelial differentiation

The p53-family member, p63 is a transcription factor that influences cellular adhesion, motility, proliferation, survival and apoptosis, and has a major role in regulating epithelial stem cells. Expression of p63 is often dysregulated in squamous cell carcinomas of the head and neck. In this study we show that p63 induces the expression of the basal epithelial transcription factor, Basonuclin 1. Basonuclin 1 is an unusual transcription factor that interacts with a subset of promoters of genes that are transcribed by both RNA polymerase-I and -II and has roles in maintaining ribosomal biogenesis and the proliferative potential of immature epithelial cells. Chromatin immunoprecipitation and reporter assays demonstrate that Basonuclin 1 is a direct transcriptional target of p63 and we also show that up-regulation of Basonuclin 1 is a common event in squamous cell carcinomas of the head and neck. These data identify a new transcriptional programme mediated by p63 regulation of the Basonuclin 1 transcription factor in squamous cell carcinomas and provide a novel link of p63 with the regulation of ribosomal biogenesis in epithelial cancer.

Nitric Oxide and Cellular Maturity Are Key Components of Pro-Inflammatory Cytokine-Induced Apoptosis of Human Fetal Lung Epithelial Cells

Inflammation is a major contributor to the pathogenesis of bronchopulmonary dysplasia (BPD). BPD is associated with prematurity of birth, sepsis, with increased production of both cytokines and nitric oxide, and with the shedding of bronchial epithelial cells. The pathological mechanisms involved in this disease remain unclear, in particular the role that epithelial maturity plays. The effects of pro-inflammatory cytokines upon immature and mature cells are examined within this study, using primary culture of human lung epithelial cells. Pro-inflammatory cytokines increase inducible nitric oxide synthase (iNOS) expression and raise NO production, irrespective of cellular maturity. Pre-incubation with 1400W, a specific iNOS inhibitor, abrogated pro-inflammatory cytokine-induced NO generation and apoptosis. However, immature fetal lung epithelial cells were uniquely sensitive to cellular injury in response to cytokine exposure. These observations suggest that pro-inflammatory cytokines, which are present within BPD, may cause apoptosis of lung epithelial cells via de novo generation of NO. Furthermore, the prematurity of lung epithelial cells may be a factor in free radical mediated pulmonary damage.