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1.
Bratisl Lek Listy ; 113(2): 92-4, 2012.
Article in English | MEDLINE | ID: mdl-22394038

ABSTRACT

BACKGROUND: Although the iron is an essential element for the physiological functions of cells, tissues and organs, it is also an important inductor of reactive oxygen species (ROS). MATERIAL AND METHODS: Three groups of human spleen with autoimmune thrombocytopenia (AITP), hereditary spherocytosis (HS) and reference samples stained by haematoxylin and eosin, Perls' reaction for nonheme Fe(III) iron and Alcian blue for glycoconjugates detection were studied. RESULTS: Positive Perls' reaction in both AITP and HS groups was seen. Higher positivity in the HS than in AITP group was observed. HS group showed a higher amount of acidic glycoconjugates deposits than AITP group. Iron overload in HS and AITP leads to overproduction of ROS. CONCLUSION: We suggest that acidic glycoconjugates deposits are involved in antioxidant defence by elimination and restriction of iron as a ROS inducer (Fig. 4, Ref. 19).


Subject(s)
Ferric Compounds/metabolism , Purpura, Thrombocytopenic, Idiopathic/metabolism , Spherocytosis, Hereditary/metabolism , Spleen/metabolism , Glycoconjugates/metabolism , Histocytochemistry , Humans , Reactive Oxygen Species/metabolism
2.
Int J Oncol ; 37(3): 707-18, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20664940

ABSTRACT

Accumulating evidence suggests that colorectal cancer (CRC) should be viewed as a heterogeneous disease, with proximal and distal CRCs showing multiple biological and clinical differences. The aim of this study was to develop a clinicopathological, molecular and protein profile for CRCs based on their region and thus providing insight into their heterogeneity. CRC patients (n=399) were evaluated for clinicopathologic and molecular features including K-RAS, BRAF and MSI status. Tumors were also screened for expression of 50 immunohistochemical markers linked to major signaling pathways involved in tumor-progression or immune response. Proximally located tumors show significantly larger tumor size, higher T-stage, higher tumor grade and more frequent mucinous histologic subtype compared to the distal colon and rectum. The frequency of BRAF mutation and MSI-high phenotype were significantly higher in proximal colon cancers. There is a significant difference in regional expression of 10 tumor-associated markers (CDX2, CD44v6, CD44s, TOPK, nuclear beta-catenin, pERK, APAF-1, E-cadherin, p21 and bcl2) and 4 immune response markers (CD68, CD163, FoxP3 and TIA-1). In multivariate analysis CD44s, CD44v6, nuclear beta-catenin and CD68 expression was found to best discriminate left- versus right-sided colon cancers. Tumor diameter, pT stage and MSI status best distinguish right-sided colon cancers from rectal cancers and pT stage and E-cadherin best discriminate left-sided colon cancers and rectal cancers. These data along with existing evidence for the presence of distinct regional embryological origin and gene expression profile are highly supportive of the concept that proximal and distal CRCs are distinct clinicopathologic entities.


Subject(s)
Biomarkers, Tumor/metabolism , Colonic Neoplasms/pathology , Neoplasm Proteins/metabolism , Rectal Neoplasms/pathology , Adult , Aged , Aged, 80 and over , Biomarkers, Tumor/genetics , Colonic Neoplasms/genetics , Colonic Neoplasms/metabolism , Female , Genes, ras , Humans , Immunohistochemistry , Male , Middle Aged , Mutation , Neoplasm Proteins/genetics , Neoplasm Staging , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Proto-Oncogene Proteins B-raf/genetics , Rectal Neoplasms/genetics , Rectal Neoplasms/metabolism
3.
J Perinatol ; 30(9): 584-9, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20220763

ABSTRACT

OBJECTIVE: To determine whether 17-alpha hydroxyprogesterone (17-OHPC) alters tumor necrosis factor-alpha (TNF-alpha) production and the expression of cyclooxygenase type 2 (COX-2) in myometrium exposed to lipopolysaccharide (LPS). STUDY DESIGN: Lower segment myometrial biopsies were obtained from non-laboring patients at term. Tissues were cultured in serum-free media with 17-OHPC (1 microM) and LPS (1 microg/ml), either alone or in combination. At 24 h, the production of tumor necrosis factor-alpha (TNF-alpha) and the expression of COX-2 was determined using enzyme linked immunosorbent assay and real-time (RT-PCR). Statistical analysis was performed using non-parametric testing. A P-value of <0.05 was considered significant. RESULT: 17-OHPC had no effect on TNF-alpha production and COX-2 expression when compared with untreated myometrial explants (P=0.61 and P=0.95). LPS induced production of TNF-alpha (P=0.03) and expression of COX-2 (P=0.02). Treatment with 17-OHPC did not block LPS-induced TNF-alpha production (P=0.37) or COX-2 expression (P=0.12). CONCLUSION: In this pilot study, 17-OHPC did not affect the production of TNF-alpha or COX-2 expression in human myometrium.


Subject(s)
Cyclooxygenase 2/metabolism , Hydroxyprogesterones/pharmacology , Myometrium/drug effects , Myometrium/metabolism , Tumor Necrosis Factor-alpha/metabolism , 17 alpha-Hydroxyprogesterone Caproate , Cells, Cultured , Cyclooxygenase 2/genetics , Female , Humans , Lipopolysaccharides , Pregnancy , RNA, Messenger/metabolism
4.
J Pathol ; 212(3): 260-8, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17516584

ABSTRACT

Tumour budding or dedifferentiation at the invasive margin of colorectal cancer (CRC) is an important prognostic marker and linked mechanistically to dysregulation of Wnt pathway signalling. Since budding is observed in only 40% of CRCs, we hypothesized that Wnt pathway dysregulation may be a necessary but insufficient explanation for budding and that buds may be destroyed selectively by tumour immune mechanisms. Twenty potential markers of tumour budding were evaluated in tissue microarrays (TMAs) obtained from the main tumour body of 1164 DNA mismatch repair-proficient CRCs and the findings were correlated with tumour budding, lymphocytic infiltration and survival. Loss of expression of E-cadherin and APAF-1 were independent predictors of budding (sensitivity 70.3% and specificity 48.2% when one or the other was lost). Peritumoral lymphocytes (PTLs) were observed more frequently in CRCs with loss of either E-cadherin or APAF-1 that were budding-negative. PTLs and tumour-infiltrating lymphocytes (TILs) were strongly correlated. The absence of TILs increased the adverse prognostic impact of E-cadherin and APAF-1 loss. Co-occurrence of E-cadherin loss, APAF-1 loss and low TIL counts in CRCs was an independent prognostic factor. The findings were verified in whole tissue sections from 88 CRCs with known KRAS mutation status (which was not associated with budding). Loss of E-cadherin and APAF-1 within the main body of CRCs are independent predictors of tumour budding. The prognostic benefit of lymphocytic infiltration may be explained by the immune destruction of budding cells.


Subject(s)
Adenoma/pathology , Apoptotic Protease-Activating Factor 1/genetics , Biomarkers, Tumor/analysis , Cadherins/genetics , Colorectal Neoplasms/pathology , Lymphocytes, Tumor-Infiltrating/pathology , Adenoma/immunology , Adenoma/mortality , Apoptotic Protease-Activating Factor 1/analysis , Cadherins/analysis , Case-Control Studies , Colon/immunology , Colorectal Neoplasms/immunology , Colorectal Neoplasms/mortality , DNA Mismatch Repair , Gene Deletion , Humans , Immunohistochemistry , Multivariate Analysis , Neoplasm Invasiveness , Neoplasm Staging , Oligonucleotide Array Sequence Analysis , Prognosis , ROC Curve , Survival Analysis
5.
J Pathol ; 212(2): 124-33, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17427169

ABSTRACT

There is increasing evidence for an alternative pathway of sporadic colorectal tumourigenesis that is associated with DNA microsatellite instability (MSI), due to methylation and loss of expression of the mismatch repair gene MLH1. Recent studies have highlighted a serrated pathway of colorectal cancer (CRC) in which serrated polyps with activating mutations in BRAF progress to CRCs with MSI following methylation and silencing of MLH1. The present study provides a novel mechanistic experimental model for these clinical observations. We investigated the role of BRAF activating mutation (BRAF-V600E) in colorectal tumourigenesis by studying the effects of forced expression of BRAF-V600E in the 'normal' colon epithelial NCM460 cell line and by targeting endogenous BRAF-V600E in MSI-High (MSI-H) colon cancer cell lines. The findings indicate that BRAF mutation in colon epithelial cells contributes to a gain in resistance towards apoptotic stimuli, which is likely to be an important characteristic of pre-malignant serrated lesions. BRAF-V600E also plays a role in the development and maintenance of transformed and invasive phenotypes in colon epithelial cells. Our findings also suggest that BRAF mutation potentiates promoter hypermethylation of the MLH1 gene promoter. Together, these results highlight BRAF as a potential target for therapeutic intervention in sporadic MSI-H colorectal cancers.


Subject(s)
Colorectal Neoplasms/genetics , Neoplasm Proteins/genetics , Proto-Oncogene Proteins B-raf/genetics , Adaptor Proteins, Signal Transducing/genetics , Apoptosis/genetics , Cell Line , Cell Line, Tumor , Cell Transformation, Neoplastic/genetics , Colon/pathology , Colorectal Neoplasms/pathology , CpG Islands/genetics , Epithelial Cells/pathology , Humans , Intestinal Mucosa/pathology , Methylation , Microsatellite Instability , Models, Biological , MutL Protein Homolog 1 , Mutation , Neoplasm Invasiveness , Nuclear Proteins/genetics , Phenotype , Promoter Regions, Genetic/genetics
6.
Histopathology ; 50(4): 453-64, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17448021

ABSTRACT

AIMS: To investigate dysregulation of the wnt signalling pathway by assessing beta-catenin expression/increasing expression and loss of cytoplasmic adenomatous polyposis coli (APC) and membranous E-cadherin in colorectal cancer (CRC) and determining the prognostic significance of these variables. METHODS AND RESULTS: Unselected, non-consecutive CRC resections (n = 1420) were subdivided into three groups: mismatch repair (MMR)-proficient, MLH1- and presumed hereditary non-polyposis colonic cancer (HNPCC). Immunohistochemical analysis of beta-catenin expression (0% versus > 0%) and increasing expression (increasing percentage-positivity) and loss of APC and E-cadherin was performed using the tissue microarray technique. In MMR-proficient CRC, increased nuclear beta-catenin expression and loss of membranous E-cadherin were independently associated with higher N stage (P = 0.03 and < 0.0001), vascular invasion (P < 0.01 and < 0.001) and worse survival (P < 0.01 and < 0.001). Additionally, there was an association between loss of membranous E-cadherin and higher T stage (P = 0.03). In MLH1- CRC, loss of membranous E-cadherin was associated with higher N stage (P = 0.05) and worse survival (P = 0.03). In presumed HNPCC CRC nuclear beta-catenin and membranous E-cadherin were not associated with tumour progression or worse survival. In all CRC subsets loss of cytoplasmic APC was not associated with clinicopathological features. CONCLUSIONS: Increasing nuclear beta-catenin expression and loss of membranous E-cadherin are independent, adverse prognostic factors in MMR-proficient and MLH1- CRC.


Subject(s)
Adenomatous Polyposis Coli Protein/physiology , Cadherins/physiology , Colorectal Neoplasms/diagnosis , Wnt Proteins/physiology , beta Catenin/physiology , Adaptor Proteins, Signal Transducing , Adenomatous Polyposis Coli Protein/biosynthesis , Cadherins/biosynthesis , Carrier Proteins/genetics , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Colorectal Neoplasms, Hereditary Nonpolyposis/diagnosis , Colorectal Neoplasms, Hereditary Nonpolyposis/metabolism , Cytoplasm/metabolism , DNA Mismatch Repair , Female , Humans , Immunohistochemistry , Intestinal Mucosa/metabolism , Intestinal Mucosa/pathology , Male , Middle Aged , MutL Protein Homolog 1 , Nuclear Proteins/genetics , Prognosis , Regression Analysis , Signal Transduction , Tissue Array Analysis , beta Catenin/biosynthesis
7.
J Clin Pathol ; 60(5): 534-9, 2007 May.
Article in English | MEDLINE | ID: mdl-16816167

ABSTRACT

BACKGROUND: Expression of mucin antigen MUC1 and down regulation of MUC2 are associated with adverse prognosis in colorectal cancer (CRC), but their prognostic significance with respect to differing DNA mis- match repair (MMR) status is poorly understood. OBJECTIVE: To determine the prognostic significance of MUC1 and MUC2 in CRC with different MMR statuses. METHODS: Using the tissue microarray (TMA) technique, a series of 1420 unselected, non-consecutive CRC resections was subdivided into three groups: (1) MMR-proficient; (2) MLH1-negative; and (3) presumed hereditary non-polyposis colon cancer (HNPCC). Immunohistochemical analysis of MUC1 and MUC2 expression (>0%) and loss (0%) was performed, and the results were correlated with clinicopathological parameters. RESULTS: In MMR-proficient CRC, MUC1 expression was more frequently found in tumours with higher tumour stage (p=0.004) and higher tumour grade (p=0.041) and loss of MUC2 was associated with higher tumour stage (p=0.028), node stage (p=0.001), presence of vascular invasion (p=0.028) and worse survival (p=0.034). In MLH1-negative CRC, MUC2 loss was associated with the presence of lymph node metastasis (p=0.028) and worse survival (p=0.015), but there was no association between MUC1 expression and clinicopathological features. In presumed HNPCC, MUC1 expression and MUC2 loss were not associated with clinicopathological parameters. CONCLUSIONS: Mucins have a prognostic significance in sporadic CRC, but not in hereditary CRC. Loss of MUC2 is an adverse prognostic factor in MMR-proficient and MLH1-negative CRC, whereas MUC1 expression is associated with tumour progression in MMR-proficient CRC only.


Subject(s)
Biomarkers, Tumor/metabolism , Colorectal Neoplasms/metabolism , DNA Mismatch Repair , Mucins/metabolism , Adult , Aged , Aged, 80 and over , Antigens, Neoplasm/metabolism , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , DNA, Neoplasm/genetics , Disease Progression , Female , Humans , Immunoenzyme Techniques , Male , Middle Aged , Mucin-1 , Mucin-2 , Neoplasm Invasiveness , Neoplasm Proteins/metabolism , Neoplasm Staging , Prognosis , Protein Array Analysis/methods , Survival Analysis
8.
Life Sci ; 79(25): 2349-56, 2006 Nov 17.
Article in English | MEDLINE | ID: mdl-16952379

ABSTRACT

Using a previously published model of human BPD this study examines whether preterm lung inflammatory cells produce transforming growth factor beta 1 (TGF-beta1), a cytokine pivotal in pathogenesis of bronchopulmonary dysplasia (BPD), and whether TGF-beta1 expression is regulated by inflammation. Lung inflammatory cells (neutrophils and macrophages) recovered in the broncho-alveolar (BAL) fluid of premature infants intubated for respiratory distress after birth expressed TGF-b1 mRNA and protein. Total and bioactive TGF-beta1 were abundantly found in the BAL fluid of the same infants. In cell culture stimulation by lipopolysaccharide (LPS) did not result in any further expression of total or bioactive TGF-beta1 by neonatal lung inflammatory cells over constitutive concentrations. In conclusion, lung inflammatory cells from premature infants are a source of TGF-beta1 but LPS does not regulate TGF-b1 production in these cells.


Subject(s)
Bronchopulmonary Dysplasia/metabolism , Gene Expression Regulation/physiology , Lung/cytology , Macrophages/metabolism , Neutrophils/metabolism , Premature Birth , Transforming Growth Factor beta1/genetics , Bronchoalveolar Lavage Fluid , Cells, Cultured , Female , Gene Expression Regulation/drug effects , Humans , Infant, Newborn , Lipopolysaccharides/pharmacology , Macrophages/drug effects , Neutrophils/drug effects , Pregnancy , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction
9.
J Pathol ; 210(2): 137-40, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16917802

ABSTRACT

Interest in the role of oncogene-induced senescence in tumorigenesis is mounting. Raf-associated senescence in cutaneous nevi has been advanced as an example of this process occurring in the context of a human tumour. In this model, conversion from a senescent nevus to a malignant melanoma is accompanied by loss of expression of p16. Serrated polyps of the colorectum may provide a further example of oncogene-induced senescence. BRAF and KRAS mutation may initiate different pathways of senescence-associated serrated neoplasia in the colorectum, the former linked to CpG island methylator phenotype (CIMP)-high (CIMP1) and microsatellite instability (MSI)-high status and the latter with CIMP-low (CIMP2) and MSI-low status. The role of methylation in both Raf- and Ras-associated pathways is to drive tumorigenesis by silencing pro-apoptotic and cell cycle inhibitory genes. Both pathways are associated with mutation of Ras-induced senescence 1 (RIS1), but the biological role of RIS1 requires further elucidation.


Subject(s)
Cell Transformation, Neoplastic/genetics , Cellular Senescence/genetics , Intestinal Polyps/genetics , Colorectal Neoplasms/genetics , Genes, cdc , Humans , Mutation , Oncogenes/genetics
10.
Gut ; 55(10): 1467-74, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16469793

ABSTRACT

BACKGROUND: Hyperplastic polyposis of the colorectum is a precancerous condition that has been linked with DNA methylation. The polyps in this condition have been distinguished from typical small hyperplastic polyps and renamed sessile serrated adenomas. Sessile serrated adenomas also occur sporadically and appear to be indistinguishable from their counterparts in hyperplastic polyposis. AIMS AND METHODS: The existence of distinguishing molecular features was explored in a series of serrated polyps and matched normal mucosa from patients with and without hyperplastic polyposis by assessing mutation of BRAF, DNA methylation in 14 markers (MINTs 1, 2 and 31, p16, MGMT, MLH1, RASSF1, RASSF2, NORE1 (RASSF5), RKIP, MST1, DAPK, FAS, and CHFR), and immunoexpression of MLH1. RESULTS: There was more extensive methylation in sessile serrated adenomas from subjects with hyperplastic polyposis (p<0.0001). A more clearcut difference in patients with hyperplastic polyposis was the finding of extensive DNA methylation in normal mucosa from the proximal colon. CONCLUSIONS: A genetic predisposition may underlie at least some forms of hyperplastic polyposis in which the earliest manifestation may be hypermethylation of multiple gene promoters in normal colorectal mucosa. Additionally, some of the heterogeneity within hyperplastic polyposis may be explained by different propensities for MLH1 inactivation within polyps.


Subject(s)
Colonic Polyps/genetics , DNA Methylation , Mutation/genetics , Proto-Oncogene Proteins B-raf/genetics , Adaptor Proteins, Signal Transducing/metabolism , Adult , Aged , Biomarkers, Tumor/metabolism , CpG Islands , DNA, Neoplasm/metabolism , Female , Genetic Predisposition to Disease , Humans , Immunohistochemistry , Male , Middle Aged , MutL Protein Homolog 1 , Nuclear Proteins/metabolism , Polymerase Chain Reaction/methods
11.
Hum Reprod ; 20(6): 1709-19, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15734750

ABSTRACT

BACKGROUND: Apoptosis occurs in late secretory and menstrual human endometrium and is thought to play an important role in endometrial physiology. Menstrual-like breakdown has been observed in vitro in endometrial explants. The purpose of this study was to assess the role of apoptosis in menstrual-like breakdown in human endometrial explants. METHODS: Human endometrial tissue was obtained during the mid-secretory phase and cultured with or without estrogen and progesterone. The occurrence of breakdown was assessed by histology. Apoptosis was determined by gel electrophoresis for the detection of DNA fragmentation and by immunohistochemistry using the M30 CytoDEATH and anti-cleaved caspase-3 (CASP3) antibodies for the detection of caspase activity. Expression of BCL2 and BAX was quantified using real-time PCR analysis. RESULTS: Apoptosis occurred in human endometrial explants at all time-points studied. Cleaved CASP3 and M30 antigen expression increased in all explants, suggesting the involvement of CASP3 in the apoptosis. Low BCL2:BAX ratios were observed in all samples when compared with pre-culture controls. Estradiol and progesterone supplementation of the culture media reduced or eliminated menstrual-like breakdown but did not affect the degree of apoptosis observed. CONCLUSIONS: The apoptosis observed in endometrium during the late secretory phase and menstrual phase does not appear to be mechanistically related to the tissue breakdown but rather may be involved in the impending remodelling that occurs in the endometrium in the transition from secretory to proliferative phase following the menses.


Subject(s)
Apoptosis/physiology , Endometrium/pathology , Menstrual Cycle/physiology , Adult , Apoptosis/genetics , Caspase 3 , Caspases/immunology , Caspases/metabolism , DNA Fragmentation , Endometrium/physiology , Enzyme Activation , Female , Gene Expression Regulation , Humans , Menstruation/physiology , Organ Culture Techniques , Proto-Oncogene Proteins c-bcl-2/genetics , bcl-2-Associated X Protein
12.
Life Sci ; 74(24): 2941-57, 2004 Apr 30.
Article in English | MEDLINE | ID: mdl-15051419

ABSTRACT

Regulation of transforming growth factor beta 1 (TGF-beta1) expression remains unclear. Inflammation has been inferred to play a major role in stimulating TGF-beta1 production since high concentrations of TGF-beta1 have been found in the lungs of patients with various diffuse inflammatory lung diseases. To establish an association between inflammation and TGF-beta1 expression, human alveolar epithelial (A549) cells were co-cultured with lipopolysaccharide (LPS), Tumor necrosis factor alpha (TNFalpha), Interleukin 1 beta (IL-1beta) and Interleukin 8 (IL-8) for 12 hours. Total and bioactive TGF-beta1 protein were then measured. A549 cells transiently transfected with a plasmid containing the TGF-beta1 promoter linked to a luciferase reported gene were then co-cultured with the same inflammatory peptides for 12 hours and TGF-beta1 promoter activity determined. Nuclear transcription factors AP-1 (c-jun) or NF-kappa (p65, p50 and p105) were over expressed in A549 cells transiently transfected with the TGF-beta1 promoter and TGF-beta1 promoter activity subsequently measured. Stimulation with inflammatory signals LPS, TNFalpha, IL-1beta, IL-8 resulted in no increase of total or bioactive TGF-beta1 activity above constitutive concentrations in vitro. TGF-beta1 promoter activity was also unchanged from baseline levels in response to the same inflammatory peptides. Expression of c-jun however led to significant increases of TGF-beta1 promoter activity over constitutive levels. In contrast p65 and p105 expression resulted in inhibition of TGF-beta1 promoter activity below baseline levels. We conclude that in a human alveolar epithelial cell line, inflammation does not regulate TGF-beta1 expression. These studies suggest that in lung pathologies such as asthma, lung fibrosis and CLD, TGF-beta1 production may involve pathways independent of inflammatory mediators LPS, TNFalpha, IL-1beta and IL-8.


Subject(s)
Gene Expression Regulation , Inflammation Mediators/physiology , Pulmonary Alveoli/metabolism , Transforming Growth Factor beta/metabolism , Cell Line , Drug Combinations , Humans , Inflammation Mediators/pharmacology , Interleukin-1/pharmacology , Interleukin-8/pharmacology , Lipopolysaccharides/pharmacology , NF-kappa B/biosynthesis , Promoter Regions, Genetic/genetics , Proto-Oncogene Proteins c-jun/biosynthesis , Pulmonary Alveoli/cytology , Pulmonary Alveoli/drug effects , Transcription Factor AP-1/biosynthesis , Transfection , Transforming Growth Factor alpha/pharmacology , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta1
13.
J Matern Fetal Neonatal Med ; 14(3): 205-11, 2003 Sep.
Article in English | MEDLINE | ID: mdl-14694976

ABSTRACT

OBJECTIVE: To determine whether the presence of the proinflammatory cytokine interleukin (IL)-1beta in the lungs of preterm infants immediately after birth was associated with maternal inflammation and could predict adverse neonatal outcome. STUDY DESIGN: Prospective evaluation of serially obtained tracheal aspirates for the presence of IL-1beta in 25 preterm infants (birth weight 595-1700 g; gestational age 24-32 weeks) with respiratory distress syndrome. The initial tracheal aspirate was obtained within 1 h after delivery. RESULTS: An initial tracheal aspirate positive for IL-1beta had a highly significant correlation with documented maternal chorioamnionitis for the given patient. In addition, the presence of IL-1beta correlated significantly with elevated total cell count (2.62 vs. 0.96 x 10(6)/ml, p = 0.0097), granulocyte count (2.12 vs. 0.22 x 10(6)/ml, p = 0.001), macrophage count (0.28 vs. 0.01 x 10(6)/ml, p = 0.02) and the presence of proinflammatory cytokines IL-6, IL-8 and tumor necrosis factor (TNF)-alpha. Preterm neonates positive for IL-1beta in their initial sample were on prolonged assisted ventilation (38 vs. 16 days, p = 0.013) and oxygen supplementation (62 vs. 40.5 days, p = 0.0462) and required prolonged hospitalization (69 vs. 46 days, p = 0.0165). CONCLUSIONS: The concentration of IL-1beta in the initial tracheal aspirate obtained from the lungs of preterm infants within the first hour of life may serve as a marker of antenatal/perinatal inflammation, probably due to maternal chorioamnionitis, and could predict an adverse clinical course and short-term outcome.


Subject(s)
Bronchoalveolar Lavage Fluid/cytology , Chorioamnionitis/immunology , Infant, Premature/metabolism , Interleukin-1/metabolism , Biomarkers/analysis , Cesarean Section/statistics & numerical data , Cytokines/metabolism , Female , Humans , Infant, Newborn , Intubation, Intratracheal , Length of Stay/statistics & numerical data , Male , Outcome Assessment, Health Care , Oxygen Inhalation Therapy , Pregnancy , Prospective Studies , Respiration, Artificial , Respiratory Distress Syndrome, Newborn/immunology , Respiratory Distress Syndrome, Newborn/metabolism
14.
Am J Physiol Lung Cell Mol Physiol ; 283(3): L510-7, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12169568

ABSTRACT

We propose that lung morphogenesis and repair are characterized by complex cell-cell interactions of endodermal and mesodermal origin, leading to (or returning back to) an alveolar structure that can effectively exchange gases between the circulation and the alveolar space. We provide the developmental basis for cell/molecular control of lung development and disease, what is known about growth and transcription factors in normal and abnormal lung development, and how endodermal and mesodermal cell origins interact during lung development and disease. The global mechanisms that mediate mesenchymal-epithelial interactions and the plasticity of mesenchymal cells in normal lung development and remodeling provide a functional genomic model that may bring these concepts closer together. We present a synopsis followed by a vertical integration of the developmental and injury/repair mechanisms.


Subject(s)
Aging/physiology , Lung/embryology , Lung/growth & development , Mesoderm/physiology , Wound Healing/physiology , Animals , Bronchopulmonary Dysplasia/etiology , Embryo, Mammalian/physiology , Embryonic and Fetal Development , Epithelium/physiology , Fibroblasts/physiology , Humans , Infant, Newborn , Muscle, Smooth/cytology , Muscle, Smooth/physiology , Transcription Factors/physiology
15.
Pediatr Res ; 50(6): 726-31, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11726731

ABSTRACT

Lung injury in preterm neonates with respiratory failure has been attributed to persistent inflammation, which is likely to involve lung macrophages (LM). The study objective was to investigate LM during the first 8 d of life from preterm infants (n = 19), using term infants (n = 11) with respiratory failure as control subjects. LM percentages from mixed-cell suspensions produced from tracheobronchial lavage were calculated. A postnatal increase in the mean LM concentration was demonstrated within the preterm group (p = 0.01), which was greater in comparison to that from the term group (p < 0.01). Regression analyses were significant for direct relationships between LM concentrations and ex vivo lipopolysaccharide-induced tumor necrosis factor-alpha and IL-10 production (r = 0.93 and r = 0.63, respectively), establishing LM as the source of these cytokines. Comparative analyses demonstrated that the ability of preterm versus term LM to produce tumor necrosis factor-alpha was nearly identical; in contrast, a trend toward diminished levels of IL-10 expression in the preterm group was observed (p = 0.06). Thus, although studies have shown that LM precursors (i.e. cord blood monocytes) produce less tumor necrosis factor-alpha in preterm versus term infants, the present data strongly suggest that this relationship does not hold postnatally with respect to terminally differentiated LM in sick neonates. Overall, the data are consistent with a pro- versus antiinflammatory imbalance that may bear functional significance on the pathogenesis of chronic lung disease.


Subject(s)
Infant, Newborn/physiology , Infant, Premature/physiology , Interleukin-10/biosynthesis , Lipopolysaccharides/pharmacology , Macrophages, Alveolar/physiology , Tumor Necrosis Factor-alpha/biosynthesis , Humans , Infant, Newborn, Diseases/physiopathology , Macrophages, Alveolar/cytology , Macrophages, Alveolar/drug effects , Regression Analysis
16.
Mol Cell Biol ; 21(21): 7380-90, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11585919

ABSTRACT

T/EBP/NKX2.1, a member of the NKX family of homeodomain-containing transcription factors, regulates the expression of a number of genes in lung and thyroid. Here we describe the isolation and characterization of a novel target gene, termed claudin-18, that is down-regulated in the lungs of T/ebp/Nkx2.1-null mouse embryos. The gene product exhibits an amino acid sequence similar to those of the claudin multigene family of proteins that constitute tight junction strands in epithelial cells. The gene was localized by fluorescence in situ hybridization to mouse chromosome 9 at region 9E3-F1 and to human chromosome 3 at region 3q21-23. The claudin-18 gene has two promoters, each with its own unique exon 1 that is spliced to common exons 2 through 5. Alternative usage of these promoters leads to production of lung and stomach-specific transcripts. The downstream lung-specific promoter contains two T/EBP/NKX2.1 binding sites responsible for trans activation of the gene by T/EBP/NKX2.1 in lung cells. Only claudin-18 was down-regulated in T/ebp/Nkx2.1-null embryo lungs among 11 claudin transcripts examined. Furthermore, the claudin-18 transcript has an alternative 12-bp insertion derived from the 5' end of intron 4, which produces a C-terminally truncated isoform in lung and stomach. Immunohistochemistry demonstrated complete membrane localization of claudin-18 with small focal dots in the lung and stomach epithelial cells. Immunogold electron microscopy analysis revealed that claudin-18 is concentrated at the cell-cell borders of epithelial cells. These unique features suggest a potentially important role for claudin-18 in the structure and function of tight junctions in lung and stomach.


Subject(s)
Alternative Splicing , Gastric Mucosa/metabolism , Homeodomain Proteins/chemistry , Homeodomain Proteins/metabolism , Lung/metabolism , Membrane Proteins/chemistry , Membrane Proteins/genetics , Membrane Proteins/metabolism , Transcription Factors/metabolism , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Blotting, Northern , Cell Nucleus/metabolism , Chromosome Mapping , Chromosomes , Chromosomes, Human, Pair 3 , Claudins , Cloning, Molecular , DNA, Complementary/metabolism , Down-Regulation , Exons , Gene Deletion , Gene Library , HeLa Cells , Humans , Immunohistochemistry , Luciferases/metabolism , Mice , Mice, Transgenic , Molecular Sequence Data , Mutation , Plasmids/metabolism , Promoter Regions, Genetic , Protein Binding , Protein Isoforms , Protein Structure, Tertiary , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Tissue Distribution , Transcription, Genetic , Transfection
17.
Life Sci ; 70(3): 253-67, 2001 Dec 07.
Article in English | MEDLINE | ID: mdl-12005259

ABSTRACT

Persistent expression of pro-inflammatory cytokines is believed to play a major role in the pathogenesis of chronic lung disease (CLD) in premature infants. Inhibition of pro-inflammatory cytokine production in the lungs of preterm newborns may result in the attenuation of CLD. Curcumin is a naturally occurring phenolic compound derived from the food spice tumeric with broad based in vitro anti-inflammatory properties. In this study lung inflammatory cells from preterm newborns at risk for the development of CLD were derived via modified broncho-alveolar lavage and stimulated ex vivo with lipopolysaccharide (LPS) (10 ng/ml). Curcumin was added to these cultures at 0, 0.5 and 20 uM concentrations. Pro-inflammatory cytokine, TNFalpha, IL-1beta and IL-8 protein was measured from the culture supernatants 12 hours post culture. For control, adult peripheral blood mononuclear cells (PBMC) were cultured under the same conditions. Both neonatal lung inflammatory cells and adult PBMC produced high levels of pro-inflammatory cytokines in response to LPS. Curcumin produced significant inhibition of IL-1beta and IL-8 but minimal inhibition of TNFalpha expression by preterm lung inflammatory cells at 20 uM concentrations. Adult PBMC expression of IL-8 was significantly inhibited by curcumin at 20 uM concentrations. Therefore, curcumin inhibits pro-inflammatory cytokine production (TNFalpha, IL-1beta and IL-8) by lung inflammatory cells ex vivo. Pathways involved with curcumin regulation of these cytokines are developmentally intact and functional in premature infants. Curcumin may be effective as a therapeutic agent in the attenuation of CLD.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Curcumin/pharmacology , Cytokines/biosynthesis , Hyaline Membrane Disease/immunology , Inflammation/immunology , Adult , Bronchoalveolar Lavage Fluid/chemistry , Bronchoalveolar Lavage Fluid/cytology , Coculture Techniques , Cytokines/analysis , Enzyme-Linked Immunosorbent Assay , Humans , Infant, Newborn , Lipopolysaccharides/pharmacology
18.
Biol Neonate ; 77(4): 217-23, 2000 May.
Article in English | MEDLINE | ID: mdl-10828572

ABSTRACT

Transforming growth factor-beta (TGF-beta) is a peptide implicated in tissue injury and repair but its role in the premature human lung remains unclear. In the present study, we used a TGF-beta responsive-promoter-luciferase construct in mink lung epithelial cells to quantify levels of biologically active TGF-beta (BA-TGF-beta) in the endotracheal aspirate (ETA) fluid from 16 extremely low birthweight neonates [6 M/10 F, mean GA 26 weeks (range 23-30), mean BW 774 g (range 555-1,075)]. ETA fluid was obtained on day 1 and then every 4 days up to 32 days. BA-TGF-beta levels were low (92 +/- 19 pg/ml) in the first 24 h of life and then increased 5- to 10-fold with peak BA-TGF-beta levels (400 +/- 50 pg/ml) on day 20-25. BA-TGF-beta levels were higher in male than female infants (p = 0.0056). Prenatal steroids decreased significantly the amount of BA-TGF-beta recovered. High initial levels of BA-TGF-beta persisted over time and were predictive of the need for oxygen therapy at home. We conclude that abundant BA- TGF-beta is present in the lungs of preterm infants and speculate that it may be involved in inflammatory and repair processes encountered in acute and chronic lung disease.


Subject(s)
Home Care Services , Infant, Low Birth Weight/metabolism , Lung/metabolism , Oxygen/therapeutic use , Transforming Growth Factor beta/metabolism , Bronchoalveolar Lavage Fluid/cytology , Cell Count , Female , Humans , Infant, Newborn , Macrophages/cytology , Male , Prognosis , Respiration Disorders/metabolism , Sex Characteristics
19.
Dev Dyn ; 217(2): 180-90, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10706142

ABSTRACT

In vitro and in vivo results are consistent with a critical role for NKX2.1, an epithelial homeodomain transcription factor in lung morphogenesis. Nkx2.1 null mutant embryos die at birth due to respiratory insufficiency caused by profoundly abnormal lungs. However, the precise role of NKX2.1 in the multistep process of lung structural morphogenesis and differentiation of various pulmonary cell types remains unknown. In the current study, we tested the hypothesis that the mutant lungs do not undergo branching morphogenesis beyond the formation of the mainstem bronchi and therefore consist solely of dilated tracheobronchial structures. To test this hypothesis, we determined the spatial and temporal expression pattern of a number of extracellular matrix (ECM) proteins and their cellular receptors, including alpha-integrins, laminin, and collagen type IV. Although laminin is expressed in the mutant Nkx2.1(-/-) lungs, expression of alpha-integrins and collagen type IV is significantly reduced or absent. In addition, examination of regionally specific expression of differentially spliced Vegf (vascular endothelial growth factor) transcripts, clearly indicates that the epithelial phenotype of the Nkx2.1(-/-) lungs is similar to the tracheobronchial epithelium. In contrast to wild-type lungs in which both Vegf1 and Vegf3 are developmentally expressed, Nkx2.1(-/-) lungs are characterized by predominant expression of Vegf1 and reduced or absent Vegf3. A similar pattern of Vegf expression is also observed in isolated tracheo-bronchial tissue. The sum of these findings suggest that at least two separate pathways may exist in embryonic lung morphogenesis: proximal lung morphogenesis is Nkx2.1 independent, while distal lung morphogenesis appears to be strictly dependent on the wild-type activity of Nkx2.1.


Subject(s)
Lung/embryology , Nuclear Proteins/physiology , Transcription Factors/physiology , Actins/biosynthesis , Animals , Antigens, CD/biosynthesis , Cadherins/biosynthesis , Collagen/biosynthesis , Endothelial Growth Factors/genetics , Epithelium , Female , Integrin alpha1 , Integrin alpha2 , Integrin alpha3 , Integrins/biosynthesis , Lung/blood supply , Lymphokines/genetics , Mice , Mice, Knockout , Morphogenesis , Muscle, Smooth , Nuclear Proteins/genetics , Phenotype , Thyroid Nuclear Factor 1 , Transcription Factors/genetics , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors
20.
Biochim Biophys Acta ; 1490(3): 213-24, 2000 Feb 29.
Article in English | MEDLINE | ID: mdl-10684967

ABSTRACT

NKX2.1 is a member of the NK2 family of homeodomain-containing transcription factors whose targeted disruption in mouse results in the absence of thyroid tissue and a severely abnormal lung phenotype. Little is known regarding the mechanisms that control tissue and temporal specificity of Nkx2.1 gene expression. The Nkx2.1 gene has been cloned from a number of species and it is composed of three exons and two introns. Two distinct DNA domains located 5' of exon I and within intron I have been found to exhibit promoter activity in lung and thyroid cells. In the current study we used deletional analysis of the 5' flanking region of exon I and identified a 300 bp TATA-less region that exhibits significant promoter activity in H441 cells. The DNA sequence of this region contains multiple palindromes, composed of G/C-rich elements. DNase I footprinting demonstrates that this promoter region interacts with nuclear factors present in H441 cells. In particular electrophoretic mobility shift assay using antibodies against the Sp family members show that both Sp1 and Sp3 as well as an as yet unknown H441-specific factor interact with the palindromic structure within this promoter region. Co-transfection studies show that this promoter region responds to Sp1 and Sp3 and mutations therein result in a significantly diminished response to these transcriptional factors. Therefore, we have identified a novel DNA structure on the Nkx2.1 gene which participates in transcription of this gene in pulmonary epithelial cells by Sp1 and Sp3 transcription factors.


Subject(s)
DNA-Binding Proteins/metabolism , DNA/metabolism , Lung/metabolism , Nuclear Proteins/genetics , Sp1 Transcription Factor/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Base Sequence , Binding Sites , Cells, Cultured , DNA/chemistry , DNA Footprinting , Epithelial Cells/metabolism , HeLa Cells , Humans , Mutagenesis, Site-Directed , Nuclear Proteins/metabolism , Point Mutation , Promoter Regions, Genetic , Sp3 Transcription Factor , Thyroid Nuclear Factor 1 , Transcription, Genetic , Transfection
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