Mucin 4 (MUC4) gene: regional assignment (3q29) and RFLP analysis.

The use of a probe (JER64) containing a mucin 4 (MUC4) cDNA insert of 1.83 kb allowed to assign by in situ hybridization, the MUC4 gene to 3q29. This probe detected RFLPs with all restriction enzymes used (BamHI, HindIII, PstI, EcoRI, and TaqI). Particularly numerous alleles were observed with PstI, EcoRI and TaqI, in a small sample of unrelated DNAs (25 digested with PstI, 8 with EcoRI and 8 with TaqI). The PIC values were 0.69, 0.63 and 0.70 for PstI, EcoRI and TaqI respectively. The polymorphisms observed of variable number of tandem repeat (VNTR) type are in relation with the presence of tandemly repeated nucleotide sequences in MUC4 gene.     

Modeling the human intestinal Mucin (MUC2) C-terminal cystine knot dimer

Intestinal mucus, a viscous secretion that lines the mucosa, is believed to be a barrier to absorption of many therapeutic compounds and carriers, and is known to play an important physiological role in controlling pathogen invasion. Nevertheless, there is as yet no clear understanding of the barrier properties of mucus, such as the nature of the molecular interactions between drug molecules and mucus components as well as those that govern gel formation. Secretory mucins, large and complex glycoprotein molecules, are the principal determinants of the viscoelastic properties of intestinal mucus. Despite the important role that mucins play in controlling transport and in diseases such as cystic fibrosis, their structures remain poorly characterized. The major intestinal secretory mucin gene, MUC2, has been identified and fully sequenced. The present study was undertaken to determine a detailed structure of the cysteine-rich region within the C-terminal end of human intestinal mucin (MUC2) via homology modeling, and explore possible configurations of a dimer of this cysteine-rich region, which may play an important role in governing mucus gel formation. Based on sequence–structure alignments and three-dimensional modeling, a cystine knot tertiary structure homologous to that of human chorionic gonadotropin (HCG) is predicted at the C-terminus of MUC2. Dimers of this C-terminal cystine knot (CTCK) were modeled using sequence alignment based on HCG and TGF-beta, followed by molecular dynamics and simulated annealing. Results support the formation of a cystine knot dimer with a structure analogous to that of HCG.      

粒细胞集落刺激因子对全身炎症反应综合征中细胞因子的影响

目的:对粒细胞集落刺激因子(rhG-CSF)在全身炎症反应综合征及其治疗中的作用和机制进行初步的探究.方法:腹腔注射脂多糖(LPS)建立全身炎症反应综合征的小鼠动物模型后,将BALB/c模型小鼠随机分为生理盐水(NS)、50 μg/m2重组粒细胞集落刺激因子(rhG-CSF)、200 μg/m2rhG-CSF、400 μg/m2 rhG-CSF四组,进行脾淋巴细胞的分离,通过RT-PCR、ELISA等方法检测共刺激分子和炎症相关因子在不同组别中的表达.结果:各RHG-CSF组的炎症相关因子表达有不同程度下调.诱生性共刺激分子(ICOS)表达下调,同时杀伤性T细胞相关抗原-4(CTLA-4)的表达量明显上调.作用最明显的是200μg/m2 rhG-CSF组.结论:rhG-CSF对于全身炎症反应综合征有一定的治疗作用且存在剂量依赖性.     

The epithelium of the middle ear in the guinea pig

Summary An electron microscopic study of aldehyde and osmium fixed normal guinea pig middle ear epithelium was made. Numerous branching microvilli occur between the cilia of the ciliated cells. The granules of the secretory cells are always surrounded by a membrane, and they vary in their content of electron dense substance. Half desmosomes are frequent in basal cells. The squamous epithelial cells of the bulla contain few microvilli and pinocytoric invaginations. In the basal part of the squamous epithelium dilations of the intercellular clefts often occur. The luminal part of the intercellular clefts are closed by multiple tight junctions.     

Pathobiological Implications of Mucin (MUC) Expression in the Outcome of Small Bowel Cancer

Mucins have been associated with survival in various cancer patients, but there have been no studies of mucins in small bowel carcinoma (SBC). In this study, we investigated the relationships between mucin expression and clinicopathologic factors in 60 SBC cases, in which expression profiles of MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC6 and MUC16 in cancer and normal tissues were examined by immunohistochemistry. MUC1, MUC5AC and MUC16 expression was increased in SBC lesions compared to the normal epithelium, and expression of these mucins was related to clinicopathologic factors, as follows: MUC1 [tumor location (p = 0.019), depth (p = 0.017) and curability (p = 0.007)], MUC5AC [tumor location (p = 0.063) and lymph node metastasis (p = 0.059)], and MUC16 [venous invasion (p = 0.016) and curability (p = 0.016)]. Analysis of 58 cases with survival data revealed five factors associated with a poor prognosis: poorly-differentiated or neuroendocrine histological type (p<0.001), lymph node metastasis (p<0.001), lymphatic invasion (p = 0.026), venous invasion (p<0.001) and curative resection (p<0.001), in addition to expression of MUC1 (p = 0.042), MUC5AC (p = 0.007) and MUC16 (p<0.001). In subsequent multivariate analysis with curability as the covariate, lymph node metastasis, venous invasion, and MUC5AC and/or MUC16 expression were significantly related to the prognosis. Multivariate analysis in curative cases (n = 45) showed that SBC with MUC5AC and/or MUC16 expression had a significantly independent high hazard risk after adjusting for the effects of venous invasion (hazard ratio: 5.6, 95% confidence interval: 1.8–17). In conclusion, the study shows that a MUC5AC-positive and/or MUC16-positive status is useful as a predictor of a poor outcome in patients with SBC.     

Mucin gene (MUC1) transfected dendritic cells as vaccine: results of a phase I/II clinical trial

Dendritic cells (DC) derived from peripheral blood monocytes are currently being investigated in clinical trials for their role in stimulating the immune system. We performed a phase I/II clinical trial using human autologous DC transfected with cDNA of the human tumor antigen mucin (MUC1) as a vaccine in 10 patients with advanced breast, pancreatic or papillary cancer. After liposomal transfection, flow cytometry testing showed that 2% to 53% of the DC expressed mucin epitopes. Patients were immunized two or three times with 1 million transfected DC injected subcutaneously (s.c.). A vaccine-specific delayed-type hypersensitivity (DTH) reaction was observed in 3 out of 10 patients. After vaccination, 4 patients showed a 2- to 10-fold increase in the frequency of mucin-specific interferon-gamma (IFN-gamma)-secreting CD8+ T cells. We demonstrated the feasibility and safety of a vaccine consisting of autologous gene-transfected DC, and that immunologic responses could be induced even in patients with pretreated and advanced disease.     

Variation in gene expression of inflammatory cytokines in leukocyte-derived cells of high-fat-diet-induced insulin-resistant rats

A high-fat diet is thought to enhance inflammation in various tissues by increasing insulin resistance. In this study, we determined the mRNA levels of inflammatory cytokines in leukocyte-derived cells in the blood of rats with high-fat-diet-induced insulin resistance. Feeding rats a high-fat diet for 77 d induced moderate insulin resistance, which was determined by increased plasma glucose and insulin concentrations, following an oral glucose tolerance test. The interleukin (IL)-1beta mRNA level was higher in the insulin-resistant rats than in control rats at the fasting stage, whereas the tumor necrosis factor (TNF)-alpha mRNA level was greatly elevated at 180 min after glucose administration in the insulin-resistant rats. The results suggest that feeding rats a high-fat diet enhances the expression of fasting IL-1beta and postprandial TNF-alpha genes in leukocyte-derived cells.     

Changes in plasma levels of inflammatory cytokines in response to paclitaxel chemotherapy

BACKGROUND: Flu-like symptoms are common, early transient side effects of paclitaxel chemotherapy. We hypothesized that these symptoms may be due to release of inflammatory cytokines in response to treatment. The objective of this study was to assess changes in plasma levels of interleukin (IL)-1beta, IL-6, IL-8, IL-10, IL-12, and TNF-alpha during chemotherapy and to correlate these changes with musculoskeletal symptoms. METHODS: Ninety patients with breast cancer were included; 70 patients received single agent paclitaxel either weekly or every 3 weeks and 20 received FAC (5-FU, doxorubicin, cyclophosphamide) chemotherapy. Fifteen healthy volunteers were included as controls. Cytokines and symptoms were measured before starting therapy, on day 3 and on the last day of one treatment cycle. RESULTS: At baseline, all subjects had measurable levels of IL-8 but only 49% had IL-12, 45% had IL-10, 32% had IL-6, and 21% had IL-1beta or TNF-alpha in their plasma. There was no difference in baseline cytokine levels between cancer patients and the healthy volunteers. Schedule-dependent transient changes in the levels of 3 cytokines were observed in the paclitaxel treated patients. In the every 3-week paclitaxel group, IL-6 and IL-8 increased whereas in the weekly paclitaxel group IL-10 increased significantly compared to baseline. Fatigue and flu-like symptoms were also worse on day 3. In the weekly paclitaxel group, increase in IL-10 level correlated positively with joint pain (p=0.003). In the every 3-week paclitaxel group, increase in IL-8 level correlated positively with flu-like symptom (p=0.008). In the FAC-treated group and among the healthy volunteers none of these cytokines increased significantly. CONCLUSIONS: Weekly paclitaxel induces transient increase in IL-10 levels whereas every 3-week higher dose treatments induce IL-8 and IL-6 in the plasma. These changes correlate with joint pain and flu-like symptoms.     

Altered expression of MUC2 and MUC5AC in response to Shigella infection, an in vivo study

Infection of mucosal epithelial cells by Shigella species leads to an intense and acute inflammatory bowel disease that is characterized by watery diarrhea and purulent discharge. Mucin production is a common defense mechanism to protect the underlying mucosa against pathogens. The molecular mechanism(s) underlying mucin induction is unknown in Shigellosis. In this study, we have evaluated the relationship between Shigella infection, the expression of MUC2 and MUC5AC and the participation of signaling molecules TNF-alpha, PKC and ERK1/2. Shigella infection up-regulated MUC2 and MUC5AC expression in 6-8 h, through activation of TNF-alpha, PKC and ERK1/2. These results confirm that, in response to Shigella infection, the normal expression pattern of MUC-2 and MUC-5AC is altered. This in vivo study brings new insights into the molecular pathogenesis of Shigellosis and new potential therapeutic targets for Shigellosis.     

Mucin 1 (MUC1) is a novel partner for MAL2 in breast carcinoma cells

Background  

The MAL2 gene, encoding a four-transmembrane protein of the MAL family, is amplified and overexpressed in breast and other cancers, yet the significance of this is unknown. MAL-like proteins have trafficking functions, but their molecular roles are largely obscure, partly due to a lack of known binding partners.     

Mucin gene expression in rat airways following infection and irritation.

Airway mucus hypersecretion occurs in response to infection and irritation and poses an important and poorly understood clinical problem. In order to gain insight into its pathogenesis, we have focused on an mRNA encoding the major mucus glycoprotein, mucin. Northern blots showed that mucin mRNA was abundant in the intestine of specific pathogen free rats whereas it was undetectable in the airways of these rats until pathogen-free conditions were suspended and rats acquired Sendai (Parainfluenza I) virus infections. Airway mucin hybridization signals in rats that were both infected with Sendai virus and exposed to SO2 were more intense than those in rats with infection alone. These results suggest that pathogen-and irritant-induced hypersecretion may be partly controlled at the level of mucin mRNA.     

Mucin 1 (MUC1) signalling contributes to increase the resistance to cell death in human bronchial epithelial cells exposed to nickel acetate

We have previously reported that nickel acetate (Ni²⁺), a well-known human carcinogenic agents, differentially affected apoptosis in two different airway epithelial cell lines derived from the human respiratory tract (A549 and Beas-2B, respectively), suggesting a potential involvement of epidermal growth factor receptor (EGFR)/Neu receptors in mediating this effect. Since ErbBs are closely associated to Mucin 1 (MUC1), a glycoprotein component of airway mucus that is overexpressed in lung tumors, we have investigated the role of this signaling system in the survival response of airway epithelial cells against Ni²⁺-induced cell death. We found that A549 cells exposed to Ni²⁺ do not show any significant increase of MUC1 levels. Conversely, Beas-2B cells exposed to equivalent concentrations of Ni²⁺ showed increased expression of MUC1 levels and this correlated with increased phosphorylation of both EGFR and of the extracellular-regulated kinase 1/2 (ERK1/2) and increase resistance to apoptosis, as indicated by cell viability assessments and DNA damage. Interestingly, suppression of MUC1 by small interfering RNA inhibited the EGFR activation in Beas-2B cells, leading to a significant decrease of survival and enhancement of DNA fragmentation and cleaved Caspase-3 expression. These results strongly suggest a role for MUC1 in Ni²⁺-induced neoplastic transformation, which likely involves the activation of the EGFR-mediated cell survival pathway, highlighting new avenues in the molecular approach to lung cancer prevention.     

The angiogenic response of the aorta to injury and inflammatory cytokines requires macrophages

The purpose of this study was to define early events during the angiogenic response of the aortic wall to injury. Rat aortic rings produced neovessels in collagen culture but lost this capacity over time. These quiescent rings responded to vascular endothelial growth factor but not to a mixture of macrophage-stimulatory cytokines and chemokines that was angiogenically active on fresh rings. Analysis of cytokine receptor expression revealed selective loss in quiescent rings of the proangiogenic chemokine receptor CXCR2, which was expressed predominantly in aortic macrophages. Pharmacologic inhibition of CXCR2 impaired angiogenesis from fresh rings but had no effect on vascular endothelial growth factor-induced angiogenesis from quiescent explants. Angiogenesis was also impaired in cultures of aortic rings from CXCR2-deficient mice. Reduced CXCR2 expression in quiescent rat aortic rings correlated with marked macrophage depletion. Pharmacologic ablation of macrophages from aortic explants blocked formation of neovessels in vitro and reduced aortic ring-induced angiogenesis in vivo. The angiogenic response of macrophage-depleted rings was completely restored by adding exogenous macrophages. Moreover, angiogenesis from fresh rings was promoted by macrophage CSF (CSF-1) and inhibited with anti-CSF-1 Ab. Thus, aortic angiogenic sprouting following injury is strongly influenced by conditions that modulate resident macrophage numbers and function.     

Hepcidin expression by human monocytes in response to adhesion and pro-inflammatory cytokines

Background

A previous urine proteomic analysis from our laboratory suggested that hepcidin may be a biomarker for lupus nephritis flare. Immunohistochemical staining of kidney biopsies from lupus patients showed that hepcidin was expressed by infiltrating renal leukocytes. Here we investigated whether inflammatory cytokines relevant to the pathogenesis of lupus nephritis and other glomerular diseases regulate hepcidin expression by human monocytes.

Methods

Human CD14+ monocytes were incubated with interferon alpha (IFNα), interferon gamma (IFNγ), interleukin-6 (IL6), interleukin-1 beta (IL1β), monocyte chemotactic factor-1 (MCP1), or tumor necrosis factor alpha (TNFα). Hepcidin expression was examined by real-time PCR and enzyme immunoassay.

Results

Monocyte hepcidin mRNA increased during adherence to the tissue culture wells, reaching a level 150-fold higher than baseline within 12 h of plating. After accounting for the effects of adhesion, monocytes showed time and dose-dependent up-regulation of hepcidin mRNA upon treatment with IFNα or IL6. One hour of incubation with IFNα or IL6 increased hepcidin mRNA 20 and 80-fold, respectively; by 24 h the mRNA remained 5- and 2.4-fold higher than baseline. IL1β, IFNγ, and MCP-1 did not affect monocyte hepcidin expression. TNFα inhibited hepcidin induction by IL6 in monocytes by 44%. After 24 h of treatment with IFNα or IL6, immunoreactive hepcidin production by monocytes increased 3- and 2.6-fold, respectively.

Conclusion

Human monocytes produce hepcidin in response to adhesion and the pro-inflammatory cytokines IFNα and IL6.

General significance

The appearance of hepcidin in the kidneys or urine during glomerular diseases may be from infiltrating monocytes induced to express hepcidin by adherence and exposure to pro-inflammatory cytokines found in the renal milieu.     

NEU1 Sialidase Regulates Membrane-tethered Mucin (MUC1) Ectodomain Adhesiveness for Pseudomonas aeruginosa and Decoy Receptor Release

Airway epithelia express sialylated receptors that recognize exogenous danger signals. Regulation of receptor responsiveness to these signals remains incompletely defined. Here, we explore the mechanisms through which the human sialidase, neuraminidase-1 (NEU1), promotes the interaction between the sialoprotein, mucin 1 (MUC1), and the opportunistic pathogen, Pseudomonas aeruginosa. P. aeruginosa flagellin engaged the MUC1 ectodomain (ED), increasing NEU1 association with MUC1. The flagellin stimulus increased the association of MUC1-ED with both NEU1 and its chaperone/transport protein, protective protein/cathepsin A. Scatchard analysis demonstrated NEU1-dependent increased binding affinity of flagellin to MUC1-expressing epithelia. NEU1-driven MUC1-ED desialylation rapidly increased P. aeruginosa adhesion to and invasion of the airway epithelium. MUC1-ED desialylation also increased its shedding, and the shed MUC1-ED competitively blocked P. aeruginosa adhesion to cell-associated MUC1-ED. Levels of desialylated MUC1-ED were elevated in the bronchoalveolar lavage fluid of mechanically ventilated patients with P. aeruginosa airway colonization. Preincubation of P. aeruginosa with these same ex vivo fluids competitively inhibited bacterial adhesion to airway epithelia, and MUC1-ED immunodepletion completely abrogated their inhibitory activity. These data indicate that a prokaryote, P. aeruginosa, in a ligand-specific manner, mobilizes eukaryotic NEU1 to enhance bacterial pathogenicity, but the host retaliates by releasing MUC1-ED into the airway lumen as a hyperadhesive decoy receptor.