Beneficial effects of early (but not late) intervention of heme oxygenase-1 on bleomycin-induced pulmonary fibrosis in mice

The aim of this study is to explore the effects of early and late intervention in heme oxygenase-1 (HO-1) expression or activity on pulmonary fibrosis in mice. Mice were divided into four groups: one control and three bleomycin hydrochloride-induced groups in which mice were administered phosphate-buffered saline (PBS), hemin or Cr (III) mesoporphyrin IX chloride (CrMP). Early intervention with hemin, an HO-1 inducer, abrogated bleomycin-induced pulmonary fibrosis (fibrotic/reparative score decrease from 21.0±2.4 to 13.8±1.7, P<0.01), and early intervention with CrMP, an HO-1 inhibitor, worsened bleomycin-induced pulmonary fibrosis (fibrotic/reparative score increase from 21.0±2.4 to 32.5±2.9, P<0.01). Elevated glutathione expression and reduced expression of TGF-β1, hydroxyproline, LDH and MDA were seen in the lungs of the early hemin intervention group compared to that seen in the PBS group (P<0.05). These results taken together show that HO-1 can prevent or ameliorate pulmonary fibrosis and oxidative stress and inflammation at an early stage of pulmonary fibrosis.     

巨噬细胞在人类肺组织纤维化中的作用

目的：探讨肺泡巨噬细胞在纤维性间质性肺病胶原纤维异常沉积中的作用。方法：应用ＨＥ 染色、Ｍａｓｓｏｎ 三色染色和免疫组织化学染色。结果：在纤维性间质性肺病组和疾病对照组之间,肺泡巨噬细胞ＴＧＦβ１ 和ｂＦＧＦ阳性表达率差异均有显著性（ Ｐ＜ ０－０５） ;肺泡巨噬细胞ＰＤＧＦα受体阳性率差异亦有显著性（Ｐ＜０－０５）。在增生性肺泡Ⅱ型上皮细胞,ＰＤＧＦα受体阳性表达率,无胶原沉积组高于胶原沉积组（ Ｐ＜ ０－０５） ;而ＴＧＦβ１ 阳性表达率胶原沉积组高于无胶原沉积组（ Ｐ＝ ０－０１）。结论：肺泡巨噬细胞参与了纤维性间质性肺病及其胶原纤维异常沉积的发生发展过程。     

Self-management for pulmonary fibrosis: Insights from people living with the disease and healthcare professionals

ObjectivePeople with pulmonary fibrosis (PF) consider self-management essential for maintaining health. This study aims to explore the needs and expectations of PF self-management from the patient and healthcare professionals (HCPs) perspectives.MethodsSemi-structured interviews were conducted with people with PF and HCPs. Purposive sampling was used to recruit participants. Thematic analysis was performed using the principles of grounded theory.Results18 individuals with PF and 15 HCPs were interviewed. Common self-management components reported included exercise, nutrition, maintaining healthy mind, avoiding infections, recognising deterioration and seeking help, managing symptoms and treatments, social support, and end-of-life planning. Both groups felt that effective self-management required individualised strategies, supports, and reliable information. People with PF identified access to personal health data and self-acceptance as part of self-management. HCPs highlighted the importance of accessible supports and managing patient expectations of disease course and treatments. Some HCPs concerned about missed detection of deterioration and suggested that self-management strategies for PF may differ to other lung diseases.ConclusionThis study identified components important for self-management in PF and provides a basis for designing a PF self-management package. Practice implications: Self-management of PF can be facilitated with individualised support from HCPs and reliable information that is accessible.     

Dysregulation of glucose metabolism is an early event in sporadic Parkinson's disease

Unlike most other cell types, neurons preferentially metabolize glucose via the pentose phosphate pathway (PPP) to maintain their antioxidant status. Inhibiting the PPP in neuronal cell models causes cell death. In rodents, inhibition of this pathway causes selective dopaminergic cell death leading to motor deficits resembling parkinsonism. Using postmortem human brain tissue, we characterized glucose metabolism via the PPP in sporadic Parkinson's disease (PD), Alzheimer's disease (AD), and controls. AD brains showed increased nicotinamide adenine dinucleotide phosphate (NADPH) production in areas affected by disease. In PD however, increased NADPH production was only seen in the affected areas of late-stage cases. Quantifying PPP NADPH-producing enzymes glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase by enzyme-linked immunosorbent assay, showed a reduction in the putamen of early-stage PD and interestingly in the cerebellum of early and late-stage PD. Importantly, there was no decrease in enzyme levels in the cortex, putamen, or cerebellum of AD. Our results suggest that down-regulation of PPP enzymes and a failure to increase antioxidant reserve is an early event in the pathogenesis of sporadic PD.     

Expression of pulmonary aquaporin 1 is dramatically upregulated in mice with pulmonary fibrosis induced by bleomycin

Introduction

Pulmonary fibrosis occurs due to fibroblast proliferation and collagen production in the lung and begins with alveolar inflammatory edema. Aquaporins (AQPs) play pivotal roles in lung fluid transport. In this study we establish the experimental model for pulmonary fibrosis in C57BL/6 mice to investigate expressions of AQP1 and AQP5 in lung tissue.

Material and methods

Mice in model groups were treated intratracheally with bleomycin with the dose of 5 mg/kg body weight. The mice were sacrificed at 1 week, 2 weeks and 3 weeks respectively. The left upper lungs were harvested for histopathologic H-E and Masson''s staining. The mRNAs of AQP1 and AQP5 were analyzed by real-time polymerase chain reaction (real-time PCR) and the proteins of AQP1 and AQP5 were analyzed by western blotting.

Results

Real-time PCR showed that AQP1 mRNA in bleomycin 1 w, 2 w, and 3 w groups increased by 377%, 880% and 823% respectively compared to that in the control group (p < 0.01). Western blotting showed that the expression of AQP1 protein in bleomycin 1 w, 2 w, and 3 w groups increased by 53%, 144%, and 141%, respectively (p < 0.05). AQP5 mRNA in bleomycin 1 w and 2 w group decreased by 78% and 66%, respectively (p < 0.05). In bleomycin 2 w and 3 w groups it decreased by 69% and 80% (p < 0.05).

Conclusions

The expression of AQP1 dramatically increased in pulmonary fibrosis. AQP1 plays an important role in the progress of pulmonary fibrosis.     

GSTP1 promoter hypermethylation is an early event in breast carcinogenesis

Promoter hypermethylation in precursor lesions of the breast cancer may be biomarkers of cancer risk and targets for cancer chemoprevention. Pi-class glutathione-S-transferases (GSTP1) is inactivated by promoter hypermethylation in invasive breast cancers. However, little is known about epigenetic silencing of GSTP1 gene by promoter hypermethylation in precursor lesions. To determine the significance of GSTP1 promoter hypermethylation in breast carcinogenesis, methylation status of GSTP1 gene was studied by nested methylation-specific polymerase chain reaction, and GSTP1 expression was studied by immunohistochemistry in invasive ductal carcinoma (IDC), ductal carcinoma in situ (DCIS), usual ductal hyperplasia (UDH), and normal breast tissue. GSTP1 promoter hypermethylation was detected in 4/24 (16.7%) of UDH, 18/49 (36.7%) of DCIS, and 14/36 (38.9%) of IDC. No hypermethylation was detected in normal breast tissues. GSTP1 promoter hypermethylation was found to be progressively elevated during breast carcinogenesis (p < 0.01). GSTP1 promoter hypermethylation was associated with loss of GSTP1 expression (p < 0.01 for UDH, p < 0.001 for DCIS and IDC). Our results suggest that GSTP1 promoter hypermethylation is an early event in breast carcinogenesis and appears to functionally silence GSTP1 expression. GSTP1 promoter hypermethylation in the precursor lesions of breast cancer may be used as a target for cancer chemoprevention.     

K-ras gene point mutation in neogenetic lesions of subpleural fibrotic lesions: Either an early genetic event in lung cancer development or a non-specific genetic change during the inflammatory reparative process

In the present study, K-ras mutation was investigated in 156 neogenetic epithelia that appeared in the lesion of subpleural fibrosis in order to elucidate the close relationship of lung cancer development with pulmonary interstitial pneumonia. The neogenetic epithelia were histologically subclassified into six types: (i) ciliated bronchial epithelium (CBE); (ii) squamous metaplastic epithelium (SME); (iii) cuboidal immature epithelium (CIE); (iv) stratified immature epithelium (SIE); (v) mucus cell epithelium (MCE); and (vi) intestinal metaplastic epithelium (IME). K-ras mutation was detected in 9.6% of neogenetic epithelia overall; 21% of CIE, 12% of SIE, 16% of SME, but not in other types of neogenetic epithelia. Immunohistochemically, CIE and SIE frequently expressed surfactant apoprotein and SME was characteristic to carcinoembryonic antigen expression. According to Ki-67 immunostain, CIE, SIE and SME are likely to grow faster than other histological types of epithelia. K-ras mutation was seen exclusively in codon 12 with predominant G to A and G to C substitutions without any G to T transversions, results which are somewhat different to previous studies in lung cancers. The present study clearly demonstrated that K-ras mutation appeared in certain histological types of neogenetic epithelia, but raised the question of whether K-ras mutation in neogenetic epithelia during the inflammatory reparative process might be an early genetic event in lung carcinogenesis.     

Analysis of macrophage migration inhibitory factor (MIF) in patients with idiopathic pulmonary fibrosis

By proteomic approach we previously characterised bronchoalveolar lavage (BAL) protein profiles of patients with idiopathic pulmonary fibrosis (IPF), sarcoidosis and systemic sclerosis. Among differently expressed proteins we identified macrophage migration inhibitory factor (MIF), a multi-function pleiotropic cytokine.This study was performed to validate our findings by a further proteomic approach and ELISA in a larger population of patients and controls. MIF expression in lung tissue was also evaluated by immunohistochemistry.MIF was identified in all 2-DE gels of IPF patients and it was significantly increased compared to controls (p < 0.05). This result was confirmed by ELISA: MIF concentrations were significantly higher in IPF patients than controls (p < 0.001) and were directly correlated with neutrophil percentages (p = 0.0095). Immunohistochemical analysis revealed enhanced expression in bronchiolar epithelium, alveolar epithelium, and fibroblastic foci.In conclusion, MIF is a pleiotropic cytokine that could be involved in the pathogenesis of IPF, being particularly abundant in BAL of these patients and mainly expressed in the areas of active fibrosis.     

PAC1 deficiency attenuates progression of atherosclerosis in ApoE deficient mice under cholesterol-enriched diet

The neuropeptide, pituitary adenylate cyclase-activating polypeptide (PACAP) is vasoactive and cytoprotective and exerts immunoregulatory functions throughout the nervous, neuroendocrine cardiovascular and immune systems in health and disease. PACAP mainly acts through PAC1 receptor signaling in neuronal communication, but the role of PAC1 in immune regulation of atherosclerosis is not known. Here, we generated PAC1^−/−/ApoE^−/− mice to test, whether PAC1^−/− influences plasma cholesterol-/triglyceride levels and/or atherogenesis in the brachiocephalic trunk (BT) seen in ApoE^−/− mice, under standard chow (SC) or cholesterol-enriched diet (CED). Furthermore, the effect of PAC1^−/−, on inflammatory, autophagy-, apoptosis- and necroptosis-relevant proteins in atherosclerotic plaques was determined. In plaques of PAC1^−/−/ApoE^−/− mice fed a SC, the immunoreactivity for apoptotic, autophagic, necroptotic and proinflammatory proteins was increased, however, proliferation was unaffected. Interestingly, without affecting hyperlipidemia, PAC1^−/− in ApoE^−/− mice remarkably reduced CED-induced lumen stenosis seen in ApoE^−/− mice. Thus, PAC1^−/− allows unchecked inflammation, necroptosis and decreased proliferation during SC, apparently priming the BT to develop reduced atheroma under subsequent CED. Remarkably, no differences in inflammation/necroptosis signatures in the atheroma under CED between PAC1^−/−/ApoE^−/− and ApoE^−/− mice were observed. These data indicate that selective PAC1 antagonists should offer potential as a novel class of atheroprotective therapeutics, especially during hypercholesterolemia.     

Enhanced oxidative stress is an early event during development of Alzheimer-like pathologies in presenilin conditional knock-out mice

Conditional double knock-out of presenilin-1 (PS1) and presenilin-2 (PS2) (PS cDKO) in forebrain of mice led to progressive memory dysfunction and forebrain degeneration. These changes in the brain recapitulated most of the neurodegenerative phenotypes of Alzheimer's disease (AD). Oxidative stress in brain tissues is intimately related to AD. In this report, we examined oxidative stress status in cerebral cortex in 2-, 4- and 7-month PS cDKO and the age- and gender-matched control mice (WT). Lipid peroxidation (MDA as the measure) and protein oxidation (protein carbonyl as the measure) were found to be significantly increased in PS cDKO mice over the age points examined, notably in those at 2-month, suggesting that oxidative stress is an early event in response to PS loss-of-function. The oxidative modification of cortical proteins was further confirmed by Oxyblot assay. The investigations into endogenous antioxidant defense (CAT, SOD and GSH-px as measures) revealed a compensatory defense against oxidative stress, particularly at the early age stage, in PS cDKO mice. The expression level of cortical glial fibrillary acidic protein (GFAP) increased in an age-related manner, in particular in 2-month PS cDKO mice, suggesting that the interaction relationship between oxidative stress and inflammatory response may be closely associated with the underlying loss-of-function pathogenesis of AD.     

EMR1, the human homolog of F4/80, is an eosinophil-specific receptor

The EGF-TM7 F4/80 is a defining marker of murine macrophage populations. Applying flow cytometric analysis using the newly generated mAb A10, and quantitative real-time PCR, we here report the surprising observation that the human ortholog of F4/80, EGF-like module containing mucin-like hormone receptor (EMR)1, is absent on mononuclear phagocytic cells including monocytes, macrophages, and myeloid dendritic cells. Unexpectedly, we found that EMR1 expression is restricted to eosinophilic granulocytes, where expression is overlapping with the eotaxin receptor CCR3 and the immunoglobulin-like lectin Siglec-8. Absence on other leukocytes, including basophils, implies that EMR1 is a highly specific marker for eosinophils in humans.     

大鼠肺纤维化形成过程中肺内一氧化氮代谢的动态变化

目的:观察大鼠肺纤维化过程中肺内一氧化氮代谢的动态变化及其与肺纤维化形成的关系。方法:气管内一次性滴注平阳霉素(5mL/kg),观察注后7、14、21、30d和70d组大鼠肺组织羟脯氨酸含量,出、入肺血NO₂^-/NO₃^-含量以及14d组肺泡巨噬细胞培养上清液中NO₂^-/NO₃^-含量和肺间质诱导型一氧化氮合酶(iNOS)免疫组化阳性细胞数量的变化。结果:7d组大鼠肺组织羟脯氨酸含量与对照组比无明显差异,14d组高于对照组(P＜0.05),21d组、30d组和70d组更为明显(均P＜0.01)。7d组、14d组出肺血NO₂^-/NO₃^-含量明显高于对照组(均P＜0.01),入肺血NO₂^-/NO₃^-含量明显低于对照组(均P＜0.01),21d组出肺血NO₂^-/NO₃^-含量的变化无明显差异(P＞0.05),入肺血仍较低(P＜0.01),30d组和70d组出、入肺血NO₂^-/NO₃^-含量与对照组无明显差异(P＞0.05)。14d组大鼠肺泡巨噬细胞培养上清液中NO₂^-/NO₃^-含量明显高于对照组(P＜0.01)。14d组大鼠肺间质iNOS免疫组化阳性细胞增多。结论:大鼠肺纤维化形成过程中,先有肺内NO生成增多,后出现肺纤维化;在肺纤维化形成后,肺内NO趋向恢复。肺内NO增多与肺泡巨噬细胞释放NO能力增加、肺内iNOS的增多有关。肺内NO的大量生成可能是促使肺纤维化形成的因素之一。     

甘草甜素抑制肺纤维化大鼠肺组织单核细胞趋化蛋白-1的表达

目的: 观察甘草甜素对博来霉素(BLM)诱导的大鼠肺纤维化的干预作用及可能机制。方法: 随机将大鼠分为对照组、肺纤维化模型组、甘草甜素干预组。气管内注入博莱霉素造成动物模型后,于当天开始每天给药,分别于7 、28 d处死,取肺组织,行嗜伊红染色、Masson染色；检测肺组织匀浆中羟脯氨酸(HYP) 的变化; RT-PCR法测肺组织单核细胞趋化因子-1 mRNA的表达,免疫组化测肺组织单核细胞趋化因子-1蛋白的表达。结果: 干预组肺纤维化程度轻于模型组; 肺组织匀浆羟脯氨酸(HYP)含量显著低于模型组( P<0.01) ;在模型组 MCP-1第7 d表达就明显升高，第28 d下降，但仍高于对照组，干预组与模型组有同一规律，但均减弱，第7、28 d与模型组(M组)比较，P<0.05。结论: 甘草甜素能减轻博莱霉素诱导的大鼠肺纤维化，这种作用可能部分是通过抑制MCP-1的表达而实现。     

TGF-β1介导的RhoA/ROCK通路在大鼠肺肌成纤维细胞分化中的调节作用

 目的：探讨转化生长因子β1 (TGF-β1)介导的RhoA/Rho相关卷曲螺旋形成蛋白激酶(ROCK)通路在调控肺成纤维细胞向肌成纤维细胞分化过程中的作用。方法：胰酶消化法获得原代培养的肺成纤维细胞,进行以下实验：(1) 观察TGF-β1诱导肺成纤维细胞不同时间后p-RhoA、ROCK、磷酸化肌球蛋白磷酸酶靶亚基(p-MBS)、血清应答因子(SRF)、α-平滑肌肌动蛋白(α-SMA)、I型和III型胶原蛋白表达变化;(2) 将细胞分为对照组、TGF-β1诱导分化组和Y-27632（ROCK通路阻滞剂）干预组,免疫细胞化学染色与Western blotting法分别观察与检测ROCK、p-MBS、SRF、α-SMA、I型和III型胶原蛋白在细胞内的定位分布与表达。结果：（1）经TGF-β1诱导24 h后,大鼠肺成纤维细胞胞体内出现大量平行或交叉排列的α-SMA抗体标记的肌丝。随着TGF-β1诱导刺激时间的延长,p-RhoA、ROCK、p-MBS、SRF、α-SMA、I型和III型胶原蛋白表达逐渐增高,其中RhoA/ROCK信号（p-RhoA、ROCK、p-MBS）、SRF和α-SMA蛋白分别在诱导后6 h、12 h和24 h达到高峰。I型胶原和III型胶原蛋白表达均在24 h达高峰,分别为诱导前的2.19和3.04倍（P<0.05）。 (2)与TGF-β1诱导分化组比较,当给予Y-27632干预后,在相应时点ROCK、p-MBS、SRF、α-SMA、I型和III型胶原蛋白表达均明显降低,差异有统计学意义（P<0.05）。结论：TGF-β1通过ROCK信号转导通路的活化,刺激了大鼠肺成纤维细胞向肌成纤维细胞分化,进而促进了胶原蛋白的合成,可能在（矽）肺纤维化形成过程中发挥着重要作用。     

Overexpression of human telomerase RNA is an early event in oesophageal carcinogenesis

 Telomerase, the ribonucleoprotein enzyme that elongates telomeres, is repressed in normal human somatic cells but is reactivated during tumour progression. The purpose of this study was to investigate the localization of human telomerase RNA (hTR) expression in human oesophageal dysplasia and cancer by using in situ mRNA hybridization (ISH) with avidin–biotin staining. Ki-67 immunoreactivity was also examined. We analysed 51 squamous cell carcinomas, 9 dysplasias and 60 normal mucosae. The integrity of the mRNA in each sample was verified by using a poly d(T)₂₀ probe. Seventy-six samples (63%) showed no mRNA degradation; these included 30 carcinomas, 7 dysplasias and 39 normal mucosae. At the single-cell level, high levels of hTR expression were found in the cytoplasm and especially in the nucleus. Most (>90%) cancer cells demonstrated high levels of hTR expression in 29 (97%) of the 30 tumours. Most dysplastic cells also showed high levels of hTR in all 7 dysplastic cases. In all 39 normal mucosae, most basal cells indicated high levels of hTR expression, which were also seen in infiltrating lymphocytes. The distribution of hTR-expressing cells was similar to that of Ki-67-positive cells. These data suggest that overexpression of hTR may be correlated with the proliferative activity that defined by Ki-67 immunoreactivity and is an early event in carcinogenesis of the oesophagus. Received: 14 December 1998 / Accepted: 20 January 1999     

Pancreatic insufficiency and pulmonary disease in German and Slavic cystic fibrosis patients with the R347P mutation

Cystic fibrosis (CF) is caused by mutations in the gene for the cystic fibrosis transmembrane conductance regulator (CFTR) that codes for a cAMP-regulated chloride channel. The R347P is a missensemutation located within the first membrane spanning domain (MSD1,) of the CFTR protein. This mutation occurs with an overall worldwide frequency of about 0.2%. The patients, originally described with this mutation were compound heterozygotes with the ΔF508 mutation and had a very mild course of CF, suggesting that R347P, similar to other missense mutations affecting the MSDl domain, causes a mild phenotype. We report here a group of 19 CF patients with the R347P mutation of German, Bulgarian, Czech, and Slovak origin, including two homozygotes. Most patients presented with early disease onset, pancreas insufficiency (PI), and early pulmonary involvement, suggesting that this mutation can lead to a severe course of CF. Most R347P alleles in the group studied share a common polymorphic haplotype. In addition, these analyses gave evidence for recurrence of the mutation in two CF patients of German and Czech origin. © 1995 Wiley-Liss, Inc.     

Immunoglobulin and complement deposits in the lungs of New Zealand Black/White mice

Summary Forty-four New Zealand Black/White (NZB/W) F1 hybrid mice were studied for evidence of immune complex deposition in the lungs and kidneys. Positive immunofluorescence was seen in the lungs of 27 mice. The pattern of lung fluorescence was granular in association with capillary walls in 16 mice and intracellular in 12. The incidence of granular fluorescence was increased with age and was seen in 80% of the lungs of mice over 13 months old.There was a correlation between capillary wall fluorescence in the lungs and the kidneys (P<0.001) and between lung cellular fluorescence and renal mesangial fluorescence (P<0.001). The role of immune complex deposition in human pulmonary disease is discussed.