Alpha-1-antitrypsin aerosolized augmentation abrogates neutrophil elastase-induced expression of cathepsin B and matrix metalloprotease 2 in vivo and in vitro

Background: Neutrophil elastase (NE) activity is increased in lung diseases such as a1-antitrypsin (A1AT) deficiency and pneumonia. It has recently been shown to induce expression of cathepsin B and matrix metalloprotease 2 (MMP-2) in vitro and in a mouse model. It is postulated that increased cathepsin B and MMP-2 in acute and chronic lung diseases result from high levels of extracellular NE and that expression of these proteases could be inhibited by A1AT augmentation therapy.

Methods: Cathepsin and MMP activities were assessed in bronchoalveolar lavage (BAL) fluid from patients with A1AT deficiency, pneumonia and control subjects. Macrophages were exposed to BAL fluid rich in free NE from patients with pneumonia following pretreatment with A1AT. MMP-2, cathepsin B, secretory leucoprotease inhibitor (SLPI) and lactoferrin levels were determined in BAL fluid from A1AT-deficient patients before and after aerosolisation of A1AT.

Results: BAL fluid from both patients with pneumonia and those with A1AT deficiency containing free NE had increased cathepsin B and MMP-2 activities compared with BAL fluid from healthy volunteers. The addition of A1AT to BAL fluid from patients with pneumonia greatly reduced NE-induced cathepsin B and MMP-2 expression in macrophages in vitro. A1AT augmentation therapy to A1AT-deficient individuals also reduced cathepsin B and MMP-2 activity in BAL fluid in vivo. Furthermore, A1AT-deficient patients had higher levels of SLPI and lactoferrin after A1AT augmentation therapy.

Conclusion: These findings suggest a novel role for A1AT inhibition of NE-induced upregulation of MMP and cathepsin expression both in vitro and in vivo.

Increased expression of fibroblast activation protein-alpha in keloid fibroblasts: implications for development of a novel treatment option.

Keloid scars are common benign fibroproliferative reticular dermal lesions with unknown etiology and ill-defined management with high rate of recurrence post surgery. The progression of keloids is characterized by increased deposition of extracellular matrix proteins, invasion into the surrounding healthy skin and inflammation. Fibroblasts are considered to be the key cellular mediators of fibrogenesis in keloid scars. Fibroblast activation protein alpha (FAP-a) and dipeptidyl peptidase IV (DPPIV) are proteases located at the plasma membrane promoting cell invasiveness and tumor growth and have been previously associated with keloid scars. Therefore, in this study we analyzed in further detail the expression of FAP-a in keloid fibroblasts compared to control skin fibroblasts. Dermal fibroblasts were obtained from punch-biopsies from the active margin of four keloids and four control skin samples. Flow cytometry was used to analyze FAP-a expression and the CytoSelect(®) 24-Well Collagen I Cell Invasion Assay was applied to study fibroblast invasion. Secretion of extracellular matrix (ECM) proteins was investigated by multiplexed particle-based flow cytometric assay and enzyme-linked immunosorbent assay. We found an increased expression of FAP-a in keloid fibroblasts compared to control skin fibroblasts (p

Global down-regulation of HOX gene expression in PML-RAR alpha(+) acute promyelocytic leukemia identified by small-array real-time PCR

Acute promyelocytic leukemia (APL) is associated with a reciprocal and balanced translocation involving the retinoic acid receptor-alpha (RARalpha). All-trans retinoic acid (ATRA) is used to treat APL and is a potent morphogen that regulates HOX gene expression in embryogenesis and organogenesis. HOX genes are also involved in hematopoiesis and leukemogenesis. Thirty-nine mammalian HOX genes have been identified and classified into 13 paralogous groups clustered on 4 chromosomes. They encode a complex net-Work of transcription regulatory proteins whose precise targets remain poorly understood. The overall function of the network appears to be dictated by gene dosage. To investigate the mechanisms involved in HOX gene regulation in hematopoiesis and leukemogenesis by precise measurement of individual HOX genes, a small-array real-time HOX (SMART-HOX) quantitative polymerase chain reaction (PCR) platform was designed and validated. Application of SMART-HOX to 16 APL bone marrow samples revealed a global down-regulation of 26 HOX genes compared with normal controls. HOX gene expression was also altered during differentiation induced by ATRA in the PML-RARalpha(+) NB4 cell line. PML-RARalpha, fusion proteins have been reported to act as part of a repressor complex during myelold cell differentiation, and a model linking HOX gene expression to this PML-RARalpha repressor complex is now proposed.

Differential expression of steroid 5 alpha-reductase isozymes and association with disease severity and angiogenic genes predict their biological role in prostate cancer

The biological role of steroid 5 alpha-reductase isozymes (encoded by the SRD5A1 and SRD5A2 genes) and angiogenic factors that play important roles in the pathogenesis and vascularization of prostate cancer (PC) is poorly understood. The sub-cellular expression of these isozymes and vascular endothelial growth factor (VEGF) in PC tissue microarrays (n=62) was examined using immunohistochemistry. The effect of SRD5A inhibition on the angiogenesis pathway genes in PC was also examined in prostate cell lines, LNCaP, PC3, and RWPE-1, by treating them with the SRD5A inhibitors finasteride and dutasteride, followed by western blot, quantitative PCR, and ELISA chip array techniques. In PC tissues, nuclear SRD5A1 expression was strongly associated with higher cancer Gleason scores (P=0.02), higher cancer stage (P=0.01), and higher serum prostate specific antigen (PSA) levels (P=0.01), whereas nuclear SRD5A2 expression was correlated with VEGF expression (P=0.01). Prostate tumor cell viability was significantly reduced in dutasteride-treated PC3 and RWPE-1 cells compared with finasteride-treated groups. Expression of the angiogenesis pathway genes transforming growth factor beta 1 (TGFB1), endothelin (EDN1), TGF alpha (TGFA), and VEGFR1 was upregulated in LNCaP cells, and at least 7 out of 21 genes were upregulated in PC3 cells treated with finasteride (25 mu M). Our findings suggest that SRD5A1 expression predominates in advanced PC, and that inhibition of SRD5A1 and SRD5A2 together was more effective in reducing cell numbers than inhibition of SRD5A2 alone. However, these inhibitors did not show any significant difference in prostate cell angiogenic response. Interestingly, some angiogenic genes remained activated after treatment, possibly due to the duration of treatment and tumor resistance to inhibitors. Endocrine-Related Cancer (2010) 17 757-770

CD69, CD25, and HLA-DR activation antigen expression on CD3+ lymphocytes and relationship to serum TNF-alpha, IFN-gamma, and sIL-2R levels in aging

Aging is associated with changes in lymphocyte subsets and unexplained HLA-DR upregulation on T-lymphocytes. We further investigated this activation, by measuring early (CD69), middle (CD25), and late (HLA-DR) T-lymphocyte activation markers on CD3+ lymphocytes, across subjects (20-100 years) together with serum tumor necrosis factor (TNF-alpha), interferon-gamma (IFN-gamma), and soluble interleukin-2 receptor (sIL-2R). HLA-DR was present as a CD3+ HLA-DR+ subset that constituted 8% of total lymphocytes, increased twofold with age and included CD4+, CD8+, and CD45RA+ phenotypes. HLA-DR was also expressed on a CD8+ CD57+ subset. The CD3+ CD25+ subset constituted 13% of lymphocytes, fell with age but was weakly associated with the CD3+ HLA-DR+ subset especially in older subjects. A small 3-5% CD3+ CD69+ subsets showed no age effect. Serum sIL-2R, TNF-alpha, but not IFN-gamma, were associated with CD3+ HLA-DR+ lymphocytes, TNF-alpha with CD8+ CD57+ count and sIL-2R and IFN-gamma with the CD3+ CD25+/CD3+ CD4+ ratio. The study confirms age-related upregulation of HLA-DR on CD3+ lymphocytes, shows some evidence for associated upregulation of CD25 on CD3+ cells in older subjects, and links serum TNF-alpha, IFN-gamma, and sIL2-R to T-lymphocyte activation.

Expression of the alpha 1b-adrenergic receptor and G protein subunits in mammalian cell lines using the Semliki Forest virus expression system.

Semliki Forest virus (SFV) vectors have been efficiently used for rapid high level expression of several G protein-coupled receptors. Here we describe the use of SFV vectors to express the alpha 1b-adrenergic receptor (AR) alone or in the presence of the G protein alpha q and/or beta 2 and gamma 2 subunits. Infection of baby hamster kidney (BHK) cells with recombinant SFV-alpha 1b-AR particles resulted in high specific binding activity of the alpha 1b-AR (24 pmol receptor/mg protein). Time-course studies indicated that the highest level of receptor expression was obtained 30 hours post-infection. The stimulation of BHK cells, with epinephrine led to a 5-fold increase in inositol phosphate (IP) accumulation, confirming the functional coupling of the receptor to G protein-mediated activation of phospholipase C. The SFV expression system represents a rapid and reproducible system to study the pharmacological properties and interactions of G protein coupled receptors and of G protein subunits.

CD14 expression on monocytes and TNF alpha production in patients with septic shock, cardiogenic shock or bacterial pneumonia.

OBJECTIVES: In patients with septic shock, circulating monocytes become refractory to stimulation with microbial products. Whether this hyporesponsive state is induced by infection or is related to shock is unknown. To address this question, we measured TNF alpha production by monocytes or by whole blood obtained from healthy volunteers (controls), from patients with septic shock, from patients with severe infection (bacterial pneumonia) without shock, and from patients with cardiogenic shock without infection. MEASUREMENTS: The numbers of circulating monocytes, of CD14+ monocytes, and the expression of monocyte CD14 and the LPS receptor, were assessed by flow cytometry. Monocytes or whole blood were stimulated with lipopolysaccharide endotoxin (LPS), heat-killed Escherichia coli or Staphylococcus aureus, and TNF alpha production was measured by bioassay. RESULTS: The number of circulating monocytes, of CD14+ monocytes, and the monocyte CD14 expression were significantly lower in patients with septic shock than in controls, in patients with bacterial pneumonia or in those with cardiogenic shock (p < 0.001). Monocytes or whole blood of patients with septic shock exhibited a profound deficiency of TNF alpha production in response to all stimuli (p < 0.05 compared to controls). Whole blood of patients with cardiogenic shock also exhibited this defect (p < 0.05 compared to controls), although to a lesser extent, despite normal monocyte counts and normal CD14 expression. CONCLUSIONS: Unlike patients with bacterial pneumonia, patients with septic or cardiogenic shock display profoundly defective TNF alpha production in response to a broad range of infectious stimuli. Thus, down-regulation of cytokine production appears to occur in patients with systemic, but not localised, albeit severe, infections and also in patients with non-infectious circulatory failure. Whilst depletion of monocytes and reduced monocyte CD14 expression are likely to be critical components of the hyporesponsiveness observed in patients with septic shock, other as yet unidentified factors are at work in this group and in patients with cardiogenic shock.

Tumour necrosis factor alpha induces rapid reduction in AMPA receptor-mediated calcium entry in motor neurones by increasing cell surface expression of the GluR2 subunit: relevance to neurodegeneration

The AMPA receptor (AMPAR) subunit GluR2, which regulates excitotoxicity and the inflammatory cytokine tumour necrosis factor alpha (TNF alpha) have both been implicated in motor neurone vulnerability in Amyotrophic Lateral Sclerosis/Motor Neurone Disease. TNF alpha has been reported to increase cell surface expression of AMPAR subunits to increase synaptic strength and enhance excitotoxicity, but whether this mechanism occurs in motor neurones is unknown. We used primary cultures of mouse motor neurones and cortical neurones to examine the interaction between TNF alpha receptor activation, GluR2 availability, AMPAR-mediated calcium entry and susceptibility to excitotoxicity. Short exposure to a physiologically relevant concentration of TNFalpha (10 ng/ml, 15 min) caused a marked redistribution of both GluR1 and GluR2 to the cell surface as determined by cell surface biotinylation and immunofluorescence. Using Fura-2 AM microfluorimetry we showed that exposure to TNFalpha caused a rapid reduction in the peak amplitude of AMPA-mediated calcium entry in a PI3-kinase and p38 kinase-dependent manner, consistent with increased insertion of GluR2-containing AMPAR into the plasma membrane. This resulted in a protection of motor neurones against kainate-induced cell death. Our data therefore, suggests that TNF alpha acts primarily as a physiological regulator of synaptic activity in motor neurones rather than a pathological drive in ALS

Dietary alpha-tocopherol affects differential gene expression in rat testes

Gene-chip technology was employed to study the effect of dietary vitamin E (VE) on gene expression in rat testes. Male albino rats were fed with either a diet deficient in VE or a standard diet containing VE. Differential gene expression was monitored at five individual time-points over a period of 14 months with all animals individually pro. led. Low VE intake resulted in the consistent upregulation of 7-dehydrocholesterol reductase and GATA binding protein 4, both involved in testosterone synthesis. Cyclin D3, important in cell cycle progression and Wilms tumor 1, related to cancer development, were also up-regulated in the vitamin E deficient animals. This study demonstrates that low dietary VE intake has long-term effects on gene expression in the testes. Our data provides insights into the possible molecular mechanisms underlying the beneficial effects of vitamin E on the male reproductive organ.

A novel alpha-galactosidase from I'ifidobacterium bifidum with transgalactosylating properties: gene molecular cloning and heterologous expression

A genomic library of Bifidobacterium bifidum (NCIMB 41171) DNA was constructed in Escherichia coli RA11r (melA(-)B(+)) and one alpha-galactosidase encoding gene was isolated. Conceptual translation combined with insertional mutagenesis analysis indicated an open reading frame (ORF) of 759 amino acid (aa) residues encoding an alpha-galactosidase (named as MelA) of 82.8 kDa. Partial purification and characterisation showed that the enzyme had an apparent native molecular mass of a parts per thousand 243 kDa and a subunit size of a parts per thousand 85 kDa. The enzyme belongs to glycosyl hydrolases 36 family with high aa sequence similarities (a parts per thousand 73%) to other known alpha-galactosidases of bifidobacterial origin. Under optimum pH conditions for activity (pH 6.0) and high melibiose concentration (40% w/v), the enzyme was able to form oligosaccharides with degree of polymerisation (DP) a parts per thousand yen3 at higher concentration than DP = 2, with a total yield of 20.5% (w/w).

Identification of hepatic molecular mechanisms of action of alpha-tocopherol using global gene expression profile analysis in rats

The recent discovery that vitamin E (VE) regulates gene activity at the transcriptional level indicates that VE may exert part of its biological effects by mechanisms which may be independent of its well-recognised antioxidant function. The objective of this study was the identification of hepatic vitamin E-sensitive genes and examination of the effects of VE on their corresponding biological endpoints. Two groups of male rats were randomly assigned to either a VE-sufficient diet or to a control diet deficient in VE for 290 days. High-density oligonucleotide microarrays comprising over 7000 genes were used to assess the transcriptional response of the liver. Differential gene expression was monitored over a period of 9 months, at four different time-points, and rats were individually profiled. This experimental strategy identified several VE-sensitive genes, which were chronically altered by dietary VE. VE supplementation down-regulated scavenger receptor CD36, coagulation factor IX and 5-alpha-steroid reductase type 1 mRNA levels while hepatic gamma glutamyl-cysteinyl synthetase was significantly up-regulated. Measurement of the corresponding biological endpoints such as activated partial thromboplastin time, plasma dihydrotestosterone and hepatic glutathione substantiated the gene chip data which indicated that dietary VE plays an important role in a range of metabolic processes within the liver. (C) 2004 Elsevier B.V. All rights reserved.

Chronic hypoxia in the human neuroblastoma SH-SY5Y causes reduced expression of the putative alpha-secretases, ADAM10 and TACE, without altering their mRNA levels

Alzheimer's disease is more frequent following an ischemic or hypoxic episode, with levels of beta-amyloid peptides elevated in brains from patients. Similar increases are found after experimental ischemia in animals. It is possible that increased beta-amyloid deposition arises from alterations in amyloid precursor protein (APP) metabolism, indeed, we have shown that exposing cells of neuronal origin to chronic hypoxia decreased the secretion of soluble APP (sAPPalpha) derived by action of alpha-secretase on APP, coinciding with a decrease in protein levels of ADAM10, a disintegrin metalloprotease which is thought to be the major alpha-secretase. In the current study, we extended those observations to determine whether the expression of ADAM10 and another putative alpha-secretase, TACE, as well as the beta-secretase, BACE1 were regulated by chronic hypoxia at the level of protein and mRNA. Using Western blotting and RT-PCR, we demonstrate that after 48 h chronic hypoxia, such that sAPPalpha secretion is decreased by over 50%, protein levels of ADAM10 and TACE and by approximately 60% and 40% respectively with no significant decrease in BACE1 levels. In contrast, no change in the expression of the mRNA for these proteins could be detected. Thus, we conclude that under CH the level of the putative alpha-secretases, ADAM10 and TACE are regulated by post-translational mechanisms, most probably proteolysis, rather than at the level of transcription.

Retinoid-Related Receptor (ROR) alpha mRNA Expression Is Altered in the Brain of Male Mice Lacking All Ligand-Binding Thyroid Hormone Receptor (TR) Isoforms

In the vertebrate brain, the thalamus serves as a relay and integration station for diverse neuronal information en route from the periphery to the cortex. Deficiency of TH during development results in severe cerebral abnormalities similar to those seen in the mouse when the retinoic acid receptor (ROR)&alpha; gene is disrupted. To investigate the effect of the thyroid hormone recep-tors (TRs) on RORalpha gene expression, we used intact male mice, in which the genes encoding the &alpha; and beta TRs have been deleted. In situ hybridization for RORalpha mRNA revealed that this gene is expressed in specific areas of the brain including the thalamus, pons, cerebellum, cortex, and hippocampus. Our quantitative data showed differences in RORalpha mRNA expression in different subthalamic nuclei between wild-type and knock-out mice. For example, the centromedial nucleus of the thalamus, which plays a role in mediating nociceptive and visceral information from the brainstem to the basal ganglia and cortical regions, has less expression of RORalpha mRNA in the knockout mice (-37%) compared to the wild-type controls. Also, in the dorsal geniculate (+72%) and lateral posterior nuclei (+58%) we found more RORalpha mRNA in dKO as compared to dWT animals. Such differences in RORalpha mRNA expression may play a role in the behavioral alterations resulting from congenital hypothyroidism.