Expression of Nerve Growth Factor in Human Pancreatic β Cells

Nerve growth factor (NGF) is an important modulator of rat pancreatic β-cell physiology in vitro. In this study, we analysed the expression of NGF, TrkA and insulin in human pancreatic islets from normal, ductal adenocarcinoma and insulinoma-afflicted samples, using double immunofluorescent labelling and confocal microscopy.

We found that in normal human pancreas, insulin and NGF are co-expressed in β cells. Moreover, similar to previous observations in rat, the high affinity NGF receptor TrkA is also expressed in β cells.

Pancreatic β cells in normal islets from adenocarcinoma and mucinous cystadenocarcinoma patients also expressed NGF. In 2 out of 15 exocrine tumour samples, NGF was detected also in the tissue surrounding the islets, while 2 out of 13 adenocarcinoma tumours expressed this growth factor.

In five insulinoma samples, we observed weaker immunofluorescent labelling of insulin and NGF in the neoplastic tissue, compared to the islets not afflicted by the tumour, which may be a consequence of increased hormone secretion rate.

We demonstrate that human β cells express TrkA and NGF. These findings are consistent with the hypothesis that NGF modulates insulin secretion through a paracrine/autocrine loop, similar to the one observed in cultured rat β cells.     

Prognostic Significance of Stromal Platelet-Derived Growth Factor β-Receptor Expression in Human Breast Cancer

This study systematically analyzes platelet-derived growth factor (PDGF) receptor expression in six types of common tumors as well as examines associations between PDGF β-receptor status and clinicopathological characteristics in breast cancer. PDGF receptor expression was determined by immunohistochemistry on tumor tissue microarrays. Breast tumor data were combined with prognostic factors and related to outcome endpoints. PDGF α- and β-receptors were independently expressed, at variable frequencies, in the tumor stroma of all tested tumor types. There was a significant association between PDGF β-receptor expression on fibroblasts and perivascular cells in individual colon and prostate tumors. In breast cancer, high stromal PDGF β-receptor expression was significantly associated with high histopathological grade, estrogen receptor negativity, and high HER2 expression. High stromal PDGF β-receptor expression was correlated with significantly shorter recurrence-free and breast cancer-specific survival. The prognostic significance of stromal PDGF β-receptor expression was particularly prominent in tumors from premenopausal women. Stromal PDGF α- and β-receptor expression is a common, but variable and independent, property of solid tumors. In breast cancer, stromal PDGF β-receptor expression significantly correlates with less favorable clinicopathological parameters and shorter survival. These findings highlight the prognostic significance of stromal markers and should be considered in ongoing clinical development of PDGF receptor inhibitors.Platelet-derived growth factor (PDGF) α- and β-tyrosine kinase receptors exert important control functions in mesenchymal cells, such as pericytes, fibroblasts and vascular smooth muscle cells during development.¹ PDGF receptor activation has also been shown to be involved in multiple dimensions of cancer growth.² The clinical relevance of these findings is enhanced by the recent approval of tyrosine kinase inhibitors with PDGF receptor inhibitory activity, eg, imatinib, sunitinib, and sorafenib.PDGF receptor-dependent growth stimulation is well documented in malignant cells of some solid tumors, such as glioblastomas,^3,4,5,6,7 dermatofibrosarcoma protuberans^8,9 and a subset of gastrointestinal stromal tumors.^10,11 Also, in hematological malignancies such as chronic myelomonocytic leukemia and idiopathic eosinophilic syndrome, PDGF α- or β-receptor signaling has been shown to be activated through translocations or deletions of the PDGF receptor genes.^12,13,14 However, in most common solid tumors PDGF receptor signaling appears to be most important for the pericytes of the tumor vessels, and for the fibroblasts of the tumor stroma.Concerning the role of PDGF β-receptor signaling in pericytes, a series of experimental studies have demonstrated that stimulation of PDGF receptors on pericytes increases pericyte coverage of vessels in a manner that is associated with increased vessel function and, in some cases, also increased tumor growth.^15,16,17 Furthermore, vascular endothelial growth factor receptor-targeted antiangiogenic approaches in experimental tumor models appear to be most efficient on immature pericyte-poor vessels.¹⁸ Finally, combinations of vascular endothelial growth factor receptor- and PDGF-receptor inhibitors have been demonstrated to exert synergistic antiangiogenic effects.^19,20Studies in experimental tumor models have demonstrated that paracrine activation of PDGF receptors on fibroblasts acts as a potent signal for tumor stroma recruitment.^21,22 Other studies with PDGF antagonists have also demonstrated direct antitumoral effects of stromal PDGF receptor inhibition,^23,24 as well as beneficial effects on tumor drug uptake.^25,26,27,28The biological effects of PDGF receptors in tumor fibroblasts and pericytes, together with the advent of drugs with PDGF receptor-inhibitory activity thus motivates a systemic characterization of the expression pattern of PDGF α- and β-receptors in human tumors. In this study we have characterized the fibroblast and pericyte expression of PDGF α- and β-receptors in lymphomas and in colon, ovarian, prostate, lung and breast cancers. Furthermore the relationship between stromal PDGF β-receptor status and prognostic parameters and survival was analyzed in breast cancer.     

Dynamic Regulation of Platelet-Derived Growth Factor Receptor α Expression in Alveolar Fibroblasts during Realveolarization

Although the importance of platelet-derived growth factor receptor (PDGFR)-α signaling during normal alveogenesis is known, it is unclear whether this signaling pathway can regulate realveolarization in the adult lung. During alveolar development, PDGFR-α-expressing cells induce α smooth muscle actin (α-SMA) and differentiate to interstitial myofibroblasts. Fibroblast growth factor (FGF) signaling regulates myofibroblast differentiation during alveolarization, whereas peroxisome proliferator-activated receptor (PPAR)-γ activation antagonizes myofibroblast differentiation in lung fibrosis. Using left lung pneumonectomy, the roles of FGF and PPAR-γ signaling in differentiation of myofibroblasts from PDGFR-α-positive precursors during compensatory lung growth were assessed. FGF receptor (FGFR) signaling was inhibited by conditionally activating a soluble dominant-negative FGFR2 transgene. PPAR-γ signaling was activated by administration of rosiglitazone. Changes in α-SMA and PDGFR-α protein expression were assessed in PDGFR-α-green fluorescent protein (GFP) reporter mice using immunohistochemistry, flow cytometry, and real-time PCR. Immunohistochemistry and flow cytometry demonstrated that the cell ratio and expression levels of PDGFR-α-GFP changed dynamically during alveolar regeneration and that α-SMA expression was induced in a subset of PDGFR-α-GFP cells. Expression of a dominant-negative FGFR2 and administration of rosiglitazone inhibited induction of α-SMA in PDGFR-α-positive fibroblasts and formation of new septae. Changes in gene expression of epithelial and mesenchymal signaling molecules were assessed after left lobe pneumonectomy, and results demonstrated that inhibition of FGFR2 signaling and increase in PPAR-γ signaling altered the expression of Shh, FGF, Wnt, and Bmp4, genes that are also important for epithelial-mesenchymal crosstalk during early lung development. Our data demonstrate for the first time that a comparable epithelial-mesenchymal crosstalk regulates fibroblast phenotypes during alveolar septation.     

uPAR Induces Expression of Transforming Growth Factor β and Interleukin-4 in Cancer Cells to Promote Tumor-Permissive Conditioning of Macrophages

Cancer cells condition macrophages and other inflammatory cells in the tumor microenvironment so that these cells are more permissive for cancer growth and metastasis. Conditioning of inflammatory cells reflects, at least in part, soluble mediators (such as transforming growth factor β and IL-4) that are released by cancer cells and alter the phenotype of cells of the innate immune system. Signaling pathways in cancer cells that potentiate this activity are incompletely understood. The urokinase receptor (uPAR) is a cell-signaling receptor known to promote cancer cell survival, proliferation, metastasis, and cancer stem cell–like properties. The present findings show that uPAR expression in diverse cancer cells, including breast cancer, pancreatic cancer, and glioblastoma cells, promotes the ability of these cells to condition co-cultured bone marrow–derived macrophages so that the macrophages express significantly increased levels of arginase 1, a biomarker of the alternatively activated M2 macrophage phenotype. Expression of transforming growth factor β was substantially increased in uPAR-expressing cancer cells via a mechanism that requires uPA-initiated cell signaling. uPAR also controlled expression of IL-4 in cancer cells via a mechanism that involves activation of ERK1/2. The ability of uPAR to induce expression of factors that condition macrophages in the tumor microenvironment may constitute an important mechanism by which uPAR promotes cancer progression.It is well established that certain chronic infections and inflammation predispose to the development of malignancy.^1–3 Once cancer develops, inflammatory cells that infiltrate the tumor may promote disease progression.^4–6 This process is mediated by bidirectional paracrine pathways involving cancer and inflammatory cells. Growth factors and cytokines released by cancer cells are immunosuppressive, and also condition inflammatory cells so that these cells release mediators that support cancer cell growth, survival, metastasis, and angiogenesis.^7–10 Inflammatory cell conditioning is prevalent in breast cancer. These tumors include large numbers of macrophages, dendritic cells, mast cells, and T cells, and the extent to which the tumor is infiltrated by these inflammatory cells correlates with the incidence of metastasis.^11–13 A high density of tumor-associated macrophages (TAMs) is also correlated with higher breast cancer tumor grade and decreased relapse-free and overall survival.^14–17Although macrophages express a wide spectrum of phenotypic properties, these cells are frequently categorized as classically activated (M1) or alternatively activated (M2).^18–21 In response to pathogens, tissue damage, and Th1 cytokines such as IFN-γ and TNF-α, M1-polarized macrophages release cytotoxic compounds and proteins, including nitric oxide, reactive oxygen species, and proinflammatory cytokines (including IL-12, IL-23, and TNF-α). M2-polarized macrophage have been classified into a number of subcategories; in many contexts, these cells demonstrate enhanced activity in the resolution of inflammation, tissue remodeling, and healing.^18–21 Arginase 1 (Arg1), which is expressed selectively by M2-polarized macrophages, diverts substrate from the enzyme systems that produce cytotoxic levels of nitric oxide.^22,23 In general, it is thought that TAMs, which have been conditioned by cancer cells to express tumor-permissive gene products, demonstrate characteristics in common with M2-polarized macrophages, although a recent report highlights phenotypic differences.^18,19,24 Cell-signaling systems in tumor cells that promote the ability of these cells to regulate macrophage phenotype remain incompletely understood.In many forms of cancer, expression of the urokinase receptor [urokinase plasminogen activator receptor (uPAR)] correlates with poor prognosis and shortened survival.^25–28 Originally, the activity of uPAR in cancer was attributed to its ability to bind the serine protease, urokinase-type plasminogen activator (uPA), and activate a cascade of extracellular proteases involved in matrix remodeling and cell migration through tissue boundaries. The current understanding, however, is that uPAR also is a cell-signaling receptor that activates diverse signaling pathways.²⁹ Although uPAR may signal autonomously when expressed at high levels, uPA binding to uPAR robustly activates cell signaling even when the cell-surface abundance of uPAR is low.^29–32 uPAR-initiated cell signaling promotes cancer cell survival, release from states of dormancy, migration, epithelial–mesenchymal transition, cancer stem cell–like properties, and metastasis independently of protease activation.^33–38Here, we show that in multiple forms of cancer, including breast cancer, pancreatic cancer, and glioblastoma (GBM), uPAR expression promotes the ability of the cancer cells to M2-polarize co-cultured macrophages. The mediators that are released selectively by uPAR-expressing cancer cells to regulate macrophage phenotype may vary across different cancer cells; however, we provide evidence that both TGF-β and IL-4 are involved. The ability of cancer-cell uPAR to promote conditioning of inflammatory cells in the tumor microenvironment is a novel mechanism by which uPAR may promote cancer progression.     

Mannose-6-Phosphate/IGF-II Receptors Mediate the Effects of IGF-1-Induced Latent Transforming Growth Factor β1 on Expression of Type I Collagen and Collagenase in Dermal Fibroblasts

We have previously shown that insulin-like growth factor-1 (IGF-1) induces the expression of latent transforming growth factor β1 (LTGF-β1) through activation of c-fos and c-jun oncogenes. In this study we investigated whether IGF-1 induced latent TGF-β1 has autocrine effects on dermal fibroblasts and described a possible mechanism. Human dermal fibroblasts were treated with either vehicle, IGF-1 alone, or IGF-1 with either anti-TGF-β1 neutralizing antibody or mannose-6-phosphate (M6P) and levels of mRNAs for TGF-β1, collagenase and the pro α1(I) chain of type I collagen were then evaluated by Northern analysis. Conditioned medium was also collected from treated and untreated cells and assayed for TGF-β1 protein by enzyme-linked immunosorbent assay. The results of the Northern analysis revealed a differential effect on the expression of the pro α1(I) chain of type I collagen and collagenase in dermal fibroblasts: mRNA for the former being significantly increased in response to IGF-1 treatment while that for collagenase was markedly suppressed. These effects of IGF-1 were blocked to a significant extent by TGF-β1 neutralizing antibody at a concentration of 0.5–2.0 μg/ml. As the TGF-β1 induced by IGF-1 is inactive in the traditionally used mink lung epithelial cell growth inhibition assay, we explored the possible role of IGF-II/M6P receptors in facilitating these autocrine effects. The results showed that the greater than two-fold increase (201.9 ± 38 vs 81.8 ± 13, p < 0.05) in mRNA for the pro α1(I) chain of type I collagen induced by IGF-1 was at least 60% inhibited by M6P in a time-dependent fashion. A direct correlation between the expression of TGF-β1 and the pro α1(I) chain of type I collagen was found in response to either IGF-1 alone or IGF-1 with M6P. Treatment of cell cultures with TGF-β1 neutralizing antibody mimicked the effect of M6P. In contrast to the effects on expression of type I collagen, the level of collagenase mRNA was markedly reduced by IGF-1 alone and was restored by the administration of M6P. The levels of TGF-β1 in conditioned medium from treated and untreated cells showed a similar pattern to that of the mRNA detected by Northern analysis. These findings suggest that IGF-1 induces latent TGF-β1 and that the matrix-modulating autocrine effects of LTGF-β1 on dermal fibroblasts are facilitated by M6P/IGF-II receptors on these cells.     

Immobilization-Induced Cartilage Degeneration Mediated Through Expression of Hypoxia-Inducible Factor-1α, Vascular Endothelial Growth Factor,and Chondromodulin-I

Immobilization results in thinning of the articular cartilage and cartilage degeneration, although the exact mechanisms are not clear yet. Hypoxia is thought to contribute to the degeneration of articular cartilage. We investigated the roles of hypoxia inducible factor (HIF)-1α, vascular endothelial growth factor (VEGF), and the newly cloned antiangiogenic factor, chondromodulin-I (ChM-1), in cartilage degeneration in immobilized joints. Male Wistar rats (n = 30, 12-week-old) were divided randomly into the control group (n = 10), immobilization group (n = 10), and continuous passive motion (CPM) group (n = 10). In the immobilization group, the ankle joints were fixed in full plantar flexion with plaster casts for 4 weeks. In the CPM group, the ankle casts were removed during the immobilization period and the ankle joints were subjected to CPM. Significant thinning of the articular cartilage was noted in the immobilization group but not in the control or CPM group. In the immobilized group, vascular channels were found in the area between the calcified cartilage zone and the subchondral bone. The densities of HIF-1α—and VEGF-immunostained cells were higher in the immobilized group than the other two groups. In contrast, low expression of ChM-1 was detected in the articular cartilage of the immobilized group compared with the control and CPM group. Our results showed that immobilization induces thinning of the articular cartilage and appearance of vascular channel, in areas with balanced expression of HIF-1α/VEGF and ChM-1.     

Inhibition of Collagen Synthesis and Regulation of Cell Motility in Vascular Smooth Muscle Cells by Suppression of Connective Tissue growth Factor Expression Using RNAInterference

1 IntroductionVascular smooth muscle cell (VSMC) hyperplasia plays an important role in both chronic and acute pathologies including atherosclerosis and restenosis. Recent studies have shown that connective tissue growth factor (CTGF) is a novel growth factor involved in the development and progression of atherosclerosis. However, previous data about the role of CTGF on the VSMC is conflicting. Hishikawa et al demonstrated that CTGF could act as a growth inhibitor in human VSMC; but …     

Transforming Growth Factor Beta (TGF-β) Potentiates the Inhibitory Effect of Retinoic Acid on Human Breast Carcinoma (MCF-7) Cell Proliferation

Abstract

Anchorage-dependent and -independent MCF-7 cell growth was dose-dependently inhibited by retinoic acid (RA) but was insensitive to TGF-β (from 1 to 100 pin). Growth of MCF-7 monolayer cultures was inhibited (50%) when exposed to 10^?6 m RA. RA was unable to completely inhibit MCF-7 cell proliferation, as concentrations above 10^?6m were rapidly cytotoxic. However, the combination of TGF-β and RA resulted an increase in RA inhibitory effect on MCF-7 monolayer growth and a 80% reduction in colony formation in soft agar. These results demonstrate that although TGF-β does not inhibit the growth of MCF-7 cells, it potentiates the antiproliferative effect of RA, suggesting that it may play a part, albeit indirect, in the regulation of MCF-7 cell growth.     

Insulin-like growth factor-2 (IGF-2) activates estrogen receptor-α and -β via the IGF-1 and the insulin receptors in breast cancer cells

The estrogen receptor (ER) is a primary target for breast cancer (BC) treatment. As BC progresses to estrogen-independent growth, the insulin-like growth factor-1 receptor (IGF-1R) and the ER interact in synergistic cross-talk mechanisms, which result in enhanced activation of both receptors' signaling cascades. Insulin-like growth factor-2 (IGF-2) is critical in BC progression and its actions are mediated by the IGF-1R. Our previous studies showed that IGF-2 regulates survival genes that protect the mitochondria and promote chemoresistance. In this study, we analyzed BC cells by subcellular fractionation, Western-Blot, qRT-PCR, and siRNA analysis. Our results demonstrate that IGF-2 activates ER-α and ER-β, and modulates their translocation to the nucleus, membrane organelles, and the mitochondria. IGF-2 actions are mediated by the IGF-1R and the insulin receptor. This novel mechanism of IGF-2 synergistic cross-talk signaling with ER-α and ER-β can promote estrogen-independent BC progression and provide new therapeutic targets for the treatment of BC patients.     

Insulin-like growth factor-2 (IGF-2) activates estrogen receptor-α and -β via the IGF-1 and the insulin receptors in breast cancer cells

The estrogen receptor (ER) is a primary target for breast cancer (BC) treatment. As BC progresses to estrogen-independent growth, the insulin-like growth factor-1 receptor (IGF-1R) and the ER interact in synergistic cross-talk mechanisms, which result in enhanced activation of both receptors' signaling cascades. Insulin-like growth factor-2 (IGF-2) is critical in BC progression and its actions are mediated by the IGF-1R. Our previous studies showed that IGF-2 regulates survival genes that protect the mitochondria and promote chemoresistance. In this study, we analyzed BC cells by subcellular fractionation, Western-Blot, qRT-PCR, and siRNA analysis. Our results demonstrate that IGF-2 activates ER-α and ER-β, and modulates their translocation to the nucleus, membrane organelles, and the mitochondria. IGF-2 actions are mediated by the IGF-1R and the insulin receptor. This novel mechanism of IGF-2 synergistic cross-talk signaling with ER-α and ER-β can promote estrogen-independent BC progression and provide new therapeutic targets for the treatment of BC patients.     

The Abnormal High Expression of B Cell Activating Factor Belonging to TNF Superfamily （BAFF） and Its Potential Role in Kidney Transplant Recipients

B cell activating factor belonging to TNF superfamily （BAFF） is a critical regulator of B cell maturation and survival In this present study, the expression characteristic of BAFF in kidney transplantation recipients was investigated, its potential significance was analyzed and peripheral blood of follow-up kidney transplant recipients was studied. Flow cytometric assay results showed that, cell-surface BAFF was significantly highly expressed on peripheral CD3＋ T lymphocytes in 〉 5 yrs group of kidney transplant recipients, compared with other groups （p 〈 0.05）. BAFF expression could be found on CD4＋ T cells and CD8＋ T cells. The BAFF mRNA levels in peripheral mononuclear cells were consistent with the protein levels. However, serum soluble BAFF levels were inter- individually different in each group. Stratified by renal function, it was found that cell-surface BAFF levels were significantly higher in those with abnormal renal function, compared with recipients with normal renal function （p 〈 0.05）. ELISA assay results showed that expression levels of cell surface BAFF were significantly correlated with anti-HLA I ＆ II antibodies. These results indicate that BAFF may be involved in the development of graft-loss and influences the long-time outcome of kidney allografl, likely mediated by interfering with immune response. Cellular ＆ Molecular Immunology. 2008;5（6）：465-470.     

Differential Changes in Expression of a and b FGF,IGF-1 and -2, and TGF-α During Seasonal Growth and Involution of Roe Deer Testis

Growth factors are involved in the regulation of testicular growth and involution of seasonal breeders. Therefore, we studied the seasonal expression of several growth factors in roe deer: aFGF, bFGF, IGF-1, IGF-2, and TGF- &#33. Total RNA from testis tissue was extracted monthly and analyzed using quantitative RT-PCR. Localization of mRNAs was examined by in situ -hybridization. Levels of expression differed by more than three orders of magnitude. Expression also showed different seasonal patterns. IGF-1, IGF-2 and bFGF were maximally transcribed during testis recrudescence in spring. In contrast, the mRNA amount of aFGF reached its maximum between July (breeding season) and January. TGF- &#33 mRNA-levels were very low and showed poor seasonal variation. Each growth factor showed its own typical expression localization in testicular tissues and cell types. The results suggest the specific role of different growth factors in the paracrine control of spermatogenesis and its seasonal regulation.     

Expression of recombinant β-tubulin alleles from Cylicocyclus nassatus (Cyathostominae)

Small strongyles (Cyathostominae) are common nematode parasites of horses that have developed resistance to the benzimidazole anthelmintics used to control their populations. Evidence suggests that the principal mechanism of resistance involves a phenylalanine-to-tyrosine mutation at codon 200 in the β-tubulin proteins that are components of microtubules. Other works, however, suggest that a phenylalanine-to-tyrosine mutation at codon 167, or alternative mechanisms, may be involved. As part of an ongoing project examining the role that these two β-tubulin mutations may play in benzimidazole resistance, we have cloned the wild-type allele and the two alleles with the phenylalanine-to-tyrosine mutations at codons 167 and 200 of the β-tubulin isotype 1 gene from the small strongyle Cylicocyclus nassatus. In this work, we describe the construction of expression vectors containing these alleles and their expression in Escherichia coli.     

Expression of Human Complement Factor H Prevents Age-Related Macular Degeneration–Like Retina Damage and Kidney Abnormalities in Aged Cfh Knockout Mice

Complement factor H (CFH) is an important regulatory protein in the alternative pathway of the complement system, and CFH polymorphisms increase the genetic risk of age-related macular degeneration dramatically. These same human CFH variants have also been associated with dense deposit disease. To mechanistically study the function of CFH in the pathogenesis of these diseases, we created transgenic mouse lines using human CFH bacterial artificial chromosomes expressing full-length human CFH variants and crossed these to Cfh knockout (Cfh^−/−) mice. Human CFH protein inhibited cleavage of mouse complement component 3 and factor B in plasma and in retinal pigment epithelium/choroid/sclera, establishing that human CFH regulates activation of the mouse alternative pathway. One of the mouse lines, which express relatively higher levels of CFH, demonstrated functional and structural protection of the retina owing to the Cfh deletion. Impaired visual function, detected as a deficit in the scotopic electroretinographic response, was improved in this transgenic mouse line compared with Cfh^−/− mice, and transgenics had a thicker outer nuclear layer and less sub–retinal pigment epithelium deposit accumulation. In addition, expression of human CFH also completely protected the mice from developing kidney abnormalities associated with loss of CFH. These humanized CFH mice present a valuable model for study of the molecular mechanisms of age-related macular degeneration and dense deposit disease and for testing therapeutic targets.Age-related macular degeneration (AMD) is a disease that progressively damages the photoreceptors and retinal pigment epithelium (RPE) in the macula, resulting in loss of vision in the center of the visual field. AMD is the leading cause of blindness in the elderly population, affecting approximately 15% of the population older than 70 years of age.^1,2 The earliest clinical signs of AMD are pigmentary changes and the appearance of drusen, focal protein- and lipid-rich extracellular deposits that form between the RPE and the Bruch membrane (BrM). Late-stage AMD is characterized by choroidal neovascularization, geographic atrophy, or both, resulting in irreversible central loss of the choriocapillaris, RPE, and photoreceptors.^3–5 Abnormalities in dark adaptation are often the earliest functional change detected in patients with AMD and reflect impairment of rod photoreceptor function.^6,7AMD is a complex disease influenced by a variety of risk factors, including age, which is the greatest.⁸ Of the genetic risk factors, a common polymorphism (T1277C) in the complement factor H (CFH) gene, resulting in a tyrosine (Y) to histidine (H) substitution at amino acid 402 (H402), has been found to be strongly associated with the development of AMD.^9–12 The same polymorphism has also been linked to dense deposit disease (DDD, also known as membranoproliferative glomerulonephritis type II).^13,14 DDD is a condition that primarily affects kidney function. The symptoms usually appear between ages 5 and 15 years and include hematuria, proteinuria, and nephrotic syndrome.^15,16CFH is a major regulator of the alternative pathway (AP) of the complement system. The AP is initiated by complement component 3 (C3) hydrolysis in which a C3 thioester interacts with water to form C3(H₂O). This occurs spontaneously and causes the AP to be continuously active. Uninhibited, C3(H₂O) reacts with complement factor B (FB) to generate C3(H₂O)B, which in the presence of complement factor D is converted to the C3 convertase C3(H₂O)Bb. This conversion allows for further cleavage of C3 to C3b and formation of the C3bBb convertase and initiates the amplification loop. Without negative regulators, this positive feedback loop will quickly deplete all available C3 in the system. CFH is the soluble negative regulator of AP activation. CFH can inhibit formation of the C3 convertase by competing with FB for binding to C3b, accelerate decay of the C3 convertase, and act as a cofactor for factor I–mediated cleavage of C3b.¹⁷The complement system is important in defending an organism against foreign invasion by lyzing alien pathogens via the formation of membrane attack complex and signaling debris clearance. However, if dysregulated, the complement system can also attack host cells, causing local inflammation and tissue damage.¹⁸ Available data support the idea that in AMD, the AP is dysregulated owing to CFH malfunction, causing local inflammation and tissue damage in the macular region of the retina. However, the cellular/molecular mechanism(s) for the risk association of CFH in AMD pathogenesis remains unclear. Consistent with a role for CFH in AMD pathogenesis, aged mice lacking CFH (Cfh^−/−) have an ocular phenotype that shares characteristics of early AMD. These characteristics include decreased electroretinogram (ERG) response, loss of visual acuity, disrupted photoreceptor organization, and structural abnormalities of the BrM.^19,20 These mice also spontaneously develop DDD-like pathologic abnormalities.²¹To further elucidate the function of CFH in AMD pathogenesis, we generated mice in which full-length human CFH is expressed instead of mouse CFH. Using this mouse model, we tested whether i) human CFH interacts with the mouse AP of the complement system, ii) human CFH protein can rescue the ocular and renal phenotype in Cfh^−/− mice, and iii) there are phenotypic differences between mice expressing the normal Y402 or AMD risk–associated H variant of CFH.     

Comparison of the Automated Mycobacteria Growth Indicator Tube System (BACTEC 960/MGIT) with Löwenstein-Jensen medium for recovery of mycobacteria from clinical specimens

We examined whether the BACTEC/Mycobacteria Growth Indicator Tube (MGIT) system alone could supplant the use of a supplemental L?wenstein-Jensen (LJ) slant for routine recovery of Mycobacterium species from clinical specimens. A total of 6,062 specimens were included in the study. Of these, 273 specimens were positive for 278 mycobacterial isolates while 15 specimens were smear positive but culture negative using both media. Further analysis showed that 143 (51.4%) of the 278 total isolates were recovered from both the MGIT and LJ media. An additional 106 isolates (38.1%) were recovered from the MGIT only, while 29 (10.4%) isolates grew only on the LJ slant. The overall sensitivities of the MGIT and LJ media were 86.5% and 59.7%, respectively, for the recovery of mycobacteria from clinical materials. This study shows that although the MGIT system demonstrates better sensitivity for the recovery of mycobacteria from clinical specimens, both media types are necessary to maximize the sensitivity of detection.     

Construction of a Mammary-specific Expression Vector of Humanα-defensin-1 (HNP-1) Gene

1 IntroductionDefensins, also called human neutrophil peptides(HNP), are small cationic peptides with broad antimicrobial activity~([1]). Human defensins are highly abundant in the cytoplasmic granules of polymorphonuclear neutrophils. Alpha-defensin-1 is an important mediator in either innate immunity or anti-infection. It can be developed to be an ideal new type antibiotic and may provide a better solution for the present situation of extensive antibiotics-resistence. It is difficult to ac…