Insulin-like growth factor-I expression is not increased in the retina of diabetic BB/W-rats

A combination of immunocytochemistry, in situ hybridization and ligand binding were used to investigate the localization of IGF-I and its receptor in the retina of diabetic and non-diabetic BB/W-rats. Immunocytochemical localization revealed the presence of IGF-I in retinal pigment epithelium, ganglion cells, Muller cell processes and in microvessels. In most sites immunoreactivity was increased in the diabetic retina compared to that of non-diabetic BB/W-rats. In microvessels, however, immunoreactivity was decreased in diabetes. In situ hybridization using an antisense IGF-I riboprobe provided evidence of IGF-I synthesis in all retinal layers with a similar grain density in diabetic and non-diabetic rats. Autoradiographic localization of IGF-I receptors, using [125I]-IGF-I binding, demonstrated a diffuse localization in all retinal layers, with an increase in diabetic animals. These findings suggest that IGF-I synthesis is not altered in the diabetic retina, and that the increased immunoreactivity of IGF-I detectable in the various layers of the retina from diabetic rats may be due to an increased uptake of blood-derived IGF-I suggested by increased receptor density in diabetic rats.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29042/1/0000075.pd

Genetically Modified Dermal Keratinocytes Express High Levels of Transforming Growth Factor-β1

In an attempt to genetically modify cultured keratinocytes with transforming growth factor-β1 (TGF-β1), which has been proven to be one of the most important cytokines involved in wound healing, two constructs were made. One, designated pG3Z:K14-TGF-β1, is a plasmid in which the expression of TGF-β1 is driven by the keratin 14 promoter. The other, designated pLin-TGF-β1, is a retroviral vector in which the retroviral 5' long-terminal repeat promoter drives expression. In both constructs, the deletion of a small fragment of the noncoding region of the TGF-β1 gene was made to differentiate the transcript from that for endogenously expressed TGF-β1. Different types of cells were transfected with the pG3Z:K14-TGF-β1 construct using the calcium phosphate method. The pLin-TGF-β1 construct was propagated in a retroviral packaging cell line and conditioned medium that contained high titers of the virus was used to transduce keratinocytes or other types of cells grown in standard culture. Northern analysis, used to evaluate the expression of TGF-β1 mRNA in the pG3Z:K14-TGF-β1 transfected keratinocyte Cl-177 cell line, showed a smaller TGF-β1 transcript compared with that endogenously expressed by dermal fibroblasts. The level of TGF-β1 protein evaluated by enzyme-linked immunosorbent assay was significantly higher in medium conditioned by either the K14-TGF-β1 transfected or the pLin-TGF-β1 transduced keratinocytes, compared with that obtained from control cells; however, the level of TGF-β1 protein was unchanged in cultures of pG3Z:K14-TGF-β1 transfected nonkeratinocyte cells such as fetal and adult fibroblasts. Using the mink lung epithelial cell growth inhibition assay, we found an increase in TGF-β1 activity in conditioned medium from the pG3Z:K14-TGF-β1 transfected cells. To evaluate possible paracrine effects of the keratinocyte derived TGF-β1, a coculture system was established with pLin-TGF-β1 transduced keratinocytes grown in the upper chamber and dermal fibroblasts in the lower chamber. The results showed that TGF-β1 released from keratinocytes diffused to the lower chamber where it stimulated collagen production by dermal fibroblasts. In summary, we demonstrate here that primary cultured keratinocytes can be genetically modified to express high levels of TGF-β1 and suggest that this offers a potential approach for the therapy of dermal lesions such as nonhealing wounds

Immunoprotective role of indoleamine 2,3-dioxygenase in engraftment of allogenic skin substitute in wound healing

Delayed wound healing can significantly impact survival of patients who suffer from severe thermal injury. In general, the use of a wound coverage, particularly with those of bilayer skin substitute, would be ideal to promote healing and prevent infection and fluid loss. Although the use of an autologous skin substitute is desirable, its preparation is time consuming and its immediate availability is impossible. To overcome this difficulty, the authors have previously demonstrated that the expression of indoleamine 2,3 dioxygenase (IDO) could function as a local immune suppressive factor in protecting allogenic fibroblasts and keratinocytes without using any immunosuppressive medication in a wound healing animal model. IDO, which is naturally expressed in the placenta by trophoblast cells during pregnancy, plays an essential role in maternal tolerance toward the fetus. The potent and selective local immunosuppressive function of IDO makes this enzyme a very promising tool for engineering a nonrejectable skin allograft. Here, the authors reviewed and discussed how the expression of IDO by the primary cells of our skin substitute can serve as a source of IDO enzyme activity and generate a tryptophan-deficient environment. Under this condition, only skin cells but not immune cells (CD4+ and CD8+ cells) would survive and protect engraftment of this engineered and shelf-ready skin substitute to be used not only as wound coverage but also as a rich source of wound healing promoting factors. Therefore, this review summarizes the body of work on immunoprotective role of IDO in engraftment of allogenic skin substitute in wound healing, which has recently been reported by the authors' research group and others. Copyright © 2012 by the American Burn Association

Manufacture and Characterization of High Q-Factor Inductors Based on CMOS-MEMS Techniques

A high Q-factor (quality-factor) spiral inductor fabricated by the CMOS (complementary metal oxide semiconductor) process and a post-process was investigated. The spiral inductor is manufactured on a silicon substrate. A post-process is used to remove the underlying silicon substrate in order to reduce the substrate loss and to enhance the Q-factor of the inductor. The post-process adopts RIE (reactive ion etching) to etch the sacrificial oxide layer, and then TMAH (tetramethylammonium hydroxide) is employed to remove the silicon substrate for obtaining the suspended spiral inductor. The advantage of this post-processing method is its compatibility with the CMOS process. The performance of the spiral inductor is measured by an Agilent 8510C network analyzer and a Cascade probe station. Experimental results show that the Q-factor and inductance of the spiral inductor are 15 at 15 GHz and 1.8 nH at 1 GHz, respectively

Epidermal Growth Factor Stimulates Proliferation of Mouse Uterine Epithelial Cells in Primary Culture

Epidermal growth factor (EGF) is one of growth factors that are thought to mediate the stimulatory effects of estrogen on the proliferation of uterine epithelial cells. The present study was attempted to obtain direct evidence for the mitogenic effects of EGF on uterine epithelial cells, and to prove that EGF and EGF receptors are expressed in these cells. Mouse uterine epithelial cells were isolated from immature female mice and cultured with or without EGF for 5 days. EGF (1 to 100 ng/ml) significantly increased the number of uterine epithelial cells, and the maximal growth (141.9+/-8.3% of controls) was obtained at a dose of 10 ng/ml. In addition, EGF (0.1 to 100 ng/ml) increased the number of DNA-synthesizing cells immunocytochemically detected by bromodeoxyuridine uptake to the nucleus. Northern blot analysis revealed that the uterine epithelial cells expressed both EGF mRNA (4.7 kb) and EGF receptor mRNAs (10.5, 6.6, and 2.7 kb) These results suggest that the proliferation of uterine epithelial cells is regulated by the paracrine and/ or autocrine action of EGF. Our previous study demonstrated the mitogenic effect of IGF-I on uterine epithelial cells. To examine whether the EGF- and IGF-I signaling act at the same level in the regulation of the proliferation of uterine epithelial cells, the cultured cells were simultaneously treated with IGF-I and EGF. IGF-I was found to additively stimulate the mitogenic effects of EGF, suggesting that the EGF-induced growth of uterine epithelial cells is distinct from IGF-l-induced growth

Mechanism Underlying Defective Interferon Gamma-Induced IDO Expression in Non-obese Diabetic Mouse Fibroblasts

Indoleamine 2,3-dioxygenase (IDO) can locally suppress T cell-mediated immune responses. It has been shown that defective self-tolerance in early prediabetic female non-obese diabetic (NOD) mice can be attributed to the impaired interferon-gamma (IFN-γ)- induced IDO expression in dendritic cells of these animals. As IFN-γ can induce IDO in both dendritic cells and fibroblasts, we asked the question of whether there exists a similar defect in IFN-γ-induced IDO expression in NOD mice dermal fibroblasts. To this end, we examined the effect of IFN-γ on expression of IDO and its enzymatic activity in NOD dermal fibroblasts. The results showed that fibroblasts from either prediabetic (8 wks of age) female or male, and diabetic female or male (12 and 24 wks of age respectively) NOD mice failed to express IDO in response to IFN-γ treatment. To find underlying mechanisms, we scrutinized the IFN- γ signaling pathway and investigated expression of other IFN-γ-modulated factors including major histocompatibility complex class I (MHC-I) and type I collagen (COL-I). The findings revealed a defect of signal transducer and activator of transcription 1 (STAT1) phosphorylation in NOD cells relative to that of controls. Furthermore, we found an increase in MHC-I and suppression of COL-I expression in fibroblasts from both NOD and control mice following IFN-γ treatment; indicating that the impaired response to IFN-γ in NOD fibroblasts is specific to IDO gene. Finally, we showed that an IFN-γ-independent IDO expression pathway i.e. lipopolysaccharide (LPS)-mediated-c-Jun kinase is operative in NOD mice fibroblast. In conclusion, the findings of this study for the first time indicate that IFN-γ fails to induce IDO expression in NOD dermal fibroblasts; this may partially be due to defective STAT1 phosphorylation in IFN-γ-induced-IDO signaling pathway

A role for the collagen I/III and MMP-1/-13 genes in primary inguinal hernia?

BACKGROUND: Abnormal collagen metabolism is thought to play an important role in the development of primary inguinal hernia. This is underlined by detection of altered collagen metabolism and structural changes of the tissue in patients with primary inguinal hernia. However, it is still unknown whether these alterations reflect a basic dysfunction of the collagen synthesis, or of collagen degradation. METHODS: In the present study, we analysed type I and type III procollagen messenger ribonucleic acid (mRNA) and MMP-1 and MMP-13 mRNA in cultured fibroblasts from the skin of patients with primary inguinal hernia, and from patients without hernia (controls) by reverse transcription polymerase chain reaction (RT-PCR) and Northern Blot. RESULTS: The results indicated that the ratio of type I to type III procollagen mRNA was decreased in patients with primary hernia, showing significant differences as compared to controls (p = 0.01). This decrease was mainly due to the increase of type III procollagen mRNA. Furthermore, RT-PCR analysis revealed that the expression of MMP-1 mRNA in patients with primary hernia is equivalent to that of controls (p > 0.05). In addition, MMP-13 mRNA is expressed neither in patients with primary hernia nor in controls. CONCLUSION: We concluded that abnormal change of type I and type III collagen mRNAs contribute to the development of primary inguinal hernia, whereas the expressions of MMP-1 and MMP-13 mRNA appears not to be involved in the development of primary inguinal hernia. Thus, the knowledge on the transcriptional regulation of collagen in patients with primary inguinal hernia may help to understand the pathogenesis of primary inguinal hernia, and implies new therapeutic strategies for this disease