Na infecção por Leishmania a resposta imune se inicia logo após a inoculação de promastigotas no indivíduo. Nesse contexto vai haver a participação de diversos fatores da resposta imune inata que vai direcionar para uma resposta imune adaptativa responsável pela evolução da doença. Um desses fatores que participa dessa interação parasito-hospedeiro é o fator de crescimento insulina-símile I (IGF-I). Foi demonstrado que o IGF-I extrínseco favorece a proliferação do parasito e progressão da infecção. No entanto, IGF-I está presente constitutivamente em macrófagos. Neste trabalho avaliamos a expressão do IGF-I, o parasitismo e a produção de óxido nítrico em macrófagos murinos infectado por Leishmania (L.) amazonensis e o efeito da inibição de IGF-I no parasitismo após o silenciamento do IGF-I por RNA de interferência. Macrófagos peritoneiais foram infectados por 2 e 4 horas com promastigotas de L. (L.) amazonensis na presença de soro fetal bovino (SFB) 5% e Albumina de Soro Bovino 0,5% (BSA) na presença ou ausência de small-interfering RNA (siRNA) de IGF-I e lipossoma (Lipo). As células foram lavadas e mantidas depois em meio de cultura por 24, 48 e 72 h. Quando o recombinante para IGF-I foi adicionado separadamente durante a incubação inicial o parasitismo aumentou em relação ao controle. Quando o siRNA foi adicionado houve diminuição na expressão de IGF-I e consequentemente diminuição no parasitismo em relação ao controle. Os resultados obtidos sugerem um papel importante de IGF-I na infecção de macrófagos peritoneais de camundongos murinos por Leishmania (l.) amazonensis. / In Leishmania infection the immune response begins soon after the inoculation of promastigotes in the individual. In this context will be the participation of several factors of the innate immune response that will direct to an adaptive immune response responsible for the evolution of the disease. One of these factors that participates in this parasite-host interaction is the insulin-like growth factor I (IGF-I). It has been shown that extrinsic IGF-I favors parasite proliferation and infection progression. However, IGF-I is constitutively present in macrophages. In this work we evaluated the expression of IGF-I, parasitism and nitric oxide production in murine macrophages infected with Leishmania (L.) amazonensis and the effect of IGF-I inhibition on parasitism after IGF-I silencing by RNA from interference. Peritoneal macrophages were infected for 2 and 4 hours with L. (L.) amazonensis promastigotes in the presence of 5% fetal bovine serum (FBS) and 0.5% Bovine Serum Albumin (BSA) in the presence or absence of IGF-I small-interfering RNA (siRNA) and liposome (Lipo). Cells were washed and then maintained in culture medium for 24, 48 and 72 h. When the recombinant IGF-I was added separately during the initial incubation the parasitism increased relative to the control. When the siRNA was added there was a decrease in IGF-I expression and consequently a decrease in parasitism in relation to the control. The results obtained suggest an important role of IGF-I in the infection of murine mouse peritoneal macrophages by Leishmania (L.) amazonensis.

Nas leishmanioses, sabe-se que tanto na resistência quanto na suscetibilidade à infecção, a resposta imune celular é considerada a mais importante. No entanto, na fase inicial, fatores inespecíficos estão sendo considerados fundamentais na determinação do curso da doença, como o fator de crescimento insulina-símile I (\"insulin-like growth factor\"-IGF-I). Em trabalhos anteriores realizados no grupo de pesquisa da Profa. Dra. Hiro Goto mostraram que IGF-I extrínseco favorece a proliferação do parasito e progressão da infecção, com diminuição na produção de óxido nítrico e ativação da arginase de macrófagos e do parasito. Sabendo que os macrófagos produzem intrinsecamente IGF-I, avaliamos o efeito do fator intrínseco no parasitismo em macrófagos de camundongos BALB/c infectados por L. (L.) infantum silenciando a expressão do RNA mensageiro (mRNA) de IGF-I na célula pela técnica de RNA de interferência (siRNA). Iniciamos avaliando a expressão do mRNA de IGF-I e do seu receptor (IGF-IR). Foi observado uma diminuição de 1,4 vezes da expressão do mRNA de IGF-I em 24 horas e um aumento de 1,5 vezes em 48 horas quando comparado com o grupo controle. Também foi observado um aumento na expressão de mRNA do receptor de IGF-I em 24 como em 48 horas nos grupos infectados quando comparado com o grupo controle. Com o silenciamento do IGF-I por siRNA, houve diminuição da expressão de mRNA do IGF-I no macrófago em torno de 71% em 24 horas e de 51% em 48 hora. Na ausência ou diminuição do IGF-I no macrófago, observou-se uma diminuição do parasitismo na infecção com promastigotas, sendo o parasitismo recuperado com a reposição de IGF-I extrínseco no sistema, atestando a importância de IGF-I na proliferação do parasito. Esses resultados reforçam a importância do IGF-I na infecção por L. infantum, sugerindo que o IGF-I está diretamente relacionado ao parasitismo. / In leishmaniasis, it is known that in both resistance and susceptibility to infection, the cellular immune response is considered the most important. However, in the initial phase, nonspecific factors are being considered as fundamental in determining the course of the disease, such as insulin-like growth factor I (IGF-I). Studies carried out in the research group of Profa. Dr. Hiro Goto showed that extrinsic IGF-I favors parasite proliferation and infection progression, with decrease in nitric oxide production and activation of arginase of macrophages and parasite. It is known that macrophages produced IGF-I (intrinsic IGF-I), thus, in the present study we have investigated the effect of the intrinsic factor in the parasitism on macrophages of BALB/c mice infected with L. (L.) infantum by silencing the expression of messenger RNA (IGF-I mRNA) in the cell by the interference RNA technique (siRNA). We started evaluating the expression of IGF-I mRNA and its receptor (IGF-IR). A 1.4-fold decrease in IGF-I mRNA expression was observed in 24 hours and a 1.5-fold increase in 48 hours as compared to the control group. An increase in mRNA expression of the IGF-IR was also observed in 24 as well as in 48 hours in the infected groups as compared to the control group. With IGF-I silencing by siRNA, there was a decrease in IGF-I mRNA expression in the macrophage around 71% in 24 hours and 51% in 48 hours. In the absence or decrease of IGF-I in the macrophage, a decrease in the parasitism in the infection with promastigotes was observed, and the parasitism recovered with the replacement of extrinsic IGF-I in the system, confirming the importance of IGF-I in the proliferation of the parasite. These results reinforce the importance of IGF-I in L. infantum infection, suggesting that IGF-I is directly related to the parasitism.

Cheung Chun Kai. / Thesis submitted in: December 2003. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 133-146). / Abstracts in English and Chinese. / Acknowledgements --- p.ii / Abstract --- p.iv / 摘要 --- p.vii / Table of Contents --- p.ix / List of Tables --- p.xv / List of Figures --- p.xvi / List of Abbreviations --- p.xxi / Chapter Chapter 1 --- Overview --- p.1 / Chapter Chapter 2 --- Literature Review --- p.3 / Chapter 2.1 --- Historical background --- p.3 / Chapter 2.2 --- Insulin-like growth factor --- p.5 / Chapter 2.2.1 --- Structure and synthesis --- p.5 / Chapter 2.2.2 --- Physiologic role and biological actions --- p.6 / Chapter 2.3 --- Insulin-like growth factor binding protein-3 --- p.8 / Chapter 2.3.1 --- Structure and synthesis --- p.8 / Chapter 2.3.2 --- Physiologic role and biological actions --- p.8 / Chapter 2.4 --- Clinical aspects --- p.10 / Chapter 2.4.1 --- Metabolic effects of IGF-1 --- p.10 / Chapter 2.4.1.1 --- Similarities between IGF-I and insulin --- p.11 / Chapter 2.4.1.2 --- Differences between IGF-I and insulin --- p.13 / Chapter 2.4.2 --- Glucose and protein metabolism --- p.14 / Chapter 2.4.3 --- Therapeutic use of IGF-I --- p.15 / Chapter 2.4.3.1 --- Type 1 diabetes mellitus --- p.16 / Chapter 2.4.3.2 --- Type 2 diabetes mellitus --- p.17 / Chapter 2.4.4 --- Side effects --- p.19 / Chapter 2.5 --- World demands --- p.21 / Chapter 2.5.1 --- Significance of large-scale production --- p.21 / Chapter 2.5.2 --- IGF-I production --- p.21 / Chapter 2.6 --- Plants as bioreactors --- p.24 / Chapter 2.6.1 --- Medical molecular farming --- p.24 / Chapter 2.6.2 --- Advantages of plant bioreactor --- p.24 / Chapter 2.6.3 --- Commercial biopharmaceutical protein --- p.25 / Chapter 2.7 --- Tobacco expression system --- p.26 / Chapter 2.7.1 --- Tobacco model plant --- p.26 / Chapter 2.7.2 --- Transformation methods --- p.26 / Chapter 2.8 --- Hypotheses and aims of study --- p.28 / Chapter Chapter 3 --- Expression of Human IGF-I and IGFBP-3 in Transgenic Tobacco --- p.30 / Chapter 3.1 --- Introduction --- p.30 / Chapter 3.2 --- Materials and methods --- p.31 / Chapter 3.2.1 --- Chemicals --- p.31 / Chapter 3.2.2 --- Plant materials --- p.31 / Chapter 3.2.3 --- Bacterial strains --- p.32 / Chapter 3.2.4 --- Codon modification of IGF-I and IGFBP-3 cDNAs --- p.32 / Chapter 3.2.5 --- Transient assay to study IGF-I or IGFBP-3 translatability --- p.39 / Chapter 3.2.5.1 --- Construction of chimeric genes for particle bombardment --- p.39 / Chapter 3.2.5.2 --- Particle bombardment of GUS fusion constructs --- p.42 / Chapter 3.2.6 --- Construction of chimeric genes for tobacco transformation --- p.44 / Chapter 3.2.6.1 --- Construction of chimeric genes with different promoters --- p.44 / Chapter 3.2.6.1.1 --- Construction of chimeric gene with CaMV 35S promoter --- p.44 / Chapter 3.2.6.1.2 --- Construction of chimeric genes with phaseolin promoter --- p.46 / Chapter 3.2.6.2 --- Construction of fusion constructs --- p.48 / Chapter 3.2.6.2.1 --- Construction of GUS fusion constructs --- p.48 / Chapter 3.2.6.2.2 --- Construction of LRP fusion constructs --- p.51 / Chapter 3.2.6.3 --- Construction of phaseolin targeting constructs --- p.56 / Chapter 3.2.6.3.1 --- Construction of phaseolin targeting constructs without AFVY --- p.56 / Chapter 3.2.6.3.2 --- Construction of phaseolin targeting constructs with AFVY --- p.60 / Chapter 3.2.6.4 --- Cloning of chimeric genes into Agrobacterium binary vector pBI 121 --- p.64 / Chapter 3.2.7 --- Confirmation of sequencing fidelity of chimeric genes --- p.66 / Chapter 3.2.8 --- Transformation of Agrobacterium by electroporation --- p.66 / Chapter 3.2.9 --- Transformation of tobacco --- p.67 / Chapter 3.2.10 --- Selection and regeneration of transgenic tobacco --- p.67 / Chapter 3.2.11 --- GUS assay --- p.68 / Chapter 3.2.12 --- Extraction of leaf genomic DNA --- p.68 / Chapter 3.2.13 --- PCR of genomic DNA --- p.69 / Chapter 3.2.14 --- Synthesis of DIG-labeled double-stranded DNA probe --- p.69 / Chapter 3.2.15 --- Southern blot analysis --- p.70 / Chapter 3.2.16 --- Extraction of total RNA from leaves or developing seeds --- p.70 / Chapter 3.2.17 --- Northern blot analysis --- p.71 / Chapter 3.2.18 --- Extraction of total protein --- p.71 / Chapter 3.2.19 --- Tricine SDS-PAGE --- p.72 / Chapter 3.2.20 --- Western blot analysis --- p.72 / Chapter 3.2.21 --- Enterokinase digestion of fusion protein --- p.73 / Chapter Chapter 4 --- Results --- p.74 / Chapter 4.1 --- Particle bombardment for transient assay --- p.74 / Chapter 4.1.1 --- Construction of GUS fusion genes for particle bombardment --- p.74 / Chapter 4.1.2 --- Transient expression of GUS fusion genes in soybean cotyledons and tobacco leaves --- p.76 / Chapter 4.2 --- Construction of chimeric genes for tobacco transformation --- p.78 / Chapter 4.3 --- "Tobacco transformation, selection and regeneration" --- p.81 / Chapter 4.4 --- Detection of GUS activity --- p.83 / Chapter 4.5 --- Detection of transgene integration --- p.84 / Chapter 4.5.1 --- Extraction of genomic DNA and PCR --- p.84 / Chapter 4.5.2 --- Southern blot analysis --- p.88 / Chapter 4.6 --- Detection of transgene transcription --- p.92 / Chapter 4.6.1 --- Extraction of total RNA --- p.92 / Chapter 4.6.2 --- Northern blot analysis --- p.92 / Chapter 4.7 --- Detection of transgene translation --- p.99 / Chapter 4.7.1 --- Extraction of total protein and Tricine SDS-PAGE --- p.99 / Chapter 4.7.2 --- Western blot analysis --- p.102 / Chapter 4.7.3 --- Enterokinase digestion of fusion protein --- p.109 / Chapter Chapter 5 --- Discussion --- p.111 / Chapter 5.1 --- Codon modification of IGF-I and IGFBP-3 cDNAs --- p.114 / Chapter 5.2 --- Transient expression of IGF-I and IGFBP-3 cDNAs --- p.116 / Chapter 5.3 --- Fusion of IGF-I and IGFBP-3 cDNA with LRP gene --- p.118 / Chapter 5.4 --- Enterokinase digestion --- p.120 / Chapter 5.5 --- Phaseolin targeting signal --- p.122 / Chapter 5.6 --- Gene silencing --- p.124 / Chapter 5.7 --- Future perspectives --- p.128 / Chapter Chapter 6 --- Conclusion --- p.131 / References --- p.133

Insulin-like growth factor I (IGF-I) is a polypeptide protein hormone similar to insulin. It plays an important role in growth and anabolic effects in life. Most circulating IGF-I is bound to high-affinity insulin-like growth factor binding protein-3 (IGFBP-3), to form a complex (IGF-I/IGFBP-3) that can treat growth hormone insensitivity syndrome (GHIS) and can lower plasma glucose in diabetic patients. Its side effects can be reduced without affecting the therapeutic efficacy. Human insulin-like growth factor binding protein 3 (hIGFBP-3) alone is an anti-tumor agent. It has been shown to have anti-proliferation effect on numerous cancer cells, such as breast, prostate and liver cancers. / Our previous study has demonstrated that recombinant hIGF-I (rhIGF-I) and hIGFBP-3 (rhIGFBP-3) could be synthesized in transgenic tobacco plant. In the present study, we propose to establish an efficient bioreactor platform for mass production of hIGF-I and hIGFBP-3 in rice, as rice grain contains 8-15% of protein by dry weight. In order to enhance rhIGF-I and rhIGFBP-3 stability and yield, and to control their glycosylation, various constructs were designed and transformed into rice by Agrobacterium-mediated transformation. Protein targeting signal sequence (KDEL) was fused to direct the target proteins to specific compartments in rice grain for glycosylation in the Golgi apparatus or for stable accumulation without complex glycan processing in the endoplasmic reticulum. These expression constructs were driven by seed-specific glutelin promoter (Gt1pro). Western blot analysis showed that the rhIGF-I and rhIGFBP-3 were successfully expressed in transgenic rice grains. Biological activity of rhIGF-I was evidenced by the induction of membrane ruffling in L6 rat skeletal muscle cells, while rhIGFBP-3 was effective in inhibiting the effect of IGF-I on membrane ruffling of L6 cell. Moreover, rhIGFBP-3 was also found to inhibit the growth of human breast cancer MCF-7 cells. Biological activity results showed that the active expression levels of rhIGF-I and rhIGFBP-3 were found to be 10 ug and 7.36 ug per 1 g of rice seed respectively. These findings suggested that both rice-produced rhIGF-I and rhIGFBP-3 were biologically active. / Cheung, Chun Kai. / "September 2007." / Adviser: Peter Tong Chun Yip. / Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4555. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 209-243). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

O nível sérico do Fator de crescimento semelhante à insulina tipo I (IGF-I) é fundamental para auxiliar no dignóstico e controle terapêutico dos transtornos relacionados à secreção do Hormônio de Crescimento (GH), bem como no diagnóstico e seguimento de outras doenças. Estabelecer valores de referência para as dosagens séricas de IGF-I por um ensaio imunoquimioluminométrico (ICMA), utilizando o sistema automatizado Immulite 2000/Diagnostic Products Corporation (DPC), e por um ensaio imunoradiométrico (IRMA), utilizando o kit comercial ACTIVE IGF-I/Diagnostic System Laboratories (DSL)-5600, numa população brasileira adulta da cidade do Rio de Janeiro. Este estudo, aprovado pelo Comitê de Ética do Instituto Estadual de Hematologia Arthur de Siqueira Cavalcanti, Rio de Janeiro, Brasil, incluiu amostras de 484 indivíduos saudáveis (251 homens e 233 mulheres) com idades entre 18 e 70 anos. As amostras foram estudadas por ICMA- Immulite 2000/DPC and IRMA- ACTIVE IGF-I/DSL-5600. Para análise dos dados foram utilizados modelos específicos para idade e sexo, após transformação dos dados de IGF-I. Foi observada uma lenta diminuição dos níveis de IGF-I com a idade usando ambos os ensaios. Os níveis de IGF-I foram signicativamente (p=0,0181) mais elevados em mulheres do que em homens, quando as amostras foram analisadas usando ICMA. Não houve diferença significativa dos níveis de IGF-I entre homens e mulheres quando as amostras foram analisadas usando IRMA. Este estudo estabeleceu valores de referência de IGF-I específicos para idade e sexo, determinados com o sistema automatizado ICMA-Immulite 2000/DPC, e valores de referência de IGF-I específicos para idade, determinados com o kit comercial IRMA- ACTIVE IGF-I/DSL-5600, em uma população adulta brasileira, da cidade do Rio de Janeiro. / Serum level of insulin-like growth factor I (IGF-I) is fundamental in order to aid in the diagnosis and follow-up of growth hormone (GH)-related disorders, as well as in the diagnosis and follow-up of other diseases. The aim of this investigation was to determine reference values for IGF-I using an automated immunochemiluminometric assay (ICMA) system Immulite 2000/Diagnostic Products Corporation (DPC); and an immunoradiometric assay (IRMA), using the commercial kit ACTIVE IGF-I/Diagnostic System Laboratories (DSL)-5600, in an adult Brazilian population of Rio de Janeiro city. The study, approved by the Ethical Committee of the Instituto Estadual de Hematologia Arthur de Siqueira Cavalcanti, Rio de Janeiro, Brazil, included samples of blood taken from 484 healthy subjects (251men, 233 women) aged from 18 up to 70. The samples were analyzed by ICMA- Immulite 2000/DPC and IRMA- ACTIVE IGF-I/DSL-5600. For statistical analysis, age and sex-specific models were fitted after transformation of IGF-I values. In adulthood, a slow age-dependent decrease was found, using both assays. IGF-I in women were significantly (p=0,0181) higher than in men when samples were analayzed using ICMA.There was no significant difference between men and women IGF-I values when samples were analayzed using IRMA. The present study established age- and sex specific IGF-I reference values, determined with the automated system: ICMA-Immulite 2000/DPC and age-specific IGF-I reference values determined with the IRMA- ACTIVE IGF-I/DSL-5600, in an adult Brazilian population of Rio de Janeiro city.

Objective: Insulin-like growth factor-I (IGF-I) has been suggested to be a prognostic marker for the development of cancer and, more recently, cardiovascular disease. These diseases are closely linked to obesity, but reports of the association of IGF-I with measures of obesity are divergent. In this study, we assessed the association of age-dependent IGF-I standard deviation scores with body mass index (BMI) and intra-abdominal fat accumulation in a large population. Design: A cross-sectional, epidemiological study. Methods: IGF-I levels were measured with an automated chemiluminescence assay system in 6282 patients from the DETECT study. Weight, height, and waist and hip circumference were measured according to the written instructions. Standard deviation scores (SDS), correcting IGF-I levels for age, were calculated and were used for further analyses. Results: An inverse U-shaped association of IGF-I SDS with BMI, waist circumference, and the ratio of waist circumference to height was found. BMI was positively associated with IGF-I SDS in normal weight subjects, and negatively associated in obese subjects. The highest mean IGF-I SDS were seen at a BMI of 22.5–25 kg/m2 in men (+0.08), and at a BMI of 27.5–30 kg/m2 in women (+0.21). Multiple linear regression models, controlling for different diseases, medications and risk conditions, revealed a significant negative association of BMI with IGF-I SDS. BMI contributed most to the additional explained variance to the other health conditions. Conclusions: IGF-I standard deviation scores are decreased in obesity and underweight subjects. These interactions should be taken into account when analyzing the association of IGF-I with diseases and risk conditions.

Objective: IGF1 is associated with metabolic parameters and involved in glucose metabolism. Low-IGF1 has been implicated in the etiology of glucose intolerance and subjects with pathological causes of either low- or high-IGF1 are at risk of diabetes. We hypothesized that both low- and high-IGF1 levels increase the risk of diabetes and aimed to assess the role of IGF1 in the risk of developing diabetes in a large prospective study. Design: An analysis of two prospective cohort studies, the DETECT study and SHIP. Methods: We measured IGF1 levels in 7777 nondiabetic subjects and assessed incident diabetes mellitus during follow-up. Results: There were 464 cases of incident diabetes during 32 229 person-years (time of follow-up in the DETECT study and SHIP: 4.5 and 5 years respectively). There was no heterogeneity between both studies (P>0.4). The hazard ratios (HRs) of incident diabetes in subjects with IGF1 levels below the 10th or above the 90th age- and sex-specific percentile, compared to subjects with intermediate IGF1 levels, were 1.44 (95% confidence interval (CI) 1.07–1.94) and 1.55 (95% CI 1.06–2.06) respectively, after multiple adjustment. After further adjustment for metabolic parameters, the HR for low-IGF1 became insignificant. Analysis of IGF1 quintiles revealed a U-shaped association of IGF1 with risk of diabetes. Results remained similar after exclusion of patients with onset of new diabetes within 1 year or with borderline glucose or HbA1c levels at baseline. Conclusions: Subjects with low- or high-IGF1 level are at increased risk of developing diabetes.

Thesis (Ph. D.)--University of Washington, 2003. / Includes bibliographical references (leaves 101-113).

Tourniquet mediated Ischemia Reperfusion (I/R) injury causes damage to skeletal muscle, often resulting in prolonged functional impairment. The current study utilizes immunohistochemistry (IHC) to determine whether the controlled release of the anabolic factor, insulin-like growth factor-I (IGF-I), from the biodegradable PEGylated fibrin gel matrix can facilitate the recovery of skeletal muscle from I/R. Treatment groups following a 2-hour tourniquet applied to the limb of 6-9 month rats, included intramuscular injections of saline, PEGylated fibrin gel (PEG-Fib) only and IGF-I conjugated to PEGylated fibrin gel (PEG-Fib-IGF). Expression of the myogenic regulatory factors MyoD and myogenin detected via IHC in the PEG-Fib-IGF group was significantly lower compared to the saline group, showing a 1.4±0.8% nuclear co-localization for MyoD and a 2.0±0.8% nuclear co-localization for myogenin at 14 days of recovery. The saline group showed higher values, 31.4±4.4% and 44.1±7.3% for MyoD and myogenin nuclear co-localization respectively. A significantly greater percentage, 88.8±3.7% of Desmin positive myofibers was seen at 14 days of recovery, while a lower percentage of fibers expressing neonatal myosin, 7.7±2.7% was seen in the PEG-Fib-IGF group compared to the saline treatment group. These results indicate that IGF-I delivered intramuscularly via PEGylated fibrin gel, functions therapeutically in skeletal muscle recovery, from I/R mediated damage. In a separate injury model that deals with volumetric muscle loss, IHC analyses were performed to test the efficacy of a novel tissue engineering strategy utilizing extracellular matrix (ECM) as a scaffold. In this model, also called the defect model, a 1.0 X 1.0 cm piece of the lateral gastrocnemius was removed and replaced with a muscle-derived ECM. The constructs were then seeded with bone marrow derived cells (BMSCs), adipose derived stem cells (ADSCs) or the peroneal nerve was relocated to the area of the ECM implant. 42 days post recovery IHC analysis was performed on the ECM implants. The quantification of desmin-positive regenerating myofibers bearing centrally located nuclei, showed significantly greater values in the top, middle and bottom region of the ECM implants that received peroneal nerve relocation, when compared to the experimental group that received the ECM implant alone. Blood vessel density increases were seen within the middle region of the ECM implant groups that received BMSC+Nerve treatment and the bottom region of the ECM implant groups that received ADSC+Nerve treatment. Thus, these results corroborate the therapeutic effect of peroneal nerve relocation, which stimulated an increase in myofiber regeneration and vascular maintenance within the construct. / text

Prostate cancer (PCa) represents the most frequently diagnosed cancer and the second leading cause of cancer death in males. Initial development and progression of the disease is mainly regulated by androgens. However, the pathology of the disease may progress to a loss of hormone dependence, resulting in rapid growth and a metastatic phenotype. Invasion and metastasis of tumour cells results from the degradation of the basement membrane (BM) and extracellular matrix (ECM). The degradation of the BM and ECM is in part mediated by a family of proteinases called the matrix metalloproteinases (MMPs). Currently more than 20 members of the MMP family have been identified and they are further divided in to sub-classes according to their protein structure. Collectively, MMPs are capable of degrading essentially all ECM components. High expression of some MMPs correlates with a malignant phenotype of various tumours. This study focused on the expression and regulation of a sub-class of MMPs called the membrane-type MMPs (MT-MMPs) in PCa. To date 6 MT-MMPs have been identified and they are characterized by a transmembrane domain, followed by a short cytoplasmic tail (MT1-, MT2-, MT3- and MT5-MMPs) or a glycosylphosphatidylinositol (GPI) moiety (MT4- and MT6-MMPs). MT-MMPs are thought to play a key role in tumour cell invasion by virtue of their ability to activate MMP-2 (a secreted MMP, which is implicated in many metastatic tumours) and their direct degradation activity on ECM components. Elevated MT-MMP expression has been shown in breast, colon, skin, stomach, lung, pancreas and brain cancers. Until very recently there had been no studies conducted on MT-MMPs in PCa. The few studies preceding or occurring in parallel with this one, have mainly reported the mRNA expression of these enzymes in PCa. Most studies have focused on MT1-MMP. Thus, at the commencement of this project there were many unexplored aspects of the expression and regulation of the broader MT-MMP family in PCa. The aims of this study were to examine: 1 a) The expression of MT-MMPs in prostate cancer cell lines using RT-PCR and western blot analysis and b) expression of MT1-MMP and MT5-MMP in BPH (benign prostatic hyperplasia) and PCa clinical tissue sections by immunohistochemistry. 2) The regulation of MT1-MMP, MT3-MMP and MT5-MMP in PCa cell lines by Concanavalin A (Con A), phorbol-12-myristate 13-acetate (PMA), dihydrotestosterone (DHT) and insulin-like growth factors I and II (IGF I and IGF II) using western blot analysis. In this study RWPE1, a transformed but non-tumorigenic prostate cell line was used as a "normal" prostate cell model, ALVA-41 and LNCaP as androgen-dependent PCa cell models and DU-145 and PC-3 as androgen-independent PCa cell models. The mRNA expression for the 6 MT-MMPs was determined by RT-PCR. The results indicate that MT1- and MT3-MMP were detected in all cell lines. This is the first study to report MT1-MMP mRNA expression in LNCaP cells and MT3-MMP mRNA in DU-145 cells. MT2-MMP mRNA was detected in only LNCaP and DU-145 cells, whilst MT5-MMP was detected in PC-3, DU-145 and LNCaP cells. nterestingly, MT2-, MT4-, MT5- or MT6-MMP mRNA expression was not detected in the "normal" cell line RWPE1, perhaps indicating an induction in gene transcription in tumour cells. MT4-MMP mRNA was only detected in the androgen-independent cell lines, indicating a potential role in the invasion and metastasis processes of the aggressive androgen-independent PCa. In this study, very low expression of MT6-MMP was detected only in LNCaP and DU-145 cells. Previously there had been no reports on the expression of MT6-MMP in the normal or cancerous prostate. Due to the mRNA of MT1-, MT3- and MT5-MMPs being the predominant MT-MMPs expressed in the current study, and the availability of suitable antibodies against them, the protein expression of these three MT-MMPs was studied by western blot analysis. MT1-, MT3- and MT5-MMP protein expression was detected in the cell lysates and conditioned medium (CM) of RWPE1, LNCaP and PC-3 cells. For each MT-MMP, various protein species were detected including putative proforms, mature (active) forms, processed or fragmented forms as well as soluble or shed forms. The presence of soluble or shed forms of MT-MMPs in the CM of cultures of "normal" and PCa cells could imply one of the following mechanisms: ectodomain shedding by either extracellular sheddases, the secretion of intracellular processed proteins without the transmembrane domain, the release of membrane vesicles containing membrane-bound enzymes, or the presence of alternatively spliced mRNA, which gives rise to MT-MMPs without a transmembrane domain. Further characterization of these various forms, including their amino acid sequence, is required to fully elucidate their structural composition. Despite the detection of the mRNA, we did not detect the cell-associated proteins of MT1-MMP and MT5-MMP and only very low expression of MT3-MMP in DU-145 cells (CM of DU-145 cells were not screened for soluble forms of the enzymes). This is the first study to report MT5-MMP expression at the protein level in prostate derived cell lines. Immunohistochemistry was carried out on benign prostatic hyperplasia (BPH) and PCa clinical tissues using MT1- and MT5-MMP antibodies to determine their cellular localisation in benign and cancer glands. MT1- and MT5-MMPs were expressed in BPH and moderate and high grade PCa. MT1-MMP expression was highest in moderate grade cancer compared to BPH and high grade cancer. MT1-MMP expression was predominantly observed in the cytoplasm of secretory epithelial cells of both benign and cancer glands, although in cancer glands, some nuclear staining was also observed. Stromal expression of MT1-MMP was only observed in high grade cancer. This study is the first to report the immunolocalization of MT5-MMP outside the brain and in kidneys of diabetic patients. MT5-MMP was predominantly expressed in the cytoplasm of the secretory cells in benign glands. In the cancer glands, staining was heterogeneous with low to intense staining, mainly in the nuclei, plasma membrane and cytoplasm of secretory epithelial cells. Stromal expression of MT5-MMP was only observed in cancer tissues, particularly in high grade cancer. To study the regulation of MT-MMPs in PCa, we treated LNCaP and PC-3 cells, with either Con A, PMA, DHT or IGF-I and -II and studied the protein expression of MT1-, MT3- and MT5-MMPs by western blot analysis. Con A and PMA have been shown to stimulate MMP expression in other cell systems. Con A treatment showed a general increase in the protein expression of MT1-, MT3- and MT5-MMPs. By far the greatest induction by Con A observed was the nearly 4 fold increase in MT5-MMP expression caused by 40μg/mL Con A treatment of PC-3 cells. PMA treatment of LNCaP and PC-3 cells appeared to increase shedding or secretion of all three MT-MMPs in to the CM. This increase in the soluble forms corresponded to a decrease in cell-associated forms in LNCaP cells. Treatment of LNCaP with DHT alone and treatment of LNCaP and PC-3 cells with IGF-I and -II alone failed to detect any change in expression of MT1-MMP. The information gathered in this study on MT-MMPs with respect to cellular localization, expression levels and regulation by growth factors or chemicals that mimic their actions, will aid in our understanding of the role of MT-MMPs in PCa. This study provides strong preliminary data for further research, particularly with respect to functional studies of MT-MMPs in PCa. Understanding the processes which govern the actions of such proteins as these will provide potential insights into development of new management and therapeutic regimens to prevent cancer progression.