Assessment of freestanding membranes prepared from Antheraea pernyi silk fibroin as a potential vehicle for corneal epithelial cell transplantation

Freestanding membranes created from Bombyx mori silk fibroin (BMSF) offer a potential vehicle for corneal cell transplantation since they are transparent and support the growth of human corneal epithelial cells (HCE). Fibroin derived from the wild silkworm Antheraea pernyi (APSF) might provide a superior material by virtue of containing putative cell- attachment sites that are absent from BMSF. Thus we have investigated the feasibility of producing transparent, freestanding membranes from APSF and have analysed the behaviour of HCE cells on this material. No significant differences in cell numbers or phenotype were observed in short term HCE cell cultures established on either fibroin. Production of transparent freestanding APSF membranes, however, proved to be problematic as cast solutions of APSF were more prone to becoming opaque, displayed significantly lower permeability and were more brittle than BMSF-membranes. Cultures of HCE cells established on either membrane developed a normal stratified morphology with cytokeratin pair 3/12 being immuno-localized to the superficial layers. We conclude that while it is feasible to produce transparent freestanding membranes from APSF, the technical difficulties associated with this biomaterial, along with an absence of enhanced cell growth, currently favours the continued development of BMSF as a preferred vehicle for corneal cell transplantation. Nevertheless, it remains possible that refinement of techniques for processing APSF might yet lead to improvements in the handling properties and performance of this material.

Transplantation of human amnion epithelial cells reduces hepatic fibrosis in immunocompetent CCl4-treated mice

Chronic liver injury and inflammation lead to hepatic fibrosis, cirrhosis, and liver failure. Embryonic and mesenchymal stem cells have been shown to reduce experimental liver fibrosis but have potential limitations, including the formation of dysplastic precursors, tumors, and profibrogenic cells. Other stem-like cells may reduce hepatic inflammation and fibrosis without tumor and profibrogenic cell formation. To test this hypothesis we transplanted human amnion epithelial cells (hAEC), isolated from term delivered placenta, into immunocompetent C57/BL6 mice at week 2 of a 4-week regimen of carbon tetrachloride (CCl4) exposure to induce liver fibrosis. Two weeks following hAEC infusion, intact cells expressing the human-specific markers inner mitochondrial membrane protein and human leukocyte antigen-G were found in mouse liver without evidence of host rejection of the transplanted cells. Human albumin, known to be produced by hAEC, was detected in sera of hAEC-treated mice. Human DNA was detected in mouse liver and also spleen, lungs, and heart of some animals. Following hAEC transplantation, CCl4-treated animals showed decreased serum ALT levels and reduced hepatocyte apoptosis, compared to controls. hAEC-treated mouse liver had lower TNF-α and IL-6 protein levels and higher IL-10 compared to animals given CCl4 alone. Compared to CCl4 controls, hAEC-treated mice showed fewer activated collagen-producing hepatic stellate cells and less fibrosis area and collagen content. Reduced hepatic TGF-β levels in conjunction with a twofold increase in the active form of the collagen-degrading enzyme matrix metalloproteinase-2 in hAEC-treated mice compared to CCl4 controls may account for the reduction in fibrosis. hAEC transplantation into immunocompetent mice leads to cell engraftment, reduced hepatocyte apoptosis, and decreased hepatic inflammation and fibrosis.

Oral contraceptives prevent the development of endometriosis in the chicken chorioallantoic membrane model

Background: Fundamental and genetic differences between women in the endometrium may cause some to develop endometriosis, whereas others (to not. Oral contraceptives (OC) may have in effect on the endometrium, rendering the development of endometriosis less likely. Study Design: Endometrium front women using CC (OCE) and menstrual endometrium (ME) from normal cycling women were transplanted onto the chicken chorioallantoic membrane (CAM), and endometriosis-like lesion formation was evalualed. Microarray gene expression profiling was performed to identify, differentially expressed genes in the endometrium front these groups. Microarray data were validated by real-time PCR. Results: Less endometriosis-like lesions were formed after transplantation of OCE than after transplantation of ME (p<.05). Most of the differentially expressed genes belong to the TGF beta superfamily. Real-time PCR validation revealed that inhibin beta A (INHBA) expression was significantly decreased in OCE its compared to ME. Conclusion: OC use affects the characteristics Of endometrium, rendering it less potent to develop into endometriosis. (C) 2008 Elsevier Inc. All rights reserved.

Biomaterial templates for the culture and transplantation of retinal pigment epithelial cells: A critical review

The idea of retinal cell transplantation as a potential treatment for age-related retinal degeneration, a leading cause of blindness in the Western world, has been around for a number of decades. To date, however, it has not been entirely successful; one of the main reasons for this is the lack of an ideal substratum for the retinal cells, specifically for the growth of retinal pigment epithelial cells prior to transplantation. This chapter reviews the reasoning behind this potential treatment, the development of animal transplantation models for human trials, the prerequisites of an ideal substratum, the past and current research on substratum materials, and the potential for future developments in this area.

Progression of nephropathy after islet of langerhans transplantation in alloxan-induced diabetic rats

We studied the effects of islet of Langerhans transplantation (IT) on the kidney lesions of rats with alloxan-induced diabetes. Forty-five inbred male Lewis rats were randomly assigned to 3 experimental groups: group Gl included 15 non-diabetic control rats (NC), group GIT included 15 alloxan-induced diabetic rats (DC), and group III included 15 alloxan-induced diabetic rats that received pancreatic islet transplantation prepared by nonenzymatic method from normal donor Lewis rats and injected into the portal vein (IT). Each group was further divided into 3 subgroups of 5 rats which were sacrificed at 1, 3, and 6 months of follow-up, respectively. Clinical and laboratorial parameters were recorded in the mentioned periods in the 3 experimental groups. For histology, the kidneys of all rats of each subgroup were studied and 50 glomeruli and 50 tubules of each kidney were analyzed using light microscopy by two different investigators in a double blind study. The results showed progressive glomerular basement membrane thickening (GBMT), mesangial enlargement (ME), and Bowman's capsule thickening (BCT) in the 3 experimental groups throughout the follow-up. These alterations were significantly more severe in DC rats at 6 months when compared to NC rats (p < 0.01). However, the degree of GBMT, ME, and BCT observed in DC rats was not statistically different from IT rats at 1, 3, and 6 months. In addition, Armanni-Ebstein lesions of the tubules (AE) and tubular lumen protein (PRO) observed in DC rats were also observed in IT rats all over the study. These lesions were never present in NC rats. We conclude that IT did not prevent progression of kidney lesions in alloxan-induced diabetic rats within 6 months after transplantation.

Long-term effects of insulin therapy, islet transplantation, and pancreas transplantation in the prevention of glomerular changes in kidneys of alloxan-induced diabetic rats

Groups of inbred alloxan-induced diabetic rats were treated with insulin (I), islets (IT), or pancreas transplantation (PT). Nondiabetic (N) and untreated diabetic (D) control groups were concurrently included. Each group was divided into five subgroups of 10 rats and killed after follow-up of 1, 3, 6, 9, and 12 months. Clinical and laboratory parameters were recorded, and kidney ultrastructural and morphometric analyses performed in each 12-month subgroup, namely glomerular basement membrane (GM) thickening, podocyte number, and number/extension of slit diaphragms (S). Rats from the I group showed poor metabolic control of diabetes compared with N group control rats. However, successfully transplanted rats (IT and PT) had complete restoration to normal levels for all metabolic parameters. GM thickening was significantly higher in diabetic compared with control rats. In contrast, the numbers of podocytes and slits as well as slit extensions were significantly decreased. Insulin therapy did not prevent any alterations upon comparison of diabetic vs control rats. Despite good metabolic control in IT rats, the degree of kidney lesion control never compared with that achieved in PT rats. In this group all glomerular changes were similar to the age-dependent lesions observed in control rats. We conclude that either IT or PT may be a good option for diabetes treatment, although pancreas transplantation seems to be the most effective treatment to control chronic complications.

Effect of pancreas transplantation on the prevention of nephropathy in alloxan-induced diabetic rats

We studied the effects of pancreas transplantation on kidney lesions of rats with alloxan-induced diabetes. Ninety inbred male Lewis rats were randomly assigned to 3 experimental groups: group NC included 30 non-diabetic control rats, group DC included 30 alloxan-induced diabetic control rats, and group PT included 30 alloxan-induced diabetic rats that received pancreas transplants from normal donor Lewis rats. Each group was further divided into 3 subgroups of 10 rats which were sacrificed at 1, 3, and 6 months of follow-up, respectively. Clinical and laboratory parameters during these periods were documented. The kidneys of 5 rats in each subgroup were studied and 50 glomeruli and tubules from each kidney were analyzed by light microscopy by two different investigators in a double-blind study. There was progressive glomerular basement membrane thickening (GBMT), mesangial enlargement (ME), and Bowman's capsule thickening (BCT) in kidneys of rats in the 3 experimental groups during follow-up. These alterations were significantly higher in DC rats (GBMT: 1.99 +/- 0.31; ME: 2.00 +/- 0.33; BCT: 1.88 +/- 0.27) when compared to NC(GBMT: 1.54 +/- 0.30; ME: 1.56 +/- 0.47; BCT: 1.36 +/- 0.35) and PT rats (GBMT: 1.49 +/- 0.29; ME: 1.57 +/- 0.36; BCT: 1.35 +/- 0.28) at 6 months (P<0.01). The extent of GBMT, ME, and BCT observed in DC rats at 1 and 3 months was not significantly different from NC and PT rats. The amount of kidney lesions in PT rats was similar to that of NC rats and lower than those of DC rats at 6 months (P<0.01). In addition, Armanni-Ebstein lesions of the tubules (AE) and tubular lumen protein (PRO) observed in DC rats were not present in NC or PT rats. We conclude that pancreas transplantation in alloxan-induced diabetic rats prevents the development of kidney lesions beginning at 6 months after transplantation.

Bone reformation and implant integration following maxillary sinus membrane elevation: An experimental study in primates

Background: Recent clinical studies have described maxillary sinus floor augmentation by simply elevating the maxillary sinus membrane without the use of adjunctive grafting materials. Purpose: This experimental study aimed at comparing the histologic outcomes of sinus membrane elevation and simultaneous placement of implants with and without adjunctive autogenous bone grafts. The purpose was also to investigate the role played by the implant surface in osseointegration under such circumstances. Materials and Methods: Four tufted capuchin primates had all upper premolars and the first molar extracted bilaterally. Four months later, the animals underwent maxillary sinus membrane elevation surgery using a replaceable bone window technique. The schneiderian membrane was kept elevated by insertion of two implants (turned and oxidized, Brånemark System®, Nobel Biocare AB, Göteborg, Sweden) in both sinuses. The right sinus was left with no additional treatment, whereas the left sinus was filled with autogenous bone graft. Implant stability was assessed through resonance frequency analysis (Osstell™, Integration Diagnostics AB, Göteborg, Sweden) at installation and at sacrifice. The pattern of bone formation in the experimental sites and related to the different implant surfaces was investigated using fluorochromes. The animals were sacrificed 6 months after the maxillary sinus floor augmentation procedure for histology and histomorphometry (bone-implant contact, bone area in threads, and bone area in rectangle). Results: The results showed no differences between membrane-elevated and grafted sites regarding implant stability, bone-implant contacts, and bone area within and outside implant threads. The oxidized implants exhibited improved integration compared with turned ones as higher values of bone-implant contact and bone area within threads were observed. Conclusions: The amount of augmented bone tissue in the maxillary sinus after sinus membrane elevation with or without adjunctive autogenous bone grafts does not differ after 6 months of healing. New bone is frequently deposited in contact with the schneiderian membrane in coagulum-alone sites, indicating the osteoinductive potential of the membrane. Oxidized implants show a stronger bone tissue response than turned implants in sinus floor augmentation procedures. © 2006 Blackwell Publishing, Inc.

Reconstruction of Mandibular Segmental Defects Using the Guided-Bone Regeneration Technique With Polylactide Membranes and/or Autogenous Bone Graft: A Preliminary Study on the Influence of Membrane Permeability

Purpose: Bone maintenance after mandibular reconstruction with autogenous iliac crest may be disappointing due to extensive resorption in the long term. The potential of the guided-bone regeneration (GBR) technique to enhance the healing process in segmental defects lacks comprehensive scientific documentation. This study aimed to investigate the influence of polylactide membrane permeability on the fate of iliac bone graft (BG) used to treat mandibular segmental defects. Materials and Methods: Unilateral 10-mm-wide segmental defects were created through the mandibles of 34 mongrel dogs. All defects were mechanically stabilized, and the animals were divided into 6 treatment groups: control, BG alone, microporous membrane (poly L/DL-lactide 80/20%) (Mi); Mi plus BG; microporous laser-perforated (15 cm2 ratio) membrane (Mip), and Mip plus BG. Calcein fluorochrome was injected intravenously at 3 months, and animal euthanasia was carried out at 6 months postoperatively. Results: Histomorphometry showed that BG protected by Mip was consistently related to larger amounts of bone compared with other groups (P ≤ .0001). No difference was found between defects treated with Mip alone and BG alone. Mi alone rendered the least bone area and reduced the amount of grafted bone to control levels. Data from bone labeling indicated that the bone formation process was incipient in the BG group at 3 months postoperatively regardless of whether or not it was covered by membrane. In contrast, GBR with Mip tended to enhance bone formation activity at 3 months. Conclusions: The use of Mip alone could be a useful alternative to BG. The combination of Mip membrane and BG efficiently delivered increased bone amounts in segmental defects compared with other treatment modalities. © 2008 American Association of Oral and Maxillofacial Surgeons.

Treatment of gingival recession with collagen membrane and DFDBA: A histometric study in dogs

In a previous study, we evaluated the findings related to the use of resorbable collagen membranes in humans along with DFDBA (demineralized freeze-dried bone allograft). The aim of this subsequent study was to histometrically evaluate in dogs, the healing response of gingival recessions treated with collagen membrane + DFDBA (Guided Tissue Regeneration, GTR) compared to a coronally positioned flap (CPF). Two types of treatment were randomly carried out in a split-mouth study. Group 1 was considered as test (GTR: collagen membrane + DFDBA), whereas Group 2 stood for the control (only CPF). The dogs were given chemical bacterial plaque control with 0.2% chlorhexidine digluconate during a 90-day repair period. Afterwards, the animals were killed to obtain biopsies and histometric evaluation of the process of cementum and bone formation, epithelial migration and gingival level. A statistically significant difference was found between groups with a larger extension of neoformed cementum (GTR = 32.72%; CPF = 18.82%; p = 0.0004), new bone (GTR = 23.20%; CPF = 09.90%; p = 0.0401) and with a smaller area of residual gingival recession in the test group (GTR = 50.69%; CPF = 59.73%; p = 0.0055) compared to the control group. The only item assessed that showed no statistical difference was epithelial proliferation on the root surface, with means of 15.14% for the GTR group and 20.34% for the CPF group (p = 0.0890). Within the limits of this study we concluded that the treatment of gingival recession defects with GTR, associating collagen membrane with DFDBA, showed better outcomes in terms of a larger extension of neoformed cementum and bone, as well as in terms of a smaller proportion of residual recessions.

Expression profiles of fetal membrane nicotinamide adenine dinucleotide phosphate oxidases (NOX) 2 and 3 differentiates spontaneous preterm birth and pPROM pathophysiologies

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Protective Effects of Human Amniotic Fluid Stem Cells in a Model of Aorta Allograft Vasculopathy in Rats

Background. Chronic allograft vasculopathy (CAV) is an important cause of graft loss. Considering the immune inflammatory events involved in the development of CAV, therapeutic approaches to target this process are of relevance. Human amniotic fluid derived stem cells (hAFSCs), a class of fetal, pluripotent stem cells with intermediate characteristics between embryonic and adult stem cells, display immunomodulatory properties. hAFSCs express mesenchymal and embryonic markers, show high proliferation rates; however, they do not induce tumor formation, and their use does not raise ethical issues. Thus, we sought to investigate the effect of hAFSC on CAV in a model of aorta transplantation. Methods. Orthotopic aorta transplantation was performed using Fisher (F344) rats as donors and Lewis rats as recipients. Rats were divided into three groups: syngeneic (SYNG), untreated F344 receiving aorta from F344 (n = 8); allogeneic (ALLO), Lewis rats receiving allogeneic aorta from F344 (n = 8); and ALLO + hAFSC, ALLO rats treated with hAFSC (10(6) cells; n = 8). Histological analysis and immunohistochemistry were performed 30 days posttransplantation. Results. The ALLO group developed a robust aortic neointimal formation (208.7 +/- 25.4 gm) accompanied by a significant high number of ED1(+) (4845 +/- 841 cells/mm(2)) and CD43(+) cells (4064 +/- 563 cells/mm(2)), and enhanced expression of a-smooth muscle actin in the neointima (25 +/- 6%). Treatment with hAFSC diminished neointimal thickness (180.7 +/- 23.7 mu m) and induced a significant decrease of ED1(+) (1100 +/- 276 cells/mm(2)), CD43(+) cells (1080 +/- 309 cells/mu m(2)), and alpha-smooth muscle actin expression 8 +/- 3% in the neointima. Conclusions. These preliminary results showed that hAFSC suppressed inflammation and myofibroblast migration to the intima, which may contribute to ameliorate vascular changes in CAV.

Upregulation of soluble and membrane-bound human leukocyte antigen G expression is primarily observed in the milder histopathological stages of chronic hepatitis C virus infection

Chronic hepatitis C virus (HCV) infection is a worldwide health problem that may evolve to cirrhosis and hepatocellular carcinoma. Incompletely understood immune system mechanisms have been associated with impaired viral clearance. The nonclassical class I human leukocyte antigen G (HLA-G) molecule may downregulate immune system cell functions exhibiting well-recognized tolerogenic properties. HCV genotype was analyzed in chronic HCV-infected patients. Because HLA-G expression may be induced by certain viruses, we evaluated the presence of HLA-G in the liver microenvironment obtained from 89 biopsies of patients harboring chronic HCV infection and stratified according to clinical and histopathological features. Overall, data indicated that HCV genotype 1 was predominant, especially subgenotype 1a, with a prevalence of 87%. HLA-G expression was observed in 45(51%) liver specimens, and it was more frequent in milder stages of chronic hepatitis (67.4%) than in moderate (27.8%; p = 0.009) and severe (36.0%; p = 0.021) stages of the disease. Altogether, these results suggest that the expression of HLA-G in the context of HCV is a complex process modulated by many factors, which may contribute to an immunologic environment favoring viral persistence. However, because the milder forms predominantly expressed HLA-G, a protective role of this molecule may not be excluded. (C) 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

Orthotopic transplantation of v-src-expressing glioma cell lines into immunocompetent mice: establishment of a new transplantable in vivo model for malignant glioma

OBJECT: The aim of this study was to develop and characterize a new orthotopic, syngeneic, transplantable mouse brain tumor model by using the cell lines Tu-9648 and Tu-2449, which were previously isolated from tumors that arose spontaneously in glial fibrillary acidic protein (GFAP)-v-src transgenic mice. METHODS: Striatal implantation of a 1-microl suspension of 5000 to 10,000 cells from either clone into syngeneic B6C3F1 mice resulted in tumors that were histologically identified as malignant gliomas. Prior subcutaneous inoculations with irradiated autologous cells inhibited the otherwise robust development of a microscopically infiltrating malignant glioma. Untreated mice with implanted tumor cells were killed 12 days later, when the resultant gliomas were several millimeters in diameter. Immunohistochemically, the gliomas displayed both the astroglial marker GFAP and the oncogenic form of signal transducer and activator of transcription-3 (Stat3). This form is called tyrosine-705 phosphorylated Stat3, and is found in many malignant entities, including human gliomas. Phosphorylated Stat3 was particularly prominent, not only in the nucleus but also in the plasma membrane of peripherally infiltrating glioma cells, reflecting persistent overactivation of the Janus kinase/Stat3 signal transduction pathway. The Tu-2449 cells exhibited three non-random structural chromosomal aberrations, including a deletion of the long arm of chromosome 2 and an apparently balanced translocation between chromosomes 1 and 3. The GFAP-v-src transgene was mapped to the pericentromeric region of chromosome 18. CONCLUSIONS: The high rate of engraftment, the similarity to the high-grade malignant glioma of origin, and the rapid, locally invasive growth of these tumors should make this murine model useful in testing novel therapies for human malignant gliomas.

[Post-traumatic ARDS--a potential indication for lung transplantation?]

Posttraumatic adult respiratory distress syndrome (ARDS) still involves significant mortality, despite progress in management concepts. Current therapeutic strategies are briefly described, including kinetic therapy, high-frequency jet ventilation and extracorporeal membrane oxygenation. In addition, a spectacular case of the first successful lung transplantation for posttraumatic ARDS after failed ECMO (extracorporeal membrane oxygenation) support is reported. This young man with severe posttraumatic ARDS developed a potentially lethal bilateral pulmonary hemorrhage under treatment with ECMO, and on the basis of this bilateral pulmonary transplantation was considered to be indicated. The patient is alive and well 2 years after the procedure.