Yes / While achieving the spatial organization of cells within 3D assembled nanofiber/cell constructs via nanofiber-enabled cell layering, the small sizes of inter-fiber pores of the electrospun nanofiber mats could significantly limit cell penetration across the layers for rapid formation of an integrated tissue construct. To address this challenge, efforts were made to improve cell-infiltration of electrospun nanofiber mats by modulating the density distribution and spatial organization of the fibers during electrospinning. Collection of collagen-containing electrospun nanofibers (300–600 nm in diameter) onto the surface of a stainless steel metal mesh (1 mm × 1 mm in mesh size) led to the periodic alternation of fiber density from densely packed to loosely arranged distribution within the same mat, in which the densely packed fibers maintained the structural integrity while the region of loose fibers allowed for cell penetration. Along with improved cell infiltration, the distinct fiber organization between dense and loose fiber regions also induced different morphology of fibroblasts (stellate vs. elongated spindle-like). Assembly of cell-seeded nanofiber sheets into 3D constructs with such periodically organized nanofiber mats further demonstrated their advantages in improving cell penetration across layers in comparison to either random or aligned nanofiber mats. Taken together, modulation of nanofiber density to enlarge the pore size is effective to improve cell infiltration through electrospun mats for better tissue formation. / NSF-IIP. Grant Numbers: 1338958, 1346430; NSF-DMR. Grant Number: 1508511; NSF-CBET. Grant Number: 1033742; and NIAMS. Grant Number: 1R21 AR056416

Long-term survival of implanted cells requires oxygen and nutrients, the need for which is met by vasculari- zation of the implant. The use of scaffolds with surface-attached heparin as anchoring points for angiogenic growth factors has been reported to improve this process. We examined the potential role of surface modification of gelatin scaffolds in promoting endothelial cell infiltration by using a unique macromolecular conjugate of heparin as a coating. Compared to other heparin coatings, this surface modification provides flexible heparin chains, representing a new concept in heparin conjugation. In vitro cell infiltration of scaffolds was assessed using a three-dimensional model in which the novel heparin surface, without growth factors, showed a 2.5-fold increase in the number of infiltrating endothelial cells when compared to control scaffolds. No additional improvement was achieved by adding growth factors (vascular endothelial growth factor and/or fibroblast growth factor-2) to the scaffold. In vivo experiments confirmed these results and also showed that the addition of angiogenic growth factors did not significantly increase the endothelial cell infiltration but increased the number of inflammatory cells in the implanted scaffolds. The endothelial cell-stimulating ability of the heparin surface alone, combined with its growth factor-binding capacity, renders it an interesting candidate surface treatment to create a prevascularized site prepared for implantation of cells and tissues, in particular those sensitive to inflammation but in need of supportive revascularization, such as pancreatic islets of Langerhans. / <p>De två sista författarna delar sistaförfattarskapet.</p>

The role of homologous recombination deficiency (HRD) in lower grade glioma (LGG) has not been elucidated, and accurate prognostic prediction is also important for the treatment and management of LGG. The aim of this study was to construct an HRD-based risk model and to explore the immunological and molecular characteristics of this risk model. The HRD score threshold = 10 was determined from 506 LGG samples in The Cancer Genome Atlas cohort using the best cut-off value, and patients with highHRDscores had worse overall survival. A total of 251 HRD-related genes were identified by analyzing differentially expressed genes, 182 of which were associated with survival. A risk score model based on HRD-related genes was constructed using univariate Cox regression, least absolute shrinkage and selection operator regression, and stepwise regression, and patients were divided into high- and low-risk groups using the median risk score. High-risk patients had significantly worse overall survival than lowrisk patients. The risk model had excellent predictive performance for overall survival in LGG and was found to be an independent risk factor. The prognostic value of the riskmodel was validated using an independent cohort. In addition, the risk score was associated with tumor mutation burden and immune cell infiltration in LGG. High-risk patients had higher HRD scores and “hot” tumor immune microenvironment, which could benefit from poly-ADP-ribose polymerase inhibitors and immune checkpoint inhibitors. Overall, this big data study determined the threshold of HRD score in LGG, identified HRD-related genes, developed a risk model based on HRD-related genes, and determined the molecular and immunological characteristics of the risk model. This provides potential new targets for future targeted therapies and facilitates the development of individualized immunotherapy to improve prognosis.

Submitted by ANA CAROLINA ORTEGAL ALMEIDA null (ortegalmedvet@gmail.com) on 2016-03-21T11:23:32Z No. of bitstreams: 1 Dissertação_Ana_Carolina_Ortegal_Almeida.pdf: 2529882 bytes, checksum: 7d592a7135492a17094d124933d025a2 (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2016-03-22T14:01:48Z (GMT) No. of bitstreams: 1 almeida_aco_me_jabo.pdf: 2529882 bytes, checksum: 7d592a7135492a17094d124933d025a2 (MD5) / Made available in DSpace on 2016-03-22T14:01:48Z (GMT). No. of bitstreams: 1 almeida_aco_me_jabo.pdf: 2529882 bytes, checksum: 7d592a7135492a17094d124933d025a2 (MD5) Previous issue date: 2016-01-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A inspeção da carcaça e órgãos nos abatedouros objetiva limitar o aproveitamento de produtos impróprios para consumo humano, protegendo a população contra doenças transmitidas pelos alimentos. O fígado é uma víscera nutritiva e bem aceita pelos consumidores, mas suas funções metabólicas o tornam susceptível a diversas lesões, fazendo com que seja condenado frequentemente na rotina de inspeção. Este estudo analisou fígados bovinos e relacionou as lesões macro e microscópicas com os sistemas de produção dos animais abatidos (sistemas intensivo, semi-intensivo e extensivo). Avaliou também a presença de lesão microscópica em fígados sem lesão macroscópica. Foram coletadas 450 amostras de fígado bovino dos diferentes sistemas de produção, as quais foram processadas para exame histológico. As alterações macroscópicas observadas nos fígados bovinos foram: fibrose capsular (32%), aderências/peri-hepatite (25,8%), teleangiectasia (24,4%), manchas pálidas ou amareladas (20,9%), hemorragia subcapsular (7,1%), abscessos (5,3%), granulomas (3,5%), congestão (1,8%) e cistos (0,9%). Os achados microscópicos foram: infiltrado inflamatório mononuclear (66,4%), macrófagos espumosos (40,9%), tumefação hepatocelular (30%), tumefação hepatocelular centrolobular (22,7%), fibrose capsular (26,4%), degeneração gordurosa (15,1%), peri-hepatite (13,8%), necrose (13,5%), teleangiectasia (12,2%), abscessos (2,9%), granulomas (1,8%), congestão (16%), hemorragia subcapsular (4,6%), infiltrado inflamatório polimorfonuclear (2,5%), infiltrado inflamatório misto (8,7%), hiperplasia ductal (4,9%) e cistos (0,9%). As lesões que apresentaram relação com os sistemas de produção (P<0,05) foram: infiltrado inflamatório mononuclear, macrófagos espumosos e tumefação hepatocelular. Os fígados de bovinos criados em sistema intensivo apresentaram mais lesões macroscópicas e os fígados de bovinos criados em sistema extensivo apresentaram mais lesões microscópicas. Somente 19,5% dos fígados sem lesão macroscópica não apresentaram nenhum tipo de lesão microscópica. Conclui-se que o sistema de produção pode influenciar na ocorrência de algumas lesões hepáticas e que fígados sem lesão macroscópica, liberados para consumo humano, podem apresentar várias lesões microscópicas importantes. / The inspection of cattle carcasses and organs at slaughterhouses aims at limiting the use of products with abnormalities that makes them improper for human consumption, thereby protecting the public from food-borne diseases. The liver is a nutritious viscera and it is well accepted by consumers. However, its metabolic functions make it susceptible to various injuries, which often condemn it during inspections routine. This study evaluated bovine livers and linked the macro and microscopic lesions found to the beef cattle production systems (intensive, semi intensive and extensive). Also evaluated the presence of microscopic lesions on livers without macroscopic findings. Some 450 samples of beef liver were collected from various production systems and were processed for histological examination. Macroscopic changes observed on the samples were: capsular fibrosis (32%), adhesions/perihepatitis (25.8%), telangiectasia (24.4%), pale or yellowish spots (20.9%), subcapsular hemorrhage (7.1%), abscesses (5.3%), granulomas (3.5%), congestion (1.8%) and cysts (0.9%). Microscopic findings were mononuclear cell infiltration (66.4%), foamy macrophages (40.9%), hepatocellular swelling (30%), centrilobular swelling (22.7%), capsular fibrosis (26.4%), fatty degeneration (15.1%), perihepatitis (13.8%), necrosis (13.5%), telangiectasia (12.2%), abscesses (2.9%), granulomas (1.8%) congestion (16%), subcapsular hemorrhage (4.6%), polymorphonuclear inflammatory infiltrate (2.5%), mixed inflammatory infiltrate (8.7%), ductal hyperplasia (4.9%) and cysts (0.9%). Those injuries related to production systems (P<0.05) were: mononuclear cell infiltration, foamy macrophages and cellular swelling. The livers of bovines raised in extensive system present more macroscopic lesions and the livers of bovines raised in intensive system present more microscopic lesions. Only 19.5% of livers without macroscopic findings did not show histopathological lesions. This study concludes that beef cattle production systems can influence the occurrence of some liver lesions and that liver without macroscopic findings, released for human consumption may have several important microscopic lesions.

Toxoplasma gondii, an obligate intracellular protozoan parasite, establishes a latent, chronic infection by forming cysts preferentially in the brain after replication of tachyzoites in various organs during the acute stage of infection. Chronic infection with T. gondii is one of the most common parasitic diseases in humans. The immune system is required for maintaining the latency of chronic infection. Reactivation of infection can occur in immunocompromised individuals, such as AIDS patients, which results in the development of life-threatening toxoplasmic encephalitis (TE). IFN-gamma-dependent, cell mediated immune responses play an essential role in preventing the reactivation of chronic infection of T. gondii in the brain. In my dissertation study, we examined the mechanisms of IFN-gamma-mediated prevention of TE by using models of reactivation of chronic infection in BALB/c mice. This strain of mouse is genetically resistant to T. gondii infection and establishes a latent chronic infection as do immunocompetent humans, and therefore provides an excellet model for this purpose. Our laboratory previously demonstrated that both T cells and IFN-gamma-producing non-T cells are required for genetic resistance of BALB/c mice against development of TE. However, the function of T cells required for the resistance is still unclear. Therefore, in the present study, we examined whether IFN-gamma production or perforin-mediated cytotoxicity of T cells play an important role in their protective activity against TE. Immune T cells were obtained from infected IFN-gamma-knockout (IFN-g-/-), perforin-knockout (PO), and wild-type (WT) BALB/c mice, and transferred into infected, sulfadiazine-treated athymic nude mice which lack T cells but have IFN-gamma-producing non-T cells. Control nude mice that had not received any T cells developed severe TE due to reactivation of infection and died after discontinuation of sulfadiazine treatment. Animals that had received immune T cells from either PO or WT mice did not develop TE and survived. In contrast, nude mice that had received immune T cells from IFN-gamma-/- mice developed severe TE and died as early as control nude mice. T cells obtained from spleens of the animals that had received either PO or WT T cells both produced large amounts of IFN-gamma following stimulation with T. gondii antigens in vitro. In addition, the amounts of IFN-gamma mRNA expressed in the brains of PO T-cell recipients did not differ from those of WT T-cell recipients. These results indicate that IFN-gamma production, but not perforin-mediated cytotoxic activity, by T cells is required for prevention of TE in genetically resistant BALB/c mice. In our attempt to identify a T cell population(s) that produces IFN-gamma in the brain and plays an important role for prevention of TE, we analyzed T cell receptor (TCR) Vb chain usage in T cells expressing IFN-gamma in the brains of infected BALB/c mice. We found T cells bearing TCR V beta8 chain to be the most frequent IFN-g-producing population in the brains of infected animals. To examine the role of IFN-gamma production by this T cell population for prevention of TE, V beta8+ immune T cells purified from spleens of infected BALB/c and IFN-g-/- mice were transferred into infected, sulfadiazine-treated athymic nude mice. After discontinuation of sulfadiazine treatment, control nude mice that had not received any T cells and animals that had received Vb8+ T cells from IFN-g-/- mice all died due to reactivation of infection (TE). In contrast, animals that had received the cells from WT mice survived. These results indicate that IFN-gamma production by Vb8+ T cells in the absence of any other T cell population can prevent reactivation of infection. Thus, V beta8+ T cells play a crucial role in genetic resistance of BALB/c mice to TE through their production of IFN-gamma. When V beta8+ immune T cells were divided into CD4+ and CD8+ subsets, a potent protective activity was observed only in the CD8+ subset whereas a combination of both subsets provided greater protection than did the CD8+Vb8+ population alone. These results indicate that CD8+ subset of V beta8+ T cells is a major afferent limb of IFN-gamma-mediated resistance of BALB/c mice against TE, although the CD4+ subset of the T cell population works additively or synergistically with the CD8+V beta8+ population. T cells need to enter into the brains of infected mice to demonstrate their protective activity against TE. This migration is mediated, in part, by endothelial adhesion molecules. Since IFN-gamma is essential for preventing reactivation of chronic infection with this parasite in the brain, we examined whether this cytokine plays an important role in expression of lymphocyte and endothelial adhesion molecules and recruitment of T cells into the brain during chronic infection with T. gondii using IFN-g-/- and WT BALB/c mice. Although the number of cerebral vessels expressing intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) increased in both WT and IFN-g-/- mice following infection, there were more VCAM-1+ vessels in brains of infected WT than infected IFN-g-/- mice; in contrast, numbers of ICAM-1+ vessels did not differ between strains. We did not detect endothelial E-selectin, P-selectin, MAdCAM-1 or PNAd in any of the brains. Significantly fewer CD8+ T cells were recruited into brains of infected IFN-g-/- than WT mice. Treatment of infected IFN-g-/- mice with recombinant IFN-gamma restored the expression of VCAM-1 on their cerebral vessels and recruitment of CD8+ T cells into their brains, confirming an importance of this cytokine for up-regulation of VCAM-1 expression and CD8+ T cell trafficking. In infected WT and IFN-g-/- animals, almost all cerebral CD8+ T cells had an effector/memory phenotype (LFA-1high, CD44high and CD62Lneg) and approximately 38% were positive for a4b1 integrin (the ligand for VCAM-1). In adoptive transfer of immune spleen cells, pre-treatment of the cells with a monoclonal antibody against a4 integrin markedly inhibited recruitment of CD8+ T cells into the brain of chronically infected wild-type mice. These results indicate that IFN-g-induced expression of endothelial VCAM-1 and its binding to a4b1 integrin on CD8+ T cells is important for recruitment of the T cells into the brain during the chronic stage of T. gondii infection. Since we found strong expression of ICAM-1 on endothelia and LFA-1 on T cells in the brains of infected mice, LFA-1/ICAM-1 interaction, in addition to a4b1 integrin/VCAM-1 interaction, may also be involved in this process. As mentioned earlier, CD8+ T cells are crucial for prevention of TE in BALB/c mice. Therefore, IFN-gamma-mediated expression of VCAM-1 and its binding to a4b1 integrin for recruitment of CD8+ T cells may play a critical role in genetic resistance of BALB/c mice to development of TE. / Ph. D.

A histiocitose de células de Langerhans (HCL) é uma neoplasia mieloide inflamatória comumente afetando pacientes pediátricos e apresenta frequentemente mutações ativadoras somáticas em genes da via MAPK, incluindo BRAF e MAP2K1. Vários estudos sugerem que as células lesionais da HCL podem recrutar e modular células inflamatórias e cujas citocinas parecem fornecer sinais recíprocos de sobrevivência celular. Para o presente estudo foram selecionados 15 casos de HCL (10 crianças, 5 adultos), sendo as amostras de tecido avaliadas através de imunoistoquímica utilizando marcadores para macrófagos (CD68 e CD163), células dendríticas maduras (CDm) (CD83 e CD208), linfócitos T regulatórios (LTregs) (CD4, CD25 e FOXP3) e linfócitos citotóxicos (LCs) (CD56, CD57, perforina e granzima B). Além disso, marcadores de células B (CD20), células T (CD3, CD8) e confirmatórios de HCL foram analisados. Todos os casos de HCL foram positivos para S100, CD1a, CD207 e CD4; enquanto que Bcl-2 e Ciclina D1 foram positivos em 13/15 (86,7%) casos. No microambiente imune intralesional, macrófagos M2 (CD68+/CD163+), seguidos por LTregs, foram as populações celulares mais predominantes. Em quantidade significativamente menor, foram observadas CDm, seguidas por escassos LCs. Considerando a população linfoide, linfócitos T CD3+ foram mais numerosos do que linfócitos B CD20+. Dentro dos linfócitos T, linfócitos T CD4+ foram mais numerosos do que linfócitos T CD8+ (p<0,05). Nossos resultados sugerem que a infiltração de células imunes na HCL, provavelmente através de mecanismos pró-tumorais, inflamatórios e/ou imunossupressores mediados por citocinas, pode promover o desenvolvimento e sobrevivência das células lesionais da HCL, fornecendo uma justificativa para a combinação de imunoterapia e terapia gênica (BRAF) na HCL / Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasia often affecting children with constitutively somatic activating mutations in MAPK pathway genes including BRAF and MAP2K1. Several studies suggest that LCH cells can recruit and modulate inflammatory cells and whose cytokines appear provide reciprocal survival signals. For the present study, 15 cases of LCH (10 children, 5 adults) were selected, and the tissue samples were evaluated through immunohistochemistry using markers for macrophages (CD68 and CD163), mature dendritic cells (mDC) (CD83 and CD208), regulatory T-cells (Tregs) (CD4, CD25 and FOXP3) and cytotoxic lymphocytes (CLs) (CD56, CD57, perforin and granzime B). Moreover, B-cell (CD20), T-cell (CD3, CD8) and LCH markers were analyzed. All LCH cases were positive for S100, CD1a, CD207 and CD4, while Bcl-2 and Cyclin D1 were positive in 13/15 cases (86.7%). In the immune microenvironment, M2-polarized macrophages (CD68+/CD163+), followed by LTregs, were the predominant cell populations. In a significantly lower amount, mDC were observed, followed by scarce CLs. Moreover, CD3+ Tcells than CD20+ B-cells were more numerous (p>0.05), the former presenting a higher number of CD4+ than CD8+ T-cells (p<0.05). Our results suggest that immune cell infiltration in LCH, probably through cytokine-mediated pro-tumoral, inflammatory and/or immunosupressive mechanisms, can promote LCH cell development and survival, providing a rationale for combining immunotherapy and BRAF-targeted therapy in LCH

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