Both tissue microenvironment and genetic changes are involved in development of cancer. We employed the Friend murine leukemia virus (F-MuLV)- induced erythroleukemia model to study the role of these parameters in induction of malignancy. The tissue microenvironment is composed of non-cellular and cellular components. In regards to the non-cellular part, we previously reported that vascular endothelial growth factor (VEGF), in combination with macrophage chemoattractant protein-5, contributes to leukemia progression in F-MuLV- infected mice. To study the influence of constitutively elevated VEGF levels on the progression of erythroleukemia, we inoculated VEGF hi/+ mice, which are heterozygous for a VEGF “hypermorphic” allele, with F-MuLV. Unexpectedly, a significant delay in erythroleukemia was observed in these mice when compared with wild-type controls. The VEGF hi/+ mice exhibited a higher natural killer (NK) cell activity, elevated B cells, and a decrease in T-cell number. Furthermore, higher erythroid progenitors (i.e. CD34+, CD36+, and TER119+ cells) were evident in the bone marrow, spleen, and peripheral blood of these mice. Also, the CFU-E levels were significantly elevated in VEGF hi/+ bone marrow cultures. We propose that a compensatory erythropoietic response combined with increased NK cell activity account for the extended survival of erythroleukemic, VEGF hi/+mice. In regards to the cellular component of tissue microenvironment we studied the role of B cells in response to F-MuLV. To test the hypothesis that virus- neutralizing antibodies are involved in providing sterilizing immunity to F-MuLV we inoculated adult female mice with F-MuLV so that their newborns are provided with anti-viral antibodies. F-MuLV challenge of these newborns did not lead to induction of erythroleukemia. Conversely, mice from a control group (newborns whose mother had not received viral inoculation) contracted erythroleukemia upon F-MuLV challenge, as shown by hepatosplenomegaly, anemia, and emergence of leukemic cells in the spleen. These results indicated the importance of anti-viral antibodies in immunity to F-MuLV and suggested that anti-F-MuLV antibodies were generated in mothers, transferred to their offspring and protected them from viral challenge. The key genetic event upon F-MuLV infection is viral integration at the Fli-1 locus. We set to identify F-MuLV integration sites in SCID mice following two observations that these mice show a delay in induction of leukemia and also they do not exhibit viral integration at the Fli-1 locus. We hypothesized that development of leukemia in these mice is due to F-MuLV integration at a region other than the Fli-1 locus. Using a GenomeWalking approach we identified a total of 15 viral integration sites in F-MuLV-infected SCID mice, with eight of them interrupting the following genes: Mex3d, Fam125b, Prdm16, Rhoq, Ahdc1, Zc3h4, Msh3, and Hcls1. Using PCR to amplify the virus- host DNA junction fragment we found that one of the viral insertion sites (chromosome 10; position 20,942,825) occurs with a frequency of 35 % and therefore is considered as a common integration site.

Background: Caveolin-1 (Cav-1), the major component of caveolae, is a multifunctional scaffolding protein that serves as a platform for the cell’s signal-transduction and plays a role in inflammation. However, its role in inflammatory bowel disease (IBD), a chronic inflammatory condition in the gastrointestinal tract, is not clear. A recent study shows that Cav-1 mediates angiogenesis in dextran sodium sulphate (DSS)-induced colitis. These results contradict our data, in which Cav-1 levels decreased significantly in 2,4,6-trinitrobenzene sulphonic acid (TNBS)–induced colitis. Methods: To test whether Cav-1 is involved in IBD pathogenesis, various models representing different dominant Th subtype responses and mimicking the immune pathologic mechanisms of different clinical IBD setting were employed: acute colitis was induced by intra-rectal administration of a single dose of TNBS in BALB/c and C57BL/6J mice, or by drinking 3% DSS water for 6 days in C57BL/6J mice. Chronic colitis was induced by administration of TNBS once a week for 7 weeks in BALB/c mice. To assess the effects of complete loss of Cav-1, Cav-1 knock-out (Cav-1-/-) and control wild-type C57BL/6J mice received a single TNBS administration. To further test the possible role of Cav-1, one of two peptides (that either mimicked (Caveolin scaffolding domain; CSD) or antagonized (Caveolin-1 binding domain; CBD1) Cav-1)) was administered intraperitoneally to mice receiving TNBS. Body weight and clinical scores were monitored. Colon Cav-1 and pro-inflammatory cytokine levels were quantified by ELISA. Inflammation was evaluated through histological analysis. Results: Cav-1 levels in mouse colon tissue were significantly decreased in TNBS-induced colitis mice when compared to normal mice and also inversely correlated with colon inflammation and cytokine levels. Furthermore, a loss of Cav-1 (Cav-1-/-) showed increased clinical and inflammatory scores and increased body weight loss. Mice receiving peptides to alter Cav-1 levels, showed surprising effects. The mimicking peptide (CSD) showed decreased Cav-1 levels, while the antagonizing peptide (CBD1) showed increased Cav-l levels. These changes in levels were associated with clinical and inflammatory scores and body weight loss that supported the TNBS-induced data. DSS-induced colitis mice showed increased disease activity index, however no significant difference in Cav-1 levels was found between colitis and normal mice. Conclusions: Cav-1 plays an important role in the protection of TNBS-induced colitis, but not in DSS-induced colitis, an entirely different result from a previous report, suggesting that enhancement of Cav-1 expression and functions may be beneficial to IBD treatment in some specific clinical settings. Further studies are warranted.

Background: Caveolin-1 (Cav-1), the major component of caveolae, is a multifunctional scaffolding protein that serves as a platform for the cell’s signal-transduction and plays a role in inflammation. However, its role in inflammatory bowel disease (IBD), a chronic inflammatory condition in the gastrointestinal tract, is not clear. A recent study shows that Cav-1 mediates angiogenesis in dextran sodium sulphate (DSS)-induced colitis. These results contradict our data, in which Cav-1 levels decreased significantly in 2,4,6-trinitrobenzene sulphonic acid (TNBS)–induced colitis. Methods: To test whether Cav-1 is involved in IBD pathogenesis, various models representing different dominant Th subtype responses and mimicking the immune pathologic mechanisms of different clinical IBD setting were employed: acute colitis was induced by intra-rectal administration of a single dose of TNBS in BALB/c and C57BL/6J mice, or by drinking 3% DSS water for 6 days in C57BL/6J mice. Chronic colitis was induced by administration of TNBS once a week for 7 weeks in BALB/c mice. To assess the effects of complete loss of Cav-1, Cav-1 knock-out (Cav-1-/-) and control wild-type C57BL/6J mice received a single TNBS administration. To further test the possible role of Cav-1, one of two peptides (that either mimicked (Caveolin scaffolding domain; CSD) or antagonized (Caveolin-1 binding domain; CBD1) Cav-1)) was administered intraperitoneally to mice receiving TNBS. Body weight and clinical scores were monitored. Colon Cav-1 and pro-inflammatory cytokine levels were quantified by ELISA. Inflammation was evaluated through histological analysis. Results: Cav-1 levels in mouse colon tissue were significantly decreased in TNBS-induced colitis mice when compared to normal mice and also inversely correlated with colon inflammation and cytokine levels. Furthermore, a loss of Cav-1 (Cav-1-/-) showed increased clinical and inflammatory scores and increased body weight loss. Mice receiving peptides to alter Cav-1 levels, showed surprising effects. The mimicking peptide (CSD) showed decreased Cav-1 levels, while the antagonizing peptide (CBD1) showed increased Cav-l levels. These changes in levels were associated with clinical and inflammatory scores and body weight loss that supported the TNBS-induced data. DSS-induced colitis mice showed increased disease activity index, however no significant difference in Cav-1 levels was found between colitis and normal mice. Conclusions: Cav-1 plays an important role in the protection of TNBS-induced colitis, but not in DSS-induced colitis, an entirely different result from a previous report, suggesting that enhancement of Cav-1 expression and functions may be beneficial to IBD treatment in some specific clinical settings. Further studies are warranted.

Universiẗat, Diss., 2007--Frankfurt (Main). / Zsfassung in dt. und engl. Sprache.

Thesis--Copenhagen. / "Résumé" (in Danish): p. [95]-97. "The translation into English by Robert Fraser." "Literature": p. [99]-103.

Impact of the chromatin remodeller SMARCAD1 on murine intestinal intraepithelial lymphocyte and white adipose tissue biology. Chromatin remodelling factors use the energy of ATP hydrolysis to drive the movement of and/or affect molecular changes to the nucleosome. One such factor, SMARCAD1 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A containing DEAD/H box 1), has been previously shown to restore heterochromatin at the replication fork in vitro. This project aimed to assess the impact of SMARCAD1 on mammalian biology, utilising an animal model in which the catalytic ATPase domain of murine SMARCAD1 had been deleted using Cre/lox technology. Preliminary results had implicated SMARCAD1 in adaptive-immunity and white adipose tissue biology, and SMARCAD1 expression in these tissues/cells was confirmed by tissue-panel western blot. This project therefore aimed to build on these results to understand better the impact of SMARCAD1 on adaptive immune development and white adipose tissue biology. In addition, fewer than expected viable Smarcad1-/- homozygous offspring were produced during Smarcad1+/- x +/- matings, which both confirmed the observation from a previous knockout model of Smarcad1, and limited the number of knockout animals available for this study. Investigation of systemic B- and T-cells in the bone marrow, thymus and spleen had previously suggested there was no significant defect in adaptive immune development in Smarcad1-/- mice, however a tissue-specific and age-related loss of intra-epithelial (IEL) T-lymphocytes was found in the small intestine by flow cytometry. Analysis by qPCR of duodenal RNA suggested that differentiation rather than inflammation may underpin any loss-of-IEL phenotype, although further examination of cell-proliferation and crypt/villus anatomy by EdU incorporation and immunofluorescence revealed no overt cell-anatomical or proliferative difference in the knockout mice. The requirement for large numbers of aged mice made further investigation of the intestinal IEL phenotype logistically prohibitive. The reduction of epididymal white adipose tissue (eWAT) size had also been observed in male Smarcad1-/- mice, and serum from these mice showed elevated triglyceride (TG) and free fatty acids (FFA) levels. Transcriptomic analysis by RNA-seq of whole-WAT revealed an elevation in macrophage-related markers in knockout mice, which was confirmed by flow cytometry. As a number of reports have implicated SMARCAD1 in stem cell biology, putative adipose stem cells were isolated from +/+ and -/- mice by FACS and used for adipogenic differentiation assays ex-vivo In parallel, mouse embryonic fibroblasts from +/- and -/- mice were also assayed for adipogenic differentiation. While no significant differences in adipogenesis were observed, Smarcad1-/- mice challenged with a (60%) high fat diet did show increased weight gain over +/+ mice, and measurements of adipocyte size and cell cycle/cell proliferation analysis suggested hyperplasia rather than defects in adipogenesis may drive any WAT-related pathology in these mice.

Regulation of the immune system and of uterine tissue homeostasis, growth, and remodelling are deeply intertwined during pregnancy and are essential for successful reproduction. Recent findings showed that tissue-resident innate lymphoid cells (ILCs) are crucial regulators of both physiology and pathology of the tissues they populate. Uterine natural killer (uNK) cells are a subtype of ILCs known to regulate trophoblast invasion, uterine vascular adaptation to pregnancy, and foetal growth. We recently described additional types of ILCs in the uterus of women and mice. However, the role of these ILCs during reproduction is unknown. Among them, group 2 ILCs (ILC2s) have been previously characterised in other tissues, in which they modulate immune cells and tissue homeostasis by producing type-2 cytokines and growth factors (i.e. IL-4, IL-5, IL-13, and Amphiregulin). Based on these premises, I hypothesized that uterine ILC2s (uILC2s) regulate uterine immune homeostasis and thus contribute to successful reproduction. To test this, I first characterised the uILC subtypes present in humans and mice at various stages of the reproductive cycle. Secondly, I addressed the functional role of uILC2s during pregnancy by taking advantage of a uILC2 knockout mouse model. My results show that uterine ILC2s represent < 1% and < 0.1% of murine and human uterine leukocytes, respectively. However, as they can quickly produce large amounts of cytokines, uILCs are capable of potently affect both other immune cells and the surrounding tissue. Indeed, I found that compared to other tissue-resident ILC2s, uILC2s produce high levels of IL-5 and Areg even in the absence of any stimulation. On the contrary, non-uterine ILC2s mainly produce IL-13, which is lowly expressed by uILC2s. To further characterize the tissuespecific properties of uILC2s, I then performed RNAseq on uILC2s isolated from virgin, midgestation, and term murine uterus, and I compared their transcriptomes with those of ILC2s from lung, intestine, and bone marrow. Interestingly, uILC2s specifically express granzymes and genes typical of regulatory T cells. Therefore, uILC2s have tissue-specific properties and are modulated during pregnancy. Furthermore, the ability of uILC2s to produce IL-5 and Areg suggests that they may be crucial in the regulation of uterine type-2 immunity. I then studied the phenotype of $Rora^{flox/flox}Il7ra^{cre/wt}$(ILC2KO) mouse models, as well as that of mice lacking the ILC2 activating cytokine IL-33 ($IL33^{cit/cit}$; IL33KO). I examined the immune microenvironment in both the myometrium and decidua in ILC2KO mice and found alterations in type-2 cytokines and myeloid cell homeostasis. In particular, in absence of ILC2s, IL-4 and IL-5 are dramatically reduced, IL-13 is absent, and decidual inflammatory cytokines IL1β and IL-6 are increased. Furthermore, uterine dendritic cells (uDC), uterine macrophages (uMac), and uterine neutrophils (uN) increase, while uterine eosinophils (uEo) are virtually absent. These results show that uILC2s regulate uterine type-2 immunity, suggesting that uILC2s could be important during pregnancy. Accordingly, I found that lack of uILC2s leads to insufficient spiral artery remodelling and restricted foetal growth. Type-2 cytokines and in particular IL-4 regulates alternative activation of Macrophages (Mac) and Dendritic Cells (DCs), which promote the development of an anti-inflammatory environment and facilitate tissue remodelling. I hypothesised that similar mechanisms occur in the uterus and that uILC2s have a central role in the polarisation of the immune response. To explore this, I studied in more detail the characteristics of uEo, uMac, and uDCs dissected from wild type and ILC2KO mice. I found a reduction in genes associated with alternative activation in uMac and uDCs in the uterus of pregnant ILC2KO mice. Additionally, I showed that uEo are the main producers of the IL-4. This demonstrates that uILC2s promote alternative activation of myeloid cell population by modulating the uterine immune microenvironment. I then assessed the role of uILC2s-dependent type-2 immunity in inflammatory pathology following a type-1 response to bacterial infection. When challenged with LPS, pregnant ILC2KO mice showed more pronounced foetal demise. Therefore, uILC2s regulate uterine type-2 immune homeostasis and this prevents inflammatory pathology. Collectively, my work advances our knowledge of the innate immune mechanisms that control physiological and pathological events during pregnancy. These findings have implications to the field of immunology of pregnancy and may lead to clinical progress in diagnosis and prevention of infection-induced abortion in human pregnancies.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Pesquisas cientÃficas buscando a utilizaÃÃo de antÃgenos de Coccidioides posadasii sÃo comuns na literatura especializada, uma vez que, sÃo instrumentos relevantes para diagnÃstico e possÃvel efeito imunoprotetor em humanos. Diante do exposto, foi realizada caracterizaÃÃo bioquÃmica de antÃgeno protÃico oriundo de C. posadasii. busca ativa de casos humanos de coccidioidomicose no Estado do CearÃ, bem como, avaliaÃÃo da resposta imunolÃgica in vivo. Para concretizar tais objetivos, a caracterizaÃÃo bioquÃmica do antÃgeno foi realizada por meio de eletroforese (SDS-PAGE e 2D-PAGE), detecÃÃo de proteases, glicoproteÃnas e sequenciamento N-terminal e foram buscados, ativamente, quadros de coccidiodiomicose em trÃs caÃadores de tatu com a respectiva descriÃÃo clÃnica e avaliaÃÃo laboratorial. Ademais, foi descrito modelo murino de coccidioidomicose com testagem de possÃvel efeito imunoprotetor do antÃgeno in-house. Quanto a anÃlise bioquÃmica, observou-se delimitaÃÃo de bandas nas faixas de 45-67 kDa e 67-97 kDa (SDS-PAGE), detecÃÃo de glicoproteÃnas, proteases e sequenciamento N-terminal demonstrando serem as bandas uma &#946;-glucosidase e uma glutamina sintetase. Nos casos clÃnicos, foram encontrados queixas pneumÃnicas, exame micolÃgico direto, cultivo de escarro, imunodifusÃo radial dupla (com antÃgeno in-house) e PCR positivos para C. posadasii. No modelo murino, o grupo infectado, na presenÃa de antÃgeno e adjuvante, apresentou, na anÃlise histopatolÃgica, menores alteraÃÃes pulmonares que os demais grupos, alÃm de maior estÃmulo linfÃide esplÃnico. No que concerne a dosagem de citocinas (IL-6, IL-12 e TNF&#945;), nÃo se observou diferenÃa significativa entre os grupos, mas uma tendÃncia à resposta imunoprotetora. O grupo infectado, sem imunoproteÃÃo, apresentou maior perda ponderal. Na anÃlise macroscÃpica, o mÃximo comprometimento foi a presenÃa de 2 granulomas, neste Ãltimo grupo. Na anÃlise dos hemogramas, a sÃrie branca demonstrou maiores diferenÃas entre os grupos. Em conclusÃo, o antÃgeno in-house se mostrou tratar-se de uma &#946;-glicosidase e uma glutamina sintetase que tenderam a estimular a imunidade celular em modelo murino. AlÃm disso, a descriÃÃo de casos humanos contribui para a difusÃo do diagnÃstico precoce da coccidioidomicose e requer investigaÃÃes laboratoriais complementares. / Scientific researches seeking to use Coccidioides posadasii antigens are common in the literature, as relevant instruments to diagnosis and possible immunoprotector effect in humans. Thus, we performed biochemical characterization of protein antigen derived from C. posadasii, an active search for human cases of coccidioidomycosis in CearÃ, as well as evaluation of immune response in vivo. To achieve these goals, the biochemical characterization of the antigen was performed by electrophoresis (SDS-PAGE and 2D-PAGE), detection of proteases, glycoproteins and N-terminal sequencing and we reported three human cases of coccidiodiomycosis in armadillo hunters with its clinical description and laboratory evaluation. Further, a murine model was described testing a possible imunoprotector effect with an in-house antigen. For biochemical analysis, it was observed delimitation bands in ranges 45-67 kDa and 67-97 kDa (SDS-PAGE), detection of glycoproteins, proteases and N-terminal sequencing demonstrating the bands being a &#946;-glucosidase and a glutamine synthetase. In clinical cases, it was found pneumonic disease, direct mycological examination, sputum culture, double radial immunodiffusion (antigen in-house) and PCR positive for C. posadasii. In the murine model, the infected group, in the presence of antigen and adjuvant, showed, histologically, lung disorders smaller than the other groups, and increased splenic lymphoid stimulus. Regarding the cytokines (IL-6, IL-12 and TNF), there was no significant difference between groups, but a trend toward immunoprotective response. The infected group, without immunoprotection, showed greater weight loss. In the macroscopic analysis, the maximum commitment was the presence of 2 granulomas in the latter group. In the analysis of blood counts, the white run showed major differences between groups. In conclusion, the in-house antigen showed that it was a &#946;-glucosidase and a glutamine synthetase which tended to stimulate cellular immunity in a murine model. Moreover, the description of human cases contributes to the spread of early diagnosis of coccidioidomycosis and requires additional laboratory investigations.

Histopathology of a murine lymph node, 9 days post infection with Coccidioides. Magnification 10X

Indiana University-Purdue University Indianapolis (IUPUI) / Neurofibromatosis type 1 (NF1) is a genetic disorder resulting from mutations in the tumor suppressor gene NF1. NF1 encodes the protein neurofibromin, which functions to negatively regulate p21Ras signaling. NF1 has a wide range of clinical manifestations, including vascular disease, which is characterized by neointima formation and subsequent vessel occlusion. Despite numerous clinical observations of NF1 vasculopathy, the pathogenesis of vascular lesion formation remains unclear. To determine the consequence of Nf1 haploinsufficiency in vascular disease, we generated an in vivo model for dissecting vascular lesion formation. In response to mechanical arterial injury, Nf1+/- mice have significantly enhanced neointima formation characterized by an accumulation of vascular smooth muscle cells (VSMCs) and excessive cellular proliferation and Ras activation. Further, using the pharmacological antagonist, imatinib mesylate, we identified that neointima formation in Nf1+/- mice was directly dependent on Ras signaling through either the platelet derived growth factor β receptor (PDGF-βR) and/or the C-kit receptor activation. These observations identify a molecular mechanism of neointima formation given that our group has previously demonstrated that Nf1+/- VSMCs have hyperactive Ras signaling through PDGF-βR activation and Nf1+/- bone marrow derived cells (BMDCs) have increased recruitment and survival in response to C-kit activation compared to WT controls. In order to dissect the cellular contribution to neointima formation, we utilized cre/lox technology and adoptive hematopoietic stem cell transfer techniques to genetically delete one allele of Nf1 in endothelial cells, VSMCs or BMDCs individually to test which cell lineage is predominant in NF1 vasculopathy. Surprisingly, in response to carotid artery injury, heterozygous inactivation of Nf1 in BMDCs alone was necessary and sufficient for neointima formation. Specifically, Nf1 haploinsufficiency in BMDCs resulted in an infiltration of macrophages into the neointima, providing evidence of “vascular inflammation” as factor in NF1 vasculopathy. Further, we demonstrate for the first time that NF1 patients have evidence of chronic inflammation determined by increased concentrations of circulating monocytes and pro-inflammatory cytokines. In sum, we provide genetic and cellular evidence of vascular inflammation in NF1 patients and Nf1+/- mice and provide a framework for understanding the pathogenesis of NF1 vasculopathy and potential therapeutic and diagnostic interventions.