Orientador: Alexandre Leite Rodrigues de Oliveira / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T17:19:57Z (GMT). No. of bitstreams: 1 Simoes_GustavoFerreira_D.pdf: 65115196 bytes, checksum: 18f51cababf3e248cceb806b8faef631 (MD5) Previous issue date: 2012 / Resumo: Atualmente, muito se sabe sobre o acometimento muscular na DMD, mas poucos estudos estão voltados para os efeitos no Sistema Nervoso Central (SNC), mais especificamente no microambiente do motoneurônio medular. Sabe-se que durante a evolução da doença, o terminal axonal, na junção neuromuscular, entra em um ciclo de denervação (retração) e reinervação (brotamento). A possibilidade de modulação do MHC I se apresenta como uma nova estratégia de influenciar positivamente o processo de plasticidade sináptica após lesões do Sistema Nervoso Periférico (SNP) e SNC. Tal modulação pode ser realizada através da utilização ou desenvolvimento de drogas específicas. O fator estimulador de colônias glanulocitárias (G-CSF) é uma glicoproteína que foi descrita há mais de vinte anos, possui aprovação do ANVISA (Agência Nacional de Vigilância Sanitária) e é comumente utilizada para tratar neutropenia, ou para transplantes de medula óssea. O GCSF possui um efeito neuroprotetor aparentemente multimodal, incluindo-se a atividade anti-apoptóptica em neurônios, regeneração da vascularização, efeito antiinflamatório e estimulação de neurogênese endógena, sendo capaz de atuar efetivamente no processo de regeneração do sistema nervoso. No presente trabalho, foram utilizados camundongos MDX. Os camundongos foram distribuídos em 4 grupos (axotomia + G-CSF; Axotomia; Controle + G-CSF e Controle), com n=10. Incluiu-se para imunoistoquímica o grupo placebo, onde os animais receberam uma dose diária de 200?m, via subcutânea, de glicose a 25%. Nossos resultados indicam que redução de sinapses nos motoneurônios alfamedulares e aumento da astrogliose circunjacente aos neurônios alfa-medulares, seja decorrente da desconexão parcial entre o orgão alvo e o corpo neuronal durante o período de ciclos de degeneração/regeneração muscular que ocorrem a partir das primeiras semanas de vida nos camundongos MDX. Estes ciclos podem repercutir retrogradamente nos corpos celulares dos motoneurônios alfa-medulares, provocando uma série de alterações denominadas cromatólise. A axotomia do nervo isquiático resulta num aumento significativo da expressão de MHC I nas duas linhagens estudadas. Contudo, nos animais MDX, este aumento é menor, comparativamente à linhagem C57BL/10. Quando tratados com G-CSF a expressão de MCH I ficou maior em relação aos grupos não tratados e, isso pode indicar um papel ativo da droga no potencial regenerativo após a lesão. Também podemos sugerir que, apesar dos animais MDX apresentarem uma menor função motora em relação aos animais controle, os resultados indicam que o tratamento com G-CSF é capaz de reduzir os efeitos inflamatórios e atuar positivamente no processo de regeneração nervosa periférica após esmagamento do nervo isquiático / Abstract: Currently, much is known about the muscular involvement in DMD, but few studies have focused on the effects on the central nervous system (CNS), specifically in the microenvironment of spinal motor neurons. It is known that during the course of the disease, the axon terminal at the neuromuscular junction, enters a cycle of denervation (retraction) and reinnervation (sprouting). The possibility of modulation of MHC I presents itself as a new strategy to positively influence the process of synaptic plasticity after injury Peripheral Nervous System (PNS) and CNS. Such modulation may be accomplished through the use or development of special drugs. The granulocyte colony-stimulating factor (G-CSF) is a glycoprotein which was first described more than twenty years, has approval from ANVISA (Agência Nacional de Vigilância Sanitária) and is commonly used to treat neutropenia, or bone marrow transplants. The G-CSF has a multimodal neuroprotective effect l, including the anti-apoptotic activity in neurons, regeneration of vascularization, anti-inflammatory effect and stimulation of endogenous neurogenesis, being able to act effectively in the process of regeneration of the nervous system. In this study, we used MDX mice. The mice were divided into 4 groups (axotomy + G-CSF; axotomy, Control + G-CSF and Control), with n = 10. Included immunohistochemistry to the placebo group, where the animals received a daily dose of 200?m, subcutaneously, glucose 25%. Our results indicate that reduction of synapses in the alpha motoneurosn and increased astrogliosis , either due to partial disconnection between the target organ and the neuronal body during the cycles of degeneration /regeneration muscle that occur from first weeks of life in MDX mice. These cycles can pass retrogradely in alpha motoneurons cell bodies, causing a series of changes called chromatolysis. The sciatic nerve axotomy results in a significant increase of MHC I expression in both strains studied. However, in MDX strain, this increase is smaller, compared to C57BL/10. After treatment with G-CSF the expression of MCH I got bigger compared to untreated groups, and this may indicate an active role in the regenerative potential of the drug after injury. Also we suggest that while the animals present MDX a smaller motor function compared to control animals, the results indicate that treatment with G-CSF is capable of reducing the inflammatory effects and act positively on peripheral nerve regeneration process after nerve crush sciatic. Also our results indicate that treatment with G-CSF is able to reduce the inflammatory effects and act positively on peripheral nerve regeneration process after nerve crush sciatic / Doutorado / Anatomia / Doutor em Biologia Celular e Estrutural

Orientador : Alexandre Leite Rodrigues de Oliveira / Tese (Doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-22T00:27:25Z (GMT). No. of bitstreams: 1 Freria_CamilaMarques_D.pdf: 9799936 bytes, checksum: 9a9057c6ab5ca87ea1bc6767c4f71490 (MD5) Previous issue date: 2013 / Resumo: Lesões nervosas periféricas e centrais levam à inflamação local e retrógrada, resultando em alterações axonais, perdas neuronais e sinápticas significativas. Juntamente a tais alterações, as células gliais tornam-se reativas, influenciando na remodelação do SNC após lesão. Os mecanismos que desencadeiam tais mudanças não são completamente compreendidos, mas é evidente que as moléculas classicamente relacionadas com o sistema imune estão envolvidas em tais eventos diretamente ou através da modulação da reatividade glial. Assim, nossa hipótese é que o controle da sinalização inflamatória após a lesão central ou periférica possa afetar indiretamente nos mecanismos endógenos de reparação no SNC, resultando em maior preservação das conexões neurais e melhor recuperação funcional. Para isso, realizamos lesões periféricas e centrais expondo os animais a diferentes microambientes de lesão a fim de investigar o papel das células gliais na sobrevivência, capacidade regenerativa axonal e estabilidade sináptica de motoneurônios medulares. Os resultados mostraram que, após lesão, a modulação da sinalização inflamatória através da administração de citocinas ou deleção de moléculas expressas na superfície das células gliais podem influenciar direta ou indiretamente na estabilidade dos circuitos neuronais, na regeneração axonal e sobrevivência neuronal. Desse modo, conclui-se que o controle da inflamação e da reatividade glial são, provavelmente, críticos para a plasticidade no Sistema Nervoso viabilizando, assim, novas estratégicas de tratamentos / Abstract: Central or peripheral lesions result in local and retrograde inflammation, leading to axonal degeneration, synaptic and/or neuronal loss. Additionally, after injury, reactive glial cells are recruited to the lesion site, influencing the plasticity of the nervous system. The mechanisms which trigger such changes are not completely understood, but evidences have shown that molecules classically related to the immune system are involved in such events directly or indirectly by glial modulation. Based on this, our hypotheses is that the control of inflammatory signaling after central or peripheral injury may indirectly affect the endogenous repair mechanisms, resulting in a greater synaptic preservation and better functional recovery. In this sense, animals were submitted to both central and peripheral lesions in order to investigate the effects of glial cells on neuron survival, axonal regeneration and synaptic plasticity. The results showed that, after lesion, the modulation of inflammatory signaling by cytokines or knocking down molecules on glial surface, directly or indirectly influence the stability of neural circuits, neuronal survival and axonal regeneration. Thus, we believe that this is important findings that may be critical to the development of new therapeutic strategies following nervous system injury / Doutorado / Clinica Medica / Doutora em Ciências

No description available.

A formulação HIVBr18, previamente desenvolvida e testada, é uma vacina de DNA que codifica 18 epítopos CD4, promíscuos e conservados do HIV-1, e que após imunização de camundongos transgênicos para diversas moléculas de HLA de classe II humanas, observou-se proliferação de linfócitos T CD4+ e CD8+ e produção de IFN-? direcionadas a múltiplos epítopos codificados pela vacina. Abordamos aqui estratégias baseadas na fusão ou combinação dos epítopos codificados pela vacina HIVBr18 à glicoproteína D (gD) do HSV-1, e também na coadministração de plasmídeos que codificam citocinas (IL-2, -12, -15 e GM-CSF) visando aumentar a imunogenicidade de HIVBr18. A sequencia de DNA que codifica os 18 peptídeos da vacina HIVBr18 foi amplificada por PCR e clonada em um plasmídeo que abrigava a sequencia da gD do HSV-1. dando origem ao plasmídeo pVAX-gDh-HIVBr18. Animais imunizados com gDh-HIVBr18 apresentaram resposta imunológica similar ao grupo que recebeu somente HIVBr18, não sendo diferente também daqueles que receberam plasmídeos gDh-HIVBr18 que sofreram alterações nas sequências para melhorar o padrão de distribuição hidrofóbica e permitir a migração da proteína de fusão para o meio extracelular. Construímos e testamos um plasmídeo bicistrônico que expressa gDh e HIVBr18 isoladamente, mas também não observamos aumento na resposta imune induzida. A coadministração com o plasmídeo HIVBr18 e plasmídeos que codificam as citocinas IL-12, IL-15 e GM-CSF, proporciona um aumento na magnitude da resposta imunológica induzida contra o pool de peptídeos codificados pela vacina, entretanto sem alteração da amplitude da resposta. Além disso, o plasmídeo de GM-CSF induziu maior número de células T CD4+ polifuncionais. Demonstramos também que a coadministração do plasmídeo que codifica GM-CSF, induz uma resposta imune celular de maior magnitude mesmo em uma condição de dose reduzida. Entretanto, observamos que esta citocina não é um bom adjuvante quando utilizamos como vetor de imunização um adenovírus que expressa os 18 epítopos / The formulation HIVBr18, previously developed and tested, is based on a DNA vaccine encoding 18 conserved and promiscuous HIV-1 CD4 epitopes and after immunization of transgenic mice for many human HLA class II molecules using this DNA vaccine, could be observed proliferation of CD4+ and CD8+ T cells and IFN-y production directed to multiple epitopes encoded by the vaccine. We intend to explore here, strategies based on fusion or combination of epitopes encoded by HIVBr18 vaccine with glycoprotein D (gD) of HSV- 1 and also the coadministration of cytokine-encoding plasmids (pIL-2, -12, -15 and pGM -CSF) aiming to enhance immunogenicity of HIVBr18. The DNA sequence of epitopes encoded by HIVBr18 vaccine was amplified by PCR and cloned into a plasmid that contained the sequence of gD, giving rise to plasmid pVAX-gDh-HIVBr18. After mice immunization, animals immunized with this construct showed similar immune response to the group that received HIVBr18, and also the group of animals that received gDh-HIVBr18 plasmid that had been modified by exchange in peptides order to assure to the molecule a better hydrophobic distribution and allow translocation to the extracellular face of cell membrane. We constructed and injected mice with a bicistronic plasmid expressing gDh and HIVBr18, simultaneously and isolated, but no increase in the magnitude of the immune response was observed. HIVBr18 coadministration with cytokine-encoding plasmids pIL-12, pIL-15 and pGM-CSF, provides an increase in the magnitude of immune response induced against the peptides encoded by the vaccine, and similar breadth. In addition, co-immunization with pGM-CSF induced greater number of polyfunctional CD4 + T cells. We also demonstrate that, even in a low dose approach coadministration of pGM-CSF induced a higher immune response than HIVBr18 alone in the same dose. However, we observed that this cytokine is not a good adjuvant when used in combination with an adenovirus that expresses the 18 HIV-1 epitopes.

A formulação HIVBr18, previamente desenvolvida e testada, é uma vacina de DNA que codifica 18 epítopos CD4, promíscuos e conservados do HIV-1, e que após imunização de camundongos transgênicos para diversas moléculas de HLA de classe II humanas, observou-se proliferação de linfócitos T CD4+ e CD8+ e produção de IFN-? direcionadas a múltiplos epítopos codificados pela vacina. Abordamos aqui estratégias baseadas na fusão ou combinação dos epítopos codificados pela vacina HIVBr18 à glicoproteína D (gD) do HSV-1, e também na coadministração de plasmídeos que codificam citocinas (IL-2, -12, -15 e GM-CSF) visando aumentar a imunogenicidade de HIVBr18. A sequencia de DNA que codifica os 18 peptídeos da vacina HIVBr18 foi amplificada por PCR e clonada em um plasmídeo que abrigava a sequencia da gD do HSV-1. dando origem ao plasmídeo pVAX-gDh-HIVBr18. Animais imunizados com gDh-HIVBr18 apresentaram resposta imunológica similar ao grupo que recebeu somente HIVBr18, não sendo diferente também daqueles que receberam plasmídeos gDh-HIVBr18 que sofreram alterações nas sequências para melhorar o padrão de distribuição hidrofóbica e permitir a migração da proteína de fusão para o meio extracelular. Construímos e testamos um plasmídeo bicistrônico que expressa gDh e HIVBr18 isoladamente, mas também não observamos aumento na resposta imune induzida. A coadministração com o plasmídeo HIVBr18 e plasmídeos que codificam as citocinas IL-12, IL-15 e GM-CSF, proporciona um aumento na magnitude da resposta imunológica induzida contra o pool de peptídeos codificados pela vacina, entretanto sem alteração da amplitude da resposta. Além disso, o plasmídeo de GM-CSF induziu maior número de células T CD4+ polifuncionais. Demonstramos também que a coadministração do plasmídeo que codifica GM-CSF, induz uma resposta imune celular de maior magnitude mesmo em uma condição de dose reduzida. Entretanto, observamos que esta citocina não é um bom adjuvante quando utilizamos como vetor de imunização um adenovírus que expressa os 18 epítopos / The formulation HIVBr18, previously developed and tested, is based on a DNA vaccine encoding 18 conserved and promiscuous HIV-1 CD4 epitopes and after immunization of transgenic mice for many human HLA class II molecules using this DNA vaccine, could be observed proliferation of CD4+ and CD8+ T cells and IFN-y production directed to multiple epitopes encoded by the vaccine. We intend to explore here, strategies based on fusion or combination of epitopes encoded by HIVBr18 vaccine with glycoprotein D (gD) of HSV- 1 and also the coadministration of cytokine-encoding plasmids (pIL-2, -12, -15 and pGM -CSF) aiming to enhance immunogenicity of HIVBr18. The DNA sequence of epitopes encoded by HIVBr18 vaccine was amplified by PCR and cloned into a plasmid that contained the sequence of gD, giving rise to plasmid pVAX-gDh-HIVBr18. After mice immunization, animals immunized with this construct showed similar immune response to the group that received HIVBr18, and also the group of animals that received gDh-HIVBr18 plasmid that had been modified by exchange in peptides order to assure to the molecule a better hydrophobic distribution and allow translocation to the extracellular face of cell membrane. We constructed and injected mice with a bicistronic plasmid expressing gDh and HIVBr18, simultaneously and isolated, but no increase in the magnitude of the immune response was observed. HIVBr18 coadministration with cytokine-encoding plasmids pIL-12, pIL-15 and pGM-CSF, provides an increase in the magnitude of immune response induced against the peptides encoded by the vaccine, and similar breadth. In addition, co-immunization with pGM-CSF induced greater number of polyfunctional CD4 + T cells. We also demonstrate that, even in a low dose approach coadministration of pGM-CSF induced a higher immune response than HIVBr18 alone in the same dose. However, we observed that this cytokine is not a good adjuvant when used in combination with an adenovirus that expresses the 18 HIV-1 epitopes.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The granulocyte-colony stimulating factor (G-CSF) is a hematopoietic cytokine that stimulates and regulates the proliferation and differentiation of neutrophils precursors cells of the bone marrow. The recombinant hormone (rhG-CSF) non-glycosylated, filgrastim, is used to treat the neutropenia induced by chemotherapy and bone marrow transplantation. The hydrophobic protein is a 175 aminoacids chain which contains an extra methionine at its N-terminus, and molecular weight of 18.8 kDa. In the present study, a capillary zone electrophoresis (CZE) method was developed and validated for the analysis of filgrastim in pharmaceuticals. The analyses were performed on a fused-silica capillary (75 μm i.d.; effective length, 72 cm) and background electrolyte consisted of 50 mM sodium tetraborate solution at pH 9.0. The capillary temperature was maintained at 15 °C and the applied voltage was 15 kV. The injection was performed using the hydrodynamic mode at 50 mbar for 6 s, with detection at 195 nm using a PDA detector. The electrophoretic separation was obtained with migration time of 21.8 and 15.8 minutes for the filgrastim and leuprorrelin acetate (internal standard), respectively, and with run time of 30 minutes. The procedure was validated by the parameters of specificity, linearity, precision, accuracy, robustness, limit of quantitation and limit of detection. The method was linear in the concentration range of 1 200 μg/mL (r2 = 0.9978) and the limit of quantitation (LOQ) was 1 μg/mL, with acceptable validation parameters. The method was applied for the analysis of pharmaceutical formulations, and the results were correlated to the reversed-phase HPLC method (RP-HPLC), size-exclusion HPLC method (SE-HPLC) and in vitro bioassay method. Therefore, the procedures represent valid alternatives which can improve the quality control, assuring the safety and therapeutic efficacy of the biological product. / O fator estimulador da colônia de granulócitos humanos é uma citocina hematopoiética que estimula e regula a proliferação e diferenciação de células precursoras de neutrófilos da medula óssea. O hormônio recombinante (rhG-CSF) sob a forma não-glicosilada, filgrastima, é usado para o tratamento de neutropenia induzida por quimioterapia e transplante de medula óssea. A proteína hidrofóbica é constituída por uma cadeia de 175 aminoácidos com uma metionina N-terminal, e massa molecular de 18,8 kDa. No presente estudo, foi desenvolvido e validado método por eletroforese capilar de zona (CZE) para determinação de filgrastima em formulações farmacêuticas. As análises foram realizadas em capilar de sílica fundida (comprimento efetivo de 72 cm e diâmetro interno de 75 μm) e solução eletrolítica composta de tetraborato de sódio 50 mM, pH 9,0. O capilar foi mantido a temperatura de 15 °C, e a tensão aplicada foi de 15 kV. O tempo de injeção foi de 6 s, com pressão de 50 mbar, e detecção por arranjo de diodos (DAD) a 195 nm. A separação eletroforética foi obtida com tempo de migração de 21,8 e 15,8 minutos para filgrastima e acetato de leuprorrelina (padrão interno), respectivamente, e tempo total de corrida de 30 minutos. O procedimento foi validado, avaliando-se os parâmetros de especificidade, linearidade, precisão, exatidão, robustez, limite de quantificação e limite de detecção. Demonstrou-se linearidade na faixa de concentração de 1 200 μg/mL (r2 = 0,9978) e limite de quantificação (LQ) de 1 μg/mL, com os demais parâmetros de validação aceitáveis. O método foi aplicado para análise de formulações farmacêuticas, e os resultados foram correlacionados com os obtidos por cromatografia líquida em fase reversa (CL-FR) e por exclusão molecular (CLEM), e pelo bioensaio in vitro. Deste modo, os procedimentos pesquisados contribuem para o estabelecimento de alternativas que aprimoram o controle da qualidade, garantindo a segurança e eficácia terapêutica do produto biológico.

Transaldolase-Mangel (TALDO) ist ein extrem seltener, angeborener Stoffwechseldefekt, von dem weltweit nur 34 Fälle bekannt sind. Der Defekt geht auf den Verlust des Enzyms Transaldolase 1 aus dem nicht-oxidativen Pentosephosphat-Weg (nicht-oxPPW) zurück und äußert sich in einem weiten Spektrum klinischer Symptome. Die schwerwiegendsten Folgen sind Leber- und Nierenmangelfunktionen, die zum sehr frühen Tod führen können. Desweiteren leiden 15 % der Patienten an wiederkehrenden Infektionen. Neutrophile Granulozyten (Neutrophile) sind die häufigsten weißen Blutkörperchen im Menschen und essentiell für die angeborene Immunantwort gegen Infektionserreger. Ich habe hier funktionale Aspekte von TALDO-Neutrophilen untersucht. Der oxidative Pentosephosphat-Weg (oxPPW) stellt das Reduktionsäquivalent NADPH bereit, welches indirekt für die Entstehung von reactive oxygen species (ROS)-abhängigen Neutrophil Extracellular Traps (NETs) verantwortlich ist. Der Beitrag des nicht-oxPPW zur ROS-abhängigen NET-Bildung ist bislang nicht bekannt. In dieser Arbeit konnte ich für Neutrophile aus drei TALDO-Patienten eine jeweils komplett abwesende Entstehung ROS-abhängiger NETs und einen deutlich verringerten oxidativen Burst nach PMA-Stimulation zeigen. Um diese Beobachtungen in einem unabhängigen Modelsystem zu bestätigen, habe ich mit Hilfe des CRISPR-Cas9-Systems, ‚knock-out‘ Mutanten von Transaldolase 1 und dessen Partnerenzym Transketolase in der Neutrophil-ähnlichen Zelllinie PLB-985 hergestellt. Die dergestalt genetisch manipulierten Zellen waren nicht mehr zu PMA-induziertem Zelltod in der Lage. Dies ist somit der erste genetische Beweis für die Abhängigkeit des oxidativen Burst und der Bildung von NETs vom nicht-oxPPW. Diese Erkenntnis trägt zum einen zum mechanistischen Verständnis der NET-Entstehung bei und liefert zum anderen eine potentielle Erklärung für einige der bei TALDO beobachteten Symptome. Desweiteren wurden einige der metabolischen Erfordernisse für die Bildung von NETs mit Hilfe von Inhibitoren untersucht. Die erhaltenen Erkenntnisse zeigen, dass das initiale Maximum des oxidativen Bursts für NET-Bildung unerheblich ist und vielmehr die ROS-Generierung nach ca. 50 Minuten entscheidende Bedeutung für diese hat. / Transdaldolase 1-deficiency (TALDO) is a rare genetic disease with only 34 described cases globally. Transaldolase 1 is part of the non-oxidative pentose phosphate pathway (PPP) and its deficiency results in many clinical symptoms including kidney and liver failure, which can lead to early child-mortality. Some of these patients suffer from recurrent infections, for example in the respiratory tract. Neutrophils are the most abundant white blood cells and essential for the innate immune defence against bacterial and fungal pathogens. The PPP generates reduced NADPH that is crucial for the generation of superoxide by the NADPH oxidase NOX2. In turn, NOX2 is essential for neutrophil extracellular trap (NET) formation. NETs occur through the neutrophil-specific cell death netosis and consist of chromatin decorated with granular proteins. Here I report that neutrophils of three TALDO patients did not make NETs. Deletion of transaldolase 1, and its partner enzyme transketolase, in the neutrophil-like PLB-985 cell line reduced ROS generation and cell death. This confirms that transaldolase 1 is required for NET formation. We present, to the best of our knowledge, the first genetic evidence that the non-oxidative PPP is required for ROS generation and NET formation. Furthermore, some of the metabolic requirements for NET formation were assessed. The obtained data indicate that the initial peak of the oxidative burst is irrelevant for NET formation but the ROS generation after 50 minutes on the contrary has crucial significance.

Non-resolving inflammation plays a critical role during the transition from renal injury towards end-stage renal disease. The glucocorticoid-inducible protein annexin A1 has been shown to function as key regulator in the resolution phase of inflammation, but its role in immune-mediated crescentic glomerulonephritis has not been studied so far. Methods: Acute crescentic glomerulonephritis was induced in annexin A1-deficient and wildtype mice using a sheep serum against rat glomerular basement membrane constituents. Animals were sacrificed at d5 and d10 after nephritis induction. Renal leukocyte abundance was studied by immunofluorescence and flow cytometry. Alterations in gene expression were determined by RNA-Seq and gene ontology analysis. Renal levels of eicosanoids and related lipid products were measured using lipid mass spectrometry. Results: Histological analysis revealed an increased number of sclerotic glomeruli and aggravated tubulointerstitial damage in the kidneys of annexin A1-deficient mice compared to the wildtype controls. Flow cytometry analysis confirmed an increased number of CD45+ leukocytes and neutrophil granulocytes in the absence of annexin A1. Lipid mass spectrometry showed elevated levels of prostaglandins PGE2 and PGD2 and reduced levels of antiinflammatory epoxydocosapentaenoic acid regioisomers. RNA-Seq with subsequent gene ontology analysis revealed induction of gene products related to leukocyte activation and chemotaxis as well as regulation of cytokine production and secretion. Conclusion: Intrinsic annexin A1 reduces proinflammatory signals and infiltration of neutrophil granulocytes and thereby protects the kidney during crescentic glomerulonephritis. The annexin A1 signaling cascade may therefore provide novel targets for the treatment of inflammatory kidney disease.

Stem cells differentiate through an organized hierarchy of intermediate cell types to terminally differentiated cell types. This process is largely guided by master transcriptional regulators, but it also depends on the expression of many other types of genes. The discrete cell types in the differentiation hierarchy are often identified based on the expression or non-expression of certain marker genes. Historically, these have often been various cell-surface proteins, which are fairly easy to assay biochemically but are not necessarily causative of the cell type, in the sense of being master transcriptional regulators. This raises important questions about how gene expression across the whole genome controls or reflects cell state, and in particular, differentiation hierarchies. Traditional approaches to understanding gene expression patterns across multiple conditions, such as principal components analysis or K-means clustering, can group cell types based on gene expression, but they do so without knowledge of the differentiation hierarchy. Hierarchical clustering and maximization of parsimony can organize the cell types into a tree, but in general this tree is different from the differentiation hierarchy. Using hematopoietic differentiation as an example, we demonstrate how many genes other than marker genes are able to discriminate between different branches of the differentiation tree by proposing two models for detecting genes that are up-regulated or down-regulated in distinct lineages. We then propose a novel approach to solving the following problem: Given the differentiation hierarchy and gene expression data at each node, construct a weighted Euclidean distance metric such that the minimum spanning tree with respect to that metric is precisely the given differentiation hierarchy. We provide a set of linear constraints that are provably sufficient for the desired construction and a linear programming framework to identify sparse sets of weights, effectively identifying genes that are most relevant for discriminating different parts of the tree. We apply our method to microarray gene expression data describing 38 cell types in the hematopoiesis hierarchy, constructing a sparse weighted Euclidean metric that uses just 175 genes. These 175 genes are different than the marker genes that were used to identify the 38 cell types, hence offering a novel alternative way of discriminating different branches of the tree. A DAVID functional annotation analysis shows that the 175 genes reflect major processes and pathways active in different parts of the tree. However, we find that there are many alternative sets of weights that satisfy the linear constraints. Thus, in the style of random-forest training, we also construct metrics based on random subsets of the genes and compare them to the metric of 175 genes. Our results show that the 175 genes frequently appear in the random metrics, implicating their significance from an empirical point of view as well. Finally, we show how our linear programming method is able to identify columns that were selected to build minimum spanning trees on the nodes of random variable-size matrices.