39 research outputs found
Analytical validation of innovative magneto-inertial outcomes: a controlled environment study.
peer reviewe
Molecular Dynamics for Synthetic Biology
Synthetic biology is the field concerned with the design, engineering, and construction of organisms and biomolecules. Biomolecules such as proteins are nature's nano-bots, and provide both a shortcut to the construction of nano-scale tools and insight into the design of abiotic nanotechnology. A fundamental technique in protein engineering is protein fusion, the concatenation of two proteins so that they form domains of a new protein. The resulting fusion protein generally retains both functions, especially when a linker sequence is introduced between the two domains to allow them to fold independently. Fusion proteins can have features absent from all of their components; for example, FRET biosensors are fusion proteins of two fluorescent proteins with a binding domain. When the binding domain forms a complex with a ligand, its dynamics translate the concentration of the ligand to the ratio of fluorescence intensities via FRET.
Despite these successes, protein engineering remains laborious and expensive. Computer modelling has the potential to improve the situation by enabling some design work to occur virtually. Synthetic biologists commonly use fast, heuristic structure prediction tools like ROSETTA, I-TASSER and FoldX, despite their inaccuracy. By contrast, molecular dynamics with modern force fields has proven itself accurate, but sampling sufficiently to solve problems accurately and quickly enough to be relevant to experimenters remains challenging.
In this thesis, I introduce molecular dynamics to a structural biology audience, and discuss the challenges and theory behind the technique. With this knowledge, I introduce synthetic biology through a review of fluorescent sensors. I then develop a simple computational tool, Rangefinder, for the design of one variety of these sensors, and demonstrate its ability to predict sensor performance experimentally. I demonstrate the importance of the choice of linker with yet another sensor whose performance depends critically thereon. In chapter 6, I investigate the structure of a conserved, repeating linker sequence connecting two domains of the malaria circumsporozoite protein. Finally, I develop a multi-scale enhanced sampling molecular dynamics approach to predicting the structure and dynamics of fusion proteins. It is my hope that this work contributes to the structural biology community's understanding of molecular dynamics and inspires new techniques developed for protein engineering
Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition)
The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer-reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state-of-the-art handbook for basic and clinical researchers
Study of the immune microenvironment in Mycosis Fungoides
Mycosis fungoides is a rare, indolent and incurable lymphoma of T cells affecting the skin. While the majority of patients have survival measured in the decades, around one third present with advanced disease and another quarter progress rapidly. Advanced disease compromises skin barrier integrity, and is amenable to few effective treatments. The malignant cell is usually a CD4+ T cell,
which in healthy individuals plays a key role in our protection against pathogens and cancer. As a clonal expansion of an existing T cell, these tumour cells already possess an idiomatic mutation in the form of the rearranged T cell receptor (TCR) gene.
Paired skin biopsies and blood was taken from consenting patients, the skin sample digested, and stained with anti-T Cell Receptor V-beta region (TCR-Vβ) antibodies to identify a clonotypic tumour population. This antibody was used to discriminate tumour and Tumour Infiltrating Lymphocytes (TIL) in a study looking at immune checkpoint markers, functionality, cytotoxicity and regulatory phenotypes. Single cell RNA sequencing (scRNA-seq) was employed with simultaneous TCR sequencing.
We found that TIL demonstrated a homogeneous exhaustion phenotype which was consistent between samples and with disease progression. The tumour population was heterogeneous, clustering into three phenotypic groups, and demonstrating high polyfunctionality, overproducing IL-4 and IL-17a, in what is likely immune evasion by deviation. The tumour did not show a
suppressive phenotype, and a mathematical model was created to understand why this may not provide an advantage to the tumour. scRNA-seq analysis found several recurrent differentially expressed genes in tumour, which may represent promising targets for therapeutic potential.
This work is the largest single cell study of the immune microenvironment in mycosis fungoides, and utilises two novel methods to discriminate tumour and TIL, adding substantially to our understanding of the aetiology and progression of mycosis fungoides. Developing new therapeutic targets in mycosis fungoides must ensure that the infiltrating T cells are retained while targeting the tumour population, and this work identifies several avenues by which this could be achieved
Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition)
The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer‐reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state‐of‐the‐art handbook for basic and clinical researchers.DFG, 389687267, Kompartimentalisierung, Aufrechterhaltung und Reaktivierung humaner Gedächtnis-T-Lymphozyten aus Knochenmark und peripherem BlutDFG, 80750187, SFB 841: Leberentzündungen: Infektion, Immunregulation und KonsequenzenEC/H2020/800924/EU/International Cancer Research Fellowships - 2/iCARE-2DFG, 252623821, Die Rolle von follikulären T-Helferzellen in T-Helferzell-Differenzierung, Funktion und PlastizitätDFG, 390873048, EXC 2151: ImmunoSensation2 - the immune sensory syste
Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition)
The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer-reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state-of-the-art handbook for basic and clinical researchers
Targeting STAT3 and STAT5 in Cancer
Every minute, 34 new patients are diagnosed with cancer globally. Although over the past 50 years treatments have improved and survival rates have increased dramatically for several types of cancers, many remain incurable. Several aggressive types of blood and solid cancers form when mutations occur in a critical cellular signaling pathway, the JAK-STAT pathway; (Janus Kinase-Signal Transducer and Activator of Transcription). Currently, there are no clinically available drugs that target the oncogenic STAT3/5 proteins in particular or their Gain of Function hyperactive mutant products. Here, we summarize targeting approaches on STAT3/5, as the field moves towards clinical applications as well as we illuminate on upstream or downstream JAK-STAT pathway interference with kinase inhibitors, heat shock protein blockers or changing nuclear import/export processes. We cover the design paradigms and medicinal chemistry approaches to illuminate progress and challenges in understanding the pleiotropic role of STAT3 and STAT5 in oncogenesis, the microenvironment, the immune system in particular, all culminating in a complex interplay towards cancer progression
Evaluation of the T cell response during the Trypanosoma cruzi infection and its relation with the infection control
El parásito protozoo intracelular Trypanosoma cruzi, causante de la enfermedad de Chagas, establece un estado crónico de infección, que en aproximadamente el 60% de los individuos cursa con ausencia de síntomas durante toda la vida; sin embargo, el resto de los individuos desarrollan alteraciones cardiacas o digestivas que los pueden llevar a la muerte (Perez-Molina et al., 2018). Aunque a ¬la fecha no se conoce con certeza el papel que juega el sistema inmune en el desenlace de la infección por T. cruzi, en modelos múridos de la enfermedad de Chagas se ha descrito que los Linfocitos T (LT) y su respuesta efectora son necesarias para el control de la parasitemia y la sobrevida de los ratones (Tarleton et al., 1994; Tzelepis et al., 2006). Además, a la fecha, si bien el mecanismo relacio¬¬nado con la patología de la enfermedad de Chagas no es claro, estudios han sugerido que la falla en el control de la infección por el parásito estaría relacionada con el desenlace de la misma. En humanos con infección crónica por T. cruzi se ha mostrado que individuos asintomáticos presentan aumento en la frecuencia de LT con menor grado de diferenciación celular, mayor capacidad multifuncional y menor expresión de receptores inhibitorios en comparación con pacientes con las formas severas de la enfermedad de Chagas (Lasso et al., 2015; Mateus et al., 2015), sin embargo, se desconoce si estos hallazgos están relacionados con el control de la infección crónica por T. cruzi.
En estudios realizados en humanos y ratones en modelos de enfermedades infecciosas se ha encontrado que un aspecto que puede contribuir a la eliminación o al control eficiente de agentes infecciosos está relacionado con la calidad de la respuesta funcional de los LT (Appay et al., 2002; Bengsch et al., 2010; Betts et al., 2006; Bhadra et al., 2011; Esch et al., 2013; Riou et al., 2012; Wherry, Teichgraber, et al., 2003). Teniendo en cuenta la historia natural de la enfermedad de Chagas, la dificultad para seguir y documentar los individuos infectados hasta por 40 años y para establecer la relación entre el control de la infección por T. cruzi con la respuesta funcional de los LT, en el presente estudio se usaron dos enfoques metodológicos para dar respuesta al siguiente interrogante: ¿cuál es la relación de la repuesta funcional de los LT con el control de la infección por T. cruzi?
En vista que reportes han mostrado que el tratamiento antiparasitario reduce la carga parasitaria en individuos y ratones con infección crónica por T. cruzi (Bustamante et al., 2008; Morillo et al., 2015) y que este podría ser útil para entender si el control de la infección por el parásito en individuos crónicamente infectados y tratados estaría relacionada con cambios en la respuesta funcional de los LT, en el primer enfoque se evaluó el efecto del tratamiento antiparasitario sobre la capacidad funcional de la respuesta de los LT de pacientes con enfermedad de Chagas en fase crónica (PCC). Por otro lado, el segundo enfoque incluyó el uso del modelo múrido experimental de infección por T. cruzi, una herramienta que ha mostrado ser útil para el estudio de la enfermedad de Chagas. Con este enfoque se determinó la respuesta funcional de los LT en el modelo experimental de infección por T. cruzi y su relación con el control de la infección evaluado por la carga parasitaria y el daño en el tejido. Para desarrollar este enfoque, primero se estableció un modelo de infección aguda y crónica con el propósito de determinar si durante la infección crónica, al igual que en los humanos, se observaba el deterioro de los LT. Posteriormente, teniendo en cuenta que una limitación para el estudio de la infección crónica por T. cruzi en humanos y modelos múridos es la cuantificación de la carga parasitaria y del daño en tejido y que se ha documentado que la variabilidad genética del parásito o las reinfecciones pueden tener un impacto en el curso y desenlace de la infección por T. cruzi (Andrade et al., 2006; Bustamante et al., 2007; Bustamante et al., 2002; Perez et al., 2018; Santi-Rocca et al., 2017), se evaluó si la respuesta de los LT se relaciona con el control de la infección en un modelo múrido de infección con cepas de dos grupos genéticos del parásito y durante la reinfección o infección mixta por cepas de T. cruzi.
Con el desarrollo de este trabajo, usando los enfoques mencionados anteriormente, se encontró que posterior al tratamiento antiparasitario de PCC se evidencia una mejor calidad de la respuesta de LT al comparar con PCC sin tratamiento antiparasitario. Esto fue revelado por mayor frecuencia de LT con un menor grado de diferenciación celular (TCM), aumento de la capacidad funcional y multifuncional de los LT y disminución de la expresión de receptores inhibitorios en LT. Por otro lado, en el modelo experimental de infección se encontró que la infección aguda con la cepa Y de T. cruzi conduce al aumento de la carga parasitaria y del infiltrado inflamatorio en tejido, aumento de células efectoras, la generación de LT multifuncionales específicos de antígeno y su regulación mediante la alta expresión de receptores inhibitorios; en contraste, ratones con infección crónica con la cepa Y del parásito presentaron bajos niveles de carga parasitaria y del infiltrado inflamatorio en tejido y deterioro de la respuesta de los LT caracterizada por aumento de células con mayor grado de diferenciación (TEM), disminuida capacidad funcional y aumento en la expresión de receptores inhibitorios. Al comparar estos resultados con los hallazgos de los ratones infectados con la cepa DA de T. cruzi, se encontró menor carga parasitaria e infiltrado inflamatorio en tejido y una respuesta de LT caracterizada por células con menor grado de diferenciación celular, mayor capacidad funcional y disminuida expresión de receptores inhibitorios durante la infección aguda y crónica por T. cruzi.
Posteriormente, en vista de que las reinfecciones detectadas en individuos naturalmente infectados pueden estar relacionadas con la progresión de la enfermedad de Chagas (Perez et al., 2014; Zicker et al., 1990) y que en modelos animales experimentales se ha mostrado que las reinfecciones conducen a la falla en el control de la infección (evidenciada por alta carga parasitaria y alto infiltrado inflamatorio en tejido) (Andrade et al., 2006; Bustamante et al., 2007; Bustamante et al., 2002; Perez et al., 2018), se evaluó si la respuesta funcional de los LT se relaciona con el control de la infección crónica por el parásito en un modelo múrido de reinfección por cepas de T. cruzi. Se encontró menor carga parasitaria en ratones infectados y reinfectados con la cepa Y (Y/Y) en comparación con ratones infectados con la cepa DA y reinfectados con la cepa Y (DA/Y). De manera interesante, los ratones infectados con Y/Y presentaron una mejor respuesta de los LT, caracterizada por aumento de LT con un menor grado de diferenciación celular (TCM) y disminución de la expresión de receptores inhibitorios en LT en comparación con los ratones DA/Y. Finalmente, un análisis de correlación mostró que hay una relación entre la calidad de la repuesta de los LT con el control de la infección en ratones sometidos a infecciones mixtas por cepas del parásito.
Por lo tanto, tomando en consideración los hallazgos mencionados anteriormente, tanto en PCC sin y con tratamiento antiparasitario como en el modelo experimental de infección y reinfección por T. cruzi, los resultados nos permiten concluir que la calidad de la respuesta de los LT se relaciona con el control de la infección por el parásito. Estos hallazgos son un precedente para entender los mecanismos relacionados con la progresión de la enfermedad de Chagas en humanos crónicamente infectados y diseñar estrategias de control y seguimiento de los individuos infectados.COLCIENCIASPontificia Universidad JaverianaChagas disease (ChD) is a chronic infection caused by Trypanosoma cruzi, a protozoan that causes potentially life-threatening illnesses. This intracellular parasite is highly diverse and has been classified, based on the genetic markers, into seven genotypes or discrete typing units (DTUs, TcI-TcVI and TcBat); however, the parasite genotypes overlap in geographic range, vectors, and clinical characteristics. Although studies have suggested that the progression of ChD is due to a deterioration in the quality of the immune response, a direct relationship between the T cell responses and the outcome of the disease has not been established. Here, different approaches were used to determine the T cell responses in chronic chagasic patients (CCPs) and mice experimentally infected with T. cruzi to investigate the relationship between the parasite control and the T cell response.
Since there is a lack of biological markers to evaluate the effectiveness of antiparasitic treatment, and little is known about the effect of the treatment on T cells from CCPs. The first approach aimed to analyze the early effects of antiparasitic treatment on CD8+ T cells from CCPs with asymptomatic clinical forms. To evaluate the CD8+ T cell subsets, expression of inhibitory receptors, and functionality of T cells in CCPs, PBMCs were isolated. The results showed that the treatment of CCPs with the asymptomatic form of the disease induces an increase in the frequency of CD8+ central memory T cells and terminal effector T cells and a decrease in the coexpression of inhibitory receptors. Also, an improved Ag-specific CD8+ T cell response exhibited by the individual production of IFNg or IL-2, and a multifunctional CD8+ T cell profile of up to four functions were detected. These findings showed that the antiparasitic treatment improved the quality of Ag-specific CD8+ T cell responses associated with a decrease in the expression of inhibitory receptors, which could serve as biomarkers for monitoring the effectiveness of antiparasitic treatment.
Given that the long natural history of ChD makes the monitoring of patients and the sequential study of the immune mechanisms underlying protection or disease pathogenesis intricate and the experimental models of T. cruzi infection have been successfully used to propose or develop new strategies to combat ChD, the second approach included the development of an experimental murine model of T. cruzi infection to investigate the T cell response and the infection control. First, it was investigated the T cell response in a murine model of acute (10 and 30 days) and chronic (100 and 260 days) ChD, characterized by parasite persistence for up to 260 days post-infection and moderate inflammation of the colon and liver of T. cruzi-infected mice. Acute ChD induced a high Ag-specific multifunctional T cell response by producing IFNg, TNFa, IL-2, granzyme B, and perforin, and a high frequency of T cells coexpressed 2B4, CD160, CTLA-4, and PD-1. In contrast, chronically infected mice with moderate inflammatory infiltrate in liver tissue exhibited monofunctional Ag-specific cells, high cytotoxic activity (granzyme B and perforin), and elevated levels of inhibitory receptors (predominantly CTLA-4 and PD-1) coexpressed on T cells. Thus, these data support our previous results showing that similar to humans, the T. cruzi persistence in mice promotes the dysfunctionality of T cells, and these changes might correlate with ChD progression.
Then, to analyzed the relationship between the parasite control and the immune response in a murine model of T. cruzi infection, it was determined the histological and parasitological outcomes and dissected the T cell responses in mice infected by two genetically different T. cruzi strains (Y or DA) or in mice with homologous (Y/Y) or heterologous (DA/Y) infection by T. cruzi strains. Chronically infected mice with low inflammatory infiltrate (for DA infected mice) or low parasitemia and parasitism (for Y/Y infected mice), showed an increase in early-differentiated T cells and a multifunctional T cell response along with a lower expression of inhibitory receptors on T cells. In contrast, chronically infected mice with high inflammatory infiltrate (for Y infected mice), or high parasitemia and parasitism (for DA/Y infected mice) showed a T cell response distinguished by an increase in late-differentiated cells, monofunctional response, and increment in the expression of inhibitory receptors on T cells.
Therefore, considering the findings mentioned above, in CCPs and the experimental model of infection and reinfection by T. cruzi, the results allow us to conclude that the quality of the T cell response is related to parasite control during the chronic T. cruzi infection. These findings are a precedent for understanding the mechanisms related to the progression of Chagas disease in chronically infected humans and design strategies for the control and follow-up of infected individuals.Doctor en Ciencias BiológicasDoctorad