Intestinal macrophages: differentiation and involvement in intestinal immunopathologies

Intestinal macrophages, preferentially located in the subepithelial lamina propria, represent the largest pool of tissue macrophages in humans. As an adaptation to the local antigen- and bacteria-rich environment, intestinal macrophages exhibit several distinct phenotypic and functional characteristics. Notably, microbe-associated molecular pattern receptors, including the lipopolysaccharide (LPS) receptors CD14 and TLR4, and also the Fc receptors for IgA and IgG are absent on most intestinal macrophages under homeostatic conditions. Moreover, while macrophages in the intestinal mucosa are refractory to the induction of proinflammatory cytokine secretion, they still display potent phagocytic activity. These adaptations allow intestinal macrophages to comply with their main task, i.e., the efficient removal of microbes while maintaining local tissue homeostasis. In this paper, we review recent findings on the functional differentiation of monocyte subsets into distinct macrophage populations and on the phenotypic and functional adaptations that have evolved in intestinal macrophages in response to their antigen-rich environment. Furthermore, the involvement of intestinal macrophages in the pathogenesis of celiac disease and inflammatory bowel diseases is discussed.

Influence of age and genetic risk on anti-tissue transglutaminase IgA titers

OBJECTIVES: False-positive results of anti-tissue-transglutaminase (tTG) IgA autoantibodies have been reported in subjects with a genetic risk for celiac disease (CD). The aims of this retrospective study were to assess the prevalence of false-positive tTG titers in patients at risk of CD compared with symptomatic children and to evaluate the influence of age and indication for testing on tTG titers. PATIENTS AND METHODS: All tTG results measured in our institution during a 33-month period were evaluated. Patients with known CD were excluded. Indications for testing were either symptoms suggestive of CD (group 1) or history of being at risk for CD (group 2). Duodenal biopsies were recommended if titers were positive (> or =10 U/mL) and offered if borderline (> or =4 to <10 U/mL). RESULTS: The final analysis included 2056 patients, 1707 belonged to group 1, and 349 to group 2. All 65 patients with positive tTG results underwent biopsy (group 1: 57, group 2: 8). Celiac disease was confirmed in 61 subjects (median titer: 107.8 U/mL, range 12.0-1748 mL, NS between group 1 and 2), whereas 4 had normal histology (10.2-25.2 U/mL). Three out of 16 patients with borderline results underwent biopsy and had normal histology. Borderline titers were more common in group 2 patients (2.6% vs 0.4%, P<0.001). Multiple regression analysis in patients with negative tTG results (n=1975) revealed that titers were independently related to age (P<0.05) and indication for testing (P<0.001). CONCLUSIONS: The influence of age and genetic predisposition/risk has to be taken into account when interpreting tTG results.

Directed differentiation and functional maturation of cortical interneurons from human embryonic stem cells

Human pluripotent stem cells are a powerful tool for modeling brain development and disease. The human cortex is composed of two major neuronal populations: projection neurons and local interneurons. Cortical interneurons comprise a diverse class of cell types expressing the neurotransmitter GABA. Dysfunction of cortical interneurons has been implicated in neuropsychiatric diseases, including schizophrenia, autism, and epilepsy. Here, we demonstrate the highly efficient derivation of human cortical interneurons in an NKX2.1::GFP human embryonic stem cell reporter line. Manipulating the timing of SHH activation yields three distinct GFP+ populations with specific transcriptional profiles, neurotransmitter phenotypes, and migratory behaviors. Further differentiation in a murine cortical environment yields parvalbumin- and somatostatin-expressing neurons that exhibit synaptic inputs and electrophysiological properties of cortical interneurons. Our study defines the signals sufficient for modeling human ventral forebrain development in vitro and lays the foundation for studying cortical interneuron involvement in human disease pathology.

Alcoholic Cirrhosis Increases Risk for Autoimmune Diseases: A Nationwide Registry-Based Cohort Study

BACKGROUND & AIMS Alcoholic cirrhosis is associated with hyperactivation and dysregulation of the immune system. In addition to its ability to increase risk for infections, it also may increase the risk for autoimmune diseases. We studied the incidence of autoimmune diseases among patients with alcoholic cirrhosis vs controls in Denmark. METHODS We collected data from nationwide health care registries to identify and follow up all citizens of Denmark diagnosed with alcoholic cirrhosis from 1977 through 2010. Each patient was matched with 5 random individuals from the population (controls) of the same sex and age. The incidence rates of various autoimmune diseases were compared between patients with cirrhosis and controls and adjusted for the number of hospitalizations in the previous year (a marker for the frequency of clinical examination). RESULTS Of the 24,679 patients diagnosed with alcoholic cirrhosis, 532 developed an autoimmune disease, yielding an overall increased adjusted incidence rate ratio (aIRR) of 1.36 (95% confidence interval [CI], 1.24-1.50). The strongest associations were with Addison's disease (aIRR, 2.47; 95% CI, 1.04-5.85), inflammatory bowel disease (aIRR, 1.56; 95% CI, 1.26-1.92), celiac disease (aIRR, 5.12; 95% CI, 2.58-10.16), pernicious anemia (aIRR, 2.35; 95% CI, 1.50-3.68), and psoriasis (aIRR, 4.06; 95% CI, 3.32-4.97). There was no increase in the incidence rate for rheumatoid arthritis (aIRR, 0.89; 95% CI, 0.69-1.15); the incidence rate for polymyalgia rheumatica decreased in patients with alcoholic cirrhosis compared with controls (aIRR, 0.47; 95% CI, 0.33-0.67). CONCLUSIONS Based on a nationwide cohort study of patients in Denmark, alcoholic cirrhosis is a risk factor for several autoimmune diseases.

Aberrant association of misfolded SOD1 with Na+/K+ATPase-α3 impairs its activity and contributes to motor neuron vulnerability in ALS.

Amyotrophic lateral sclerosis (ALS) is an adult onset progressive motor neuron disease with no cure. Transgenic mice overexpressing familial ALS associated human mutant SOD1 are a commonly used model for examining disease mechanisms. Presently, it is well accepted that alterations in motor neuron excitability and spinal circuits are pathological hallmarks of ALS, but the underlying molecular mechanisms remain unresolved. Here, we sought to understand whether the expression of mutant SOD1 protein could contribute to altering processes governing motor neuron excitability. We used the conformation specific antibody B8H10 which recognizes a misfolded state of SOD1 (misfSOD1) to longitudinally identify its interactome during early disease stage in SOD1G93A mice. This strategy identified a direct isozyme-specific association of misfSOD1 with Na+/K+ATPase-α3 leading to the premature impairment of its ATPase activity. Pharmacological inhibition of Na+/K+ATPase-α3 altered glutamate receptor 2 expression, modified cholinergic inputs and accelerated disease pathology. After mapping the site of direct association of misfSOD1 with Na+/K+ATPase-α3 onto a 10 amino acid stretch that is unique to Na+/K+ATPase-α3 but not found in the closely related Na+/K+ATPase-α1 isozyme, we generated a misfSOD1 binding deficient, but fully functional Na+/K+ATPase-α3 pump. Adeno associated virus (AAV)-mediated expression of this chimeric Na+/K+ATPase-α3 restored Na+/K+ATPase-α3 activity in the spinal cord, delayed pathological alterations and prolonged survival of SOD1G93A mice. Additionally, altered Na+/K+ATPase-α3 expression was observed in the spinal cord of individuals with sporadic and familial ALS. A fraction of sporadic ALS cases also presented B8H10 positive misfSOD1 immunoreactivity, suggesting that similar mechanism might contribute to the pathology.

Enfermedad celíaca y su relación con la cavidad bucal

El objetivo del siguiente trabajo es describir las características de la enfermedad celíaca, y sus expresiones más frecuentes en la cavidad bucal. Las manifestaciones de esta patología pueden ser divididas en formas típicas y atípicas. Las formas atípicas saben mostrar alteraciones extra-intestinales entre las cuales los signos orales suelen constituir las señales iniciales de la enfermedad y podrían orientar al diagnóstico precoz de la misma, favoreciendo a implementar las medidas preventivas adecuadas.

Developmental abnormalities and age-related neurodegeneration in a mouse model of Down syndrome

To study the pathogenesis of central nervous system abnormalities in Down syndrome (DS), we have analyzed a new genetic model of DS, the partial trisomy 16 (Ts65Dn) mouse. Ts65Dn mice have an extra copy of the distal aspect of mouse chromosome 16, a segment homologous to human chromosome 21 that contains much of the genetic material responsible for the DS phenotype. Ts65Dn mice show developmental delay during the postnatal period as well as abnormal behaviors in both young and adult animals that may be analogous to mental retardation. Though the Ts65Dn brain is normal on gross examination, there is age-related degeneration of septohippocampal cholinergic neurons and astrocytic hypertrophy, markers of the Alzheimer disease pathology that is present in elderly DS individuals. These findings suggest that Ts65Dn mice may be used to study certain developmental and degenerative abnormalities in the DS brain.

Neuronal overexpression of mutant amyloid precursor protein results in prominent deposition of cerebrovascular amyloid

Transgenic mice that overexpress mutant human amyloid precursor protein (APP) exhibit one hallmark of Alzheimer’s disease pathology, namely the extracellular deposition of amyloid plaques. Here, we describe significant deposition of amyloid β (Aβ) in the cerebral vasculature [cerebral amyloid angiopathy (CAA)] in aging APP23 mice that had striking similarities to that observed in human aging and Alzheimer’s disease. Amyloid deposition occurred preferentially in arterioles and capillaries and within individual vessels showed a wide heterogeneity (ranging from a thin ring of amyloid in the vessel wall to large plaque-like extrusions into the neuropil). CAA was associated with local neuron loss, synaptic abnormalities, microglial activation, and microhemorrhage. Although several factors may contribute to CAA in humans, the neuronal origin of transgenic APP, high levels of Aβ in cerebrospinal fluid, and regional localization of CAA in APP23 mice suggest transport and drainage pathways rather than local production or blood uptake of Aβ as a primary mechanism underlying cerebrovascular amyloid formation. APP23 mice on an App-null background developed a similar degree of both plaques and CAA, providing further evidence that a neuronal source of APP/Aβ is sufficient to induce cerebrovascular amyloid and associated neurodegeneration.

Expression of the C terminus of the amyloid precursor protein alters growth factor responsiveness in stably transfected PC12 cells.

The amyloid precursor protein (APP) is a molecule centrally involved in Alzheimer disease pathology, but whose normal function is still poorly understood. To investigate the consequences of increased intracellular production of various regions of APP on cellular physiology, we stably transfected PC12 cells with the C-terminal 100 amino acids of the human APP. In eight transfected clones that express the APP(C100) protein, exposure to nerve growth factor (NGF) did not promote differentiation. Transfectants continued to divide and failed to elaborate extensive neurites, whereas control PC12 cells, mock-transfected PC12 cells, and a nonexpressing transfected cell line did develop neurites and stopped dividing after NGF stimulation. Unlike NGF treatment, treatment with basic fibroblast growth factor profoundly accelerated neurite outgrowth in transfected cells. Also, a dramatic increase in a tyrosine phosphatase activity was noted. Expression and accumulation of APP C100 protein in PC12 cells results in an abnormal response to growth factor stimulation.

Innovative analytical strategies for the development of sensor devices and mass spectrometry methods

Il lavoro presentato in questa tesi di Dottorato è incentrato sullo sviluppo di strategie analitiche innovative basate sulla sensoristica e su tecniche di spettrometria di massa in ambito biologico e della sicurezza alimentare. Il primo capitolo tratta lo studio di aspetti metodologici ed applicativi di procedure sensoristiche per l’identificazione e la determinazione di biomarkers associati alla malattia celiaca. In tale ambito, sono stati sviluppati due immunosensori, uno a trasduzione piezoelettrica e uno a trasduzione amperometrica, per la rivelazione di anticorpi anti-transglutaminasi tissutale associati a questa malattia. L’innovazione di questi dispositivi riguarda l’immobilizzazione dell’enzima tTG nella conformazione aperta (Open-tTG), che è stato dimostrato essere quella principalmente coinvolta nella patogenesi. Sulla base dei risultati ottenuti, entrambi i sistemi sviluppati si sono dimostrati una valida alternativa ai test di screening attualmente in uso per la diagnosi della celiachia. Rimanendo sempre nel contesto della malattia celiaca, ulteriore ricerca oggetto di questa tesi di Dottorato, ha riguardato lo sviluppo di metodi affidabili per il controllo di prodotti “gluten-free”. Il secondo capitolo tratta lo sviluppo di un metodo di spettrometria di massa e di un immunosensore competitivo per la rivelazione di prolammine in alimenti “gluten-free”. E’ stato sviluppato un metodo LC-ESI-MS/MS basato su un’analisi target con modalità di acquisizione del segnale selected reaction monitoring per l’identificazione di glutine in diversi cereali potenzialmente tossici per i celiaci. Inoltre ci si è focalizzati su un immunosensore competitivo per la rivelazione di gliadina, come metodo di screening rapido di farine. Entrambi i sistemi sono stati ottimizzati impiegando miscele di farina di riso addizionata di gliadina, avenine, ordeine e secaline nel caso del sistema LC-MS/MS e con sola gliadina nel caso del sensore. Infine i sistemi analitici sono stati validati analizzando sia materie prime (farine) che alimenti (biscotti, pasta, pane, etc.). L’approccio sviluppato in spettrometria di massa apre la strada alla possibilità di sviluppare un test di screening multiplo per la valutazione della sicurezza di prodotti dichiarati “gluten-free”, mentre ulteriori studi dovranno essere svolti per ricercare condizioni di estrazione compatibili con l’immunosaggio competitivo, per ora applicabile solo all’analisi di farine estratte con etanolo. Terzo capitolo di questa tesi riguarda lo sviluppo di nuovi metodi per la rivelazione di HPV, Chlamydia e Gonorrhoeae in fluidi biologici. Si è scelto un substrato costituito da strips di carta in quanto possono costituire una valida piattaforma di rivelazione, offrendo vantaggi grazie al basso costo, alla possibilità di generare dispositivi portatili e di poter visualizzare il risultato visivamente senza la necessità di strumentazioni. La metodologia sviluppata è molto semplice, non prevede l’uso di strumentazione complessa e si basa sull’uso della isothermal rolling-circle amplification per l’amplificazione del target. Inoltre, di fondamentale importanza, è l’utilizzo di nanoparticelle colorate che, essendo state funzionalizzate con una sequenza di DNA complementare al target amplificato derivante dalla RCA, ne permettono la rivelazione a occhio nudo mediante l’uso di filtri di carta. Queste strips sono state testate su campioni reali permettendo una discriminazione tra campioni positivi e negativi in tempi rapidi (10-15 minuti), aprendo una nuova via verso nuovi test altamente competitivi con quelli attualmente sul mercato.

Doença celíaca num doente com síndrome poliglandular autoimune tipo III : caso clínico

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Sensibilidade ao glúten não-celíaca

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Characterization of heparin-binding site of tissue transglutaminase:its importance in cell surface targeting, matrix deposition, and cell signaling

Tissue transglutaminase (TG2) is a multifunctional Ca2+ activated protein crosslinking enzyme secreted into the extracellular matrix (ECM), where it is involved in wound healing and scarring, tissue fibrosis, celiac disease and metastatic cancer. Extracellular TG2 can also facilitate cell adhesion important in wound healing through a non-transamidating mechanism via its association with fibronectin (FN), heparan sulphates (HS) and integrins. Regulating the mechanism how TG2 is translocated into the ECM therefore provides a strategy for modulating these physiological and pathological functions of the enzyme. Here, through molecular modelling and mutagenesis we have identified the HS binding site of TG2 202KFLKNAGRDCSRRSSPVYVGR222. We demonstrate the requirement of this binding site for translocation of TG2 into the ECM through a mechanism involving cell surface shedding of HS. By synthesizing a peptide NPKFLKNAGRDCSRRSS corresponding to the HS binding site within TG2, we also demonstrate how this mimicking peptide can in isolation compensate the RGD-induced loss of cell adhesion on FN via binding to syndecan-4, leading to activation of PKCa, pFAK-397 and ERK1/2 and the subsequent formation of focal adhesions and actin cytoskeleton organization. A novel regulatory mechanism for TG2 translocation into the extracellular compartment that depends upon TG2 conformation and the binding of HS is proposed.