Caracterización de los cambios en el inmunofenotipo asociados a la aparición de la encefalopatía hepática mínima en pacientes cirróticos

Resumen dela tesis doctoral Alba Mangas Losada Introducción: Los pacientes con cirrosis hepática presentan frecuentemente encefalopatía hepática mínima (EHM) con déficits cognitivos, de atención y coordinación motora que disminuyen su calidad de vida y aumentan el riesgo de accidentes. No se conocen los mecanismos que conducen a estas alteraciones ni se dispone de tratamientos para mejorarlas. Se sabe, por estudios anteriores, que los pacientes con EHM tienen un estado de inflamación mayor que es el que lleva, junto con una mala detoxificación del amonio, a provocar los déficits cognitivos. Esta tesis, por tanto, se centra en el estudio del sistema inmune y la inflamación de los pacientes cirróticos con EHM y los cambios específicos en el inmunofenotipo asociados a la aparición de la EHM. Proponemos la hipótesis de que la aparición de la EHM se asociaría a algún cambio cualitativo específico en la inflamación periférica. El objetivo de este trabajo fue caracterizar los cambios en la inflamación periférica asociados a la EHM. Se analizaron por inmunofenotipificación y análisis de perfil de citocinas, en pacientes cirróticos sin o con EHM y controles sin enfermedad hepática los siguientes parámetros: a) subgrupos de monocitos: clásicos (CD14 ++ CD16-), intermedios (CD14 ++ CD16 +) y no clásicos (CD14 + CD16 ++); B) el subconjunto celular CD4 + CD28- pro-inflamatorio y los linfocitos T naive y de memoria ; C) activación de linfocitos T y B midiendo los niveles de marcador de activación temprana CD69 e IgG; D) diferenciación de linfocitos T CD4 + a diferentes subtipos: Th1, Th2, Th17, Th22, iTreg o Tfh y e) perfil de citocinas séricas: IL-6, IL-10, TNFα, CCL20, CXCL13, IL-15, CX3CR1 , TGFbeta, IL - 21, IL - 17, IL - 12, IL - 18, IL - 1beta e IL - 22. Descripción de los medios utilizados para la realización de la actividad: Metodología: Pacientes y controles. Los estudios se realizarán en controles sanos, en pacientes cirróticos sin EHM y en pacientes cirróticos con EHM diagnosticada utilizando la batería PHES de tests psicométricos. Se incluirán en el estudio pacientes que no hayan tenido EH clínica y se encuentren compensados, bien tras un episodio de descompensación por ascitis o hemorragia digestiva o por encontrarse en fase B de Child-Pugh. Se aplicará como criterio de exclusión que el paciente presente alguna otra enfermedad neurológica, psíquica, descompensación diabética, alcoholismo o adicción activa a las drogas. El procedimiento a seguir en cada individuo será el siguiente: 1) Información y recogida del consentimiento informado (Declaración de Helsinki) 2) Recogida de datos epidemiológicos y de la enfermedad hepática: etiología 3) Recogida de valores analíticos 4) Determinación de la presencia de EHM. Se determinará mediante la batería de tests psicométricos Psychometric Hepatic Encephalopathy Score (PHES). Se considerará que un individuo presenta EHM si su puntuación es -4 ó inferior. (Weissenborn et al., 2001; Ferenci et al., 2002). 5) Evaluación de atención selectiva y sostenida y de coordinación motora: Los sujetos realizarán los siguientes tests: Test Stroop, test de atención “d2” y test de coordinación bimanual y visomotora. 6) Evaluación de la inflamación y de parámetros relacionados con el sistema inmunológico en sangre. Posible identificación de biomarcadores periféricos para diagnosticar la presencia de EHM y de alteraciones neuropsicológicas específicas en pacientes cirróticos. Relación entre el sistema periférico y el deterioro cognitivo visto en pacientes con EHM. Resultados obtenidos: Las principales alteraciones asociadas específicamente con EHM, no presentes en los pacientes sin MHE, son: 1) aumento de la activación de todos los subtipos de linfocitos T CD4 +, como se indica por el aumento de la expresión de CD69; 2) una mayor cantidad de linfocitos CD4 + CD28-T, asociados con niveles aumentados de CX3CL1 (fractalkina) y de IL-15; 3) aumento de la diferenciación de linfocitos T CD4 + a Th folicular y Th22; 4) aumento de la activación de linfocitos B y IgG sérica. Conclusión: Este estudio ha identificado algunas alteraciones del sistema inmune que están asociadas específicamente con la aparición de las alteraciones neurológicas en pacientes cirróticos con EMH. Discusión: La encefalopatía hepática (EH) es una consecuencia del fallo hepático y conlleva un amplio rango de alteraciones neuropsiquiátricas, desde ligeros cambios en la personalidad o alteraciones en el ciclo sueño-vigilia, hasta alteraciones en la función cognitiva, en la coordinación motora y en el grado de consciencia. Entre el 30 y el 50% de los pacientes con cirrosis que no muestran signos clínicos de EH presentan encefalopatía hepática mínima (EHM) con un leve deterioro cognitivo. Los pacientes con EHM pueden presentar diversos déficits cognitivos (percepción visuoespacial, atención, concentración) y motores (enlentecimiento psicomotor) que no son detectables en un examen neurológico rutinario pero se ponen de manifiesto cuando se realizan test psicométricos específicos (Amodio et al., 2004). Los pacientes cirróticos presentan hiperamonemia, que alcanza niveles similares en los grupos sin y con EHM. El amonio es capaz de atravesar la barrera hematoencefalica (BHE) induciendo alteraciones en el sistema nervioso central. Se ha demostrado que la hiperamonemia crónica altera la vía del GMPc en cerebro y que este es responsable de algunos tipos de deterioro cognitivo (Felipo, 2013). La inflamación agrava las alteraciones neurológicas producidas por la hiperamonemia en pacientes cirróticos y que además, determina el grado de severidad de la EH en pacientes cirróticos con EH avanzada (Shawcross et al., 2004). En esta tesis se ha realizado un amplio estudio de citocinas en pacientes cirróticos, demostrando que los niveles de IL-6, IL-21, IL-17, IL-10, IFNγ, IL-18, CCL20, TNFα, CXCL13, IL-15 y CX3CL1 en suero están aumentados tanto en los cirróticos con o sin EHM, pero los pacientes con EHM muestran un aumento mayor que los pacientes sin EHM, sugiriendo una respuesta inmunológica más potente en los pacientes con EHM. En el caso del TGFβ, los pacientes cirróticos muestran una disminución en suero independiente de la presencia de EHM. Además, los niveles de IL-12, IL-1β e IL-22 muestran un incremento exclusivo de los pacientes con EHM, que no vemos en paciente sin EHM, sugiriendo que el aumento de estas interleucinas se produce en estadios más tardíos del desarrollo de la EHM. Además, la IL-6, IL-21, TGFβ, IL-10, IL-18, IL-13, CCL20, TNFα, CXCL13, IL-15 y CX3CL1 correlacionan significativamente con el grado de EHM (según puntuación del PHES), indicando que la inflamación es un contribuyente esencial al deterioro neurológico en la EHM. Todos estos datos concluyen claramente que la inflamación tiene un papel fundamental en el desarrollo del deterioro neurológico, cognitivo y motor, en los pacientes cirróticos que presentan EHM. El hecho de que la inflamación esté relacionada con más alteraciones neurológicas que la hiperamonemia, indica que sería el grado de inflamación el que tendría más peso en la determinación de la aparición del deterioro neurológico. En cuanto al estudio del inmunofenotipo, observamos una serie de cambios en los pacientes con EHM. En primer lugar, atendemos a los monocitos representados por los marcadores de superficie CD14 y CD16, con mayor o menos expresión en función de si son clásicos, intermedios o no clásicos. Hay una disminución de la población clásica (CD14++CD16-) y un aumento de la población intermedia (CD14++CD16+) en los pacientes con EHM. Esto va a provocar, por una parte, que haya mayor producción de citocinas proinflamatorias por parte de los monocitos/macrófagos intermedios que va a propiciar un ambiente proinflamatorio que condicionará la diferenciación de los diferentes subtipos de linfocitos CD4. Por otra parte, estos mismos macrófagos M1 van a actuar como CPA con los linfocitos, de manera que pueden activar a los linfocitos naive. Observamos un aumento de los linfocitos autoreactivos CD4+CD28- en los pacientes con EHM. Esta población celular va a ser atraída hacia gradientes de CX3CL1 ayudados por la IL-15 (vemos que ambas citocinas están aumentadas en EHM). El CX3CL1 puede ser producido en células endoteliales y las IL-15 en células de tejido nervioso, entre otras. Proponemos la primera hipótesis de infiltración de CD4+CD28- a través de la BHE, promovida por el aumento de CX3CL1 e IL-15 en el medio. Vemos un cambio de los linfocitos naive hacia los de memoria en ambos tipos de pacientes. Por el contrario, observamos un mayor número de células positivas para el marcador de activación CD69, exclusivo en pacientes con EHM, en varios tipos celulares, como los linfocitos CD4, tanto CD28+ como CD28-, en los CD4 naive, en los CD4 de memoria y en los linfocitos B. A partir del aumento de ciertas citocinas proinflamatorias en suero de pacientes con EHM, como el TNFα, la IL-6 y la IL-1β, que tienen sus receptores en células endoteliales, proponemos nuestra segunda hipótesis, la transmisión de señales a través de los receptores de interleucinas en células endoteliales. Tras desencadenar la cascada de trasmisión de señales, causarían un efecto directo sobre las células neurales al otro lado de las BHE. Los linfocitos CD4 naive se diferencian en varias subpoblaciones en función de ciertos factores que condicionan el medio. Las dos subpoblaciones principales aumentadas en pacientes con EHM son los Th22 y los Tfh, corroborado también con el aumento de las citocinas que producen. La IL-21 favorece a la diferenciación de los Th17 e inhibe las iTreg, pero su principal función es la activación de los linfocitos B. Por ello, vemos una mayor producción de IgG en los pacientes con EHM y la posible formación de centros germinales. Además, la IL-22, la IL-17 y la IL-21 favorecen la secreción de CXCL13, desde estos centros germinales, cuya función es atraer a los linfocitos B y los Thf hacia los mismos. A partir de estos datos establecemos nuestras dos últimas hipótesis, la infiltración de Tfh y la posible formación de órganos linfoides terciarios dentro del cerebro de pacientes con EHM, que estaría propiciada por los propios Tfh infiltrados. A partir de aquí, sería necesaria la continuidad de estos estudios para caracterizar mejor la contribución de los cambios inmunológicos en las alteraciones neurológicas y su posible utilidad en el diagnóstico temprano de la EHM en pacientes cirróticos.Introduction: Patients with cirrhosis of the liver often have minimal hepatic encephalopathy (MHE) with cognitive deficits, attention and motor coordination that decrease their quality of life and increase the risk of accidents. The mechanisms that lead to these alterations are not known, nor are treatments available to improve them. It is known from previous studies that patients with MHD have a higher inflammation state which leads, along with poor ammonium detoxification, to cause cognitive deficits. This thesis, therefore, focuses on the study of the immune system and inflammation of cirrhotic patients with MHE and the specific changes in the immunophenotype associated with the onset of MHE. We hypothesize that the onset of MHE would be associated with some specific qualitative change in peripheral inflammation. The aim of this study was to characterize the changes in peripheral inflammation associated with MH. The following parameters were analyzed by immunophenotyping and cytokine profile analysis in cirrhotic patients with or without HMV and controls without hepatic disease: a) monocyte subgroups: classic (CD14 + CD16-), intermediate (CD14 + CD16 +) And non-classical (CD14 + CD16 ++); B) the CD4 + CD28-pro-inflammatory cell subset and the naive and memory T lymphocytes; C) activation of T and B lymphocytes by measuring levels of early activation marker CD69 and IgG; D) differentiation of CD4 + T lymphocytes at different subtypes: Th1, Th2, Th17, Th22, iTreg or Tfh and e) cytokine profile: serum IL-6, IL-10, TNFα, CCL20, CXCL13, IL-15, CX3CR1, TGFbeta, IL-21, IL-17, IL-12, IL-18, IL-1beta and IL-22. Description of the means used to carry out the activity: Methodology: Patients and controls. The studies will be performed in healthy controls, in cirrhotic patients without MHE and in cirrhotic patients with MHE diagnosed using the PHES battery of psychometric tests. Patients who have not had clinical HB and are compensated, either after an episode of ascites decompensation or gastrointestinal bleeding or Child-Pugh B-phase, will be included in the study. As an exclusion criterion, the patient will present some other neurological, psychic, diabetic decompensation, alcoholism or active addiction to drugs. The procedure to be followed in each individual will be as follows: 1) Information and collection of informed consent (Declaration of Helsinki) 2) Collection of epidemiological data and liver disease: etiology 3) Collection of analytical values 4) Determination of the presence of EHM. It will be determined using the Psychometric Hepatic Encephalopathy Score (PHES) battery of psychometric tests. An individual will be considered to have MHE if their score is -4 or less. (Weissenborn et al., 2001; Ferenci et al., 2002). 5) Evaluation of selective and sustained care and motor coordination: The subjects will perform the following tests: Stroop test, "d2" care test and bimanual and visomotor coordination test. 6) Evaluation of inflammation and parameters related to the immune system in blood. Possible identification of peripheral biomarkers to diagnose the presence of MHE and specific neuropsychological alterations in cirrhotic patients. Relationship between the peripheral system and the cognitive impairment seen in patients with MHD. Results obtained: The major alterations associated specifically with MHD, not present in patients without MHE, are: 1) increased activation of all CD4 + T lymphocyte subtypes, as indicated by increased CD69 expression; 2) increased CD4 + CD28-T lymphocytes, associated with increased levels of CX3CL1 (fractalkin) and IL-15; 3) increased differentiation of CD4 + T lymphocytes to follicular Th and Th22; 4) increased activation of B lymphocytes and serum IgG. Conclusion: This study has identified some alterations of the immune system that are associated specifically with the appearance of neurological alterations in cirrhotic patients with EMH. Discussion: Hepatic encephalopathy (HE) is a consequence of hepatic failure and carries a wide range of neuropsychiatric alterations, ranging from slight changes in personality or alterations in the sleep-wake cycle, to alterations in cognitive function, motor coordination and Degree of consciousness. Between 30 and 50% of patients with cirrhosis who do not show clinical signs of HD have minimal hepatic encephalopathy (MHE) with a slight cognitive impairment. Patients with MHD may present with various cognitive deficits (visuospatial perception, attention, concentration) and motor (psychomotor slowing) that are not detectable on a routine neurological examination but are revealed when specific psychometric tests are performed (Amodio et al., 2004 ). Cirrhotic patients have hyperammonemia, which reaches similar levels in the groups without and with MH. Ammonium is able to cross the blood-brain barrier (BHE) inducing alterations in the central nervous system. Chronic hyperammonemia has been shown to alter the cGMP pathway in the brain and that it is responsible for some types of cognitive impairment (Felipo, 2013). Inflammation aggravates the neurological alterations produced by hyperammonemia in cirrhotic patients and also determines the severity of HD in cirrhotic patients with advanced HD (Shawcross et al., 2004). In this thesis a large study of cytokines has been performed in cirrhotic patients, showing that IL-6, IL-21, IL-17, IL-10, IFNγ, IL-18, CCL20, TNFα, CXCL13, IL- 15 and CX3CL1 in serum are increased both in cirrhotic patients with or without MHD, but patients with MHD show a greater increase than patients without MHD, suggesting a more potent immune response in patients with MH. In the case of TGFβ, cirrhotic patients show a decrease in serum independent of the presence of MH. In addition, levels of IL-12, IL-1β and IL-22 show a unique increase in patients with HMD, which we do not see in patients without HMD, suggesting that the increase of these interleukins occurs in later stages of the development of The EHM. In addition, IL-6, IL-21, TGFβ, IL-10, IL-18, IL-13, CCL20, TNFα, CXCL13, IL-15 and CX3CL1 correlate significantly with the degree of MHD (according to PHES score) Indicating that inflammation is an essential contributor to neurological impairment in MNE. All these data clearly conclude that inflammation plays a key role in the development of neurological, cognitive and motor impairment in cirrhotic patients presenting with MH. The fact that the inflammation is related to more neurological alterations than hyperammonemia indicates that it would be the degree of inflammation that would have the greatest weight in determining the occurrence of neurological deterioration. Regarding the study of the immunophenotype, we observed a series of changes in the patients with MH. First, we deal with the monocytes represented by CD14 and CD16 surface markers, with greater or lesser expression depending on whether they are classical, intermediate or non-classical. There is a decrease in the classical population (CD14 + CD16-) and an increase in the intermediate population (CD14 + CD16 +) in patients with MS. This will cause, on the one hand, a greater production of proinflammatory cytokines by the intermediate monocytes / macrophages that will promote a proinflammatory environment that will condition the differentiation of the different subtypes of CD4 lymphocytes. On the other hand, these same M1 macrophages will act as CPA with the lymphocytes, so that they can activate naive lymphocytes. We observed an increase in CD4 + CD28 autoreactive lymphocytes in patients with HMD. This cell population will be attracted to CX3CL1 gradients aided by IL-15 (we see that both cytokines are increased in EHM). CX3CL1 can be produced in endothelial cells and IL-15 in nerve tissue cells, among others. We propose the first hypothesis of infiltration of CD4 + CD28- through BBB, promoted by the increase of CX3CL1 and IL-15 in the medium. We see a change from naive lymphocytes to memory lymphocytes in both types of patients. In contrast, we observed a greater number of cells positive for the CD69 activation marker, exclusive in patients with MHE, in several cell types, such as CD28 lymphocytes, both CD28 + and CD28-, naive CD4, CD4 memory And in B lymphocytes. From the increase in certain serum proinflammatory cytokines in patients with HMD, such as TNFα, IL-6 and IL-1β, which have their receptors on endothelial cells, we propose our second hypothesis, the transmission of signals through the Receptors on endothelial cells. After triggering the signal transmission cascade, they would have a direct effect on the neural cells on the other side of the BBB. The naive CD4 lymphocytes differ in several subpopulations depending on certain factors that condition the medium. The two major subpopulations increased in patients with MHE are Th22 and Tfh, also corroborated by the increase in the cytokines they produce. IL-21 favors the differentiation of Th17 and inhibits iTreg, but its main function is the activation of B lymphocytes. Therefore, we see a greater production of IgG in patients with MHD and the possible formation of germinal centers. In addition, IL-22, IL-17 and IL-21 favor the secretion of CXCL13, from these germinal centers, whose function is to attract the B lymphocytes and the Thf to them. From these data we establish our last two hypotheses, the infiltration of Tfh and the possible formation of tertiary lymphoid organs inside the brain of patients with MHE, which would be facilitated by the infiltrated Tfh themselves. From here, it would be necessary to continue these studies to better characterize the contribution of immunological changes in neurological disorders and their possible utility in the early diagnosis of MHE in cirrhotic patients

Learning and Memory Impairments in Patients with Minimal Hepatic Encephalopathy are Associated with Structural and Functional Connectivity Alterations in Hippocampus

Patients with minimal hepatic encephalopathy (MHE) show mild cognitive impairment associated with alterations in attentional and executive networks. There are no studies evaluating the relationship between memory in MHE and structural and functional connectivity (FC) changes in the hippocampal system. This study aimed to evaluate verbal learning and long-term memory in cirrhotic patients with (C-MHE) and without MHE (C-NMHE) and healthy controls. We assessed the relationship between alterations in memory and the structural integrity and FC of the hippocampal system. C-MHE patients showed impairments in learning, long-term memory, and recognition, compared to C-NMHE patients and controls. Cirrhotic patients showed reduced fimbria volume compared to controls. Larger volumes in hippocampus subfields were related to better memory performance in C-NMHE patients and controls. C-MHE patients presented lower FC between the L-presubiculum and L-precuneus than C-NMHE patients. Compared to controls, C-MHE patients had reduced FC between L-presubiculum and subiculum seeds and bilateral precuneus, which correlated with cognitive impairment and memory performance. Alterations in the FC of the hippocampal system could contribute to learning and long-term memory impairments in C-MHE patients. This study demonstrates the association between alterations in learning and long-term memory and structural and FC disturbances in hippocampal structures in cirrhotic patients

New points of CDK regulation in meiotic recombination

Resumen del trabajo presentado en el Spanish Meiosis Meeting, celebrado de forma virtual, del 6 al 8 de julio de 2021Meiotic recombination is a key step of meiosis. Besides generating genetic diversity, this process is crucial for crossover formation between homologous chromosomes that guarantees their proper segregation during meiosis I. Given the biological relevance of meiotic recombination, multiple mechanisms exist to control its progression and coordination within cell cycle. Previous work determined that CDK activity regulates meiotic recombination initiation in S. pombe, since deletion of several cyclins decrease DSB formation in a natural hotspot. Moreover, deletion of crs1 and cig2 give rise to opposite effects in recombination outcome, decreasing or increasing, respectively, CO formation despite the defect in DSB levels. These results suggest that particular CDK-cyclin complexes may play an additional role in DSB processing and repair, but the substrates they could phosphorylate and control remain unknown. The Swi5-Sfr1 mediator complex is an auxiliary factor that promotes recombination by enhancing Rad51 and Dmc1 recombinase activity and stabilizing the presynaptic filament. The Sfr1 subunit is a good CDK substrate candidate because of its N-terminal domain contains 7 potential CDK phosphorylation consensus sequences. We have found that Sfr1 is phosphorylated during prophase I in synchronous meiosis, and this phosphorylation is, at least partly, CDK-dependent. Moreover, the phosphomimetic mutant sfr1-7D presents decreased levels of GC and CO, indicating that phosphorylation of these residues could reduce its activity to promote meiotic recombination. These results propose a new CDK regulation mechanism for the Swi5-Sfr1 mediator complex, where Sfr1 phosphorylation at late prophase would reduce its activity in order to inhibit recombination prior to chromosome segregations

Peripheral inflammation induces neuroinflammation that alters neurotransmission and cognitive and motor function in hepatic encephalopathy: Underlying mechanisms and therapeutic implications.

Several million patients with liver cirrhosis suffer minimal hepatic encephalopathy (MHE), with mild cognitive and coordination impairments that reduce their quality of life and life span. Hyperammonaemia and peripheral inflammation act synergistically to induce these neurological alterations. We propose that MHE appearance is because of the changes in peripheral immune system, which are transmitted to brain, leading to neuroinflammation that alters neurotransmission leading to cognitive and motor alterations. We summarize studies showing that MHE in cirrhotic patients is associated with alterations in the immune system and that patients died with HE show neuroinflammation in cerebellum, with microglial and astrocytic activation and Purkinje cell loss. We also summarize studies in animal models of MHE on the role of peripheral inflammation in neuroinflammation induction, how neuroinflammation alters neurotransmission and how this leads to cognitive and motor alterations. These studies identify therapeutic targets and treatments that improve cognitive and motor function. Rats with MHE show neuroinflammation in hippocampus and altered NMDA and AMPA receptor membrane expression, which impairs spatial learning and memory. Neuroinflammation in cerebellum is associated with altered GABA transporters and extracellular GABA, which impair motor coordination and learning in a Y maze. These alterations are reversed by treatments that reduce peripheral inflammation (anti-TNFα, ibuprofen), neuroinflammation (sulphoraphane, p38 inhibitors), GABAergic tone (bicuculline, pregnenolone sulphate) or increase extracellular cGMP (sildenafil or cGMP). The mechanisms identified would also occur in other chronic diseases associated with inflammation, aging and some mental and neurodegenerative diseases. Treatments that improve MHE may also be beneficial to treat these pathologies

The PHES battery does not detect all cirrhotic patients with early neurological deficits, which are different in different patients.

BACKGROUND AND AIMS:The psychometric hepatic encephalopathy score (PHES) is the "gold standard" for minimal hepatic encephalopathy (MHE) diagnosis. Some reports suggest that some cirrhotic patients "without" MHE according to PHES show neurological deficits and other reports that neurological alterations are not homogeneous in all cirrhotic patients. This work aimed to assess whether: 1) a relevant proportion of cirrhotic patients show neurological deficits not detected by PHES; 2) cirrhotic patients with mild neurological deficits are a homogeneous population or may be classified in sub-groups according to specific deficits. METHODS:Cirrhotic patients "without" (n = 56) or "with" MHE (n = 41) according to PHES and controls (n = 52) performed psychometric tests assessing attention, concentration, mental processing speed, working memory and bimanual and visuomotor coordination. Heterogeneity of neurological alterations was analysed using Hierarchical Clustering Analysis. RESULTS:PHES classified as "with" MHE 42% of patients. Around 40% of patients "without" MHE according to PHES fail two psychometric tests. Oral SDMT, d2, bimanual and visuo-motor coordination tests are failed by 54, 51, 51 and 43% of patients, respectively. The earliest neurological alterations are different for different patients. Hierarchical clustering analysis shows that patients "without" MHE according to PHES may be classified in clusters according to the tests failed. In some patients coordination impairment appear before cognitive impairment while in others concentration and attention deficits appear before. CONCLUSIONS:PHES is not sensitive enough to detect early neurological alterations in a relevant proportion of cirrhotic patients. Oral SDMT, d2 and bimanual and visuo-motor coordination tests are more sensitive. The earliest neurological alterations are different in different cirrhotic patients. These data also have relevant clinical implications. Patients classified as "without MHE" by PHES belonging to clusters 3 and 4 in our study have a high risk of suffering clinical complications, including overt HE and must be diagnosed and clinically followed

Reduced resting state connectivity and gray matter volume correlate with cognitive impairment in minimal hepatic encephalopathy.

Minimal hepatic encephalopathy (MHE) is associated with cognitive alterations and changes in connectivity. We assessed the relationship of the abnormalities of resting-state functional connectivity (rs-FC) and gray matter (GM) volume with different cognitive alterations and biochemical parameters associated to MHE.Thirty-nine cirrhotic patients (26 without and 13 with MHE) and 24 controls were widely cognitive assessed with a battery of psychometric tests. Atrophy was determined using Voxel-Based Morphometry and rs-FC was assessed by independent component analysis. Receiver operating characteristic (ROC) curves was performed to assess the diagnostic utility of rs-FC and GM reduction for the discrimination of patients with and without MHE. Blood ammonia, cGMP, and levels of pro-inflammatory interleukins were measured.MHE patients showed significant decrease of GM volume and lesser degree of rs-FC in different networks related to attention and executive functions as compared to controls and patients without MHE. There is a progressive reduction in rs-FC in the default mode network with the progression of cognitive impairment. MHE patients showed GM reduction in the right frontal lobe, right insula and right cerebellum compared to patients without MHE. Alterations in GM volume and rs-FC correlated with the scores of different cognitive tests.Decreased cognitive performance is associated by reduced rs-FC and GM atrophy in MHE patients. These changes could have predictive value for detecting MHE

Dendrogram showing the clustering of all individuals in main groups and sub-groups.

<p>All subjects included in the study have been arranged according to the similarity-dissimilarity of their performance in the combination of tests indicated using hierarchical clustering analysis. This analysis provides clusters including subjects more closely related between them than subjects assigned to a different cluster. These clusters are visualized in the dendrogram shown. The first column shows the number identifying each subject, which have been coloured according to their classification by the PHES: control (black), without MHE (red) or with MHE (green). These colours are repeated in the second column to facilitate its identification. The tests failed by each individual are indicated by blue colour in the corresponding box. The following tests have been included: PHES, critical flicker frequency (CFF), the congruent, neutral and incongruent tasks of the Stroop test, Bimanual and visuo-motor coordination, d2 test, TOT: total correctly processed and d2-CON: concentration, measured with the d2 test; Oral SDMT test, Digit Span and Letter-number test.</p

Performance in the PHES and in each of its tests.

<p>Performance in the PHES and in each of its tests.</p