Metabolic regulation of the HBV-specific T cell function.

Chronically HBV infected subjects are more than 260 million worldwide; cirrhosis and liver cancer represent possible outcomes which affect around 700,000 patients per year. Both innate and adaptive immune responses are necessary for viral control and both have been shown to be defective in chronic patients. Metabolic remodeling is an essential process in T cell biology, particularly for T cell activation, differentiation and survival. Cellular metabolism relies on the conversion of nutrients into energy to support intracellular processes, and to generate fundamental intermediate components for cell proliferation and growth. Adaptive immune responses are the central mechanisms for the resolution of primary human infections leading to the activation of pathogen-specific B and T cell functions. In chronic HBV infection the anti-viral immune response fails to contain the virus and leads to persistent hepatic tissue damage which may finally result in liver cirrhosis and cancer. This T cell failure is associated with metabolic alterations suggesting that control of nutrient uptake and intracellular utilization as well as correct regulation of intracellular metabolic pathways are strategic for T cell differentiation during persistent chronic infections. This review will discuss some of the main features of the T cell metabolic processes which are relevant to the generation of an efficient antiviral response, with specific focus on their clinical relevance in chronic HBV infection in the perspective of possible strategies to correct deregulated metabolic pathways underlying T cell dysfunction of chronic HBV patients

Donor/recipient mixed chimerism does not predict graft failure in children with β-thalassemia given an allogeneic cord blood transplant from an HLA-identical sibling

Background Donor/recipient mixed chimerism has been reported to be associated with an increased risk of graft failure in patients with β-thalassemia given a bone marrow transplant. We investigated the relationship between the degree of mixed chimerism over time and clinical outcome of children undergoing cord blood transplantation for β-thalassemia.Design and Methods Twenty-seven consecutive children given a cord blood transplant from a related donor were analyzed by short tandem repeat polymerase chain reaction and their chimerism results were compared with those of 79 consecutive patients who received a bone marrow transplant from either a relative (RD-BMT, n=42) or an unrelated donor (UD-BMT, n=37). Cord blood and bone marrow recipients received comparable preparative regimens.Results All cord blood recipients engrafted and displayed mixed chimerism early after transplantation; 13/27 converted to full donor chimerism over time, while 14 maintained stable mixed chimerism; all patients are alive and transfusion-independent. Twenty-four of the 79 bone marrow-recipients (12 UD- and 12 RD-BMT) exhibited full donor chimerism at all time points examined, 4/79 (2 UD- and 2 RD-BMT) did not engraft and 51/79 (23 UD- and 28 RD-BMT) displayed mixed chimerism at the time of hematologic reconstitution. Forty of 51 bone marrow recipients with mixed chimerism converted to full donor chimerism (17 UD- and 23 RD-BMT), 3/51 maintained stable mixed chimerism (1 UD- and 2 RD-BMT), while 8/51 (5 UD- and 3 RD-BMT) progressively lost the graft, and became transfusion-dependent again.Conclusions Mixed chimerism is a frequent event and does not predict the occurrence of graft failure in children with β-thalassemia given a cord blood transplant from a relative

Brain response to a humanoid robot in areas implicated in the perception of human emotional gestures

BACKGROUND: The humanoid robot WE4-RII was designed to express human emotions in order to improve human-robot interaction. We can read the emotions depicted in its gestures, yet might utilize different neural processes than those used for reading the emotions in human agents. METHODOLOGY: Here, fMRI was used to assess how brain areas activated by the perception of human basic emotions (facial expression of Anger, Joy, Disgust) and silent speech respond to a humanoid robot impersonating the same emotions, while participants were instructed to attend either to the emotion or to the motion depicted. PRINCIPAL FINDINGS: Increased responses to robot compared to human stimuli in the occipital and posterior temporal cortices suggest additional visual processing when perceiving a mechanical anthropomorphic agent. In contrast, activity in cortical areas endowed with mirror properties, like left Broca’s area for the perception of speech, and in the processing of emotions like the left anterior insula for the perception of disgust and the orbitofrontal cortex for the perception of anger, is reduced for robot stimuli, suggesting lesser resonance with the mechanical agent. Finally, instructions to explicitly attend to the emotion significantly increased response to robot, but not human facial expressions in the anterior part of the left inferior frontal gyrus, a neural marker of motor resonance. CONCLUSIONS: Motor resonance towards a humanoid robot, but not a human, display of facial emotion is increased when attention is directed towards judging emotions. SIGNIFICANCE: Artificial agents can be used to assess how factors like anthropomorphism affect neural response to the perception of human actions

Chromosome anomalies in bone marrow as primarycause of aplastic or hypoplastic conditions andperipheral cytopenia: disorders due to secondaryimpairment of RUNX1 and MPL genes

Background Chromosome changes in the bone marrow (BM) of patients with persistent cytopenia are often considered diagnostic for a myelodysplastic syndrome (MDS). Comprehensive cytogenetic evaluations may give evidence of the real pathogenetic role of these changes in cases with cytopenia without morphological signs of MDS. Results Chromosome anomalies were found in the BM of three patients, without any morphological evidence of MDS: 1) an acquired complex rearrangement of chromosome 21 in a boy with severe aplastic anaemia (SAA); the rearrangement caused the loss of exons 2-8 of the RUNX1 gene with subsequent hypoexpression. 2) a constitutional complex rearrangement of chromosome 21 in a girl with congenital thrombocytopenia; the rearrangement led to RUNX1 disruption and hypoexpression. 3) an acquired paracentric inversion of chromosome 1, in which two regions at the breakpoints were shown to be lost, in a boy with aplastic anaemia; the MPL gene, localized in chromosome 1 short arms was not mutated neither disrupted, but its expression was severely reduced: we postulate that the aplastic anaemia was due to position effects acting both in cis and in trans, and causing Congenital Amegakaryocytic Thrombocytopenia (CAMT). Conclusions A clonal anomaly in BM does not imply per se a diagnosis of MDS: a subgroup of BM hypoplastic disorders is directly due to chromosome structural anomalies with effects on specific genes, as was the case of RUNX1 and MPL in the patients here reported with diagnosis of SAA, thrombocytopenia, and CAMT. The anomaly may be either acquired or constitutional, and it may act by deletion/disruption of the gene, or by position effects. Full cytogenetic investigations, including a-CGH, should always be part of the diagnostic evaluation of patients with BM aplasia/hypoplasia and peripheral cytopenias

RANKL Cytokine: From Pioneer of the Osteoimmunology Era to Cure for a Rare Disease

Since its identification, the RANKL cytokine has been demonstrated to play a crucial role in bone homeostasis and lymphoid tissue organization. Genetic defects impairing its function lead to a peculiar form of autosomal recessive osteopetrosis (ARO), a rare genetic bone disease presenting early in life and characterized by increased bone density due to failure in bone resorption by the osteoclasts. Hematopoietic stem cell transplantation (HSCT) is the only option for the majority of patients affected by this life-threatening disease. However, the RANKL-dependent ARO does not gain any benefit from this approach, because the genetic defect is not intrinsic to the hematopoietic osteoclast lineage but rather to the mesenchymal one. Of note, we recently provided proof of concept of the efficacy of a pharmacological RANKL-based therapy to cure this form of the disease. Here we provide an overview of the diverse roles of RANKL in the bone and immune systems and review the clinical features of RANKL-deficient ARO patients and the results of our preclinical studies. We emphasize that these patients present a continuous worsening of the disease in the absence of a cure and strongly wish that the therapy we propose will be further developed

Analisi immunoistochimica del rimodellamento strutturale e della distribuzione dei fenotipi staminali nel pancreas umano diabetico

I dati epidemiologici evidenziano che l’incidenza del diabete mellito, entro il 2025, raggiungerà il 5,4% della popolazione mondiale adulta e che la maggior parte delle morti sarà attribuibile a patologie cardiovascolari ad esso associato. Il diabete potrebbe rappresentare una condizione di partenza per stabilire se la patologia d’organo sia primitivamente dovuta a modificazioni nelle proprietà funzionali delle cellule staminali, delle cellule parenchimali o di entrambe le popolazioni. La condizione di iperglicemia cronica caratteristica del diabete si associa ad un danneggiamento a lungo termine, disfunzione, fino all’insufficienza multiorgano, attraverso complicanze micro- e macro-vascolari che colpiscono cuore, cervello, vasi sanguigni periferici, retina, reni e sistema nervoso periferico. La variabilità della risposta tissutale all’iperglicemia causata dal diabete rende difficile, però, attribuire una causa univoca dello scompenso multiorgano associato alla malattia. Esiste una letteratura significativa riguardante i difetti funzionali dei progenitori endoteliali sia midollari che circolanti (EPCs), e numerose osservazioni del nostro gruppo hanno documentato la presenza di microangiopatia nel midollo e una alterazione delle nicchie osteoblastiche dovuta ad un difetto del sistema nervoso simpatico nel diabete. L’ipotesi che si vuole dimostrare nel corso di uno studio molto più ampio è che, il diabete, oltre all’insulto metabolico diretto alle cellule parenchimali, possa anche determinare uno scompenso funzionale delle cellule staminali progenitrici residenti e circolanti, tali da portare ad alterazioni dell’omeostasi e disfunzione di organi bersaglio. Poiché in letteratura sono scarsi i dati che descrivono in un organo complesso quale il pancreas, la distribuzione sia di cellule organo-specifiche (alfa e beta cellule) sia di cellule caratterizzanti le nicchie staminali in corso di diabete, ci siamo proposti di studiare morfologicamente e morfometricamente le componenti strutturali dell’organo al fine di descrivere possibili alterazioni indotte dalla malattia. La casistica comprende 35 campioni di pancreas umano di cui 15 provenienti da pazienti normoglicemici e 20 da pazienti con diabete mellito di tipo 2. Dopo aver quantificato la componente fibrotica del tessuto mediante colorazione tricromica di Masson, avvalendoci di tecniche di immunoistochimica, è stato effettuato il riconoscimento di cellule alfa secernenti glucagone e cellule beta secernenti insulina. E’ stata, poi, effettuata una valutazione mediante immunofluorescenza della distribuzione della rete vascolare, riconoscendo i profili positivi ad actina muscolare liscia (arterie), CD34 (venule e capillari) e D2-40/podoplanina (vasi linfatici). Mediante tecniche di immunofluorescenza abbiamo identificato la presenza e la localizzazione di cellule che presentassero fenotipi di staminalità adulta (c-kit, CD133, CD34 e CD44) in assenza di altri marcatori di differenziazione. Dal momento che i mastociti esprimono alti livelli di c-kit abbiamo effettuato una doppia colorazione per Tryptase. Infine, su un numero limitato di campioni di pancreas ottenuti da pancreasectomia, abbiamo isolato e caratterizzato le cellule endoteliali e le abbiamo testate sottoponendole a diverse concentrazioni di glucosio, paragonandole alle cellule HUVEC, cellule usate come controllo nello studio di popolazioni endoteliali. La prima valutazione, volta alla descrizione della distribuzione delle varie componenti strutturali ha evidenziato che circa il 60% del pancreas è occupato dal tessuto parenchima funzionale (esocrino ed endocrino), 18% da fibrosi mentre per il 22% da stroma vascolare; percentuali che si modificano in presenza di diabete con una riduzione della componente funzionale ed un aumento della deposizione di collagene, soprattutto di tipo interstiziale e perivascolare. Per quanto riguarda la porzione endocrina, nel diabete le isole risultano essere di minore dimensione e con una interessante riduzione sia di cellule positive per insulina sia di quelle positive per glucagone. Contrariamente, è stata ritrovata una popolazione di cellule singole positive per i due marcatori distribuite nel parenchima pancreatico che, invece, è risultata arricchita nei pazienti con diabete. La valutazione della rete vascolare documenta una riduzione della densità venulare e capillare nelle sole isole di Langerhans diabetiche mentre la densità di arteriole -SMApos è ridotta nel parenchima, ma con un aumento dello spessore della parete arteriolare nei soggetti diabetici. Tuttavia, la riduzione del parenchima funzionale, come conseguenza del diabete mellito, tende a depletare il rapporto vasi /cellule in confronto con il parenchima di controllo. Anche per i vasi linfatici si documenta un trend di riduzione nei campioni con presenza di patologia diabetica. Si è quindi proceduto al riconoscimento delle cellule progenitrici, costituite da una popolazione di cellule positive per c-kit, recettore di stem cell factor. Queste cellule c-kitpos rappresentano lo 0,058±0,015% della popolazione totale nei pancreas normoglicemici, risultando prevalentemente (>50%) localizzate all’interno degli isolotti; nei campioni diabetici, invece, la percentuale si riduce drasticamente e in maniera più evidente nel parenchima pancreatico. Il fenotipo staminale CD133 mostra invece un andamento opposto, con un incremento anche se non significativo nei campioni patologici. È interessante notare che le cellule progenitrici positive per CD34 sono risultate significativamente ridotte (p<0,05) nei pancreas dei pazienti diabetici confrontati con i controlli normoglicemici. Per quanto riguarda lo studio in vitro, una volta isolate le cellule endoteliali dai pezzi a fresco di pancreas umano, è stata condotta un’analisi funzionale sul comportamento di queste cellule in condizioni di iperglicemia, volte a mimare il microambiente diabetico. Inaspettatamente, il saggio di vitalità effettuato a 12 e 72ore ha mostrato un aumento della proliferazione delle cellule endoteliali pancreatiche (PanEC) nel medium condizionato con i più alti livelli di glucosio (22mM) mentre le HUVEC risentono dell’effetto tossico dell’iperglicemia. I saggi di Wound Healing e angiogenesi su matrigel hanno mostrato un rallentamento della migrazione e della tubulogenesi nella nostra popolazione di interesse (PanEC) in tenore di alto glucosio. Questo studio offre le basi per indagini successive volte a comprendere l’impatto del diabete mellito sul riarrangiamento strutturale e sul possibile coinvolgimento di cellule progenitrici nei meccanismi di disregolazione dell’omeostasi tissutale e funzionale. Pur non avendo stabilito se queste modificazioni siano una causa o una conseguenza del danneggiamento multiorgano tipico del diabete, il nostro approccio potrebbe offrire nuovi spunti nella comprensione della angiopatia diabetica tessuto-specifica.Epidemiologic data show that the incidence of diabetes mellitus will reach 5.4% of the adult world population by 2025 and that the majority of deaths will be attributable to the associated cardiovascular diseases. Diabetes mellitus may represent a unique condition to establish whether organ pathology is primarily due to changes in the functional properties of stem or parenchymal cells or both. Chronic hyperglycemia is associated with long-term damage, dysfunction, and failure of different organs, especially the eyes, kidneys, nerves, heart, and blood vessels. The variability in tissue response to chronic hyperglycaemia makes it difficult to sustain a unified hypothesis to dissect the mechanism of diabetes associated multiorgan damage. A significant literature exists on the functional impairment of endothelial progenitors’ cells (EPCs) and several observations by our group have documented diabetic microangiopathy in the bone marrow and demonstrated an alteration of osteoblastic stem niches by changes in the sympathetic autonomic nervous system. The present study is on line with the view that diabetes, is directed not only to parenchymal cells, but can also affect stem cell populations, in terms of distribution, number and function. Alterations in stem cell homeostatic control lead to dysfunction of target organs. Robust data on the structural changes in organ-specific cells (alpha and beta cells) and stem cells phenotypes in the course of diabetes have been poorly described. Thus, we set out to study morphologically and morphometrically the structural components of the organ in order to describe alterations induced by the disease. Our study includes 35 samples of human pancreas, 15 from normoglycemic patients and 20 from patients with type 2 diabetes mellitus. After the quantification of the fibrotic component by Masson's trichrome staining, alpha-secreting glucagon and beta-secreting insulin cells were immunohistochemically detected. The immunofluorescence evaluation of the vascular network distribution was performed, by the identification of smooth muscle actin positive arteries, CD34 positive venules and capillaries and D2-40/podoplanine labelled lymphatic vessels. Using immunofluorescence techniques, we identified the presence and localization of adult stem cells phenotypes (c-kit, CD133, CD34 and CD44) in the absence of other differentiation markers. Since mast cells express high levels of c-kit we performed a double staining for Tryptase. Finally, on a limited number of pancreatic samples obtained from pancreatectomy, endothelial cells were isolated, characterized and exposed to different glucose concentrations. HUVEC were employed as control. The first evaluation, aimed at describing the distribution of the various structural components, showed that nearly 60% of the pancreas is occupied by the functional parenchymal tissue (exocrine and endocrine), 18% by fibrosis, while 22% is occupied by vascular stroma. Diabetic tissue showed a reduction of the functional component and an increase in collagen deposition especially in the interstitial and perivascular location. In diabetes the endocrine islands resulted of smaller size and with an interesting reduction of both insulin and glucagon positive cells. In contrast, a population of individual cells positive for the two markers distributed in the pancreatic parenchyma was found and was enriched in patients with diabetes. Evaluation of the vascular network documented a reduction of venular and capillary density in diabetic Langerhans islets while the density of arterioles -SMApos was reduced in the parenchyma, although the thickness of the arteriolar wall was increased in diabetic subjects. However, the reduction of the functional parenchyma, as a consequence of diabetes mellitus, tended to deplete the vessel/cell ratio in comparison with non-diabetic control parenchyma. Lymphatic vessels also showed a trend to decrease in diabetic samples. We candidate c-kit, the receptor of the stem cell factor (SCF), as one of the markers to identify the population of stem and progenitor cells resident in the pancreas. c-kitpos cells represented 0.058±0.015% of the total population in the normoglycemic pancreas, resulting predominantly (>50%) located within the islets; on the other hand, in diabetic samples the percentage was drastically reduced and to amore extent in pancreatic parenchyma. CD133pos stem cell phenotype showed an opposite trend, although without statistical significance. It is interesting to note that progenitor cells positive for CD34 were significantly reduced (p<0.05) in the pancreas of diabetic patients compared to normoglycemic controls. Regarding the in vitro study, once the endothelial cells were isolated from fresh human pancreas, a functional analysis was carried out. We tested the behavior of these cells in hyperglycemia, mimicking the diabetic microenvironment. Unexpectedly, the vitality test performed after 12 and 72h of treatment showed an increase in the proliferation of pancreatic endothelial cells in conditioned medium with the highest levels of glucose (22mM) while HUVECs were affected by the toxic effect of hyperglycemia. Wound healing and angiogenesis assay showed an inhibition of migratory and tubulogenesis capacity in PanECs in high-glucose medium. This study provides the basis for subsequent investigations on human pacreas to understand the impact of diabetes mellitus on the structural rearrangement and on the possible involvement of progenitor cells in the mechanisms of dysregulation of tissue and functional homeostasis. Although we did not establish whether these structural alterations were the consequence, or a cause of diabetes associated multiorgan damage, our approach may offer new insights on the understanding of the diabetic paradox of a tissue specific angiopathy

Comparison of octreotide and hyoscine butylbromide in controlling gastrointestinal symptoms due to malignant inoperable bowel obstruction

In advanced cancer patients with inoperable bowel obstruction, the administration of antisecretive and antiemetic drugs has proved to be effective in controlling gastrointestinal symptoms caused by bowel obstruction. However, controlled studies concerning the most effective antisecretive drug are lacking. The aim of this randomized controlled study was to determine whether octreotide or hyoscine butylbromide was the more effective antisecretive drug for use in states of inoperable bowel obstruction. Eighteen patients with inoperable bowel obstruction randomly received octreotide 0.3 mg daily (n = 9) or hyoscine butylbromide (HB) 60 mg daily (n = 9) s.c. The following parameters were measured: episodes of vomiting, nausea, drowsiness, continuous and colicky pain, using a Likert scale corresponding to a numerical value: (none 0, slight 1, moderate 2, severe 3) recorded before starting the treatment (T0) and 24 h (T1), 48 h (T2) and 72 h after (T3), and the mean daily amounts of fluids administered i.v. or s.c. during the period of study. Three patients dropped out of the study because data were incomplete. Octreotide treatment induced a significantly rapid reduction in the number of daily episodes of vomiting and intensity of nausea compared with HB treatment at the different time intervals examined. No relevant changes were found in dry mouth, drowsiness and colicky pain. Lower levels of hydration were associated with nausea regardless of the treatment. At the doses used in this study, octreotide was more effective than HB in controlling gastrointestinal symptoms of bowel obstruction. Further studies are necessary to understand the role of hydration more clearly in such a clinical situation

Role of octreotide, scopolamine butylbromide, and hydration in symptom control of patients with inoperable bowel obstruction and nasogastric tubes: A prospective randomized trial

Bowel obstruction may be an inoperable complication in patients with end-stage cancer. Scopolamine butylbromide (SB) and octreotide (OCT) have been successfully used with the aim of reducing gastrointestinal (GI) secretions to avoid placement of a nasogastric tube (NGT); however, there have been no comparative studies concerning the efficacy of these drugs. Furthermore, there is little information about the role played by parenteral hydration in symptom control of these patients. In a prospective trial that involved all 17 inoperable bowel-obstructed patients presenting to our services with a decompressive NGT, patients were randomized to OCT 0.3 mg/day or SB 60 mg/day for 3 days through a continuous subcutaneous infusion. Clinical data, survival time, and the time interval from the first diagnosis of cancer to the onset of inoperable bowel obstruction were noted. The intensity of pain, nausea, dry mouth, thirst, dyspnea, feeling of abdominal distension, and drowsiness were assessed by means of a verbal scale before starting treatment with the drugs under study (T0) and then daily for 3 days (T1, T2, T3). Moreover, daily information was collected regarding the quantity of GI secretions through the NGT, the oral intake of fluids, the quantity of parenteral hydration, and the analgesic therapy used. The NGT could be removed in all 10 home care and in 3 hospitalized patients without changing the dosage of the drugs. OCT significantly reduced the amount of GI secretions at T2 (P = 0.016) and T3 (P = 0.020). Compared to the home care patients, the hospitalized patients received significantly more parenteral hydration (P = 0.0005) and drank more fluids (P = 0.025). There was no difference in the daily thirst and dry mouth intensity in relation to the amount of parenteral hydration or the treatment provided (OCT or SB). Independent of antisecretory treatment, the patients receiving less parenteral hydration presented significantly more nausea (T0 P = 0.002; T1 P = 0.001; T2 P = 0.003; T3 P = 0.001) and drowsiness at T3 (P < 0.05). Pain relief was obtained in all 17 patients and only two patients required an increase in the morphine dose at T1. All patients with inoperable malignant bowel obstruction should undergo treatment with antisecretory drugs so as to evaluate the possibility of removing the NGT. When a more rapid reduction in GI secretions is desired, OCT should be considered as the first choice drug. Parenteral hydration over 500 ml/day may reduce nausea and drowsiness. (C) U.S. Cancer Pain Relief Committee, 2000