Lucia Piceni Sereni

Metabolic effects of liver transplantation in cirrhotic patients.

To assess whether liver transplantation (LTx) can correct the metabolic alterations of chronic liver disease, 14 patients (LTx-5) were studied 5+/-1 mo after LTx, 9 patients (LTx-13) 13+/-1 mo after LTx, and 10 patients (LTx-26) 26+/-2 months after LTx. Subjects with chronic uveitis (CU) and healthy volunteers (CON) were also studied. Basal plasma leucine and branched-chain amino acids were reduced in LTx-5, LTx-13, and LTx-26 when compared with CU and CON (P < 0.01). The basal free fatty acids (FFA) were reduced in LTx-26 with respect to CON (P < 0.01). To assess protein metabolism, LTx-5, LTx-13, and LTx-26 were studied with the [1-14C]leucine turnover combined with a 40-mU/m2 per min insulin clamp. To relate changes in FFA metabolism to glucose metabolism, eight LTx-26 were studied with the [1-14C]palmitate and [3-3H]glucose turnovers combined with a two-step (8 and 40 mU/m2 per min) euglycemic insulin clamp. In the postabsorptive state, LTx-5 had lower endogenous leucine flux (ELF) (P < 0.005), lower leucine oxidation (LO) (P < 0.004), and lower non-oxidative leucine disposal (NOLD) (P < 0.03) with respect to CON (primary pool model). At 2 yr (LTx-26) both ELF (P < 0.001 vs. LTx-5) and NOLD (P < 0.01 vs. LTx-5) were normalized, but not LO (P < 0.001 vs. CON) (primary and reciprocal pool models). Suppression of ELF by insulin (delta-reduction) was impaired in LTx-5 and LTx-13 when compared with CU and CON (P < 0.01), but normalized in LTx-26 (P < 0.004 vs. LTx-5 and P = 0.3 vs. CON). The basal FFA turnover rate was decreased in LTx-26 (P < 0.01) and CU (P < 0.02) vs. CON. LTx-26 showed a lower FFA oxidation rate than CON (P < 0.02). Tissue glucose disposal was impaired in LTx-5 (P < 0.005) and LTx-13 (P < 0.03), but not in LTx-26 when compared to CON. LTx-26 had normal basal and insulin-modulated endogenous glucose production. In conclusion, LTx have impaired insulin-stimulated glucose, FFA, and protein metabolism 5 mo after surgery. Follow-up at 26 mo results in (a) normalization of insulin-dependent glucose metabolism, most likely related to the reduction of prednisone dose, and, (b) maintenance of some alterations in leucine and FFA metabolism, probably related to the functional denervation of the graft and to the immunosuppressive treatment.

Metabolic effects of a corticosteroid-free immunosuppressive regimen in recipients of pancreatic transplant

Background. A corticosteroid (CS)-free immunosuppressive regimen may be considered less diabetogenic than treatments including CSs principally after pancreas transplantation. Methods. To test whether a CS-free immunosuppressive treatment is metabolically superior to a regimen including CSs, we prospectively studied 19 CS-free simultaneous pancreas and kidney (SPK) transplant recipients (body mass index=22±1 kg/m2; cyclosporine dose=400±19 mg/kg/day; azathioprine dose=77±8 mg/day; basal plasma C-peptide=1.3±0.12 ng/mL) and 12 matched CS-treated SPK transplant recipients (prednisone dose=9±1 mg/day; basal C-peptide=2.2±0.2 ng/mL) by means of the 6,6-2H2-glucose infusion and the euglycemic insulin clamp (1 mU/kg/min, insulin infusion rate). In addition, six renal transplant recipients receiving a CS-free regimen were also studied as a control group. Results. In the postabsorptive state, CS-treated SPK transplant recipients demonstrated comparable plasma glucose levels but higher plasma insulin levels than CS-free SPK transplant recipients. Plasma triglyceride levels were significantly higher in CS-treated SPK patients than in CS-free SPK patients (1.16±0.16 mg/dL vs. 0.88±0.08; P <0.05). High-density lipoprotein and apoprotein A1 levels were similar in both groups. No difference was observed in pyruvate, lactate, &bgr;-OH-butyrate, and basal endogenous glucose production in all three groups of patients studied. During euglycemic hyperinsulinemia, the inhibition of endogenous glucose production and the stimulation of tissue glucose disposal were not statistically different among the three groups. Conclusions. SPK recipients receiving chronic low-dose CS maintenance therapy do not present a lower glucose disposal than CS-free recipients. Nonetheless, this is obtained at the expense of a higher endogenous insulin secretion, which can cause an alteration of the triglyceride profile.

B-cell reconstitution after lentiviral vector–mediated gene therapy in patients with Wiskott-Aldrich syndrome

Background Wiskott-Aldrich syndrome (WAS) is a severe X-linked immunodeficiency characterized by microthrombocytopenia, eczema, recurrent infections, and susceptibility to autoimmunity and lymphomas. Hematopoietic stem cell transplantation is the treatment of choice; however, administration of WAS gene–corrected autologous hematopoietic stem cells has been demonstrated as a feasible alternative therapeutic approach. Objective Because B-cell homeostasis is perturbed in patients with WAS and restoration of immune competence is one of the main therapeutic goals, we have evaluated reconstitution of the B-cell compartment in 4 patients who received autologous hematopoietic stem cells transduced with lentiviral vector after a reduced-intensity conditioning regimen combined with anti-CD20 administration. Methods We evaluated B-cell counts, B-cell subset distribution, B cell–activating factor and immunoglobulin levels, and autoantibody production before and after gene therapy (GT). WAS gene transfer in B cells was assessed by measuring vector copy numbers and expression of Wiskott-Aldrich syndrome protein. Results After lentiviral vector-mediated GT, the number of transduced B cells progressively increased in the peripheral blood of all patients. Lentiviral vector-transduced progenitor cells were able to repopulate the B-cell compartment with a normal distribution of B-cell subsets both in bone marrow and the periphery, showing a WAS protein expression profile similar to that of healthy donors. In addition, after GT, we observed a normalized frequency of autoimmune-associated CD19+CD21−CD35− and CD21low B cells and a reduction in B cell–activating factor levels. Immunoglobulin serum levels and autoantibody production improved in all treated patients. Conclusions We provide evidence that lentiviral vector-mediated GT induces transgene expression in the B-cell compartment, resulting in ameliorated B-cell development and functionality and contributing to immunologic improvement in patients with WAS.

Assessment of insulin sensitivity based on a fasting blood sample in men with liver cirrhosis before and after liver transplantation.

Background. Insulin resistance is a key factor in the pathogenesis of hepatogenous diabetes and influences the prognosis of chronic liver diseases. In vivo assessment of insulin resistance in humans is expensive; therefore, surrogate indices based on a fasting plasma glucose and insulin concentrations (HOMA-IS, QUICKI) were proposed. This study aimed to test whether these simple indices are reliable measures of insulin sensitivity in patients with liver cirrhosis before and after liver transplantation (LTx). Methods. HOMA-IS and QUICKI were compared with insulin sensitivity as assessed with the gold standard technique (insulin clamp) in 20 patients with liver cirrhosis, in 36 patients after LTx, and in 25 matched healthy subjects (predominantly men). To test whether these indices may be applied also in prospective studies, 10 patients with liver cirrhosis were studied longitudinally before and 2 years after LTx. Results. Both HOMA-IS and QUICKI were associated with insulin sensitivity in patients with liver cirrhosis (r =0.63, P =0.005 and r =0.60, P =0.009) and in LTx patients (r =0.41, P =0.02 and r =0.46, P =0.05). Both were able to detect the improvement of insulin sensitivity after LTx in the patients studied prospectively. Conclusions. HOMA-IS and QUICKI are simple reliable tools to assess insulin sensitivity in clinical and epidemiologic investigations of chronic liver disease before and after LTx.

Protein, glucose and lipid metabolism in the cancer cachexia: A preliminary report

To the EditorCancer cachexia is a complex syndrome character-ized by weight loss with depletion of both skeletalmuscle and adipose tissue mass, associated withextreme weakness, dry and wrinkled skin. Cancercachexia is associated with protein degradation ofhuman skeletal muscle with a slow or fast progres-sion rate.The mechanism of wasting occurring in malignantdiseases is multifactorial. The causes of the cancercachexia syndrome may include decreased foodintake [1], increased faecal and urinary nutrientlosses, and malfunction of metabolic pathways inboth the host and the tumour. Also the site and themass of the tumour may be involved in causinganorexia due to excessive metabolites consumption.In addition metabolic alterations induced by thetumour itself, through production of specific factors(several cytokines, especially TNF-a, INF-g, andIL-6), may be interfering with normal metabolism[2].Glucose intolerance is one of the earliest meta-bolic alterations recognized in patients with cancer[3]. The whole body glucose turnover is increased,while glucose utilization is impaired due to insulinresistance. Increased gluconeogenesis from lactateand alanine is also a typical alteration of glucosemetabolism in cancer cachexia [4]. Insulin resis-tance does not appear to be a consequence ofmalnutrition, but seems to be related to the tumouritself [5].Depletion of muscle protein masses is typical incancer cachexia with increased tumour proteinaccumulation [2]. The majority of the authors havefound increased whole body protein turnover [6],while the results on protein synthesis (enhanced,decreased) and on the relative resistance to insulin[7] are not consistent.As the progressive loss of muscle mass is the mostprominent phenotypic feature of cancer cachexia, westudied amino acids metabolism in order to help todevelop more effective therapeutic strategies forpreventing cancer-related muscle wasting.With this purpose we assessed insulin action onprotein, lipid and glucose metabolism in patientsaffected by cancer with a recent relevant weight loss,utilizing the classical technique of the euglycemichyperinsulinemic clamp which is the golden stan-dard procedure for the determination of insulinaction.All patients were recruited in the Department ofMedicine of S. Raffaele Hospital, Milano, Italy. Fivepatients (3 males and 2 woman, age: 619

IL-10 Critically Modulates B Cell Responsiveness in Rankl−/− Mice

The immune and the skeletal system are tightly interconnected, and B lymphocytes are uniquely endowed with osteo-interactive properties. In this context, receptor activator of NF-κB (RANK) ligand (RANKL) plays a pivotal role in lymphoid tissue formation and bone homeostasis. Although murine models lacking RANK or RANKL show defects in B cell number, the role of the RANKL–RANK axis on B physiology is still a matter of debate. In this study, we have characterized in detail B cell compartment in Rankl−/− mice, finding a relative expansion of marginal zone B cells, B1 cells, and plasma cells associated with increased Ig serum levels, spontaneous germinal center formation, and hyperresponse to CD40 triggering. Such abnormalities were associated with an increased frequency of regulatory B cells and augmented B cell–derived IL-10 production. Remarkably, in vivo IL-10-R blockade reduced T cell–triggered plasma cell differentiation and restrained the expansion of regulatory B cells. These data point to a novel role of the RANKL–RANK axis in the regulation of B cell homeostasis and highlight an unexpected link between IL-10 CD40 signaling and the RANKL pathway.

Platelets in Wiskott‐Aldrich syndrome: Victims or executioners?

Microthrombocytopenia is the clinical hallmark of WAS, a rare X‐linked immunodeficiency that is characterized by eczema, autoimmunity, and cancer susceptibility. This disease is caused by mutations in the WAS gene, which is expressed in hematopoietic cells and regulates actin cytoskeleton remodeling thereby modulating various cellular functions, including motility, immunologic synapse assembly, and signaling. Despite extensive studies that have provided great insight into the relevance of this molecule to innate and cellular immunity, the exact mechanisms of microthrombocytopenia in WAS are still unknown. This review focuses on the recent progress made in dissecting the pathogenesis of platelet defects in patients with WAS and their murine counterparts. In parallel, we will provide an overview of the state‐of‐the art platelets as immune modulators at the interface between hemostasis and the immune system, which suggests that these cells may have a direct role in the pathogenesis of immune dysregulation in WAS.

In vivo chronic stimulation unveils autoreactive potential of Wiskott-Aldrich syndrome protein-deficient b cells

Wiskott–Aldrich syndrome (WAS) is a primary immunodeficiency caused by mutations in the gene encoding the hematopoietic-specific WAS protein (WASp). WAS is frequently associated with autoimmunity, indicating a critical role of WASp in maintenance of tolerance. The role of B cells in the induction of autoreactive immune responses in WAS has been investigated in several settings, but the mechanisms leading to the development of autoimmune manifestations have been difficult to evaluate in the mouse models of the disease that do not spontaneously develop autoimmunity. We performed an extensive characterization of Was−/− mice that provided evidence of the potential alteration in B cell selection, because of the presence of autoantibodies against double-stranded DNA, platelets, and tissue antigens. To uncover the mechanisms leading to the activation of the potentially autoreactive B cells in Was−/− mice, we performed in vivo chronic stimulations with toll-like receptors agonists (LPS and CpG) and apoptotic cells or infection with lymphocytic choriomeningitis virus. All treatments led to increased production of autoantibodies, increased proteinuria, and kidney tissue damage in Was−/− mice. These findings demonstrate that a lower clearance of pathogens and/or self-antigens and the resulting chronic inflammatory state could cause B cell tolerance breakdown leading to autoimmunity in WAS.

Autonomous role of Wiskott-Aldrich syndrome platelet deficiency in inducing autoimmunity and inflammation

Background Wiskott‐Aldrich syndrome (WAS) is an X‐linked immunodeficiency characterized by eczema, infections, and susceptibility to autoimmunity and malignancies. Thrombocytopenia is a constant finding, but its pathogenesis remains elusive. Objective To dissect the basis of the WAS platelet defect, we used a novel conditional mouse model (CoWas) lacking Wiskott‐Aldrich syndrome protein (WASp) only in the megakaryocytic lineage in the presence of a normal immunologic environment, and in parallel we analyzed samples obtained from patients with WAS. Methods Phenotypic and functional characterization of megakaryocytes and platelets in mutant CoWas mice and patients with WAS with and without autoantibodies was performed. Platelet antigen expression was examined through a protein expression profile and cluster proteomic interaction network. Platelet immunogenicity was tested by using ELISAs and B‐cell and platelet cocultures. Results CoWas mice showed increased megakaryocyte numbers and normal thrombopoiesis in vitro, but WASp‐deficient platelets had short lifespan and high expression of activation markers. Proteomic analysis identified signatures compatible with defects in cytoskeletal reorganization and metabolism yet surprisingly increased antigen‐processing capabilities. In addition, WASp‐deficient platelets expressed high levels of surface and soluble CD40 ligand and were capable of inducing B‐cell activation in vitro. WASp‐deficient platelets were highly immunostimulatory in mice and triggered the generation of antibodies specific for WASp‐deficient platelets, even in the context of a normal immune system. Patients with WAS also showed platelet hyperactivation and increased plasma soluble CD40 ligand levels correlating with the presence of autoantibodies. Conclusion Overall, these findings suggest that intrinsic defects in WASp‐deficient platelets decrease their lifespan and dysregulate immune responses, corroborating the role of platelets as modulators of inflammation and immunity. Graphical abstract Figure. No Caption available.