Giancarlo Labbadia

PD-L1 negatively regulates CD4+CD25+Foxp3+ Tregs by limiting STAT-5 phosphorylation in patients chronically infected with HCV

CD4+CD25+Foxp3+ Tregs suppress autoimmune responses. In addition, they limit T cell responses during chronic infection, thereby minimizing T cell-dependent immunopathology. We sought to investigate how Tregs are regulated in the livers of patients chronically infected with HCV, where they control the balance between an adequate protective immune response and suppression of immunopathology. We found that, despite accumulating and proliferating at sites of infection in the livers of patients chronically infected with HCV, Tregs were relatively less expanded than CD4+CD25+Foxp3- effector T cells. The relative lower expansion of intrahepatic Tregs coincided with their upregulation of programmed death-1 (PD-1). PD-1 expression inversely correlated with both Treg proliferation and clinical markers of immune suppression in vivo. Consistent with the possibility that PD-1 controls Tregs, blockade of the interaction between PD-1 and programmed death-1 ligand 1 (PD-L1) enhanced the in vitro expansion and function of Tregs isolated from the livers of patients chronically infected with HCV. Blockade of the interaction between PD-L1 and B7.1 also improved the proliferation of these cells. Interestingly, both PD-1 and phosphorylated STAT-5 were overexpressed in intrahepatic Tregs in a parallel fashion in steady disease conditions, and in an alternate-fluctuating fashion during the course of severe hepatitis reactivation. Notably, PD-L1 blockade upregulated STAT-5 phosphorylation in Tregs ex vivo. These data suggest that PD-L1 negatively regulates Tregs at sites of chronic inflammation by controlling STAT-5 phosphorylation.

Role of anti-cyclic citrullinated peptide antibodies in discriminating patients with rheumatoid arthritis from patients with chronic hepatitis C infection-associated polyarticular involvement

This study was performed to assess the utility of anti-cyclic citrullinated peptide (anti-CCP) antibodies in distinguishing between patients with rheumatoid arthritis (RA) and patients with polyarticular involvement associated with chronic hepatitis C virus (HCV) infection. Serum anti-CCP antibodies and rheumatoid factor (RF) were evaluated in 30 patients with RA, 8 patients with chronic HCV infection and associated articular involvement and 31 patients with chronic HCV infection without any joint involvement. In addition, we retrospectively analysed sera collected at the time of first visit in 10 patients originally presenting with symmetric polyarthritis and HCV and subsequently developing well-established RA. Anti-CCP antibodies and RF were detected by commercial second-generation anti-CCP2 enzyme-linked immunosorbent assay and immunonephelometry respectively. Anti-CCP antibodies were detected in 23 of 30 (76.6%) patients with RA but not in patients with chronic HCV infection irrespective of the presence of articular involvement. Conversely, RF was detected in 27 of 30 (90%) patients with RA, 3 of 8 (37.5%) patients with HCV-related arthropathy and 3 of 31 (9.7%) patients with HCV infection without joint involvement. Finally, anti-CCP antibodies were retrospectively detected in 6 of 10 (60%) patients with RA and HCV. This indicates that anti-CCP antibodies can be useful in discriminating patients with RA from patients with HCV-associated arthropathy.

Liver vitamin D receptor, CYP2R1, and CYP27A1 expression: relationship with liver histology and vitamin D3 levels in patients with nonalcoholic steatohepatitis or hepatitis C virus

Evidence suggests an association between low serum 25‐hydroxy‐vitamin D3 [25(OH)D3] levels and the presence and prognosis of liver disease. Vitamin D receptor (VDR) has been widely detected in the liver, but its expression in the course of liver disease has never been investigated. We evaluated the hepatic expression of VDR along with that of vitamin D 25‐hydroxylases in patients with nonalcoholic steatohepatitis (NASH) or chronic hepatitis C (CHC) and its relationship with hepatic histological features and serum 25(OH)D3 levels. We evaluated 61 patients (25 NASH and 36 CHC) who had undergone liver biopsy for clinical purposes and 20 subjects without liver disease. Serum 25(OH)D3 was measured via colorimetric assay. Expression of VDR, CYP2R1, and CYP27A1 was evaluated via immunohistochemistry in hepatocytes, cholangiocytes, and liver inflammatory cells. Parenchymal and inflammatory cells from liver biopsies of patients with NASH and CHC expressed VDR, CYP2R1, and CYP27A1. In NASH patients, VDR expression on cholangiocytes was inversely correlated with steatosis severity (P < 0.02), lobular inflammation (P < 0.01), and nonalcoholic fatty liver disease score (P < 0.03). Moreover, expression of CYP2R1 in hepatocytes correlated strongly with VDR positivity on liver inflammatory cells. In CHC subjects, fibrosis stage was associated with low hepatic CYP27A1 expression, whereas portal inflammation was significantly higher in patients with VDR‐negative inflammatory cells (P < 0.009) and low VDR expression in hepatocytes (P < 0.03). Conclusion: VDR is widely expressed in the liver and inflammatory cells of chronic liver disease patients and its expression is negatively associated with the severity of liver histology in both NASH and CHC patients. These data suggest that vitamin D/VDR system may play a role in the progression of metabolic and viral chronic liver damage. (HEPATOLOGY 2012;56:2180–2187)

Mutations in the ANGPTL3 gene and familial combined hypolipidemia: a clinical and biochemical characterization.

CONTEXT Familial combined hypolipidemia causes a global reduction of plasma lipoproteins. Its clinical correlates and metabolic implications have not been well defined. OBJECTIVE The objective of the study was to investigate the genetic, clinical, and metabolic characteristics of a cohort of subjects with familial combined hypolipidemia. DESIGN The design of the study included candidate gene screening and the comparison of the clinical and metabolic characteristics between carrier and noncarrier individuals. SETTING The study was conducted in a general community. SUBJECTS Participants in the study included individuals belonging to nine families with familial combined hypolipidemia identified in a small town (Campodimele) as well as from other 352 subjects living in the same community. MAIN OUTCOMES MEASURES Serum concentrations of lipoproteins, Angiopoietin-like 3 (Angptl3) proteins, and noncholesterol sterols were measured. RESULTS The ANGPTL3 S17X mutation was found in all probands, 20 affected family members, and 32 individuals of the community. Two additional frame shift mutations, FsE96del and FsS122, were also identified in two hypocholesterolemic individuals. Homozygotes for the ANGPTL3 S17X mutation had no circulating Angptl3 and a marked reduction of all plasma lipids (P < 0.001). Heterozygotes had 42% reduction in Angptl3 level compared with noncarriers (P < 0.0001) but a significant reduction of only total cholesterol and high-density lipoprotein cholesterol. No differences were observed in the plasma noncholesterol sterols between carriers and noncarriers. No association between familial combined hypolipidemia and the risk of hepatic or cardiovascular diseases were detected. CONCLUSIONS Familial combined hypolipidemia segregates as a recessive trait so that apolipoprotein B- and apolipoprotein A-I-containing lipoproteins are comprehensively affected only by the total deficiency of Angptl3. Familial combined hypolipidemia does not perturb whole-body cholesterol homeostasis and is not associated with adverse clinical sequelae.

Severe Hypertriglyceridemia-Related Acute Pancreatitis

Acute pancreatitis is a potentially life‐threatening complication of severe hypertriglyceridemia. In some cases, inborn errors of metabolism such as lipoprotein lipase deficiency, apoprotein C‐II deficiency, and familial hypertriglyceridemia have been reported as causes of severe hypertriglyceridemia. More often, severe hypertriglyceridemia describes various clinical conditions characterized by high plasma levels of triglycerides (>1000 mg/dL), chylomicron remnants, or intermediate density lipoprotein like particles, and/or chylomicrons. International guidelines on the management of acute pancreatitis are currently available. Standard therapeutic measures are based on the use of lipid‐lowering agents (fenofibrate, gemfibrozil, niacin, Ω‐3 fatty acids), low molecular weight heparin, and insulin in diabetic patients. However, when standard medical therapies have failed, non‐pharmacological approaches based upon the removal of triglycerides with therapeutic plasma exchange can also provide benefit to patients with severe hypertriglyceridemia and acute pancreatitis. Plasma exchange could be very helpful in reducing triglycerides levels during the acute phase of hyperlipidemic pancreatitis, and in the prevention of recurrence. The current evidence on management of acute pancreatitis and severe hypertriglyceridemia, focusing on symptoms, treatment and potential complications is reviewed herein.

Lipoprotein(a) serum levels in patients affected by chronic obstructive pulmonary disease

A recent study has suggested that symptoms of chronic bronchitis predict the risk of coronary disease independently of the known major cardiovascular risk factors. High serum levels of lipoprotein(a) (Lp(a)) have also been considered as an independent risk factor for coronary heart disease. Therefore, the aim of the present study was to investigate the behaviour of Lp(a) in patients affected by chronic obstructive pulmonary disease (COPD). Serum levels of total-cholesterol (TC), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), triglycerides, apolipoprotein (Apo) B-100, and Lp(a) were measured in 90 COPD patients and in 90 normal subjects matched for age, sex and smoking habit. COPD patients showed lower serum levels of Apo B-100 (P<0.0001) and Lp(a) (P<0.003) compared to controls. Conversely, TC, HDL-C, LDL-C and triglycerides were similar between patients and controls. No significant differences were found in Apo B-100 and Lp(a) levels of patients either undergoing different therapeutic regimens, or with different smoking habits. A significant correlation between Apo B-100 and Lp(a) (rho=0.433, P<0. 0001) was also observed. In conclusion, COPD patients do not show an atherogenetic lipid pattern and their increased risk of coronary disease could be attributable to different factors, such as the ongoing hypercoagulability state often associated with COPD.

Ultrasound evaluation of liver fibrosis: preliminary experience with acoustic structure quantification (ASQ) software.

PurposeThe aim of our study was to assess the diagnostic accuracy of acoustic structure quantification (ASQ) ultrasound software in estimating the degree of hepatic fibrosis compared to Fibroscan and liver biopsy.Materials and methodsSeventy-seven patients with chronic viral hepatitis B and C underwent standard ultrasound examination, ASQ, Fibroscan and liver biopsy. ASQ analysis was conducted by placing a single region of interest (ROI) on each image captured, and calculating mode, average and standard deviation. The sonographic technique was developed through a preliminary evaluation of 20 healthy volunteers.ResultsThe area under the receiver operating characteristic (AUROC) curve for the diagnosis of cirrhosis (F≥4) with ASQ was 0.77, whereas for the diagnosis of any degree of fibrosis (F≥1) it was 0.71. The AUROC for the diagnosis of cirrhosis (F≥4) with Fibroscan was 0.98, while for the diagnosis of any degree of fibrosis (F≥1) it was 0.94. The difference between the AUROC was statistically significant (p<0.05).ConclusionsASQ is a promising new ultrasound software programme which offers encouraging results in the diagnosis of both liver cirrhosis (F=4) and fibrosis (F≥1). However, to date it has not attained the same level of diagnostic performance as Fibroscan.RiassuntoObiettivoScopo del nostro studio è stato valutare l’accuratezza diagnostica del software ecografico acoustic structure quantification (ASQ) nella stima del grado di fibrosi epatica, ponendolo a confronto con Fibroscan e con la biopsia epatica.Materiali e metodiSono stati arruolati 77 pazienti affetti da epatite virale cronica B e C, che sono stati sottoposti ad esame ecografico standard, ASQ, Fibroscan e biopsia epatica. L’analisi ASQ è stata condotta posizionando una singola regione di interesse (ROI) per immagine, su tutte le immagini acquisite, calcolando moda, media e deviazione standard. La tecnica ecografica è stata messa a punto attraverso una valutazione preliminare su 20 volontari sani.RisultatiL’area sottesa alla curva receiver operating characteristic (AUROC) per diagnosi di cirrosi (F≥4) con ASQ è risultata 0,77 mentre per diagnosi di fibrosi di qualsiasi grado (F≥1) è stata 0,71. L’AUROC per la diagnosi di cirrosi (F≥4) con Fibroscan è risultata 0,98 mentre per la diagnosi di fibrosi di qualsiasi grado (F≥1) è stata 0,94. La differenza tra le due AUROC è risultata statisticamente significativa (p<0,05).ConclusioniL’ASQ è un nuovo e promettente software ecografico che presenta risultati incoraggianti sia nella diagnosi di cirrosi epatica (F=4) che nella diagnosi di fibrosi (F≥1). Attualmente tuttavia ancora non sono state raggiunte le performance diagnostiche del Fibroscan.

Anti-lysobisphosphatidic acid antibodies in patients with antiphospholipid syndrome and systemic lupus erythematosus

Lyso(bis)phosphatidic acid (LBPA) is a novel antigenic target in anti‐phospholipid syndrome (APS) and antibodies directed against LBPA (aLBPA) have been detected in sera from APS patients. In this study we first evaluated aLBPA in comparison with the most widely used methods (i.e. anticardiolipin [(aCL)‐enzyme‐linked immunosorbent assay (ELISA)] and antibeta‐2‐glycoprotein‐I antibodies (aβ2‐GPI‐ELISA) utilized to detect antiphospholipid antibodies in patients with primary or secondary APS, systemic lupus erythematosus, chronic HCV infection and healthy subjects. We then assessed the relationship between aLBPA, lupus anticoagulant (LAC) and the main clinical manifestations of APS. Finally, we evaluated the presence of ‘pure’ (i.e. β2‐GPI‐independent) aLBPA in patients with APS and controls. The results indicate that aLBPA as well as aβ2‐GPI display higher specificity but lower sensitivity for APS compared to aCL. Moreover, serum aLBPA correlate closely with aCL and aβ2‐GPI in APS patients and are strictly associated with LAC positivity. We demonstrate that β2‐GPI binds to LBPA with affinity similar to CL, and antibodies able to react with phosholipid‐protein complex exist; however, ‘pure’ aLBPA can also be detected in sera of APS patients. Altogether these data confirm that LBPA may be an antigenic target in APS and that aLBPA are serological markers of APS with similar sensitivity and specificity compared to aβ2‐GPI. However, the clinical utility of aLBPA detection alone or in combination with aCL and/or aβ2‐GPI remains to be elucidated in larger and longitudinal studies.

Soluble P-Selectin and Proinflammatory Cytokines in Patients with Polygenic Type IIa Hypercholesterolemia

Plasma soluble P-selectin (sP-selectin), β-thromboglobulin (β-TG), von Willebrand Factor (vWF), prothrombin factor 1+2 (F1+2), IL-6 and IL-1β levels were analyzed in 35 consecutive patients with polygenic type IIa hypercholesterolemia (HC) and 35 age- and sex-matched healthy subjects. sP-selectin (p < 0.005), β-TG (p < 0.05) and IL-1β (p < 0.02) levels were higher in HC patients than healthy subjects whereas no significant difference was observed for vWF. sP-selectin directly correlated with β-TG (p < 0.05) and IL-1β levels (p < 0.005), but not with the other variables analyzed. A direct correlation was observed between F1+2 and IL-6 (p < 0.05), total cholesterol (p < 0.05) or LDL cholesterol (p < 0.05). We conclude that HC is associated with an increase of plasma sP-selectin levels, and that sP-selectin may be considered as a marker of in vivo platelet activation in type IIa polygenic HC. The correlations observed among the variables analyzed in the study suggest that proinflammatory cytokines might play a role in the prothrombotic state often associated with HC.

Clinical characteristics and plasma lipids in subjects with familial combined hypolipidemia: a pooled analysis

Angiopoietin-like 3 (ANGPTL3) regulates lipoprotein metabolism by modulating extracellular lipases. Loss-of function mutations in ANGPTL3 gene cause familial combined hypolipidemia (FHBL2). The mode of inheritance and hepatic and vascular consequences of FHBL2 have not been fully elucidated. To get further insights on these aspects, we reevaluated the clinical and the biochemical characteristics of all reported cases of FHBL2. One hundred fifteen FHBL2 individuals carrying 13 different mutations in the ANGPTL3 gene (14 homozygotes, 8 compound heterozygotes, and 93 heterozygotes) and 402 controls were considered. Carriers of two mutant alleles had undetectable plasma levels of ANGPTL3 protein, whereas heterozygotes showed a reduction ranging from 34% to 88%, according to genotype. Compared with controls, homozygotes as well as heterozygotes showed a significant reduction of all plasma lipoproteins, while no difference in lipoprotein(a) [Lp(a)] levels was detected between groups. The prevalence of fatty liver was not different in FHBL2 subjects compared with controls. Notably, diabetes mellitus and cardiovascular disease were absent among homozygotes. FHBL2 trait is inherited in a codominant manner, and the lipid-lowering effect of two ANGPTL3 mutant alleles was more than four times larger than that of one mutant allele. No changes in Lp(a) were detected in FHBL2. Furthermore, our analysis confirmed that FHBL2 is not associated with adverse clinical sequelae. The possibility that FHBL2 confers lower risk of diabetes and cardiovascular disease warrants more detailed investigation.