Objectives To establish risks for development of hepatocellular carcinoma (HCC) in children with biliary atresia (BA), the most common chronic liver disease of childhood. Study design In our tertiary ...referral center database we have identified children with BA who had development of or have been incidentally found to have HCC. Their demographic, clinical, radiologic, and histologic features were analyzed. Results Between 1990 and 2008, 387 infants were diagnosed with BA at our center. Of these, three (0.8 %) who underwent operation at a median age of 68 (range 66 to 71) days had development of a histologically proven HCC detected at a median age of 2.1 (range 1.8 to 4.9) years. Another two, referred later, were diagnosed with HCC on their liver explants at ages 1.1 and 17.75 years, respectively. Overall, two had elevated serum levels of alpha-fetoprotein. All five children underwent successful liver transplantation at a median age of 2.1 years (range 1.1 to 17.75) and remain well after a median of 2.5 (range 2 to 5.7) years. Conclusion HCC develops in a small percentage of children with BA. Serum alpha-fetoprotein levels and ultrasound screening are helpful but not absolute markers of the malignant change. In the absence of the extrahepatic involvement, liver transplantation represents an effective treatment.
IntroductionConcordance for autoimmune hepatitis (AIH) is rare within families, though non-hepatic autoimmune disorders are frequent among first degree relatives (FDR) of AIH patients. While a defect ...in immunoregulation has been demonstrated in AIH patients, the mechanism preventing the development of AIH in FDR, who share genetic background, remains to be elucidated.AimTo investigate multiple immunoregulatory systems in AIH patients and their FDR.Method44 children with AIH (33 AIH-1 and 11 AIH-2, median age 13.5 yrs, 23 females), 65 FDR from 34 families 23 fathers, 47 yrs (38–58); 28 mothers, 44 yrs (24–53) and 14 siblings, 7 females, 13 yrs (5–24) and 42 healthy subjects HS, 36 yrs (22–54), 37 females were studied. Frequency of conventional CD4posCD25pos regulatory T-cells (Tregs), CD4posCD25highCD127neg True Tregs, CD4posPD-1pos and CD8posCD28neg T cells, CD3negCD56pos natural killer (NK) cells, CD3posTCRVa24pos/TCRVb11pos invariant NKT cells (iNKT) was defined by flowcytometry. Tregs and CD4posPD-1pos T cells were purified from PBMCs using immunomagnetic beads. CD25neg and PD-1neg cells (responders) were co-cultured for 5 days with cells with regulatory potential and their proliferation was measured by 3H-thymidine incorporation.ResultsConventional Tregs were lower in patients (10.5%±1.1) than FDR (15.9%±1.1, p=0.001) and HS (14.7%±1.7, p=0.04), while ‘True’ Tregs were similar in all (6.0%±0.6, 6.3%±0.4 and 6.2%±0.5). CD4posPD-1pos T cells were lower in patients (1.7%±0.2) and in FDR (1.9%±0.2) than in HS (3.0%±0.2, p<0.0001 for patients and p=0.0007 for FDR). NK cells were lower in patients (8.6%±1.2) than in FDR (15.8%±1.2, p=0.0004) and HS (12.3%±0.9, p=0.02). CD8posCD28neg T cells in patients tended to be lower (8.8%±1.46) than in FDR (12.43%±1.56, p=0.18) and HS (13.1%±1.82, p=0.11). The frequency of iNKT cells was similar in all groups. ‘True’ Tregs decreased CD25neg cell proliferation by 13.6% in patients, 28.8% (p=0.007) in FDR and 36.9% (p<0.0001) in HS, while CD4posPD-1pos T cells decreased similarly PD-1neg cell proliferation in patients (25.4%), FDR (22.5%) and HS (26.8%).ConclusionA numerical impairment of CD4posPD-1pos T cells in patients and their FDR suggests that these defects are genetically determined and account for family clustering of autoimmune disorders. A numerical impairment of conventional Tregs and functional impairment of CD4posCD25highCD127neg ‘True’ Tregs, confined to patients with AIH, may be crucial to loss of liver tolerance.
IntroductionChanges in HBsAg plasma levels during antiviral therapy with pegylated interferon (IFN) predict response in adults with chronic hepatitis B (CH-B). Whether pre-treatment HBsAg plasma ...levels correlate with liver relaxed circular (RC) HBV DNA and covalently closed circular (ccc) DNA is controversial. To date, no information is available on HBsAg plasma level behaviour and response to treatment prediction in tolerant patients with infancy-acquired CH-B treated with lamivudine (LAM) and IFN.AimTo investigate whether HBsAg plasma levels predict response to LAM + IFN treatment in tolerant children with infancy-acquired CH-B, and to determine their association with plasma HBV DNA levels during treatment and with pre-treatment liver RC HBV DNA and cccDNA.MethodPatients: 23 children (8 males, median age 10.2 yrs) with infancy-acquired CH-B (all HBeAg positive), treated for 52 weeks lead-in LAM (3 mg/kg/d) for 9 weeks; LAM plus IFN-α (5 MU/m2 TIW) from week 9 for 44 weeks, were divided according to treatment response: 5 responders (R = anti-HBs seroconversion) and 18 non-responders (NR).Methods: Plasma HBsAg and HBV DNA levels were measured before (treatment week 0, TW0), during (TW9, TW28, TW52) and after (follow-up week, FUW24) therapy by Abbott ARCHTECT_ assay and real-time TaqMan PCR both log10 IU/ml. Baseline liver RC HBV DNA and cccDNA was quantified by real-time TaqMan PCR copies/ng genomic DNA. Results are presented as median.ResultsBaseline HBsAg levels were lower in R than NR (4.36 vs 4.74, p=0.02), but similar in R and NR at the end of LAM lead-in therapy (TW9) (4.34 vs 4.66, p=0.1). During IFN add-on therapy, at TW28 (2.34 vs 4.33) and TW52 (0 vs 4.08) levels were markedly lower in R than NR, the difference persisting at FUW24 (0 vs 4.51) (p<0.01 for all). Plasma HBV DNA levels were similar at baseline in R and NR (8.94 vs 8.98), but decreased significantly in R compared to NR at TW9 (4.91 vs 5.48), TW28 (3.42 vs 4.39), TW52 (1.57 vs 4.07) and FUW24 (0.27 vs 7.75) (p<0.01 for all). There was a strong positive correlation between plasma HBsAg and HBV DNA levels at TW28 (r=0.6, p<0.01) and TW52 (r=0.64, p<0.01). Baseline liver RC HBV DNA (43 800 vs 52 300 copies/ng genomic DNA) and cccDNA (41 vs 49 copies/ng genomic DNA) were similar in R and NR, with no correlation between liver RC HBV DNA or cccDNA and baseline HBsAg.ConclusionLower baseline HBsAg plasma levels and a sharp decrease of plasma HBV DNA levels at TW9 (LAM lead-in) followed by declining plasma HBsAg levels from TW28 (IFN add-on) heralds HBsAg clearance and response to treatment in tolerant children with CH-B.
IntroductionAutoimmune diseases are frequent among first degree relatives (FDR) of patients with autoimmune hepatitis (AIH), but concordance for AIH is rare. A numerical and functional impairment of ...CD4posCD25pos regulatory T cells (Tregs) is described in AIH patients, but no study has addressed Treg status in their FDR.AimTo define whether the defect of Tregs in AIH is inherited and is associated with disease predisposing HLA genes.Method44 children with AIH (33 AIH-1 and 11 AIH-2, median age 13.5 yrs, 23 females), 65 FDR from 34 families 23 fathers, 47 yrs (38–58); 28 mothers, 44 yrs (24–53) and 14 siblings, 7 females, 13 yrs (5–24) and 42 healthy subjects HS, 36 yrs (22–54), 37 females were studied. Tregs were purified from PBMCs using immunomagnetic beads and their phenotype and frequency was assessed by flowcytometry. CD25neg cells were used as responders in co-culture with Tregs and their proliferation was measured by 3H-thymidine incorporation. HLA genotyping was performed by PCR using gene specific primers.ResultsThe frequency of the disease predisposing gene HLA DR3 was significantly higher in patients (71%) and their FDR (56%) than in HS (23%, p<0.0001 and p<0.005). The frequency of homozygous DR3 was higher in patients (29%) than in FDR (9%, p=0.015) and HS (0%, p=0.001). In patients the frequency of HLA A1-B8-DR3 haplotype (42%) was higher than in FDR (27%, p=0.15) and HS (16%, p=0.02). The frequency of conventional CD4posCD25pos Tregs was lower in patients (6.2%±0.5) than in FDR (9.3%±0.7, p=0.0016) and HS (9.7%±0.8, p=0.001). Though the frequency of CD4posCD25highCD127neg ‘True’ Tregs was similar among patients, FDR and HS (6.0%±0.6, 6.3%±0.4 and 6.2%±0.5), their suppressive function was lower in patients (13.6% reduction of CD25neg cell proliferation) than in FDR (28.8%, p=0.007) and HS (36.9%, p<0.0001). Among subjects positive for HLA DR3, the frequency of conventional Tregs was lower in patients (5.6%±0.5) than in FDR (8.4%±0.97, p=0.01) and in HS (9.1%±1.4, p=0.006). Among subjects positive for HLA A1-B8-DR3 haplotype, the frequency of Tregs was lower in patients (6.0%±0.7) than in FDR (9.1%±1.5, p=0.045) and HS (9.8%±1.8, p=0.02).ConclusionA numerical and functional impairment of Tregs in AIH patients is associated with possession of HLA disease predisposing genes, and in particular HLA DR3 homozygosity. Possession of DR3 was not associated to a similar immune regulatory impairment in FDR and HS, suggesting that a gene dose effect contributes to the impairment of immunoregulation and to the development of AIH.
IntroductionAutoimmune hepatitis (AIH) is a severe inflammatory liver disorder characterised by hypergammaglobulinaemia, seropositivity for autoantibodies and interface hepatitis on histology. Though ...the mechanisms leading to immune-tolerance breakdown have not been fully elucidated, a numerical and functional defect of circulating CD4posCD25high regulatory T-cells (T-regs) plays a key role in permitting effector lymphocytes to attack hepatocytes. We have identified a cohort of children with concomitant AIH and systemic lupus erythematosus (SLE), a multi-system autoimmune disease in the context of which most studies have reported numerical and functional T-reg impairment.AimTo define the phenotypic and functional profile of T-regs in children with AIH and SLE (AIH/SLE).Method9 AIH/SLE patients (8 females; median age: 13.9 years), 16 AIH patients (13 females; median age: 13.3 years) and 9 healthy subjects (HS, 6 females; median age: 34 years) were studied. T-reg phenotype was determined by flow cytometry after cell incubation with anti-CD4, CD25 and CD127 monoclonal antibodies. Frequency of FOXP3pos and IFNγ, IL-4, IL-17 and IL-9-producing cells was assessed by intracellular staining. T-reg suppressor function was evaluated as reduction of cell proliferation, measured by 3H-thymidine uptake, in co-culture experiments where CD4posCD25highCD127negT-regs were added to CD25neg target cells.ResultsThe number of T-regs in AIH/SLE patients (7.06±1.1) tended to be higher than in AIH (4.88±1.1; p=0.1) and was similar to HS (7.03±0.6). While the proportion of FOXP3pos cells within T-regs did not differ, that of CD4posCD25highCD127pos cells was higher in AIH/SLE (2.4±0.99) than in AIH (0.46±0.2; p=0.019) and HS (0.63±0.3; p=0.05). The frequency of IFNγ-producing cells within T-regs was higher in AIH/SLE (6.75±1.7) than in AIH (4.8±1.9; p=0.019) and HS (3.5±0.82; p=0.05); conversely that of IL-4-producing cells within T-regs was lower in AIH/SLE (0±0) than in AIH (0.86±0.3; p=0.02) and HS (0.9±0.47; p=0.06). The frequency of IL-17 and IL-9-producing cells was negligible and did not differ among the groups. Addition of CD127negT-regs, while decreasing CD25neg cell proliferation by 31.6% in HS (p=0.08) and by 12.3% (p=0.056) in AIH, did not affect the proliferation of CD25neg cells isolated from AIH/SLE patients.ConclusionCompared to AIH and HS, T-regs from AIH/SLE patients are more activated, are skewed towards a Th1 cytokine profile and are functionally defective. These data suggest that more severe alteration of T-reg phenotype and function may predispose to multiple autoimmune manifestations.
Hepatitis B virus (HBV) is a noncytopathic virus, and the recognition of infected hepatocytes by HBV-specific CD8 cells has been assumed to be the central mechanism causing both liver damage and ...virus control. To understand the role of cytotoxic T cells in the pathogenesis of HBV infection, we used functional assays that require T cell expansion in vitro and human histocompatibility leukocyte antigen (HLA)-peptide tetramers that allow direct ex vivo quantification of circulating and liver-infiltrating HBV-specific CD8 cells. Two groups of patients with persistent HBV infection were studied: one without liver inflammation and HBV replication, the other with liver inflammation and a high level of HBV replication. Contrary to expectation, a high frequency of intrahepatic HBV-specific CD8 cells was found in the absence of hepatic immunopathology. In contrast, virus-specific T cells were more diluted among liver infiltrates in viremic patients, but their absolute number was similar because of the massive cellular infiltration. Furthermore, inhibition of HBV replication was associated with the presence of a circulating reservoir of CD8(+) cells able to expand after specific virus recognition that was not detectable in highly viremic patients with liver inflammation. These results show that in the presence of an effective HBV-specific CD8 response, inhibition of virus replication can be independent of liver damage. When the HBV-specific CD8 response is unable to control virus replication, it may contribute to liver pathology not only directly but by causing the recruitment of nonvirus-specific T cells.
Cerebrospinal fluid (CSF) levels of interleukin (IL)-6, IL-11 and leukaemia inhibitory factor (LIF) were evaluated in 43 patients with Alzheimer's disease (AD) and 24 patients with frontotemporal ...lobar degeneration (FTLD) as compared with 30 agematched controls (CON), and correlated with clinical and demographic data and with CSF biomarkers amyloid beta (Aβ)42, total tau and tau phosphorylated at position 181 (P-tau). CSF IL-11 mean levels were significantly increased in AD and FTLD as compared with CON (6.5 ± 4.6 and 6.6 ± 5.1 versus 3.1 ± 3.3 pg/ml,
P
= 0.009). IL-6 mean levels did not differ between patients and CON (
P
> 0.05),whereas LIF levels were not detectable in patients or in CON. In AD patients, a significantly positive correlation between MMSE scores and IL-11 CSF concentration was observed (r = 0.344,
P
= 0.028). No correlations with CSF Aβ42, total tau and P-tau were found. IL-11, but not IL-6 levels are increased in AD and FTLD, and the highest peaks were observed in patients with a less severe degree of cognitive deterioration, therefore suggesting a role of this cytokine in early phases of neurodegeneration.
•Anionic beta cyclodextrin retains drug into the layer-by-layer system•Anionic beta cyclodextrin controls drug release from the layer-by-layer system•The effective drug delivery system is readily ...tunable to different physiological scenarios•The drug delivery system shows antibacterial effect against Staphylococcus aureus•An extended effect in bacteria reducing on coating was maintained up to 30 days
This study demonstrated a drug-delivery system with anionic beta cyclodextrin (β-CD) complexes to retain tetracycline (TC) and control its release from multilayers of poly(acrylic acid) (PAA) and poly(l-lysine) (PLL) in a ten double layers (PAA/PLL10) coating onto titanium. The drug-delivery capacity of the multilayer system was proven by controlled drug release over 15 days and sustained released over 30 days. Qualitative images confirmed TC retention within the layer-by-layer (LbL) over 30 days of incubation. Antibacterial activity of TC/anionic β-CD released from the LbL was established against Staphylococcus aureus species. Remarkably, PAA/PLL10/TC/anionic β-CD antibacterial effect was sustained even after 30 days of incubation. The non-cytotoxic effect of the multilayer system revealed normal human gingival fibroblast growth. It is expected that this novel approach and the chemical concept to improve drug incorporation into the multilayer system will open up possibilities to make the drug release system more applicable to implantable percutaneous devices.
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