A simple rule to personalize standard dual therapy across all genotypes in naive chronic hepatitis C patients: the TT4 randomized trial.

BACKGROUND: Rapid and early virological responses to peginterferon-alpha and ribavirin are predictive of sustained virological response (SVR) in hepatitis C virus (HCV) infection. We aimed at finding a simple rule to determine the shortest duration of dual therapy for all HCV genotypes, obtained by multiplying time to Initial Viral Response, IVR (first undetectable HCV-RNA) by 4 (Tailored Therapy-4, or TT4). METHOD: 267 naïve HCV-infected patients with compensated liver disease were randomized (2:1) to the TT4 (n=180) or current standard-of-care (SoC, n=87) and received peginterferon-alpha plus ribavirin. Patients with HCV-RNA decrease ≤2log10 at week 12 or detectable HCV-RNA at week 24 discontinued treatment. RESULTS: Both groups had comparable baseline characteristics, SVR rates were similar in the whole population (60.6% vs. 60.9%) and within each genotype subgroup (G1: 46.6% vs. 55.6%; G2: 90.2% vs. 94.4%; G3: 74.1% vs. 58.3%; G4: 45.8% vs. 33.3%). Relapse rate was higher in G1-TT4 than G1-SoC. Treatment duration in SVR patients was shorter in TT4 compared to SoC, both overall [25±15 vs. 36±12.1 weeks], and for subgroups: G1 [35.3±16.7 vs. 47.3±2.6 weeks], G2 [18.3±7.5 vs. 24±2.8 weeks], G3 [15.2±8.7 vs. 22.8±3 weeks] and G4 [26.9±13 vs. 48 weeks]. CONCLUSIONS: In HCV-naive patients, TT4-rule treatment yields similar SVR rates compared to SoC but with shorter treatment duration and remarkable cost reduction.

Upregulation of the inhibitory receptor ILT4 in monocytes from septic patients.

Sepsis-induced immune dysfunction is a complex phenomenon that involves both innate and adaptive responses. Upregulation of the inhibitor receptor named immunoglobulin like transcript 4 (ILT4) is crucial to the tolerogenic function of monocytes. Here, ILT4 expression, endotoxin-induced IL-12 and IL-10 production and CD86 expression were investigated in circulating monocytes from 16 patients with severe sepsis and 16 age and sex matched controls. We found that monocytes from patients with severe sepsis express significantly higher levels of ILT4 than monocytes from controls. Upregulation of ILT4 expression appeared to be induced by soluble factors present in the serum of septic patients and directly correlated with the degree of organ dysfunction. ILT4(+) monocytes from septic patients also displayed an alteration in the cytokine response to endotoxin stimulation characterized by reduced IL-12 production and increased IL-10 production, and a reduced expression of the costimulatory molecule CD86. In conclusion, the increased ILT4 expression and IL-10 production and the decreased CD86 expression and IL-12 production indicate that during sepsis monocytes undergo substantial modulation of the surface and cytokine phenotype. These phenotypic changes may interfere with the antigen presenting cell activity of monocytes, which may contribute to the impairment of adaptive immune responses that takes place during sepsis.

Leptin attenuates ischemia-reperfusion injury in the rat liver.

Leptin is an adipocytokine that reduces ischemic damage in several organs including brain and heart. STAT3 activation is a key step for the attainment of leptin effects in various tissues. We evaluated the possible effect of leptin on liver viability and STAT3 activation, in a rat model of ischemia-reperfusion injury. Rat livers, flushed and stored with Belzer solution (4° C for 24 h), were warmly reperfused (3.5 ml/min/g liver for 1 h at 37° C with O(2) ) with Krebs-Ringer bicarbonate. Treatment group underwent an identical protocol with the adjunct of Leptin (10 ng/ml). Liver effluent was harvested to assess LDH and AST output. Liver tissue was used for pSTAT3 expression (western blot and immunostaining), optical microscopy, TUNEL, and Cell Death Detection assays. The pSTAT3 expression was enhanced by administration of leptin. In parallel, LDH and AST output were reduced (P = 0.04 and P = 0.02 for LDH and AST, respectively). Optical microscopy, TUNEL, and Cell Death Detection assay results demonstrated increased viability in livers treated with leptin in comparison with others (Optical microscopy P = 0.02; TUNEL P = 0.01; Cell death Detection assay P = 0.003). In conclusion, cold storage and reperfusion with leptin reduce liver ischemia-reperfusion injury. This effect is associated with an increased expression of pSTAT-3.

Vitamin D3 modulates T lymphocyte responses in hepatitis C virus-infected liver transplant recipients.

BACKGROUND: Aim of the present study was to investigate whether 1,25(OH)(2)D(3) (Vitamin D3) modulates T lymphocyte functions in patients transplanted for hepatitis C virus-related cirrhosis. METHODS: Sixteen patients and ten healthy subjects were investigated. T lymphocytes were activated in vitro in the presence or absence of Vitamin D3 and then the proliferative response and IFN-γ and TNF-α production were assessed. RESULTS: Vitamin D3 potently reduced T-lymphocyte proliferation in a dose-related fashion. Similarly, FACS analysis and ELISA testing demonstrated that Vitamin D3 significantly decreased the response frequency and the response intensity of IFN-γ and TNF-α production in the whole CD3-positive T lymphocyte population as well as in "naive" CD4+ CD45RA+ and "memory" CD4+ CD45RO+ T lymphocyte subsets. The inhibitory effect of Vitamin D3 on T-cell proliferation and cytokine production was not different between patients and controls. No toxic effects were exerted by Vitamin D3 even at the higher concentration used (10nM). Finally, no statistically significant correlation was found between 25(OH)D serum levels and the proliferative response or cytokine production of T lymphocytes from transplanted patients. CONCLUSIONS: This study demonstrates that in patients transplanted for hepatitis C virus-related cirrhosis Vitamin D3 modulates T lymphocyte activation, and provides a rationale for the evaluation of this compound as an immunosuppressive agent in liver-transplanted patients.

Maintenance ribavirin monotherapy delays fibrosis progression in liver transplant recipients with recurrent hepatitis C at high risk of progression.

BACKGROUND: Fibrosis in liver transplant recipients with recurrent HCV is fast, yet, different patterns of progression are recognized. AIMS: To investigate histological findings associated with maintenance ribavirin monotherapy in patients with recurrent HCV transplanted > or =4 years earlier. METHODS: 14 recipients at high risk of progression (fibrosis progression rate >0.33 units/year and/or persistently elevated ALT) were assigned to receive ribavirin for 3 years. 11 patients at lower risk of progression (FPR < or =0.33 units/year and normal ALT) as controls. Biopsies were obtained yearly since transplant and 7 consecutive biopsies were evaluated. RESULTS: Improved necroinflammation (reduction > or =2 grading) was observed in 7 treated with ribavirin and 3 untreated patients, while 1 and 3 patients worsened respectively. Fibrosis improved (reduction >1 staging) in 2 ribavirin-treated patients, unchanged in 10 and worsened (increase > or =1 staging) in 2. Fibrosis progression decreased from 0.48+/-0.27 observed during the 3-year pre-treatment period to 0.04+/-0.31 units/year (p=0.003) during the 3 years of ribavirin. Among untreated fibrosis remained unchanged in 1 and worsened in 10 (p<0.001), yearly fibrosis progression rate increasing from 0.15+/-0.17 units/year to 0.42+/-0.39 units/year (p=0.10). CONCLUSIONS: Maintenance ribavirin monotherapy delays fibrosis progression in high risk patients, offering an alternative strategy for those failing to respond to conventional treatment.

Proinflammatory modulation of the surface and cytokine phenotype of monocytes in patients with acute Charcot foot.

OBJECTIVE: Despite increased information on the importance of an inappropriate inflammatory response in the acute Charcot process, there has been no previous attempt to define the specific pathways that mediate its pathogenesis. Here, the role played by monocytes was analyzed. RESEARCH DESIGN AND METHODS: The immune phenotype of peripheral monocytes was studied by fluorescence-activated cell sorter analysis comparing patients with acute Charcot (n = 10) in both the active and recovered phase, diabetic patients with neuropathy (with or without osteomyelitis), and normal control subjects. RESULTS: When compared with diabetic control subjects and healthy subjects, monocytes from acute Charcot patients showed a proinflammatory immune phenotype characterized by increased production of proinflammatory cytokines, reduced secretion of anti-inflammatory cytokines, increased expression of surface costimulatory molecules, and increased resistance to serum withdrawal-induced apoptosis. In addition, the pattern of circulating cytokines confirmed activation of proinflammatory cytokines. No modulation of the monocyte phenotype was documented in diabetic control subjects and healthy subjects, thus indicating that the proinflammatory alterations of monocytes are specific and causative of acute Charcot. CONCLUSIONS: Together, these data provide evidence for the role of proinflammatory changes in the immune phenotype of monocytes in the pathogenesis of acute Charcot. These alterations may explain the abnormally intense and prolonged inflammatory response that characterizes this disorder and may represent a potential therapeutic target for specific pharmacological interventions.

1Alpha,25-dihydroxyvitamin D3 inhibits CD40L-induced pro-inflammatory and immunomodulatory activity in human monocytes.

CD40 ligand (CD40L) stimulation induces proinflammatory and immunomodulatory activity in monocytes. Here, we report on the effects of the steroid hormone 1alpha,25-dihydroxyvitamin D3 (1,25D3) on human blood monocytes that have been stimulated with the CD40L ligand. Co-treatment of CD40L-stimulated monocytes with 1,25D3 resulted in reduced production and secretion of tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta, as well as in reduced expression of the surface co-stimulatory molecules CD80 and CD86. In addition, costimulation of CD4+ T lymphocytes by monocytes co-treated with CD40L and 1,25D3 resulted in reduced cell proliferation and diminished interferon (IFN)-gamma but enhanced IL-10 production by CD4+ T cells. Finally, 1,25D3 interfered with the ability of CD40L to rescue monocytes from apoptosis induced by serum withdrawal. These findings suggest that 1,25D3 may regulate the interaction of monocytes with T cells or other cell types that express CD40L, thus influencing the outcome of the immune or inflammatory response.

Downregulation of CD40 ligand response in monocytes from sepsis patients.

It has been suggested that a defective adaptive immune response contributes to septic immunosuppression. Here, the response of monocytes to CD40 ligand (CD40L) for patients with sepsis due to infection with gram-negative organisms has been analyzed. Compared to cells from controls, monocytes from septic patients showed significantly reduced production of tumor necrosis factor alpha, interleukin-1beta (IL-1beta), and IL-12 and were unable to acquire high levels of CD80 and CD86 molecules. These alterations were observed at the onset of sepsis and persisted at day 7. However, the ability of monocytes to respond to CD40L stimulation was partially but significantly restored in cells from patients who recovered from sepsis. In addition, costimulation of autologous CD4+ T lymphocytes by CD40L-activated monocytes from septic patients failed to induce cell proliferation and gamma interferon production. Finally, the ability of CD40L to rescue monocytes from apoptosis was severely impaired. We conclude that downregulation of the CD40L response may be an appropriate model for the monocyte alteration observed during septic immunosuppression and may help in the development of novel therapeutic strategies.

TUDCA prevents cholestasis and canalicular damage induced by ischemia-reperfusion injury in the rat, modulating PKCalpha-ezrin pathway.

Cholestasis, induced by liver ischemia-reperfusion injury (IRI), is characterized by dilatation of bile canaliculi and loss of microvilli. Tauroursodeoxycholic acid (TUDCA) is an anti-cholestatic agent, modulating protein kinase C (PKC) alpha pathway. PKC reduces ischemic damage in several organs, its isoform alpha modulates ezrin, a key protein in the maintenance of cell lamellipoidal extensions. We evaluated the effects of TUDCA on cholestasis, canalicular changes and PKCalpha-ezrin expression in a rat model of liver IRI. Livers flushed and stored with Belzer solution or Belzer + 10 mm TUDCA (4 degrees C for 6 h) were reperfused (37 degrees C with O(2)) with Krebs-Ringer bicarbonate + 2.5 micromol/min of Taurocholate or TUDCA. Bile was harvested for bile flow assessment. Liver tissue was employed for Electron Microscopy (EM) and for PKCalpha and ezrin immunoblot and immunofluorescence. The same experiments were conducted with the PKCalpha inhibitor Go-6976. TUDCA-treated livers showed increased bile flow (0.25+/-0.17 vs. 0.042+/-0.02 microl/min/g liver, P<0.05) and better preservation of microvilli and bile canalicular area at EM. These effects were associated with increased PKCalpha and ezrin expression (P=0.03 and P=0.04 vs. control respectively), as also confirmed by immunofluorescence data. PKCalpha inhibition abolished these TUDCA effects. TUDCA administration during IRI reduces cholestasis and canalicular damage in the liver modulating PKCalpha-ezrin pathway.

Percutaneous cryoablation of small hepatocellular carcinoma with US guidance and CT monitoring: initial experience.

The purpose of this study was to retrospectively determine the safety and effectiveness of percutaneous cryoablation, monitored with computed tomography (CT) and ultrasonographic (US) guidance, for the treatment of hepatocellular carcinoma (HCC). Four patients with small HCCs underwent one percutaneous cryoablation treatment session monitored with CT and US guidance. All patients underwent pretreatment blood chemistry testing and imaging evaluation. We treated lesions with simultaneous insertion of multiple 17-G cryoprobes (two or three) and defined technical success when the extension of a visible iceball was beyond 5 mm from the tumor margin. Intralesional enhancement or tumoral size increase was defined as local progression compared with that on images obtained immediately after ablation. We evaluated complications and follow-up (at 1, 3, and 6 months). All patients survived without short- or long-term complications. Cryoablation was technically successful in all patients at the end of the procedure. During follow-up two patients developed disease recurrence. One patient developed local tumor progression on the margin of the lesion; the other, a new HCC. In the case of local tumor progression a new elevation of alpha-fetoprotein (alphaFP) levels occurred at first follow-up control. In the other case levels of alphaFP remained stable during the first 3 months after the procedure, then demonstrated a progressive increase in alphaFP levels beginning at the fourth month, without tumor evidence during CT control at 3 months. We conclude that percutaneous cryotherapy with US guidance and CT monitoring is a feasible, safe, and effective for treatment of HCC. If local ablative procedures of hepatic lesions are to be performed, percutaneous cryoablation, not laparotomic, should be discussed as an alternative therapeutic measure. Longer follow-up should provide proof of the effectiveness of this technique.

Lipopolysaccharide desensitizes monocytes-macrophages to CD40 ligand stimulation.

Polymicrobial sepsis induces the suppression of macrophage function as determined by a reduction of pro-inflammatory cytokine production upon re-exposure to lipopolysaccharide (LPS) in vitro. Here, we examined whether macrophages were refractory to only LPS or if they were unable to respond to other stimuli such as CD40 ligand (CD40L). Monocytic cells exposed in vitro to LPS showed a dose-dependent reduction of their ability to produce interleukin-12 and tumour necrosis factor-alpha upon subsequent CD40L stimulation, as compared to cells stimulated with CD40L alone. Similarly, LPS interfered with the up-regulation of CD40, CD80 and CD86 induced by CD40L in monocytic cells. The effect of LPS on the response of monocytes to CD40L was similar whether these cells were directly exposed to LPS or cocultured with LPS-pretreated cells, indicating that soluble factors released by LPS stimulation could mediate tolerance to CD40L. We also show that the functional alterations induced by LPS in monocytes can be reversed by indomethacin, thus suggesting a role for inducible cyclooxygenase in mediating the LPS-induced hyporesponsive state of monocytes to CD40L. In conclusion, we propose that in vitro CD40L tolerance may be an appropriate model of monocyte alteration observed during septic immunosuppression and may help in the development of novel therapeutic strategies.

Increased depressive ratings in patients with hepatitis C receiving interferon-alpha-based immunotherapy are related to interferon-alpha-induced changes in the serotonergic system.

There is now evidence that repeated administration of interferon-alpha (IFN-alpha) to patients with chronic active hepatitis and cancers induces depressive symptoms. There is also evidence that induction of the cytokine network modulates the serotonergic system and that major depression is related to activation of the cytokine network and disturbances in the serotonergic metabolism. The aims of this study were to examine the effects of IFN-alpha-based immunotherapy on the development of depressive symptoms in relation to its effects on plasma tryptophan and kynurenine and serum serotonin (5-HT). Eighteen patients affected by chronic active hepatitis C were treated with IFN-alpha (3-6 million units subcutaneously three to six times a week for 6 months) and had measurements of the previous parameters before starting immunotherapy and 2, 4, 16, and 24 weeks later. Severity of depression and anxiety were measured with the Montgomery-Asberg Depression Rating Scale (MADRS) and the Hamilton Rating Scale for Anxiety (HAM-A) scale, respectively. Immunochemotherapy with IFN-alpha (1) significantly increased the MADRS and HAM-A scores and serum kynurenine concentrations and (2) significantly reduced plasma tryptophan and serum 5-HT concentrations. IFN-alpha-based immunotherapy significantly increased the kynurenine per tryptophan quotient, which estimates the activity of indoleamine 2,3-dioxygenase, the major tryptophan-catabolizing enzyme, which is induced by IFNs. There are significant relationships between the IFN-alpha-induced changes in the MADRS score and serum kynurenine (positive) and 5-HT (negative) concentrations. Immunotherapy with IFN-alpha significantly increases the severity of depressive symptoms. The latter is related to changes in the serotonergic system, such as depletion of serum 5-HT and induction of the catabolism of tryptophan to kynurenine. It is suggested that the IFN-alpha-induced changes in the serotonergic turnover could play a role in the development of IFN-alpha-induced depressive symptoms.