SCCA-IC serum levels are predictive of clinical response in HCV chronic hepatitis to antiviral therapy: a multicentric prospective study.

The combination of pegylated interferon (Peg-IFN) and ribavirin is currently the gold standard therapy in patients with HCV chronic infection. The duration of therapy, as well as the therapeutic dosage, depend on the genotype. Identification of the genotype and rapid virological response (RVR) are widely accepted as the most important predictors of clinical outcome during antiviral therapy but to optimize cost-benefits and to reduce possible side effects, further prognostic factors are needed. Squamous cell carcinoma antigens immunocomplex (SCCA-IC) has been reported to be increased in the serum of patients with liver cancer. In this multicentric prospective study, we investigated the serum levels of SCCA-IC in 103 patients with HCV chronic infection. Serum HCV-RNA was detected before the beginning of treatment, after 4, 12, 24 or 48 weeks, and at week 24 during follow-up. RVR, early virological response and sustained virological response (SVR) were assessed following the international guidelines. SCCA-IC levels were higher in responders (238 AU, interquartile difference 130-556 AU) and decreased significantly to 125 AU (70-290 AU). The mean baseline value in nonresponders was 149 AU (86.5-306.5 AU), but after 4 weeks of treatment the serum levels decreased to 115 AU (80-280 AU): the profile of reduction was different between patients with or without a positive SVR. Logistic regression with SVR as dependent variable identified as significant independent variables: the reduction in SCCA-IC after 1 month (OR = 4.82; 95% CI 1.39-16.67; P = 0.131) and a genotype other than 1 (OR = 0.094; 95% CI 0.21-0.42; P = 0.002); sex and age were also significant factors influencing SVR. SCCA-IC seems to be a reliable independent prognostic marker of therapeutic effectiveness in anti-HCV positive patients undergoing antiviral therapy.

Interleukin-28B (IL-28B) single-nucleotide polymorphisms and interferon plus ribavirin treatment outcome in Italian chronically HCV-infected patients.

To determine the single or combined effect of both rs12979860 and rs8099917 SNPs on HCV treatment response, these variants were genotyped in samples from a cohort of 170 patients infected with different HCV genotypes (HCVGT). The favourable rs12979860 CC genotype was found only in patients with sustained or rapid virological responses (SVR/RVR) and at significantly high proportions in HCVGT1/4 SVR patients. A significant association was also found between the rs8099917 TT genotype and SVR in both HCVGT1/4 and HCVGT2/3 groups of patients. In contrast, we found that there was significantly more of the rs8099917 GG genotype in nonresponders (NR) than in SVR patients which suggests a good association of the minor homozygote GG with the lack of treatment response. The combination of rs12979860/rs8099917 CC/TT favourable genotypes was found only in SVR patients and matched the frequency observed for their rs12979860 CC genotypes alone. By contrast, the inverse unfavourable correlate rs12979860/rs8099917 TT/GG genotype was seen more in NR than in SVR patients as observed for the single GG genotype. This study confirms the impact of both rs12979860 and/or rs8099917 IL-28B SNPs on treatment-induced clearance of HCV-RNA and demonstrates that the rs12979860 CC genotype is stronger than rs8099917 TT genotype in predicting a positive treatment response in HCVGT1/4 patients. The unfavourable rs8099917 GG genotype seems to be more important in predicting the failure of treatment response independently from HCV genotype.

Apoptotic effects of a chimeric plant virus carrying a mimotope of the hepatitis C virus hypervariable region 1: role of caspases and endoplasmic reticulum-stress.

The role of apoptosis in the persistence of hepatitis C virus (HCV) infection is controversial. Moreover, conflicting data on the modulation of this process by HCV proteins have been provided. We evaluated the susceptibility of peripheral lymphocytes from patients with chronic hepatitis C to apoptosis both spontaneous and after incubation with a chimeric Cucumber mosaic virus (CMV) carrying 180 copies of the synthetic R9 mimotope obtained from more than 200 hypervariable region-1 sequences of HCV. Resting T lymphocytes were found to be sensitized to apoptosis as a result of chronic HCV infection. The plant virus-derived vector R9-CMV displayed a strong pro-apoptotic effect associated with activation of both caspase-8 and -9, indicating the involvement of both extrinsic and intrinsic apoptotic pathways. A parallel R9-CMV-mediated activation of endoplasmic reticulum-stress was suggested by the significant induction of BiP/GRP78, GADD153 and caspase-12. These data contribute to define the complex HCV/host interaction, and open new prospects for developing a plant-derived antigen-presenting system to strengthen host defences against persistent pathogens.

Cucumber mosaic virus as the expression system for a potential vaccine against Alzheimer's disease.

A primary therapeutic goal in Alzheimer's disease (AD) is to reduce the quantity of amyloid ß protein (Aß) present in the brain. To develop an effective, safe system for vaccination against Alzheimer's disease, the plant virus Cucumber mosaic virus (CMV) was engineered genetically to express Aß-derived fragments that stimulate mainly humoral immune responses. Six chimeric constructs, bearing the Aß1-15 or the Aß4-15 sequence in positions 248, 392 or 529 of the CMV coat protein (CP) gene, were created. Viral products proved to be able to replicate in their natural host. However, only chimeric Aß1-15-CMVs were detected by Aß1-42 antiserum in Western blot analysis. Experimental evidence of Immunoelectron microscopy revealed a complete decoration of Aß1-15-CMV(248) and Aß1-15-CMV(392) following incubation with either anti-Aß1-15 or anti-Aß1-42 polyclonal antibodies. These two chimeric CMVs appear to be endowed with features making them possible candidates for vaccination against Alzheimer's disease.

Perihepatic lymphadenopathy and the response to therapy in chronic hepatitis C patients().

INTRODUCTION: To determine whether ultrasound (US) findings of perihepatic lymphadenopathy in patients with chronic hepatitis C (HCV) are predictive of failure to achieve sustained virological responses (SVR) to treatment with pegylated interferon (PEG-INF) alpha and ribavirin. MATERIALS AND METHODS: The study population included 89 patients with HCV treated with PEG-INF and ribavirin in 2003-2007. US scans and liver biopsies were performed at baseline; US was repeated during treatment and 6 months after its completion. Patients with SVRs at the 6-month follow-up were classified as responders; all others (those with no response or shorter responses) were considered nonresponders. RESULTS: Baseline US revealed perihepatic lymphadenopathy in 21.3% of the patients, and 31.6% of these patients were responders. In the responder subgroup, 16% of the patients experienced reductions in lymph node volume during treatment, whereas in the nonresponders, lymph node volume increased during treatment. In patients with no lymphadenopathy at baseline US, no lymph nodes were observed at US after the end of treatment, in either the responders or nonresponders. CONCLUSIONS: US is becoming increasingly important in the follow-up of chronic HCV patients. In our study, patients with hilar lymphadenopathy were usually nonresponders, and at the end of treatment their lymph node volume was significantly greater than that observed in the responder subgroup.

The use of different Peg-interferon alpha-2b regimens plus ribavirin in HCV-1b-infected patients after rapid virological response does not affect the achievement of sustained virological response.

In patients with chronic hepatitis C, rapid virological response (RVR) at week 4 of treatment seems to be strongly associated with a high probability of achieving a sustained virological response (SVR). The aim of this study was to investigate the outcome of different pegylated interferon-alpha2b (Peg-IFN-alpha2b) dosages plus ribavirin (RBV) in patients with RVR. Forty-five naïve patients chronically infected with hepatitis C virus (HCV)-1b started Peg-IFN-alpha2b (1.5 microg/kg/week) in combination with weight-based RBV doses (800-1200 mg/day). Thirty-one patients (68.9%) attained RVR at week 4 of therapy, while four further patients showed negative HCV-RNA values for the first time at week 12 and were considered early virological responders (EVR). The 31 RVR patients were randomized to receive either RBV plus 1.5 microg/kg/week (17 pts) or 1.0 microg/kg/week (14 pts) of Peg-IFN-alpha2b for the remaining 44 weeks. The two groups were matched for age, sex, baseline alanine aminotransferase levels, viral load and fibrosis score. After 6 months of post-treatment follow-up, the prevalence of SVR was 94.1% (16/17) among RVR patients treated with 1.5 microg/kg/week and 92.8% (13/14) in RVR patients treated with 1.0 microg/kg/week (P = not significant). A high-baseline viral load (P = 0.01) and bridging fibrosis/cirrhosis (P = 0.02) negatively influenced the likelihood of achieving RVR. On the contrary, the ability of RVR patients to achieve SVR did not correlate with these baseline characteristics in either of the treatment group. Finally, the SVR rate among EVR patients who responded after more than 4 weeks of treatment was significantly lower than among RVR patients (1/4 = 25%vs 29/31 = 93.5%; P = 0.0058), because of a high prevalence of post-treatment relapse among patients with EVR.

Rationale for new drugs targeting the tissue microenvironment in patients with HCC.

A better knowledge of the mechanisms underlying hepatocellular carcinoma (HCC) growth and spread is essential to improve the available treatment options. So far, the only therapies available for HCC are mainly based on tumor-destructive approaches, whereas no therapies are available to consolidate these invasive therapies or to cure the tumor. The fact that HCC develops on cirrhotic liver strongly limits the use of common anti-cancer drugs, but the need to find new therapies is strongly felt by clinicians. A large body of evidence suggests that the tissue microenvironment represents a potential target for therapies. Consistently, biological therapies such as inhibitors of the epithelial growth factor receptor (EGFR), are currently under investigation. Unfortunately, there is a discrepancy between the very promising experimental data and the results obtained in patients, although limited sample sizes and advanced stage of the disease could be important factors hampering a reliable judgment of the efficacy of such drugs. Nevertheless, a better identification of the molecular pathways involved in drug effectiveness as well as in HCC tumor progression indicates that the tissue microenvironment likely harbors the solution to the problem. In this review the role and the rationale of using biological drugs to target the microenvironment is discussed, taking into consideration new experimental advances in the field.

Tyrosine kinase inhibitors: a potential approach to the treatment of hepatocellular carcinoma.

The increasing number of patients with hepatocellular carcinoma (HCC) and the highly unfavourable prognosis of the disease are two important reasons why more effort needs to be devoted to investigating other therapies able to block or reduce tumor progression and cancer metastasis. The underlying cirrhosis on which HCC develops limits the use of common chemotherapies, mainly because of their toxicity. Recently, great attention has been paid to a family of molecules that inhibits the tyrosine kinase (TK) receptors, because of their relevant role in activating intracellular pathways responsible for several biological activities of the HCC cells. In particular, proliferation, invasion, survival, apoptosis, are regulated by Erk1/2 and Akt pathways, that can be considered for this reason as potential therapeutic targets. Therefore, the idea is to fight HCC by blocking the molecular mechanisms exploited by the cancer to develop and to metastasize. The epithelial growth factor and the vascular endothelial growth factor receptors (EGFR and VEGFR, respectively) have been identified as the major targets for these new therapies. In this review the biological role of both growth factors in HCC will be discussed, together with the use of anti-EGFR and anti-VEGFR. The preliminary results obtained in vitro or in "in vivo" experimental models have been very promising, whereas the few studies conducted in patients have been not comparably satisfactory. This could be because of the limited number of patients and of their advanced stage of HCC, nevertheless the possibilities of using this family of drugs should be further explored, together with aspects contributing to a better understanding of the molecular mechanisms driving HCC progression.

Endometriosis is characterized by an impaired localization of laminin-5 and alpha3beta1 integrin receptor.

Endometriosis is an estrogen-correlated benign disease characterized by a marked ability of endometrial-like cells to invade and proliferate outside uterine cavity, resembling for some invasive aspect the cancer growth. The molecular mechanisms regulating endometrial cell invasiveness are mostly unknown, although interactions between extracellular matrix (ECM) proteins and their transmembrane receptors, integrins, are likely to play a central role. In particular, laminin (Ln)-5 could be closely involved, as it is in cancer. We have investigated the expression of Ln-1, Ln-5, and collagen IV (Coll IV) ECM proteins and their receptors, alpha3beta1 and alpha6beta4 integrins, in atrophic, proliferative, and secretive endometrium and in endometriosis. The results show that Ln-5, but not Ln-I and Coll IV, is altered in secretive endometrium as well as in endometriosis tissues. No alterations are observed in atrophic or proliferative endometrium. Consistently, the polarization of both integrin subunits alpha3 and beta1, but not alpha6 and beta4, is altered in secretive endometrium and endometriosis tissues, but not in atrophic and proliferative endometrium. These results seem to suggest that Ln-5 and alpha3beta1 could be involved in the invasive mechanism of endometriosis. The altered expression of Ln-5, by upregulating matrix metalloproteases activity, suggest an invading process similar to that of many cancer processes.

Cucumber mosaic virus as a presentation system for a double hepatitis C virus-derived epitope.

Chimeric plant viruses are emerging as promising vectors for use in innovative vaccination strategies. In this context, cucumber mosaic virus (CMV) has proven to be a suitable carrier of the hepatitis C virus (HCV)-derived R9 mimotope. In the present work, a new chimeric CMV, expressing on its surface the HCV-derived R10 mimotope, was produced but lost the insert after the first passage on tobacco. A comparative analysis between R10- and R9-CMV properties indicated that R9-CMV stability was related to structural features typical of the foreign insert. Thus, in order to combine high virus viability with strong immuno-stimulating activity, we doubled R9 copies on each of the 180 coat protein (CP) subunits of CMV. One of the chimeras produced by this approach (2R9-CMV) was shown to systemically infect the host, stably maintaining both inserts. Notably, it was strongly recognized by sera of HCV-infected patients and, as compared with R9-CMV, displayed an enhanced ability to stimulate lymphocyte IFN-gamma production. The high immunogen levels achievable in plants or fruits infected with 2R9-CMV suggest that this chimeric form of CMV may be useful in the development of oral vaccines against HCV.

Clinical outcomes of bosentan in pulmonary arterial hypertension do not correlate with levels of TIMPs.

BACKGROUND: Matrix metalloproteinases (MMP) and their inhibitors, tissue inhibitors of metalloproteinases (TIMP), are involved in tissue inflammation and fibrotic processes. Treatment with bosentan has been shown to improve the clinical outcome of patients with pulmonary arterial hypertension (PAH) with and without association with systemic sclerosis (SSc), and also to modulate the serum levels of matrix metalloproteases-9. We measured TIMP-1 and TIMP-2 in the serum of patients with SSc with and without PAH treated with long-term bosentan compared with healthy donors (HD). MATERIALS AND METHODS: Serum samples from HD (n = 16) and patients with SSc (n = 35), including patients with SSc without PAH (n = 23) and patients with PAH (n = 12), were analyzed using enzyme-linked immunosorbent assays (ELISAs) for total TIMP-1 and TIMP-2. RESULTS: Both mean TIMP-1 and TIMP-2 levels were significantly increased in patients with SSc compared with HD, but no differences were observed between patients with SSc with and without PAH. In the eight bosentan-treated patients, TIMP-1 and TIMP-2 levels did not change during 1 year of treatment, while bosentan increased the 6-min walking distance by 136 meters after 1 year, as well as clinical outcomes. CONCLUSIONS: Increased levels of TIMP-1 and TIMP-2 in patients with SSc compared with HD suggest that the inhibition of proteolysis allows the accumulation of ECM proteins. As bosentan does not stimulate TIMPs, it appears to favour proteolytic imbalance and to increase the turnover of ECM proteins.

New frontiers in biomarkers for hepatocellular carcinoma.

Hepatocellular carcinoma represents the fifth most frequent cancer in the world; it commonly occurs on cirrhotic liver. Prognosis and survival are still poor, mainly because of diagnosis at a late stage and/or recurrence of the disease. For this reason, surveillance strategies are widely used to screen for early occurrence of cancer in populations at risk. Alpha-foetoprotein is so far the only serological marker available, but its diagnostic accuracy is unsatisfactory because of low sensitivity despite reliable specificity. For this reason, new biomarkers for the diagnosis of hepatocellular carcinoma are in strong demand by clinicians. In this review, we will focus on new biomarkers currently under investigation, but also on still newer, very promising biomarkers that seem to significantly improve diagnosis.

Laminin-5 with transforming growth factor-beta1 induces epithelial to mesenchymal transition in hepatocellular carcinoma.

BACKGROUND & AIMS: How hepatocellular carcinoma (HCC) cells acquire the ability to invade surrounding tissue is unknown, but epithelial mesenchymal transition (EMT) likely plays a role. We investigate how transforming growth factor (TGF)-beta1 and extracellular matrix protein Laminin-5 (Ln-5) induce EMT and cancer invasion. METHODS: Snail, Slug, E-cadherin, beta-catenin and Ln-5 were investigated on HCC tissues and on HCC cell lines. RESULTS: We show that in HCC but not in peritumoral tissue of the same HCC patients, Ln-5, Snail, and Slug are up-regulated, E-cadherin is down-regulated and beta-catenin is translocated into the nuclei. In vitro, HCC "invasive" cells, partially EMT-transformed, show low levels of E-cadherin. In presence of Ln-5, Snail, and Slug are up-regulated, E-cadherin is down-regulated, beta-catenin is translocated into the nuclei, and cells undergo a dramatic morphological change, becoming scattered and undergoing a complete EMT. This effect is reversed by anti-alpha3 but not by anti-alpha6 integrin blocking antibody. HCC "noninvasive" cells are not EMT-transformed, and have constitutively high levels of E-cadherin. In presence of Ln-5, cells undergo partial EMT, Snail, and Slug are up-regulated, E-cadherin is down-regulated but cells do not scatter. However, the presence of both Ln-5 and TGF-beta1 completes the EMT process, beta-catenin is translocated into the nuclei, cells scatter and become invasive, recalling the "invasive" cells. In this case, too, the effect is reversed by anti-alpha3 integrin blocking antibody. CONCLUSIONS: Our study shows that Ln-5 and TGF-beta1 cooperatively induce EMT in HCC, suggesting the microenvironment as a potential target for new biological therapies.

Immunological and molecular aspects of liver fibrosis in chronic hepatitis C virus infection.

Chronic C hepatitis represents a major health problem worldwide, mainly because progression of the tissue damage leads to the development of cirrhosis and hepatocellular carcinoma. In this review we discuss the molecular mechanisms underlying the development of liver fibrosis. In particular we consider some immunologic aspects that regulate the interaction between HCV and the host immune defense. Reflections are made about the roles played by the host capacity to respond to the viral infection during therapy and the consequences of the deposition of extracellular matrix (ECM) proteins leading to the development of fibrosis. The involvement of inflammatory cytokines in regulating the proteolytic remodeling of the liver and the ECM turn-over is essential for the activation of hepatic stellate cells (HSCs), that have an important role in the progression of liver fibrosis. Finally, we analyze one of the aspects involved in the activation of the HSCs, namely the proteolytic remodeling of the surrounding environment.

Immunogenic properties of a chimeric plant virus expressing a hepatitis C virus (HCV)-derived epitope: new prospects for an HCV vaccine.

A vaccine against Hepatitis C virus (HCV) is urgently needed due to the unsatisfactory clinical response to current therapies. We evaluated the immunological properties of a chimeric Cucumber mosaic virus (CMV), a plant virus engineered to express on its surface a synthetic peptide derived from many HVR1 sequences of the HCV envelope protein E2 (R9 mimotope). Evidence was obtained that the chimeric R9-CMV elicits a specific humoral response in rabbits. Furthermore, in patients with chronic HCV infection, purified preparations of R9-CMV down-modulated the lymphocyte surface density of CD3 and CD8, and induced a significant release of interferon (IFN)-gamma, interleukin (IL)-12 p70 and IL-15 by lymphomonocyte cultures. Finally, an R9 mimotope-specific CD8 T-cell response, as assessed by intracellular IFN-gamma production, was achieved in the majority of the patients studied. Our results open up new prospects for the development of effective vaccines against HCV infection. Moreover, the wide edible host range of CMV makes the production of an edible vaccine conceivable.

Infliximab therapy does not modify MMP-2 and MMP-9 serum concentrations in chronic arthritis.

OBJECTIVE: Matrix metalloprotease-2 (MMP-2) and matrix metalloprotease-9 (MMP-9) play a key role in tissue remodelling after processes such as joint destruction in rheumatoid arthritis. Their expression may reflect the disease activity and they could therefore represent a useful marker to assess the efficacy of therapy. In this study MMP-2 and MMP-9 serum were evaluated in patients with chronic arthritis during therapy with the anti-TNFalpha mAb, infliximab. METHODS: Fifty patients with chronic arthritis, 26 with rheumatoid arthritis and 24 with undifferentiated chronic arthritis, were recruited and treated with infliximab (3 mg/kg). Serum concentrations of MMP-2 and MMP-9 were serially measured by gelatine zymography at baseline and after two and fourteen weeks of infliximab therapy. DAS-28 and ACR response criteria were applied to assess disease activity and clinical improvement. Twenty-four healthy donors were included in the study as controls. RESULTS: Although therapy with infliximab induced a statistically significant reduction of the DAS-28 score and improvement of the ACR clinical response, MMP-2 and MMP-9 serum concentrations were not modulated during therapy with infliximab. CONCLUSIONS: Our study provides further evidence that blocking TNFalpha by infliximab is a powerful tool in the management of chronic arthritis. Nevertheless, infliximab does not seem to be able to modify the serum expression of MMP-2 and MMP-9, probably because modification of these enzymes is restricted to the site of joint inflammation and serum detection can not truly mirror the local situation. Additional soluble factors correlating with joint damage should be investigated as possible markers for monitoring anti-TNFalpha therapy.

Matrix metalloproteinase imbalance in muscle disuse atrophy.

Muscle atrophy commonly occurs as a consequence of prolonged muscle inactivity, as observed after cast immobilization, bed rest or space flights. The molecular mechanisms responsible for muscle atrophy are still unknown, but a role has been proposed for altered permeability of the sarcolemma and of the surrounding connective tissue. Matrix metallo-proteinases (MMPs) are a family of enzymes with proteolytic activity toward a number of extracellular matrix (ECM) components; they are inhibited by tissue inhibitors of MMPs (TIMPs). In a rat tail-suspension experimental model, we show that after fourteen days of non-weight bearing there is increased expression of MMP-2 in the atrophic soleus and gastrocnemius and decreased expression of TIMP-2. In the same experimental model the expression of Collagen I and Collagen IV, two main ECM components present in the muscles, was reduced and unevenly distributed in unloaded animals. The difference was more evident in the soleus than in the gastrocnemius muscle. This suggests that muscle disuse induces a proteolytic imbalance, which could be responsible for the breakdown of basal lamina structures such as Collagen I and Collagen IV, and that this leads to an altered permeability with consequent atrophy. In conclusion, an MMP-2/TIMP-2 imbalance could have a role in the mechanism underlying muscle disuse atrophy; more studies are needed to expand our molecular knowledge on this issue and to explore the possibility of targeting the proteolytic imbalance with MMP inhibitors.

Laminin-5 offsets the efficacy of gefitinib ('Iressa') in hepatocellular carcinoma cells.

Prognosis and survival of patients with hepatocellular carcinoma (HCC) is still very poor, and no therapies are currently available to inhibit tumour growth and metastases. Recently, we reported that the expression of an extracellular matrix component (ECM), namely Laminin-5 (Ln-5), is directly related to poor prognosis in HCC patients. The aim of our study is to investigate the preclinical effect of gefitinib in an in vitro HCC model. We found that the IC(50) of gefitinib in HCC cells ranged from 0.7 to 10.0 muM, whereas Ln-5 inhibited the activity of gefitinib in a dose-dependent manner. Complete inhibition of phosphorylated (p)-EGFR (epidermal growth factor receptor) was obtained within 6 h exposure to gefitinib and complete restoration of the receptor status was obtained after 24 h. A downstream effect yields a decrease in p-Akt and p-Erk 1/2. The addition of exogenous Ln-5 has no effect on p-EGFR, whereas it restores p-Erk 1/2 and p-Akt. Consistently, Ln-5 induces recovery of HCC cells from Gefitinib-induced apoptosis. In conclusion, gefitinib inhibits HCC cell growth and we report for the first time that Ln-5, but not other ECM molecules, reduces the ability of gefitinib to inhibit cell growth via Akt. As patients with HCC have different Ln-5 expression levels, these results may help to better understand which patients might benefit from gefitinib treatment.